Pre Formulation

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PHYSICO-CHEMICAL FACTORS
SEMINAR ON PHYSICOCHEMICAL FACTORS UNDER PREFORMULATION STUDY
GUIDED BY: DR.R.K.PARIKH
PRESENTED BY: NITU-K- CHANGOIWALA M.PHARM - I ROLL NO - 04 YEAR-2009-10
DEPT. OF PHARMACEUTICS & PCEUTICAL TECHNOLOGY L.M. COLLEGE OF PHARMACY, ABAD-09.
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CONTENTS:I. PHYSICAL CHARACTERISTICS

A. BULK CHARACTERISTIC
1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) Particle Size & Surface Area. Polymorphism. Crystallinity. Hygroscopicity. Flow properties & Bulk density. Compressibility. Drug-Excipient Compactibility. Electrostatic charge. Osmolarity. Rheology. Wettability.
B.
1)
SOLUBILITY ANALYSIS
Aqueous Solubility. a) Intrinsic Solubility. b) Dissociation Constant. Solubilization. Partition Coefficient. Thermal effect. Common ion effect. Dissolution.
2) 3) 4) 5) 6)
C. STABILITY ANALYSIS
1) 2) Solid State Stability. Solution State Stability.
II.
CHEMICAL CHARACTERISTICS
1) 2) 3) 4) 5) 6) 7) 8) Oxidation. Hydrolysis. Photolysis. Racemization. Polymerization. Isomerization. Decarboxylation. Enzyme Decomposition.
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What Is PREFORMULATION?
It is defined as phase of research and development in which preformulation scientist characterize physical & chemical properties of new drug molecule in order to develop safe, effective, and stable dosage form.
DIRECT BENEFITS:
Gives direction for development of formulation in choice of dosage form,excipients,composition,physical structure. Helps in adjustment of Pharmacokinetics and biopharmaceutical properties. Support for process development of drug substance (yield,filtration..). Produce necessary and useful data for development of analytical methods.
According to USFDA it can be characterized as: Melting point (hot stage microscopy). IR spectroscopy. XRD. Thermal analytical technique. Solid state Raman spectroscopy. Crystalline index of refraction. Phase solution analysis. Solution calorimetery. Comparative intrinsic dissolution rate.
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FLOW CHART FOR PREFORMULATION STUDY:
Receive drug substance

Obtain all available information If not available, do the literature search.
Determine physical property of the API. Macroscopic and Microscopic examination Determine polymorphs, solvates and hydrates.
If poor bioavailability test results due to solubility, pKa, P, etc. make new salt or ester If satisfactory Select most stable, active form for bioavailability testing. Check lot to lot uniformity Check API stability with excipients Prepare worksheet and final preformulation report and issue to product development dept.
Determine their solubility, partition coefficient, pKa, dissolution rate.
Stability testing at normal and exaggerated condition.
[B] SOLUBILITY ANALYSIS

AQUEOUS SOLUBILITY
A drug must possess aqueous solubility for therapeutic efficacy in physiological pH range of 1 to 8 at 37 C. Poor solubility (<10mg/ml) may result into bioabsorption problems. If solubility of drug is less than 1 mg/ml it indicates the need for a salt, particularly if the drug will be formulated as a tablet or capsule. In the range 1-10 mg/ml serious consideration should be given to salt formation. There are 2 fundamental properties mandatory for a new compd. [a] Intrinsic Solubility (Co). [b] Ionization Constant (pKa).
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[a] INTRINSIC SOLUBILITY(Co):The solubility of weakly acidic & weakly basic drug as a function of pH can be predicted with the help of eqn. S = So {1 + (K1 / [H+])} -------------- for weak acids. S = So {1 + ([H+] / K2)} -------------- for weak bases. where, S = Solubility at given pH. So = Intrinsic solubility of the neutral form. K1 = Dissociation constant of weak acid. K2 = Dissociation constant of weak base. The intrinsic solubility should ideally be measured at 2 temperatures: a) 4 C To ensure physical & chemical stability. b) 37 C To support biopharmaceutical evaluation.
Method to determine solubility

(1) Equilibrium solubility method (2) Turbidometric solubility method (3) Nephlometric solubility method (4) Ultrafiltration LC/MS solubility method (5) Direct solubility method (6) NRTL SAC method [ JPS VOL-97 NO-5 May-2008] (7) COSMO SAC method [Chemical Abstracts;July-2008 147(05)-568563h]
Solubility parameter is used to design dry suspension of cefaclor as a dual pack system. (IJPS):
BASED ON SOLUBILITY PARAMETER ONE CAN DECIDE WHETHER PARTICULAR SOLUTE WILL SOLUBILIZE IN A GIVEN SOLVENT OR NOT. SOL. PARAMETER OF CEFACLOR VARIES GREATLY WITH WATER & CEFACLOR IS HIGHLY LIPOPHILIC SO IT IS INSOL. IN WATER WHEN WATER WAS MIXED WITH CO-SOLVENT PEG IN THE RATIO 80:20 THE SOL. PARAMETER OF THE MIXTURE WAS FOUND SIMILAR TO THAT OF CEFACLOR & THEREFORE IT GETS EASILY SOLUBILIZED IN IT DUAL PACK SYSTEM IS PREFERRED BECAUSE CEFACLOR BEING A CEPHALOSPORIN CLASS ANTIBIOTIC IS HIGHLY UNSTABLE IN WATER SO
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IONIZATION CONSTANT (pKa):75 % of all drugs are weak bases, 25 % are weak acids and only, 5 % are nonionic amphoteric or alcohol.
The unionized forms are more lipid soluble & more rapidly absorbed from g.i.t. The relative conc. of unionized & ionized form of weakly acidic or basic drug in a solution at a given pH can be calculated using the Henderson-Hasselbalch equation:pH = pKa + log [unionized form] / [ionized form] ---- for weak bases. pH = pKa + log [ionized form] / [unionized form] ---- for weak acids.
Uses of these equations:1) 2) 3) 4)
To determine pka. To predict solubility at any pH provided that Co & pKa are known. To facilitate the selection of suitable salt forming compounds. It predicts the solubility & pH properties of the salts.
Limitation:To fail outside the pH limits of 4-10 or when the solution is very dilute.
Method to determine pka:1) 2) 3) 4) 5) Potentiometric method. Conductivity method. Dissolution rate method. Liquid-Liquid partition method. Spectrophotometric method.
SOLUBILIZATION
Many different approaches have been developed to improve drug solubility: 1) Micronization:Eg. Griseofulvin shows increased solubility by reducing particle size. 2) Change in pH:-
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Eg. Solubility of Nimesulide increases as pH is increased. [Chemical Abstracts, 133(6); August 2000: 79182g] Eg.Arginine increases solubility of coumarins. [Chemical Abstracts, April 2009; 150 : 290306j] Eg. Etoposide formulation is difficult because of its poor solubility & labile chemical stability so its most stable formulation is Etoposide loaded emulsion (ELE) at pH 4-5. [JPS July 2007; 96(7): 1791] 3) Cosolvency:Addition of a water miscible solvent can often improve the solubility of a weak electrolyte or nonpolar compound in water by altering the polarity of the solvent. The choice of suitable cosolvent is limited for Pceutical use because of possible toxicity & irritancy. Ideally suitable blends should possess values of dielectric constant between 25-80. Commonly used cosolvents are ethanol, sorbitol, glycerin, propylene glycol, dimethylacetamide (DMA), DMSO, etc. 4) Solubilization by surfactant:Eg. Gelucire 44/14 is a surface active excipient that can solubilize poorly soluble drugs. [JPS June 2004; 93(6): 1471] Eg. Anionic & cationic surfactants exhibited dramatically higher solubilization for gliclazide, while nonionic surfactants showed significantly lower solubilizing ability. [JPS April 2003; 92(6): 839] 5) Complexation:Eg. The Complexation of iodine with 10-15% polyvinylpyrolidone (PVP) can improve aqueous solubility of active agent. 6) Formation of Inclusion Compound:Eg. The aqueous solubility & chemical stability of Quercetin can be improved via Complexation with -cyclodextrin. Eg. The enhancement of solubilization increased 300 fold for Nimodipine at a polymer conc. 10% by use of water soluble dendrimer based on polyglycerol. (Chemical Abstracts, July 2007; 147(5): 101548u) Eg. Enhanced solubilty of oxicams through inclusion of - cyclodextrin and its dvts.
(CA-VOL151 Sep2009:107806f)
7) Chemical Modification:Many poorly soluble drugs modified into salt form (water soluble). 8) Use of Metastable polymorphs:Eg. B form of Chloramphenicol palmitate is more water soluble than A & C forms.
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PARTITION COEFFICIENT: The gastrointestinal membranes are largely lipoidal in character hence the lipid solubility of a drug is an imp. factor in the assessment for its absorption potential. When a solute is added to two immiscible liquids it will distribute itself between the two phases in a fixed ratio, which is referred to as partition or distribution coefficient. It is independent of concentration of dilute solution of given solute species. Various organic solvents used in determination of partition coefficient include Chloroform, ether, amyl acetate, etc. Solubility parameter of n-octanol (=10.24) lies midway in the range for major drugs (=8-12). Thus in formulation development the n-octanol-water partition coefficient is commonly used. P= (Conc. of drug in octanol) / (Conc. of drug in water) --- For unionizable drugs. P= (Conc. of drug in octanol) / (1-)*(Conc. of drug in water) --- For ionizable drugs. where = degree of ionization.
P > 1 Lipophilic drug. P < 1 Hydrophilic drug.

The value of P at which maximum activity of controlled release dosage forms is observed is approximately 1000:1 in octanol/water.
Methods to determine P:a) Shake Flask Method. b) Chromatographic Method (TLC, HPLC). c) Counter Current & Filter Probe method.
Applications of P:Measure of Lipophilic character of molecules. Recovery of antibiotics from fermentation broth. Extraction of drug from biological fluid for therapeutic monitoring. Absorption of drug from dosage forms. (Ointments, Suppositories, Transdermal patches). Study of distribution of flavoring oil between oil & water in emulsion.
THERMAL EFFECT: Effect of temperature on the solubility of drug can be determined by measuring heat of solution. (Hs). ln S = -Hs/R*T + C.
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where, S = Molar solubility at temperature T (K). R = Gas constant. Heat of solution represents the heat released or absorbed when a mole of solute is dissolved in a large quantity of solvent. Mostly solution process is endothermic (Hs = +ve) & thus increasing the solution temperature increase the drug solubility. Typical temp. range should include 5C, 25C, 37C & 50C. Importance: Determination of temperature effect on solubility helps in predicting storage condition & dosage form designing.
COMMON ION EFFECT:Addition of common ion reduces the solubility of slightly soluble electrolyte. The salting out results from the removal of water molecules as solvent due to the competing hydration of other ions. So weakly basic drug which are given as HCl salts have decreased solubility in acidic solution. Eg. Chlortetracycline, Papaverine, Bromhexine, Triamterene, etc. The reverse process salting in arises with larger anions. (Eg. Benzoate, salicylate) which can open the water structure. These hydrotropes increase the solubility of poorly water soluble compounds. To identify a common ion interaction the IDR (Intrinsic dissolution rate) of HCl salt should be compared between a) Water & water containing 1.2% W/V NaCl. b) 0.05 M HCl & 0.9% NaCl in 0.05 M HCl. Both saline media contains 0.2 M Cl which is typically encountered in fluids in vivo.
DISSOLUTION
The absorption of solid drugs administered orally can be understood by following flowchart.
Dissolution Solid drugs in GI fluid Solution of drug in GI fluid
Absorption Drugs in systemic circulation
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In many instances, dissolution rate in the fluids at the absorption site is the rate limiting step in the absorption process. Dissolution rate can affect - Onset of action. - Intensity of action. - Duration of response. - Control the overall Bioavailability of drug form. Dissolution is to be considered of 2 types:
[1] Intrinsic dissolution The dissolution rate of solid in its own solution is adequately described by NoyesWhitney equation:
dC/dt = AD (Cs-C) / hv
where, dc/dt = Dissolution rate. A = Surface area of dissolving solid. D = Diffusion coefficient. C = Concentration of drug in solution. h = Thickness of diffusion layer (at the solidliquid interface). v = Volume of dissolution medium. Cs = Solute concentration in the diffusion layer. This equation helps to the preformulation scientist in predicting if absorption would be dissolution rate limited or not.
Method to determine intrinsic dissolution:Rotating disk method or Woods apparatus: This method allows for the determination of dissolution from constant surface area, obtained by compressing powder into a disc of known area with a die-punch apparatus.
[2] Particulate dissolution

This method determines the dissolution of solids at different surface area. A weighed amount of powder sample from a particular sieve fraction is introduced in the dissolution medium. Agitation is usually provided by a constant speed propeller. It is used to study the influence on dissolution of particle size, surface area & mixing with excipients.
STABILITY ANALYSIS
Development of a drug substance into a suitable dosage form requires the Preformulation stability studies of drug under the following categories:[1] Solid state stability. [2] Solution state stability. PAPER-910101 L.M. COLLEGE OF PHARMACY 10
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[1] Solid state stability

Solid state reactions are much slower & more difficult to interpret than solution state reactions because of reduced no. of molecular contacts between drug & excipient molecules & occurrence of multiple reactions.
Techniques for solid state stability studies:[JPS April 2007;

96(4):777] Solid State NMR Spectroscopy. (SSNMR) Powder X-ray diffraction. (PXRD) Fourier Transform IR. (FTIR) Raman Spectroscopy. Differential Scanning Calorimetry. (DSC). Thermo gravimetric Analysis. (TGA). Dynamic Vapor Sorption. (DSV).
[2] Solution State Stability

The primary objective is identification of conditions necessary to form a solution. These studies include the effects of - pH. - Temperature. - Light. - Oxygen. Cosolvent. - Ionic Strength. Aq. Solution for injection pH 3 containing Irinotecan HCl, phosphate buffer & WFI was stably prepared by dissolving camptothecins without resorting to heating in the course of production. [Chemical Abstracts, Feb. 2007; 146(9): 169430j] Chitosan hygrogel can change reversibly well at different pH & ionic strength of solution. [Chemical Abstracts, Sep. 2007; 147(10): 219725c]
Solution Stability investigations usually commence with probing experiments to confirm decay at the extremes of pH & temperature. If the results of this solution stability studies dictate the compound as sufficiently stable, liquid formulation can be developed.
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Stress conditions used in Preformulation stability assessment:Test SOLID Heat (C) Moisture Uptake Physical Stress
AQUEOUS SOLUTION
Condition
4, 20, 30, 40, 40/75 % RH, 50 & 75. 30,40,60,75 & 90 % RH at RT. Ball milling.
pH Light Oxidation
1,3,7,9 & 11 at RT & 37C. Reflux in 1M HCl & 1M NaOH. UV (254 &366 nm) & Visible (south facing window) at RT. Sparing with oxygen at RT, UV may accelerate breakdown.
CHEMICAL CHARACTERISTICS
I. OXIDATION
It is a very common pathway for drug degradation in liquid & solid formulation. Oxidation occurs in two ways:1. Auto oxidation. 2. Free radical chain process.
Auto oxidation: It is defined as a reaction of any material with molecular oxygen which produces free radicals by hemolytic bond fission of a covalent bond. These radicals are highly unsaturated & readily take electron from other substance causing oxidation. For auto oxidation to occur in solid molecular oxygen must be able to diffuse through the crystal lattice to liable sites. Hence crystal morphology & packing are important parameters for determining oxidation kinetics.
Free radical chain process:a) INITIATION
Activation RH Light, Heat R + H
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R + O2 RO2 + RH
RO2 RCOOH + R
c) HYDROPEROXIDE DECOMPOSITION
RCOOH
d) TERMINATION
RO + OH
RO2 + X RO2 + RO2
Inactive product Inactive product
Functional groups having high susceptibility towards oxidation:- Alkenes. - Substituted aromatic groups. (Toluene, phenols, anisole). - Ethers. - Thioethers. - Amines.
Factors affecting oxidation process:1. Oxygen concentration. 2. Light. 3. Heavy metals particularly those having two or more valence state. (Eg. Copper, iron, nickel, cobalt). 4. Hydrogen & Hydroxyl ion. 5. Temperature.
Prevention of oxidation:1) Reducing oxygen content. Oxidative degradation of drug takes place in an aqueous solution, so the oxygen content can be decreased by boiling water. 2) Storage in a dark & cool condition. 3) Addition of an antioxidant. PAPER-910101 L.M. COLLEGE OF PHARMACY 13
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a) Reducing agent. b) Chain inhibitors of radical induced decomposition.
ANTIOXIDANT

Oil Soluble
Water Soluble
Free radical acceptor Oxidized itself & prevent & inhibit free radical oxidation of drug. chain process. EXAMPLES Hydroquinone Sodium metabisulphate Propyl gallate Sodium bisulphite Butylated Hydroxy Acetyl cysteine, Anisole (BHA) Ascorbic acid Butylated Hydroxy Sodium thiosulfate, Toluene (BHT) Sulphur dioxide Lecithin Thioglycolic acid - Tocopherol Thioglycerol
4) Addition of chelating agent. It forms complexes with trace amount of heavy metal ion & inactivate their catalyzing activity. Eg. EDTA, Citric acid, Tartaric acid. 5) Adjustment of pH. To optimum stability in order to reduce oxidation potential of the system. 6) Changing solvent. Solvent other than water may have catalyzing effect on oxidation reaction when used in combination with water or alone. Eg. Aldehydes, ethers, ketones may influence free radical reaction.
II. HYDROLYSIS
It involves nucleophilic attack of labile groups. Eg. Lactam > Ester > Amide > Imide. When this attack is by a solvent other than water then it is known as solvolysis. It generally follows 2nd order kinetics as there are 2 reacting species, water and API. In aqueous solution, water is in excess, the reaction is 1st order.
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Conditions that catalyze the breakdown:(1) (2) (3) (4) (5) (6) (7) (8) Presence of hydroxyl ion. Presence of hydride ion. Presence of divalent ion. Heat. Light. Ionic hydrolysis. Solution polarity & ionic strength. High drug concentration.
Prevention of hydrolysis:1) pH adjustment. Most of the potent drugs are weakly acidic or weakly basic, which are more soluble when ionized so their instability will increase. Remedy:- Formulate the drug solution close to its pH of optimum stability. - Addition of water miscible solvent in formulation. - Optimum buffer concentration to suppress ionization. 2) Addition of surfactant. Nonionic, cationic & anionic surfactant stabilizes the drug against base catalysis. 3) Salts & esters. The solubility of pceuticals undergoing ester hydrolysis can be reduced by forming less soluble salts or ester of drug. Eg. Phosphate ester of Clindamycin. 4) Store with dessicants. 5) By use of complexing agent.
III. PHOTOLYSIS
Mechanism of photodecomposition:Electronic configuration of drug overlaps with spectrum of sunlight or any artificial light, & thereby energy is absorbed by electron & it goes to the excited state. They are unstable & release the acquired energy & come to the ground state & decompose the drug.
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Photosensitization means molecule or excipient which absorbs energy but do not participate themselves directly in the reaction but pass the energy to other that will cause cellular damage by inducing radical formation.
Photosensitizer

Energy transfer
Electron transfer
Convert oxygen from its ground state to singlet excited state.
Generate superoxide molecule, which is an anion radical & acts as a powerful oxidizing agent.
Photodecomposition pathway
(1) N-Dealkylation.
Eg. Diphenhydramine, Chloroquine, Methotrexate.

(2) Dehalogenation.
Eg. Chlorpropamide, Furosemide.

(3) Dehydrogenation of Ca
channel blocker. Eg. Solution of Nifedipine Nitrosophenylpyridine (with loss of water). Rapidly yellow color Brown.
++
(4) Decarboxylation in anti-inflammatory agents.
Eg. Naproxen, Flurbiprofen, Benzoxaprofen.

(5) Oxidation.
Eg. Chlorpromazine & other phenothiazines give N- & S- oxides in the presence of sunlight.
(6) Isomerization & cyclization.
Eg. Noradrenaline, Doxepine.

(7) Rearrangement.
Eg. Metronidazole Oxidiazine Yellow color.
Examples: Aq. solution of Lincomycin was irradiated with UV light in homogenous & heterogenous systems. Lincomycin disappeared in both systems but the presence of TiO2 noticeably accelerated the degradation of antibiotic in comparison with direct
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pyrolysis. The degradation pathways involved S- & N- demethylation & propyl dealkylation. [Chemical Abstracts, April 2007; 146(18): 365263w] The photodegradation behaviour of bisphenol C studied in monochromatic UV irradiation (= 254 nm) indicated that phtotodegradation reaction rate constant of bisphenol C in aq. soln. with - cyclodextrin is higher than that wihtout cyclodextrin, mainly due to lower bond energy between some atoms in bispheol C molecule after inclusion interaction with - cyclodextrin. [Chemical Abstracts, Aug. 2007; 147(7): 149559a]
Prevention of Photodecomposition:1) Suitable packing.
Eg. Yellow-green glass gives the best protection in U.V. region while Amber confers considerable protection against U.V. radiation but little from I.R.
2) Anti-oxidant.
Eg. Photodegradation of Sulphacetamide solution may be inhibited by an antioxidant such as Sodium thiosulfate or sodium metabisulphate.
3) Protection of drug from light.
[Eg. Nifedipine is manufactured under Na light].

4) Avoiding sunbath. [Eg. Sparfloxacin]. 5) Photostabilizer (light absorber).
Colorant - Curcumine, Azorubine. Pigments - Iron oxide, Titanium dioxide.
6) Coating.
Pigments like TiO2 / ZnO. Eg. Photostabilization of Sulphasomidine Tab. by film coating containing U.V. absorber (Oxybenzone) to protect color & photolytic degradation. [JPS Feb. 1978; 67(2): 196]
IV. RACEMIZATION
The interconversion from one isomer to another can lead to different Pcokinetic properties (ADME) as well as different Pcological & toxicological effect. Eg. L-epinephrine is 15 to 20 times more active than D-form, while activity of racemic mixture is just one half of the L-form. [JPS April 2004; 93(4): 969] It follows first order kinetics. It depends on temperature, solvent, catalyst & presence or absence of light.
V. POLYMERIZATION
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It is a continuous reaction between molecules. More than one monomer reacts to form a polymer. Eg. Darkening of glucose solution is attributed to polymerization of breakdown product [5- (hydroxyl methyl) furfural]. Eg. Polymerization of HCHO to para-HCHO which crystallizes out from the solution.
VI. ISOMERIZATION
It is the process involving change of one structure to another having same empirical formula but different properties in one or more respects. Its occurrence is rare. Examples:-. (1) Tetracycline & its dvts. can undergo reversible Isomerization at pH range 2-6. (2) Trans-cis Isomerization of Amphotericin B. (3) Isomerization of tetrahydrouridine. [JPS October 2003; 92(10): 2027]
VII. DECARBOXYLATION
Evolution of CO2 gas from COOH group containing drugs. Eg. Solid PAS undergoes pyrolytic degradation to m-aminophenol & CO2. The reaction which follows 1st order kinetics is highly pH dependent & is catalysed by hydronium ions.
VIII. ENZYME DECOMPOSITION

Chemical degradation due to enzymes induced by drug results into decomposition. Remedy: - Use of buccal tab. - Use of pro-drug. (L-dopa).
o ACCORDING TO WHO:
Hydrolysis and Oxidation are the most common pathways for API degradation in the solid-state and in solution. Photolysis and trace metal catalysis are secondary processes of degradation. the rate of
Temperature affects each of the above chemical degradation pathways; degradation increases with temperature.
It is well understood that pH, particularly extremes, can encourage hydrolysis of API when ionised in aqueous solution. This necessitates buffer control if such a dosage form is required.
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SUMMARY: The preformulation evaluation of new drug substances has become

an integral part of the development process. Preformulation studies, properly carried out, have a significant part to play in anticipating formulation problems and identifying logical paths in both solid and liquid dosage form. A thorough understanding of the physicochemical properties of drug substances provides the development pharmacist with the data that are essential in designing stable & efficacious dosage forms. Comparing physico chemical property with each drug candidate within a therapeutic group, the preformulation scientist can assist the synthetic chemist to identify optimum molecule, pharmacologist to suit the vehicle for electing desired pcological response and the bulk pharmacist to select and produce best salt with proper pcle size and morphology for subsequent processing.
STUDY QUESTIONS
[1] Enlist physical, chemical & pharmaceutical factors affecting preformulation? Discuss chemical decomposition factors with special reference to photodecomposition and methods to retard it? [April 2006] [2] Enlist different chemical degradation pathways in preformulation studies? Explain the various factors influencing degradation pathway? How are the drug subs stabilized against chemical degradation? [March 2005] [3] How is the photo degradation study carried out as per current guidance documents? [Sept.2004] [4] Describe various factors to be considered in preformulation studies? [Sept.2005] [5] Explain any two of the following characteristics that are to be considered before dosage form design? Oxidation Photo degradation Hydrolysis [Jan.2003] Different means of arresting hydrolysis of Active Pceutical Materials? [May 2003] COMMENT: [6] IS PREFORMULTION STUDIES APPLICABLE TO NEW DRUGS ONLY? [7] How do we control humidity for lab scale purpose? [8] Occurrence of Degradation pathways according to WHO? (10)Techniques for the physico-chemical determination acc. to US-FDA?
REFERENCES:
1. Pharmaceutics- The science of Dosage Form Design by M. E. Aulton. (2nd edition): pg.113 2. The Science & Practice of Pharmacy by Remington.(19th edition): pg.1447
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3. The Theory & Practice of Industrial Pharmacy by Leon Lachman, Herbet A. Lieberman, Joseph L. Kaing.(3rd edition): pg.171 4. Modern Pharmaceutics by G. S. Banker & C. T. Rhodes. (4th edition): pg.211 5. Pharmaceutical Dosage Forms by Leon Lachman, H. A. Lieberman; Vol.1: pg.1 6. Pharmaceutical Dosage Forms & Delivery Systems by H.C. Ansel, L.V.Allen, N.G.Popvich; (7th edition): pg.64 7. The Pharmaceutical Codex by Walter Lund (12th edition) pg. 178 8. Encyclopedia of Pharmaceutical Technology; Volume 12: pg.421 9. Encyclopedia of Pharmaceutical Technology; Volume 18: pg.161 10. Chemical Abstracts, August 2000; 133(6): 79182g. 11. Chemical Abstracts, Feb. 2007; 146(9): 169430j. 12. Chemical Abstracts, April 2007; 146(18): 362563w. 13. Chemical Abstracts, July 2007; 147(5): 101548u. 14. Chemical Abstracts, August 2007; 147(7): 149559a. 15. Chemical Abstracts, Sep. 2007; 147(10): 219725c. 16. Journal of Pharmaceutical Sciences Feb. 1978; 67(2): 196. 17. Journal of Pharmaceutical Sciences October 2003; 92(10): 2027. 18. Journal of Pharmaceutical Sciences April 2003; 92(4): 839. 19. Journal of Pharmaceutical Sciences April 2004; 93(4): 969. 20. Journal of Pharmaceutical Sciences June 2004; 93(6): 1471. 21. Journal of Pharmaceutical Sciences April 2007; 96(4): 777. 22. Journal of Pharmaceutical Sciences July 2007; 96(7): 1719 23. Pceutical preformulation &formulation, A practical guide by Mark gibson 24. Chemical Abstracts, April 2009; 150 : 290306j 25. Chemical Abstracts, Vol 145, 314991m 26. Chemical Absracts Vol 151 Sep2009:107806f 27. Indian Journal Of Pharmaceutical Sciences
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SEMINAR ON Hygroscopicity , Powder Rheology & Compaction Properties

AS PART OF PREFORMULATION STUDY
GUIDED BY : Dr. R. K. PARIKH
PRESENTED BY:SANJAY C. MODI M-PHARM- I YEAR-2009-10 ROLL NO. -7
DEPT. OF PHARMACEUTICS AND TECHNOLOGY L.M.COLLEGE OF PHARMACY AHMEDABAD-09.
(1) HYGROSCOPICITY:CONTENTS :A. INTRODUCTION B. METHOD OF DETERMINATION C. IMPORTANCE OF MEASUREMENT D.METHOD OF IMPROVEMENT (A) INTRODUCTION :
Hygroscopicity: - It is the tendency of material to absorb moisture from atmosphere & get dynamic equilibrium with water in the atmosphere. Deliquescent: - It is the hygroscopic substance which absorb moisture from air and they can be liquefied by partially or wholly forming solution. Efflorescent: - a substance which loses water to form a lower hydrate or become anhydrous is termed as efflorescent.
(B) METHOD OF DETERMINATION :

To carry out study, sample of compound are accurately weighed
into container and placed at various humid condition for period of upto 2 weeks. o If Weight gain Deliquescent or Hygroscopic o If Weight loss Efflorescent
Also determined by TGA, GC, & KF titration Versaperm has deviced a WVTR meter that can measure the
permeability of package to moisture.so that humidity can be accurately controlled.
CLASSIFICATION OF SUBSTANCES :Essentially CLASS Below %RH 90 80 60 40-50 Moisture content (%) 0 0 LT 5% Higher After 1 Week Above %RH 90 80 80 90 Moisture content (%) LT 20% LT 40% LT 50% MT 30%
(I)-Non hygroscopic (II)- Slightly hygroscopic (III)-Moderately hygroscopic (IV)-Very hygroscopic
(C) IMPORTANCE OF MEASUREMENT :

It affects the chemical stability of drug. It also affects the flow property. Hygroscopic compounds have poor flowability so that it causes weight variation problems. Moisture in cohesive material causes solid bridges and liquid bridges formation between the particles, which ultimately form hard cake. Hygroscopic compounds are generally sticky so that also affects the compaction. (e.g. picking & sticking) It is important for aerosols containing powders. Moisture content should be below 300 ppm. Higher moisture level generally results into particle agglomeration.
(D) METHODS OF IMPROVEMENT :

For granulation of hygroscopic material, use non-aqueous solvent. For efflorescent material ,use anhydrous salt. Add finely powdered adsorbants like MgO or Mg carbonate. Perform the entire tableting operation under controlled humidity condition.eg-Very hygroscopic products are stored less than 40% RH. Make complex to form dry physical form suitable for tabletting. i.e. make clathrate of hygroscopic benzalkonium chloride with urea.
New smart excipient:
Store hygroscopic compound with desiccants in well closed container. Example: Starch is hygroscopic,but on pregelatinization it exhibits lower propensity for moisture, thus providing excellent stabilization for moisture sensitive materials.
Galen IQ, a range of multifunctional excipient by German company palatlinit It is based on hydrogenated isomaltulose,also known as isomalt Combined advantage of mannitol, sorbitol, lactose, MCC Low Hygroscopicity, at 25C temperature hardly absorb water untill 85% RH. This low hygroscopic nature combined with anti caking property makes easy mixing, agglomeration or tableting and helps in elimination of costly packing. E.g. Relationship between powder characteristics & hygroscopicity of granule preparation by different methods : Method with low hygroscopic granules are dry granulating & 2 swinging granulating method. (C.A. : Vol : 151, No.7,17th AUGUST, 2009, P. no . 156034)
(2) POWDER RHEOLOGY:CONTENTS:A. INTRODUCTION B. IMPORTANCE OF FLOW PROPERTY STUDIES C. FACTOR INFLUENCING FLOW PROPERTY D.IMPROVEMENT OF POWDER FLOW PROPERTY E. MEASUREMENT OF FLOW PROPERTY (A) INTRODUCTION
The flow properties of powder plays an important role in dosage form manufacturing process. When limited amounts of drugs are available these can be evaluated simply by measurement of bulk density and angle of repose. These are extremely useful derived parameters to assess the impact of changes in drug powder properties as new batches become available.
(B) IMPORTANCE OF FLOW PROPERTY STUDIES:1.Weight uniformity 2.Content uniformity 3.Hardness 4.Disintegration 5.Speed of production 6.Scientific design of formulations and processing equipment
(C) FACTORS INFLUENCING FLOW PROPERTY :1.Particle size & Size distribution 2.Particle shape 3.Moisture 4.Electrostatic effects 5.Powder cohesion and storage compaction 6.Effect of temperature
1. PARTICLE SIZE & SIZE DISTRIBUTION :

If size (or) dimensions of particles altered Particles shape changes Size of Particle (m) More than 250 Less than 100 flow of particles changed Less than 10 Flow Property Free flowing Poor flow Resist flow
Size distribution is carried out by using proper amounts of fines.
2. PARTICLE SHAPE & SURFACE MORPHOLOGY :

Spherical shape is the best shape which give maximum flow. Irregular shape may cause bridging in hopper.
3. MOISTURE :
The effect of moisture on flowability of particles varies from powder to powder. The particles become cohesive due to moisture absorption. Absorbed moisture in solids can exist in two forms : 1. unbound state 2. As a part of crystal structure
4.
ELECTROSTATIC EFFECTS :
The charged material show poorer flow than uncharged material. Particles acquire static charge by : Grinding Attrition collision mixing sieving Moisture Electro static charge may be positive or negative. +ve charge particles plastic surfaces -ve charge particles metal or glass surfaces
5. POWDER COHESION & STORAGE COMPACTION :

When solid remains at rest or stored in a hopper or bin , it can become more cohesive and gives poor flow. Flow chacteristics depends on : Intrinsic cohesiveness of the material Temperature of storage Load levels of Hopper and bin Time of storage Vibratory forces
(D) METHODS OF IMPROVING FLOW PROPERTIES OF POWDERS :1: By addition of glidant :- Effect depend on particle size of material to which they are added. E.g..Magnesium stearate, Talc, Colloidal silicone are effective in finer particles, Corn starch is more effective in coarser particles. 2: By size reducing or addition of fines:- both should be up to optimum level. 3: By wet granulation:- gives regular spherical shape and also reduces static charge . 4: By removing static charge:- increase flow property. 5: By making more denser:- as density increases,flow property increases. 6: By addition of flow activator like MgO:- here MgO dose not act as flow activator directly but it increases flow by absorbing moisture which affect the flow property. 7: For hygroscopic and moist powder:- use silicon treated powder eg. Silicon coated TALC or NaHCO3. 8: Alteration of process :-use force feeder, use vibrating hopper.
(E) MESUREMENT OF FLOW PROPERTIES :1) 2) 3) 4) 5) 6) 7) Bulk Density Tapped Density Carrs Compressibility Index Hausner Ratio Angle of Repose Shear Cell Determination Kava, Keta, Kuno parameter
1. Bulk density : Bulk density of a compound varies substantially with the method of crystallization, milling, or formulation. Bulk density measurement :The bulk density of a powder is dependent on particle packing and changes as the powder consolidates. A consolidated powder is likely to have a greater arch strength than a less consolidated one and may therefore be more resistant to powder flow. The ease with which a powder consolidates can be used as an indirect method of quantifying powder flow. It is determined by pouring presieved (40-mesh) bulk drug into a graduated cylinder via-a large funnel and measuring the volume and weight.
2. Tapped density : It is determined by placing a graduated cylinder containing an known mass of drug or formulation on a mechanical tapper apparatus, which is operated for a fixed numbers of taps(about1000)untill the powder bed volume has reached a minimum volume. using the weight of a drug in the cylinder and this minimum volume,the tapped density is calculated.
3.Carrs Index: Neumann and carr developed a simple test to evaluate flowability of a powder by comparing the poured (fluff)density and tapped density of a powder and the rate at which it packed down. Tapped Density - Bulk Density % Compressibility = ------------------------------------------ X 100 (Carrs index) Tapped Density % COMPRESSIBILTY RANGE 5-15 12-16 18-21 23-28 28-35 35-38 >40 FLOW DESCRIPTIONS
Excellent (free flowing granules) Good ( free flowing powder granules) Fair to passable ( powder granules) Poor ( very fluid powder) Poor ( fluid cohesive powder) Very poor ( fluid cohesive powder) Extremely poor ( cohesive powder)
4. HAUSNER
RATIO:-
Hausner predict the flow properties of powder by using interparticle friction. This is a simple index that can be determined on small quantities of powder. Hausner ratio = tapped density /poured density HAUSNER RATIO < 1.25 > 1.25 TYPE OF FLOW Good flow Poor flow
Typical values of Bulk Density,Tapped Density, Hausner Ratio & Carrs index :Lactose -product Bulk Tapped Hausner ratio Carrs index Lactochemdensity density Domo Coarse crystals Crystals Extra fine crystals Powder Fine powder Extra fine powder Super fine powder 0.75 0.74 0.73 0.64 0.61 0.45 0.47 0.88 0.86 0.86 0.89 0.84 0.74 0.74 1.2(good flow) 1.2(good flow) 1.2(good flow) 1.3(poor flow) >1.3(poor flow) >1.3(poor flow) >1.3(poor flow) 15(excellent) 15(excellent) 15(excellent) >25(poor) >25(poor) >25(poor) >25(poor)
5. Angle Of Repose : Angle of repose is defined as the angle of the free surface of a pile or heap of powder to the horizontal plane. Characterize the flow properties of solids. If material is not cohesive If material is cohesive Flows well Low heap Poor low High heap
Angle Of Repose is measured by the equation : Angle of Repose = = tan-1 ( h / r ) where, h = height of conical heap & r = radius of horizontal plane of powder Angle Of Repose as an indicatior of powder flow properties :Angle of Type of flow repose(degrees) () <25 Excellent 25-30 Good 30-40 Passable >40 Very poor REPOSOGRAPH : It is a stable instrument which at best can only indicate comparative flow properties. The formation of sharp cone would mean poor flow property while a good spread would indicate a superior flow property. Relationship between angle of repose, carrs index of a powder and its flow characteristics:-
When only small quantities of powder are available, an alternative is to determine the angle of spatula by picking up a quantity of powder on a spatula and estimating the angle of the triangular section of the powder heap viewed from the end of the spatula.this is obviously crude but is useful during preformulation,when only small quantities of drug are available. METHODS OF DETERMINATION :TYPE OF ANGLE OF REPOSE Static Angle of Repose METHOD Fixed Height Cone Fixed Based Cone Tilting Table Rotating Cylinder Rotating drum Ledge Type Crater Type Platform Type
Dynamic Angle of Repose Drained Angle of Repose
6. SHEAR CELL DETERMINATIONS : Characterize the flowability from the behavior of powder in a shear cell The powder bed was subjected to shear and its applied load for shear also noted Graph plotted between shear stress and applied load Flow factor can be obtained by determining the reciprocal of slope. FLOW FACTOR TYPE OF FLOW > 10 Free flow 4 10 1.6 - 4 < 1.6 7. Easy flow Cohesive Very cohesive
Kava, Keta, Kuno parameter :It is the recent parameter that is introduced by Japanese scientists. It is used for the measurement of flow property of crystals.
NEW MEASUREMENT SYSTEM TO EVALUATE POWDER FLOWABILITY BASED ON VIBRATIONAL CAPILLARY METHOD :
Evaluates flowability of micrometer sizes particles under actual flow condition. The amplitude and frequency of vibration is controlled by computer and mass of powder discharged from vibrating capillary tube is measured by digital balance. The mass flow rate is measured by digital processing. [Chemical Abstract : Vol :146,Jan. 2007, P.No.:9806]
(3)COMPACTION PROPERTIES:CONTENTS:A. DEFINITION B. DIFFERENT STAGES OF POWDER COMPACTION C. METHOD FOR IMPROVEMENT D.EFFECT OF COMPACTION ON DIFFERENT FACTORS E. MOISTURE AND COMPRESSION F. EVALUATION OF COMPACTION A. DEFINITION: COMPACTION :- Compaction of powder is term used to describe
the situation in which material are subjected to some level of mechanical force. COMPRESSION :- Compression is reduction in the bulk volume of the material as a result of displacement of the gaseous phase. CONSOLIDATION :- Consolidation is an increase in mechanical strength of material resulting form particle particle interactions
B. DIFFERENT STAGES OF POWDER COMPACTION :-
The Characteristics Of Material :1. PLASTICITY Plastic material are capable of permanent deformation, also exhibit a degree of brittleness (fragmentability) But plastic material will get bonding after Viscoelastic deformation. 2. FRAGMENTABILITY If material is fragmentable, neither lubricant mixing time nor dwell time affecting the tablet strength. 3. ELASTICITY E.g. PCM, MCC, ASPIRIN,etc. If material is elastic, it rebound when compression force is released. Elastic material may lead to capping & lamination They require wet massing to induce plasticity or plastic tableting material. 4. PUNCH FILMING [STICKING]: This may lead to chipping of tablet.
A new directly compressible excipient:

c*pharmamannidex DC is mannitol based. permit higher dose of active ingredient. Helpful for those companies that are looking for animal derivative to non animal derivative excipient. non hygroscopic, non carcinogenic, chemically stable, suitable for diabetics. high compressibility, high binding capacity, low friability. excellent diluent, binder, ideal for chewable tablet.
C. METHOD FOR IMPROVEMENT : If material is Elastic, it can be improved By plastic tableting matrix (like MCC). By wet massing to induce plasticity. By precompression. If material is Sticky, it can be improved By change in salt form. By using high excipient ratios. By using abrasive inorganic excipient. By wet massing. By addition of colloidal silica as a polishing agent. By addition of magnesium stearate up to 2%.
If material is Plastic, it can be improved by addition of fragmentable excipients (like lactose,calcium phosphate)
D. EFFECT OF COMPACTION ON DIFFERENT FACTORS : Compression force affects surface area,granule density,porosity, hardness and disintegration time of pharmaceutical tablets. Surface area increased to a maximum and then decreased. Porosity decrease and density increased as a linear function of the logarithm of the compression force. As the compression increase the tablet hardness and fracture resistance also rise.
E. MOISTURE & COMPACTION : Moisture is essential for the formation of the tablet.(2-4%) Moisture increases the tensile strength of the tablet by increasing contact area for bonding. Moisture decreases particle surface energy & thus decreases adhesion of the tablet to the die wall. In case of MCC, moisture present within the pores ,that facilitate the flow during the compaction. Lack of moisture leads to lamination because of elastic recovery. Excessive moisture produces capillary state of powder aggregation and thus surface tension effects are insignificant to have better compaction. Reported e.g. is that of Naproxen tablet which help of lactose. When moisture as more then 2% ,hardness of tablet decreased (at both low & high pressure).
F. EVALUTION OF COMPACTION:1. Strain index (SI) :- Measures internal strain associated with a powder when compacted. 2. Bonding index (BI) :-Ability of material to bonds. 3. Brittle fracture index (BFI) :- Measures brittleness of material. Higher is the BI index, stronger is the tablet. Higher is the SI index, softer is the tablet.
e.g.Silicified MCC as a multifunctional pharmaceutical excipient: having high compressibility,high intrinsic flow, enhanced lubrication efficiency & improved Blending properties. (C.A.: VOL.-151,No.6,10th AUGUST, P.NO.-131556)
REFERENCES : Michael .E. Aulton, Pharmaceutics- the science of dosage form design,135 ,207,second edition,Elsevier Limited. Leon Lachman et al, Theory and Practice of Industrial Pharmacy, 184-186, Third edition, Varghese publishing house, India. www.quantachrome.com Stephen. A.howard, Flow properties of solids, Encyclopedia of pharmaceutical technology by markel dekker,2nd edition, volume-2, page no-1264 -1283 N.K. Jain, Flow ,cohesiveness and compressibility, Professional pharmacy by vaallabh prakashan, 5th edition,1998 pg no-145-147 Harry G. Brittain, Physico chemical properties, ,Physical characterization of pharmaceutical solids by markel dekker.inc,1995 pg.no-281-301 CHEMICAL ABSTRACT: VOL: 151,No.7,17th AUGUST, 2009, P.NO.156034 CHEMICAL ABSTRACT : VOL :146, Jan. 2007, P.NO.: 9806 CHEMICAL ABSTRACT : VOL:151,No.6,10th AUGUST, 2009,P.NO.131556
CHIRALITY
(AS PREFORMULATION ASPECTS)
GUIDED BY: DR.R.K. PARIKH
PRESENTED BY: HIMANSHU K MANAVADARIYA M.PHARM-1 ROLL NO:02 YEAR:2009-10
DEPARTMENT OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY

L. M. COLLEGE OF PHARMACY, AHMEDABAD, 380009.
CONTENTS
1. INTRODUCTION 2. TERMS 3. NOMENCLATURE OF CHIRAL COMPOUND 4. IMPORTANT OF CHIRALITY 4.1 STERIC ASPECTS OF PHARMACOKINETICS 4.1.1 DRUG ABSORPTION 4.1.2 DRUG DISTRIBUTION 4.1.3 DRUG BIOTRANSFORMATION 4.1.4 DRUG ELIMINATION 4.2 STERIC ASPECTS OF DRUG ACTION 4.3 CHIRAL IMPURITIES 4.4 ADVANTAGES BY THE USE OF SINGLE ENANTIOMER AS ADRUG 5. SPECIFIC REQUIREMENTS FOR CHIRAL DRUG DEVELOPMENT 6. APPLICATION OF CHIRALITY IN FORMULATION AND DEVELOPEMENT 6.1 ACCEPTANCE AND REJECTION OF API 6.2 SELECTION OF ADJUVANTS 6.3 IN VITRO DISSOLUTION STUDIES AND IN VIVO STUDIES 6.4 STABILITY STUDY 6.5 NDA/ANDA APPLICATION
1. INTRODUCTION
Chiral drugs are a subgroup of drug substances that contain one or more chiral centers. More than one-half of marketed drugs are chiral. It is well established that the opposite enantiomer of a chiral drug often differs significantly in its pharmacological, toxicological, pharmacodynamic, and pharmacokinetic properties. Therefore from the points of view of safety and efficacy, the pure enantiomer is preferred over the racemate in many marketed dosage forms. However, the chiral drug is often synthesized in the racemic form, and it is frequently costly to resolve the racemic mixture into the pure enantiomers. Currently, then, most chiral drugs, including some blockbuster drugs, such as fluoxetine hydrochloride and omeprazole,are still marketed as racemates. However, the recent trend is toward marketing more single-enantiomer drugs.In addition, a racemic switch, which involves the development of a pure enantiomer of a drug that is already marketed as a racemate, is actively pursued by many companies to improve its therapeutic effiacy and to extend patent protection.The decision whether to market the racemate or the enantiomer of a chiral drug is mainly based on pharmacology, toxicology, and economics.
30%
39% achiral drug one chiral centre 18% recemic drug multichiral centre
13%
Classification of new drug by stereochemistry 2. DEFINITIONS CHIRAL

Molecules that are not superimposable on their mirror images are chiral. Chirality is necessary and sufficient condition for the existence of enantiomers. That is to say: a compound whose molecules are chiral can exist as enantiomers; a compound whose molecules are achiral cannot exist as enantiomers.
A chiral carbon is one to which four different groups are attached.
Example, Lactic acid
STEREOISOMER
It is three dimensional arrangement of atom around the chiral center.The particular types of isomer that differ from each other only in the way the atoms are oriented in space are called as stereoisomers.
2-METHYL-1-BUTANOL
ENANTIOMER
Enantiomers are pairs of configurational isomers that are mirror images of each other and yet are not superimposable. Each enantiomer is homochiral, meaning that all the molecules have exactly the same configuration.
Ephedrine (-) Ephedrine (+)
DISTEREOISOMER
Diastereomers are pairs of compounds that contain more than one chiral center, not all of which are superimposable and are not mirror emages.
MESO COMPOUND
A compound whose molecules are superimposable on their mirror images even though they contain chiral centres.
RACEMAT
A mixture of equimolar amount of enantiomers, and is heterochira meaning that the molecules have different chiralities.
EUTOMERS
Refers to enantiomers with higher pharmacological affinity or activity.
DISTOMER
Refers to enantiomers with higher pharmacological affinity or activity.
EUDISMIC INDEX (EI)

The logaritham of ratio of activity of eutomer to distomer.
RACEMIZATIONS
Conversion of enantiomer in to its racemate.
CHIRAL INVERSION
Conversion one enantiomer into its mirror image.
RRRRRR RRRRRR RRRRRR RRRRRR R a. enantiomer (homochiral)
RSRSRS RSRSRS RSRSRS RSRSRS b. racemate (hetrochiral)
RRSSSR SSRRSS RRSSRS RSRSRR c.pseudoracemate
They can be differentiated by spectroscopic method, X-ray diffraction and melting phase diagram (hot stage microscopy or DSC).
3.NOMENCLATURE OF CHIRAL COMPOUND

Chiral substances can be classified as follows:
OPTICAL ROTATION: On basis of plane polarized light:

DEXTRO ( d ) or ( + ) and LEVO ( l ) or ( - ). This method is based upon a physical property of the molecule but does not provide information about absolute configuration. CIRCULAR DICHROISM: differential absorption of left and right circularly polarized rotation. There are differences in absorption of the left and right handed components of circularly polarized light by an non racemic sample. Example:Chloramphenicol has two chiral centers so four possible stereoisomers. i.e, ( + ) chloramphenicol solublise in ethanol and ( - ) chloramphenicol solublise in ethyl acetate.
BY FISHER AND ROSANOFF: Nomenclature of carbohydrate was on

of D ( + ) or L ( - ) Glyceraldehyde and same for amino acids as D ( + ) or L ( - ) Serine.
But this system inconvenient for molecules containing more than one chiral center.
R AND S NOMENCLATURE:
In 1956 cahn, Ingold and Pregold devised a system for stereoisomers referred as the Sequence Rule System (CIP system). In this system chiral center are ranked according to their atomic number. R: CLOCKWISE and S: ANTICLOCK WISE.
Z AND E ISOMERS: According to that in double bonded compounds the substituents

can be oriented on the same side or opposite sides of the double bond.
Substituents are, on same side than Z-isomer or on opposite side than E-isomer.
4. IMPORTANT OF CHIRALITY
4.1 STERIC ASPECTS OF PHARMACOKINETICS
The macromolecules (proteins) of body can distinguish between isomers
leading to stereoselective difference in absorption, tissue and protein binding, biotransformation and renal excretion. Slight spatial differences in stereoisomers can have marked effects on the degree of association and interaction with protein and enzyme and enzyme system.
4.1.1 Drug absorption

The passive gastrointestinal absorption of enantiomeric drugs would be expected to be similar since the physical properties of partitioning and solubility of enantiomers are the same.Drugs that interact with and are absorbed by carrie transport system have a potential for stereoisomeric effects on the rate of enantiomer absorption. Stereoisomers with structural similarities to endogenous entities and nutrients display differences in permeability rates across the g.i. membrane and hence in bioavailability. L-dopa, which is absorbed by an amino acid transport system, passes the g.i. wall at a rate 4 to 5 times that of D-enantiomer. The L-methotrexate is absorbed by active processes in the g.i. tract and the d-isomer is reportedly absorbed by passive absorption. The crystalline from of racemates may not be the same as the crystal structures of the individual stereoisomers and may be a source of differences in rates of dissolution between racemic and single enantiomeric dosage forms.
4.1.2 Drug distribution

The interaction of enantiomer with a plasma protein yields a diastereomeric association. (+)-Oxazepam hemisuccinate has 30 to 50 higher association constants for albumin than its (-) isomer. The S isomer of warfarin is bound to a greater extent to albumin than R isomer. A1 acid glycoprotein binds S-propranolol (87.3 %) to a slightly higher degree than R-propranolol (83.8%) where as human albumin binds R-propranolol more strongly than S-form.
4.1.3 Drug biotranformation

The intrinsic hepatic clearance of S-warfarin is reported to be approximately two fold greater than that of R-warfarin and reflects differences in metabolic pathways of the two stereoisomers. A novel method for assessing inhibition of ibuprofen chiral inversion and its application in drug discovery*. The ibuprofen is a phenyl acetic acid derivative undergoes chiral inversion in body from R(-) isomer to S(+) isomer and its interesting to know that only S(+) isomer is responsible for anti inflammatory activity rather than R(-) isomer. (*Ref: C.A:147(2) JULY; 2007; 3868a)
4.1.4 Drug elimination

The tubular secretory contribution to drug renal clearance has the potential of producing stereoselective renal elimination in the handling of stereoisomers. The renal clearance of S-prenylamine is approximately 2.4 times higher than that of the R isomer. L-disopyramide has a renal clearance of 29 to 86% higher than that of D-isomer The analgesic activity of propoxyphene is due mainly to the d enantiomer. However, the racemate is more potent than an equimolar dose of d-propoxyphene due to a reduced clearance of the latter caused by the presence of l enantiomer in the racemate.
4.2 STERIC ASPECTS OF DRUG ACTION

The following possibilities exist for the distribution of pharmacological activity between enantiomers of a chiral drug molecule. Equipotent enantioner. eg.flecainide and cyclophosphamide All pharmacologic activity resides in one enantiomer with the other stereoisomer being inactive (stereospecificity). A classical example is -methyldopa in which all the desired antihypertensive activity is confined to the S isomer. This basis of stereospecificity of action has been related to the prodrug nature of -methyldopa, which is bioactivated stereoselectively in vivo to (1R, 2S)--methylnorepinephrine, a presynaptic 1 sympathomimetic involved in the anti-hypertensive effect. Both enantiomers have similar qualitative pharmacological activity but they exhibit significant quantitative (e.g. potency) differences (stereoselectivity). The pharmacologic behaviors of warfarin is classical example of stereoselectivity of drug action. The anticoagulant potency of S-warfarin in vivo is from two-to five fold higher than that of its R enantiomer. Enantiomers of a chiral drug differ in their therapeutic and toxicologic profiles. The therapeutic antiparkinson effects of DOPA reside in the L-isomer, while the D isomer shows granulocytopenic effects. The S enantiomers of the profen derivatives (e.g. naproxen) possess significantly greater intrinsic anti-inflammatory potency, but evidence suggests that the R-profens are more toxic by production of xenobiotic lipids.
Enantiomers possess different pharmacological properties but mutually beneficial actions form the therapeutic standpoint.
In the sulindac the Z isomer is the more potent anti-inflammatory agent and is used in pharmaceutical formulations. Although not availab commercially, E-sulindac has greater theoretical utility for the treatment of preeclampsia because of its selective effects on NAD-linked prostaglandin dehydrogenase, which conceivably could lead to a more favorable balance of vasoconstrictive and vasodilatory metabolites of arachidonic acid.
4.3 CHIRAL IMPURITIES

Chiral impurities during pharmaceutical processing include: The opposite enantiomer in a single isomer Excess enantiomer in a racemic compound A diastereomer in a homicidal or a racemic crystal. The presence of small amounts of opposite enantiomer may significantly reduce the apparent solubility of th enantiomer, because the racemic compound will form in the solution and may precipitate from the solution. For example, the solubility of ()- dexclamol hydrochloride is five times that of (_)-dexclamol hydrochloride. In ephedrine and pseudoephedrine studies demonstrated that traces (as low as 0.0025 mole fraction) of the enantiomeric impurity might cause significant changes in the physicochemical properties of the pure enantiomer. Similarly incorporation of excess enantiomers (1.5 to 3 mole fraction) resulted in significant changes in the thermodynamic property and gave rise large variations (27%)of IDR (intrinsic dissolution rate) of the racemic compound.
4.4 ADVANTAGES BY THE USE OF SINGE ENANTIOMER AS A DRUG

Separating unwanted pharmacodynamic side effects from toxic effects if these reside exclusively in one enantiomer. Expose the patient to less body load and thus reduce metabolic/renal/hepatic drug load. Easier assessment of physiology, disease, and drug co-administration effects. Reduce drug interactions. Avoid enantiomerenantiomer drug interactions. Avoid bioinversion. Easier assessment of efficacy and toxicity through pharmacokinetic or pharmacodynamic monitoring of the stereochemically pure active enantiomer.
5.SPECIFIC REQUIREMENTS FOR CHIRAL DRUG DEVELOPMENT

Development of enantiomeric assay.
Synthesis of individual enantiomer. Safety evaluation of the individual enantiomer. Pharmacokinetic of individual enantiomers. Resolution of the individual enantiomer. Bulk drug: enantiomeric purity Chiral conversion
In pharmaceutical development, the relevant expansion of the specification of a chiral drug substances is its stereochemistry in terms of optical purity, batch-to-batch variations and the confirmation of enantiomeric stability in formulation both on storage and in vivo. The decision to develop a single enantiomer is made only if it gives genuine therapeutic benefit and becomes economically feasible. In other cases, a racemate switch from a single enantiomer may extend a patent on a chiral drug initially marketed as a racemate, because a stereochemically pure compound derived from the available racemate will be treated as a new drug.
6. APPLICATION OF CHIRALITY IN F AND D

F & D scientists now have to focus their attention on chirality. The applications of chirality from the viewpoint of F & D are schematically shown in figure. ACCEPTANCCE OR REJECTION OF API INTRINSIC DISSSOLUTION ECONOMICAL CONSIDERATION SELECTION OF ADJUVANTS
FORMULATION AND DEVELOPMENT SCIENTISTS

NDA/ANDA APPLICATION STABILITY STUDY
IN VITRO DISSOLUTION TESTS AND IN VIVO STUDIES
Figure 1, Application of chirality in formulation and
development
6.1 ACCEPTANCE AND REJECTION OF API

The Chiraly pure drugs should be quantitatively analyzed for the presence of absence of chiral impurities besides the routine determination of related impurities.
Tao and Cooks recently reported that quantitative chiral analysis could be done by tandem MS. The method is rapid and requires very little sample. Lou recently reported that it would be useful to determine enantiomers and other structurally similar drug impurities using one rather than two analytical methods. The pharmaceutical companies should accept or reject a sample of API based n the ratio of the active/inactive enantiomers in the non-racemic mixtures. This ratio may change from one vendor to another. Most pharmaceutical companies do not consider this fact at the time of purchase/ the success or failure of the formulation and development team may depend upon this ratio. The in vitro dissolution test and in-vitro/in-vivo correlation (IVIVC) are the two major areas where the differences may be observed if an eye is not kept on Chirality when API is purchased. The intrinsic dissolution rate of chirally pure API may prove to be a useful quality control parameter.
6.2 SELECTION OF ADJUVANTS

Formulation and development scientists add a number of adjuvants with diversified purposes in pharmaceutical formulations. Adjuvants are added to facilitate manufacturing, for functionality improvement, and to improve appearance and stability. The adjuvants may be either chiral or non-chiral in nature. The type and amount of the adjuvant may determine the functionality of the dosage form, especially the drug release rate. The characteristics of adjuvant such as solubility, compressibility, surface area, porosity, etc/ are considered today in F & D. However, very little attention is given to the nature of adjuvant from the viewpoint of Chirality. A few references are cited below to show the effect of adjuvant type on drug release. Solinis et al. studied the release of salbutamol and ketoprofen enantiomers from hydroxypopylmethylcellulose (HPMC) K100M matrices containing two types of cellulose derivatives. The authors concluded that
stereoselectivity is dependent on the amount of chiral excipients in the formulation. Srichana and Suedee examined the in vitro dissolution of salbutamol from matrix tablets containing various chiral excipients such as gamma cyclodextrin, heptakis (2,6 di-o-methyl)-beta cyclodextrin, sulfobutyl betacyclodextrin, HPMC and egg albumin. The formulation containing gamma cyclodextrin provided significant stereoselectivity throughout the dissolution profile. The release of eutomer R-salbutamol was higher than that of the distomer S-salbutamol from the gamma cyclodextrin tablets. Suedee et al. monitored enantioselective in vitro release of propranolol. The influence of the method of polymer synthesis, drug to polymer ratio, pH, and temperature on the release of eutomer can be controlled via means of formulation. The distomer was retained in the dosage form. Stereoselective interaction of ibuprofen was evaluated with chiral excipients such as HP-beta-cyclodextrin, tartaric acid, sucrose, HPMC, methylcellulose and a non-chiral excipients citric acid. The co precipitates showed higher dissolution rate. The presence of chiral excipients did not cause stereoselective release of the drug.
The objective may be achieved by preparing a bi-layer tablet. Applications of interaction between API and chiral excipients may be explored in the areas of sustained release buccal dosage form and colon drug delivery system. Teamwork between an expert analyst in chiral science and an F &D scientist may open up many avenues in formulation development work.
6.3 IN VITRO DISSOLUTION STUDIES AND VIVO STUDY

Crystals of both enantiomer & racemic compound are having different molecular arrangement. Due to the difference between the crystal lattice of both forms, the solubility of pure enantiomers may be different from the racemic compound. It was proved by the experiment that the initial dissolution rate of racemic propranolol HCl was three times greater than that of enantiomers in distilled water. While performing dissolution testing for stereo-selective drugs, (I) we must concerned about the amount of a particular enantiomer released from the dosage form (II) We should not see the surplus amount of drug (R form + S form) release from the dosage form. Now a days, it is possible to do this because of the development of analytical tools like capillary electrophoresis*, simulated moving bed chromatography etc. Comparative study of chiral separation of ofloxacin enantiomer by capillary electrophoresis using neutral cyclodextrin. (*Ref: C.A. 147(8) AUG; 2007; 173958a) For the drugs where only one of the forms (R or S) is active, the use of a
stereo selective dissolution test is recommended for the calculation of t50 or Q30.A single point dissolution specification such as t50 (time required for 50% API dissolution) or Q30 (amount of API dissolved in 30 min) is routinely employed as a quality control release test. After establishing the similarity of in vitro dissolution testing between a reference Solubility Permeability Chiral inversion* and a test Class product in different dissolution media, in vivo testing in man (bioequivalence testing) is ordered. It is a hard fact that many experimental formulations fail to establish an IVIVC.
Table 1-Extension of Biopharmaceutics Drug Classification System
Biopharmaceutics classification system (BCS) guidance of US FDA classifies The drugs in four classes considering the solubility and permeability of drugs. Once the drug meant for oral use dissolves in gastro-intestinal fluid and subsequently permeates through the membrane, it enters into general circulation. One should remember that some drugs might undergo chiral conversion in blood. In such cases, the pharmacological action will depend upon the amount of unchanged active enantiomers reaching the receptor. Enantioselective analysis should be adopted in cases where there is the possibility of chiral conversion in blood. The third dimension can be added to BCS, i.e. chiral conversion for the drugs where only one form (R pf S) is active and the other form is inactive (table 1). The drugs that fall under class 1B will show superior action as compared to class 1A. Polli reported that one intent of in vitro- in vivo relationship (IVIVR) is to learn about the relative contribution of dissolution to a products overall absorption kinetics. Here also, chiral-specific dissolution can be used.
6.4
I II III IV
High Low High Low
High High Low Low
A B A B A B A B
High Low High Low High Low High Low
STABILITY STUDY
In the FDAs policy statement for the development of new stereomeric drugs, it is mentioned that the stability protocol for enantiomeric drug substances and drug products should include a method or methods capable of assessing the stereochemical integrity of the drug substance and drug product. However, once it has been demonstrated that stereochemical conversion does not occur, stereoselective tests might not be needed.The stability study program must address the issue of chiral inversion and racemization. Thalidomide undergoes facile base-catalysed chemical racemization in aqueous media. Shelf life of pharmaceutical formulation is fixed after considering 90% or 95% drug degradation under stated conditions of drug storage. This could be the amount of inactive enantiomers (R of S) degraded. We should not be surprised if the FDA starts assigning shelf life on the basis of degradation of active enantiomers since that seems to be more realistic.
6.5 NDA/ANDA APPLICATION

The current applicable guidance documents shall be kept in mind while preparing the applications. We should also keep in mind that increasing the desired activity should not accompanied by a rise in the untoward effect. FDA requires toxicology testing on the racemate. The data of stereoselective dissolution testing should be submitted for the enantiomers that exhibit different action (e.g, Methyl phenyl propyl barbituric acid). There are endless opportunities and It is difficult to address all relevant issues in this communication. The current guidance documents should be consulted at the time of submitting an application.
6.6 ECONOMICAL CONSIDERATION

The significant expenses associated with the development and manufacture of Stereochemically pure drugs will add to their cost and there may be economic justifi cations for acceptance or rejection of a new therapeutic entity. It may not be economically feasible to pay an increased amount for only slightly increased effi cacy.
LIST OFFICIAL CHIRAL DRUGS

Sr. no 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Drugs Dextroamphetamine Dextromethorphan Official in USP USP, BP Sr. no 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. Drugs Levomenthol Levomeproprazine Levomethadone Livamisol Levonantradol Levonordefin Levonorgesterol Levoorphanol Levophan Levophed barbiturate Levoprolactine levopropizine Levopropoxyphene Levopropylhexidine Levorenin Levoresin Levorterenol Levoterenol Levothoid Levothyroxin Official in MartindaleEP, BP MartindaleEP, BP MartindaleEP IP, BP MartindaleEP MartindaleEP MartindaleEP, IP, BP MartindaleEP, BP MartindaleEP MartindaleEP MartindaleEP MartindaleEP MartindaleEP MartindaleEP MartindaleEP MartindaleEP IP BP MartindaleEP MartindaleEP, IP, BP
Dextromoramide MartindaleEP Dextropropoxyphene MartindaleEP, BP Esmoprazole Levobunolol Hcl MartindaleEP Levobupivacaine Levocabastine Levocalamine Levocarit Levocarnil Levocarnitine Levodiphenopyrine Levodopa Levodopum Levodromaran Levofloxacine Levoglutamine Levomaprolol Levomenol MartindaleEP MartindaleEP MartindaleEP MartindaleEP MartindaleEP MartindaleEP MartindaleEP, IP, BP MartindaleEP MartindaleEP MartindaleEP MartindaleEP MartindaleEP MartindaleEP
REFERENCES
1. Williams Lemke.Foyes principle of medicinal chemistry. Ed.5. p=49-54. 2. Morrison Boyd. Organic chemistry. Ed.6. p=133. 3. Relationship between physical properties and crystal structures of chiral drug Z.jane Li and David J.W.Grant. October 1997, Volume-86,Number 10. 4. Encyclopedia of pharmaceutical technology. Volume-8. p=281. 5. A.J.Romero and C.T.Rhodes, Chirality, 3, 1 (1991).
6. Overview on Chirality and Application of Stereo-selective Dissolution testing in the Formulation and Development work by Mukesh C.Gohel. www.dissolutiontech.com 7. www.harrisononline.com 8. Indian pharmacopoeia 1996. 9. British pharmacopoeia 1993. 10. United state pharmacopoeia 2000.
STUDY QUESTIONS:
1. Application of chirality in F & D. Sept, 2006/2007 2. Define chiral and enlist such product. Discuss application of Chirality from F & D viewpoint giving suitable example. March 2006 (1st Int.) 3. Describe steric aspects of drug action. 4. Describe steric aspects of pharmacokinetics. 5. Enlist the chiral impurities during pharmaceutical processing and how they can affect the performance of the drug with an example. 6. Write a novel method for assessing inhibition of ibuprofen chiral inversion and its application in drug discovery.
Paper 910101 M.Pharm-(2009-10)
Chapter 1 Preformulation
POLYMORPHISM
POLYMORPHISM
(AS A PART OF PREFORMULATION STUDY)
Presented by Himaxi Rajput M.pharm I(09-10) Roll no.-9 Guided by Dr. R.K.Parikh
Department of pharmaceutics and pharmaceutical technology L.M.College of pharmacy, Ahmedabad - 09.
DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
POLYMORPHISM
List of contents
1. 2. 3. 4. 5. 6. 7. 8. 9. Definition Need to study polymorphism Properties Types of polymorphism How to differentiate them Pseudopolymorphism Methods to identify polymorphism Parameters to be cared by preformulator Solubility, dissolution behaviour and bioavailability of polymorph 10.Factor affecting polymorphism 11.Effect of polymorphism on bioavailability 12.Conclusion 13.References
DEFINATION:Polymorphism: Elements can exist in two or more different forms, known as

allotropes of that element .eg. Carbon: diamond in cubic (tetrahedral lattice arrangement) graphite in sheets of a hexagonal lattice. Similar phenomenon in compounds, scientifically referred to as polymorphism The term polymorphism was coined by AGUIAR ETAL in 1967. THUS IT IS DEFINED AS THE ABILITY OF SUBSTANCE TO EXIST AS TWO OR MORE CRYSTALLINE PHASES THAT HAVE DIFFERENT ARRRANGEMENTS OR CONFIRMATIONS OF THE MOLECULES IN THE CRYSTAL LATTICE. Since 1967 a series of review articles has dealt with polymorphism and its pharmaceutical application but still there is confusion in the terminology used to identify it.
NEED TO STUDY POLYMORPHISM:Polymorphs show the same properties in the liquid or gaseous state but they behave differently in the solid state. Different polymorphs of a compound are in general different in structure and properties in the same manner as the crystals of two different compounds. Furthermore polymorphism is remarkably common particularly within certain structural groups. For eg. DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10) CLASS Barbiturates Steroids Sulphonamides
POLYMORPHISM
%OF POLYMORPHISM 63 67 40
The effect of polymorphism on bioavailability is the most important consequence for drug substances if the bioavailability is mediated via dissolution. The oldest known example is chloramphenicol palmitate. Others are noviobiocine, griseofulvine, carbamazepine, aspirin and ampicilline. The polymorphism of the excipients may also play an important role in bioavailability. Thus, investigating the polymorphic behavior of drugs and excipients is an important part of the preformulation work. One latest example is of HYDOISOINDOLIN, a tachykinine receptor antagonist. Its stable polymorphic form is developed which is having better pharmacokinetic and pharmacodynamic criteria. * *(C.A. Vol-148, Number 23, June 9, 2008)
PROPERTIES OF POLYMORPHS:Polymorphs show the same properties in liquid or gaseous state but they behave differently in solid state. Polymorphs differ from each other with respect to physical properties like Melting and sublimation temperature Vapour pressure Solubility and dissolution rate Stability Optical and electrical properties Crystal habit Hygroscopicity Heat capacity Solid state reactions Conductivity Compression characteristics
TYPES OF POLYMORPHISM:-
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POLYMORPHISM
Phase transition: the process of transformation of one polymorph into another, which may also occur on storage or during processing, is called phase transition. ENANTIOTROPHS:- If one form stable over certain pressure and temperature range, while the other polymorph is stable over different pressure and temperature range. eg, sulfur MONOTROPHS:- only one polymorph is stable at all temperature below the melting point, with all other polymorph being unstable. glyceryl stearate, chloramphenicol palmitate. Both enantiotropism and monotropism are important properties of polymorphs.
TRANSITION TEMPERATURE:Temperature at which both stable and metastable forms exist in equilibrium with each other.
{Relationship between Gibbs free energy and temperature} In case of monotropy higher melting form is always thermodynamically stable form. In case of enantiotropy lower melting form is thermodynamically stable at the temperature below the transition temperature and higher melting form is stable at the temperature above the transition temperature.
HOW TO DIFFERENTIATE BETWEEN ENANTIOTROPIC AND MONOTROPIC SYSTEM?

Polymorphs can be differentiated by vapor pressure versus temperature curve and solubility versus temp curve. Here form 1 is stable at tempT1 and if it exist in form 1&2, the phenomena is called enantiotropism. In case of enantiotrophism transition temperature of both the forms are same. DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
POLYMORPHISM
A different situation exists if compound exists as form 1&3. Such phenomenon is referred as monotropism. Here transition temperature of both the forms are different since form 3 is
relatively unstable than form 1.
DIFFERENCE MONOTROPY
BETWEEN
ENANTIOTROPY
AND
Enantiotropic pair Reversible phase transition metastable stable
Monotopic pair Irreversible phase transition Metastable stable
lower melting form is higher melting form is always thermodynamically stable below the thermodynamically stable form transition temperature and higher melting form is stable above the transition temperature Transition is endothermic (heat of Transition is exothermic (heat of fusion rule) fusion rule) Higher m.p. has lower heat of fusion Higher m.p. has higher heat of fusion
Paper 910101 M.Pharm-(2009-10)
POLYMORPHISM
PSEUDOPOLYMORPHISM.
The term pseudo means false. Phenomenon in which solvent molecules get incorporated into crystal lattice of solid are known as solvates. This solvates exist in different crystal form called pseuopolymorphs and the phenomenon is called as pseudopolymorphism. Also known as hydrates when water is solvent. E.g. when the potent synthetic estrogen ethynylestradiol is crystallized from the solvents acetonitrile, methanol, chloroform and saturated with water four different crystalline solvates are formed. How to differentiate pseudopolymorphs from true polymorphs? Pseudopolymorphs can be differentiated from true polymorphs by observing melting behavior in silicon oil using hot stage microscopy. Here in this technique psrudopolymorphs evolve gas(steam or solvent vapors)causing bubbling of the oil. While true polymorphs merely melts, forming second globular phase.
METHODS TO IDENTIFY POLYMORPHISM: Optical crystallography: it is used in the identification of polymorphs .Crystal exist in isotropic and anisotropic forms . When isotropic crystals are examined the velocity of light is same in all directions while anisotropic crystals have 2 or 3 different light velocities or refractive indices. Video Recording Systems have made it possible to record the events visualized during the heating and cooling stages .The polarizing microscope fitted with a hot and cold stage is very useful for investigating polymorph .It is useful to know , 1. The degree of stability of metastable form 2. Transition Temperature 3. Melting Point 4. Rates of Transition under various thermal and physical conditions . 5. Whether to persue polymorphism as a route to an improved dosage form . Hot stage microscopy: Using this technique fluid phase transformation as a function of temperature is observed. Generally silicon oil hot stage microscopy is used for detection of pseudo polymorphs X-ray diffraction method: Using Braggs equation:n=2dsin where d=distance for different planes of crystal, =wavelengthof x-ray used ,=angle of incoming beam, n=order of spectrum X-ray diffraction-a}powder x-ray diffractionBasically focusing on packing pattern of the atom.b}exact relative location of atom in crystal is determined.
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POLYMORPHISM
NMR technique: In this technique, powder sample must be rotated at a special angle with respect to magnetic field. FTIR technique: It has been used to quantify binary mixtures of polymorphs. In identification of polymorphs , only solid samples (as mineral oil mulls & KBr pellets) can be used . o In solutions polymorphs of a compound have identical spectra . o Advantages: - Rapid. Technique is qualitative & quantitative. Dilatometry: Measure change in volume caused by thermal or chemical effect.Using dilatometry the melting behaviour of Theobroma Oil was studied .Extremely accurate but tedious , time consuming and not widely used . Microcalorimetry: Used to characterize thermodynamic properties of different molecules. Thermal methods: a}DSC[Differential scanning calorimetry] b}DTA[Differential thermal analysis]c} TGA [thermal gravimetric analysis] This method measures heat loss or gain from physical or chemical chages occurring in sample which is recorded as a function of temperature as substance is heated at uniform scale. Advantage: I Thermodynamic parameter can be evaluated. II Heat of Transition from one polymorph to the other. Melting point determination NOTE: DSC and M.P. determination are often useful technique, but only when substance undergoing investigation heated through phase transition without decomposition.
PARAMETERS TO BE CHECKED BY PREFORMULATOR WHILE DOING POLYMORPHISM STUDY:1. 2. 3. 4. 5. 6. 7. 8. 9. No of polymorphs Relative degree of stability Presence of glassy state Stabilization of metastable form Temperature stability range Solubility of each polymorph Method of preparation Effect of micronization Excipients incompatibility
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Stability characteristics:
If a compound exist polymorphism, one of the form will be more stable (physically)then the other form. Depending upon relative stability there are two forms of polymorphs: 1)stable form having lowest energy state, highest melting point and least aqueous solubility.2)Metastable form having higher energy state, lower melting point and higher aqueous solubility.
Ostwald Rule of Stages (Step Rule):Statement :- It is not the most stable state with the lowest amount of free energy that is initially formed but the least stable lying nearest in free energy to the original state.
Relative solubility of polymorphs:-In order to asses the relative increase in

solubility of polymorphs with respect to another, a simple solubility ratio can be defined:Solubility ratio =solubility of metastable form/solubility of stable form Examples Compound glybuzole(37) Quinolone dvt Tetracycline Fluconazole(ll/l) Aspirin Carbamazepine Lamivudine(7) Piroxicam(ll/lll) Indomethacine Methyl prednisolone(41) Etoposide(28) Succynil sulfathiazole Niclosamide(A/H) Solubility ratio 1 1 1 1.1 1.2 1.2 1.2 1.3 1.4 1.7 1.9 12.7 22.9
From above example, we can say that solubility ratio is higher than one just because of relative higher solubility of metastable form, that leads to increase inapparent solubility. The theoretical estimation of solubility can be done using following equation:DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10) Log Xic=Sm(Tm-T)/2.303Rt
POLYMORPHISM
Where Xic=ideal solubility Sm=entropy of melting Tm=temperature R=gas constant (1.98cal/k.mol) or (8.28J/k.mol) But solubility of metastable form may never reach to theoretical solubility due to crystallization of most stable form so data may not be consistent if we use above equation.
Dissolution behavior of the polymorphs:-the absorption rate and bioavailability

of drug administered orally is controlled by many factors among which dissolution rate is one of the most important. Therefore physicochemical state such as polymorphism or amorphism of drugs affect bioavailability of pharmaceutical preparation. This is due to the fact that, as the thermodynamic activity of polymorph is lower there is lower apparent solubility and thus absorption is also less. Order of dissolution rate: Amorphous>metastable> stable
FORMATION OF METASTABLE POLYMORPHS:Preparation of metastable polymorphs requires, 1. Supersaturating conditions for the metastable form. 2. Crystallization of the metastable state before the stable polymorph forms. 3. Stable conditions for the metastable polymorph so that conversion to the stable form is prevented.
STABILITY OF METASTABLE FORM : The difference in melting point ( mp )between polymorphs is measure of the metastable polymorph stability. If mp<10c --neither is significantally more stable. If mp is 25-500clower melting species will be difficult to crystallize and will revert rapidly. If mp is 1-250cunstable form can be obtained easily before solid-solid transition.
FACTORS AFFECTING POLYMORPHISM:(A)Temperature and humidity:Storage conditions affect physicochemical reactions which are accelerated at higher temperature.(arhenious theory). Humidity acts as catalyst on the solid surface. Therefore both are the important factors for the prefomulator scientist to consider.eg. DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
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1. Chlortetracycline hydrochloride has two different polymorphs form: and . Alpha form is stable up to 82% RH while beta form is hygroscopic. 2. Zanoterone: The solid-state degradation rate of form IV (hemihydrate A) is found to be greater than that of form III at 40C/25% RH and 40C/75% RH. At 40C/75% RH, the rate of degradation is 4-fold higher for form IV vs. form II. 3. Polymorphic transformation of phenylbutazone and cocoa butter occur after heating. 4. Recent studies done on paracetamol.It consist of form-1 (monoclinic) and form-2 (orthorhombic) At high temp(500k)form-II is stable 5. Leflunomide Form I and Form II.Here Form I : Stable below transition temperature. (127 c)
(B)Photostability:Generally light sensitive drugs are protected from the photolytic degradation by packing them suitably in light resistant container. However the bulk powder of the stable crystalline forms resists photochemical degradation and does not require light resistant system. But still there are fewer reports: 1. For eg:- Photostability of two polymorphs of tamoxifen citrate upon irradiation by visible light and u.v. light investigated using chromatography and spectroscopy. The surface color of pellets prepared with either crystal forms turned from white to brown but the extent of the color change in cross section of form A pellets was deeper than that of form B pellets. So form A exhibit higher degree of Photo-instability relative to that of form B. 2. Acetametacin: , :- Stable and :- Unstable
(C) Effect of solvent:Solvent can bring dramatic change in growth mechanism and morphology. Growth kinetic of crystal growing from solution was determined by two important factors: Degree of molecular roughness Nature of absorption of the solvent from surface.
(D) Effect of grinding:Since the physicochemical properties of the same drug polymorphs are affected by mechanical energy .It is very important tool to be taken under consideration by preformulator scientist. Grinding process reduces particle size, so increasing specific surface area and that why direct effect on dissolution rate and bioavaibility of the preparation. During grinding process solid state polymorphic transformation in to non crystalline or metastable form is caused by mechanical action. eg. Prasterone sulfate dihydrate Here dihydrate form is more stable than anhydrous form. With increasing grinding time compound become unstable because grinding weakened DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
POLYMORPHISM
bonding crystals and water molecules participating in hydrolysis process of the drug. The grinding process can easily induce the polymorphic transition of famotidine from form B to form A and this was accelerated by thermal effect.
(e)Effect of tablet compression:The mechanical stability and compaction behaviour of the polymorphic form of drug during tableting are very important in practice. Eg. Polymorphic transformations of phenylbutazone in which form 3 is converted to 2 form2 at > 2000kg/cm .
EFFECT OF POLYMORPHISM ON BIOAVAILABILITY:If the absorption of active ingredient in drug through G.I.T. is dissolution rate dependent then polymorphism is an important preformulation tool. Here successful utilization of polymorph having significant greater thermodynamic activity (solubility)may provide good therapeutic blood level from otherwise inactive drugs.eg. novobiocin, identified in two different forms : crystalline and amorphous. In tablet or capsule formulation novobiocin is used as sodium salt which is active orally but unstable chemically while insoluble form is stable chemically and orally inactive.(unabsorbable)
HIGH THROUGHPUT CRYSRTALLIZATION:A high-throughput (HT) crystallization system uses a combinatorial approach to solid form generation, where large arrays of conditions and compositions are processed in parallel. Screen 1000s of crystallization conditions. Small amount of API is required. Variety of solvents, additives, conditions necessarily generates large set of data. Solid form discovery in highly polymorphic form. A fully integrated HT crystallization system consists of experimental design and execution software, robotic dispensing and handling hardware, automated high-speed micro-analytical tools, integrated cheminformatics analysis software. Systems designed to carry out these experiments generally consist of both hardware and software components that drive and track experimentation, and permit data storage, retrieval and analysis. Such systems should be designed to be flexible and scalable to ensure that a variety of experimental procedures can be carried out either serially or concurrently. Thus, the system can be employed at various stages of drug development, where differences exist in the quality and quantity of compound available. While it is highly desirable to have the ability to mine and model experimental data, and to use the subsequent knowledge to guide further experiments, not all HTcrystallization systems are equipped with these features.
Paper 910101 M.Pharm-(2009-10)
POLYMORPHISM
CONCLUSION:If it appears that polymorphism is occurring or is likely to occur in the samples supplied for preformulation wok then a cooperative study with the bulk chemists should determine the most stable form chemically and physically. Differences in the solubility and melting point must also be assessed and then a decision can be made to determine which form to progress through to the next stage of formulation. Small difference in the stability but higher solubility of a relatively metastable form may lead to a preferential choice of a polymorph other than stable form but this is unlikely and is not encouraged by regulatory authorities. Risk associated with using the metastable form is that it will convert back to the stable form during the products life, and give a consequent change in properties. As polymorphism can have such serious consequences for the bioavailabilities of drugs with low aqueous solubility, it is essential that manufacturers check for the existence of polymorphism and ensure that they use the same appropriate polymorphic form every time they make a product.
REFERENCES:1) The theory and practice of industrial pharmacy By- Leon Lechmann -Joseph L Kanig 2) Biopharmaceutics and pharmacokinetics By-Dm Bhramankar -Sunil Jaiswal 3) Physical pharmacy by Alfred Martin 4) Pharmaceutics:-The science of dosage from design, by-Michael E Aulton. 5) 6) 7) 8) Encyclopedic of pharmaceutical technology Vol 2 Encyclopedic of pharmaceutical technology Vol 12 Encyclopedic of pharmaceutical technology Vol 15 Encyclopedic of pharmaceutical technology Vol 18
9) Ansels pharmaceutical dosages form and drug delivery system VIII edition. 10) 11) 12) 13) JPS vol. 96-9 sept.2007 Remington- the science and practice of pharmacy Drug stability second edition by Jens T carstensen Chemical abstract vol-146 no-8 2007
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14) 15) 16) 17)
Chemical abstract vol-147 no-12 2007 Chemical abstract vol-147 no-162007 Chemical abstract vol-147 no-18 2007 Chemical abstract vol-148 no-23 2008
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Chapter-1
CRYSTALLINITY & PARTICLE SIZE
SEMINAR ON
CRYSTALLINITY & PARTICLE SIZE,PARTICLE SIZE DISTRIBUTION

AS A PART OF PREFORMULATION STUDIES
GUIDED BY: Dr.R. K. Parikh
PRESENTED BY:Jignasha R. Bhuria M. Pharm Sem-1 Batch:-2009-2010 Roll no:-05
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CONTENTS:1. INTRODUCTION 2. CLASSIFICATION OF CHEMICAL COMPOUNDS 2.1 AMORPHOUS 2.2 POLYMORPHS 2.3 SOLVATES 2.4 CLATHRATES 3. COMPARISON OF CRYSTALLINE AND AMORPHOUS FORM 4. COMPARISON OF SOLUBILITY OF CRYSTAL, SOLVATE & HYDRATE 5. CRYSTAL STRUCTURE AND MORPHOLOGY 5.1 TYPES OF CRYSTALS 5.2 CRYSTAL SYSTEM 5.3 CRYSTAL HABIT 6. CRYSTALLIZATION 6.1 DEFINATION 6.2 CRYSTALLIZATION PROCESS 7. ANALYTICAL METHOD FOR CHARACTERIZATION OF CRYSTAL FORMS 8. IMPORTANCE OF CRYSTALLINITY IN PREFORMULATION STUDIES 9. LATEST TECHNIQUE DEVELOPMENTS FOR CRYSTALLIZATION 9.1 SPHERICAL CRYSTALLIZATION 9.2 CONTROLLED CRYSTALLIZATION 9.3 AMORPHOUS FORM STABILLIZATION 9.4 SUPER CRITICAL FLUID CRYSTALLIZATION
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1. INTRODUCTION
1.1 WHAT IS A CRYSTAL? A crystal is a solid in which the constituent atoms, molecules, or ions are packed in a regularly ordered, repeating pattern extending in all three spatial dimensions. The interatomic distance in a crystal of any material are constant .It can be used for the identification of material the crystal shape independent of size.eg.sugar crystal 1.2 ADVANTAGE OF CRYSTAL Better appearance Easy of filtering and washing Solubity ,density will be same no batch varition Crystal are always pure &attractive Easy to store,not form cake on storage.
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2. CLASSIFICATION OF CHEMICAL COMPOUND:-
CHEMICAL COMPOUND
HABIT
INTERNAL STRUCTURE
CRYSTALLIN E
SSSSSSTUCT URE
AMORPHOUS
SINGLE ENTITY POLYMORPH S
MOLECULA R ADDUCTS
NON-STOICHIOMETRIC INCLUSIONS
STOICHIOMETRIC SOLVATES (HYDRATE)
CHANNEL
LAYER CLATHRATE (CAGE)
2.1 AMORPHOUS COMPOUND: They have atoms or molecules randomly placed as in a liquid. They are typically prepared by:Lyophilization. E.g. Fluprednisolone in tert-butanol. Rapid quenching of chloramphenicol palmitate solution in hydrophilic solution. Rapid quenching of melted chloramphenicol palmitate in the refrigerator to -10
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Precipitation is also used to prepare the amorphous prompt insulin zinc suspension. Amorphous forms are usually of higher thermodynamic energy than corresponding crystalline forms, so solubilities as well as dissolution rates are generally greater but due to high energy they are unstable and tend to revert back to a stable form. This is particularly true for formulations like aqueous suspensions. In case of amorphous novobiocin suspension it slowly converts to a crystalline form and thus becomes less and less absorbable and finally looses therapeutic effect. The best agents found were methylcellulose, polyvinylpyrollidone, and several alginic acid derivatives such as sodium alginate and propylene glycol algin.
2.1.1 Characterization of amorphous solids:The only positive way to differentiate amorphous from crystalline solids is by means of X-ray powder diffraction. This technique gives very diffuse reflections of amorphous compounds, where the d distances, the distance between parallel planes in which the atoms of the crystal lie, cannot be determined as is done with crystalline solids.
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2.2 POLYMORPHS:Many drug substances can exist in more than one crystalline form with different space-lattice arrangements. This phenomenon is known as polymorphism and the different crystalline forms as polymorphs. Drugs like barbiturates and steroid hormones have polymorphic forms. 2.3 SOLVATES (PSEUDOPOLYMORPHISM):During crystallization from a solution , crystal separating may consist of pure component or be a molecular compound .The molecular compound may contain two or more constituents that have completely satisfied classical Valance force and are crystallize together as new single crystalline entity. Solvates are molecular complexes that have incorporated the crystallizing solvent molecule in their specific lattice position and in fixed stoichiometry. When the solvent incorporated in the solvate is water, it is called a hydrate. A classical method for distinguishing solvates from polymorphs involves observation of the melting behaviour of crystals embedded in silicon oil usingHOT STAGE MICROSCOPY, where upon heating, bubbles of
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solvent are generated by solvates. In case of polymorphs, no such generation occurs.
Example :- Steroid Estradiol form solvates with 30 solvent Hydrocortisone acetate Fluprednisolone Antibiotics Erythromycin Grmicidin Ampicillin Chlormphenicol Sulphanilamide METHOD USED TO IDENTIFICATION OF SOLVATES 1.Thermogravimetric analysis : Thermograms of different solvates shown different steps of desolvation which corresponds at a certain interval of temp. and pressure to the stable solvates . 2.Differential thermal analysis : Identify different hydrates of Phenobarbital , theophylline and other organic pharmaceuticals . 3.Differential scanning calorimetric : Identify the chloroform solvates of Griseofulvin and gas evaluation analysis can be done simultaneously with differential scanning calorimetry. 4.X-ray diffraction : This is used to differentiate fluprednisolon polymorph from solvate. APPLICATION OF SOLVATES
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Disolution :- The dissolution rate of solvate is many time greater than the anhydrous form on the other hand dissolution rate of hydrate is less than anhydrous form .The effect of the concentration of solvating solvent in dissolution medium when measuring dissolution of the pentanol solvate of fludrocortisone acetate , the dissolution rate was retarded by the addition of pentanol in dissolution medium . Bioavailability :- The difference seen in ampicillin bioavailability were due to the hydration of ampicillin since they found that solubility of two phase in dilute Hcl at 370 . It suggest that difference in bioavailability are related to formulation factor rather than when hydration state of the raw material . 2.4 CLATHRATES:A clathrate is a single-phased solid with two distinct components: the host and the guest. The guest is retained in the closed cavities provided by the crystalline structure of the host. Thus it is a non-stoichiometric molecular adduct. The major classes of clathrates are hydroquinone clathrates, water clathrates, phenol clathrates etc. 2.4.1PHARMACEUTICAL APPLICATIONS OF CLATHRATES: PURIFICATION- Benzene was purified of one of its usual contaminants thiophene by clathrate formation. Although both form clathrates with monoaminenickel cyanide, benzene is more firmly held in cage structure, so it is preferentially clathrated and separated from solution by filtration. SEPARATION OF RARE GASES- Argon is separated from neon by adjusting the pressure conditions in which hydroquinone-argon clathrate is formed, while neon do not form. SEPARATION OF OPTICAL ISOMERS- Inclusion complexing substance that will separate optical isomers is tri-o-thymotide. STORAGE OF INERT GASES- They are used for convinient storage of inert gases like hydroquinone or to introduce such gases into fairly inaccessible locations. The gas can be released by heating or dissolving the clathrates.
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MODE OF ACTION OF ANESTHETICS- Non-hydrogen bonding anesthetics work primarily due to clathrate formation of molecules of anesthetic agent with water contained in the neurons and around the neural network.
3. COMPARISON OF THE MECHANICAL PROPERTIES OFTHE COMPACTS OF THE CRYSTALLINE ANDAMORPHOUS FORMS OF A DRUG SUBSTANCE:-
AMORPHOUS FORM FORM Least ductile (highest indentation hardness value) Form compacts with lower tensile strength. Compacts have high brittleness value. Require lower compression stress to form compacts.
CRYSTALLINE More ductile (Low indentation hardness value) Form compacts with high tensile strength. Compacts have low brittleness value. Higher compression stress is required.
4. COMPARISON OF SOLUBILITY OF CRYSTAL, SOLVATE AND HYDRATE:Amorphous form is always more soluble than a corresponding crystalline form. The dissolution rates of hydrates are less than corresponding anhydrous crystalline form. E.g gluthethimide, theophylline, caffeine, succinyl sulphathiazole, phenobarbitol.
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The dissolution rates for organic solvates are higher than corresponding pure crystaline form. E.g. 1,4-dioxane solvate of nifedipine shows better solubility than dihydrate form. So organic solvates should be preferred in place of pure crystals which solves both problems, solubility and stability, but only if ICH guidelines about limits of organic residues permit MECHANISM OF CRYSTAL GROWTH The formation of crystal nuclei can be consider as a process that determine the size of the product .Control of the crystallization process to obtain a consitent and uniform crystal form, habit,density and size distribution is particularly critical for drug substance to be utilize in suspension and powdered . Eg. When the crystallization of sterile ceftazidine pentahydrate or modify to significantly increase the density to reduce the volume of the fill dose. The rate of dissolution increase significantly .Many dry solid parenteral product such as the cephalosporin are prepared by the sterile crystallization techanique. To obtain a uniform product from lot to lot , strict adherence to procedure develop for particular crystallization must be followed . Control of pH Rates of addition Solvent concentration & purity Temperature & mixing rate Each crystallization procedure has to design to ensure sterility and minimize particulate contamination .Eg. Changing the absolute ethyl alcohol instead of 95 % ethanol during washing procedure can desroy the crystalline structure . The size distribution of dispersed system may increase during aging to three principle mechanism : Ostwald ripening
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Polymorphic transformation Temperature cycling Ostwald ripening For Kelvin equation , ln s/s = 2y V/rRt Where, s = Solubility of infinitely large particle s = Solubility of small particle of radius r y = Surface tension V = Molar volume of solid Polymorphs exhibit different equilibrium solubility For Example Phenylbutazone identified by X-ray diffraction. The difference in solubility is driving force of crystal growth of suspension as the particle of more soluble form of polymorph go into solution and reprecipitate as less solution ie. More stable Temperature cycling may lead to crystal growth depending on temperature Solubility is directly related to temperature so slighst rise in temperature lead to increase equilibrium solubility. Precipitation occur to release the upersaturation and crystal growth occur.
HOW THE CRYSTAL HABIT IS ARISES?

It is possible to change the external shape of the crystal by changing the crystallization condition.With any crystalline material , the largest face is always slowest growing.the reason for that can be seen from the figure. If the drug is deposited on the two faces of the hexagonal crystal habit, then the first consequences is that the face where drug is deposited actually becomes a smaller part of the crystal, whereas the
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other faces become larger. Eventually the fastest growing faces will no longer exist. The growth on different faces will depend on the relative affinities of the solute for the solvent and the growing faces of the crystal.
5. CRYSTAL STRUCTURE & MORPHOLOGY:A crystal structure is a unique arrangement of atoms in a crystal. A crystal structure is composed of a motif, a set of atoms arranged in aparticular way, and a lattice Any crystal is characterized by its internal structure and habit. Habit is the description of the outer appearance of a crystal whereas the internal structure is the molecular arrangement within the solid. 5.1 Crystals are of two types: Irregularly shaped crystals known as anhedral or allotriomorphic. Definite shaped crystals bound by plane faces known as euhedral or idiomorphic. Anhedral crystals, although irregularly shaped, have a regular arrangement of building units which may be proved by X-ray diffraction.
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5.1.1 crystal can be classified by the type of bonds between two particles in a crystal. Metallic crystals having strong metallic bond. Ionic crystals having electrostatic ionic bond. Crystals having strong carbon covalent bond. Crystals bonded by vanderwaal forces. 5.2 CRYSTAL SYSTEM :- (INTERNAL STRUCTURE) For all crystals there are seven basic or primitive unit cells, as shown in Fig.1. We represent the side lengths as a, b and c and the angles as (between sides b and c), (between sides a and c) and (between sides a and b). The structure have atoms or molecules at each corner of unit cell. It is also possible to find unit cells with atoms or molecules at the centre of the top and bottom faces (end-centered), at the centre of every face (facecentered) or with a single atom in centre of crystal (body-centered). The most symmetric system is cubic system. The other six systems, in order of decreasing symmetry, are hexagonal, tetragonal, rhombohedral (also known as trigonal), orthorhombic, monoclinic and triclinic. Thus there are fourteen types of unit cell and we call these the Bravais lattices. For drugs there are only three common types of unit cell: triclinic, monoclinic and orthorhombic. We can identify the various planes of crystal using the system of Miller indice Cubic: sodium chloride Hexagonal: iodoform Tetragonal: urea Orthorhombic: iodine Monoclinic: sucrose Triclinic: boric acid
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Crystal system
Lattices:
triclinic
Simple
base-centered
monoclinic
orthorhombic
Simple
base-centered
body-centered
hexagonal
rhombohedral (trigonal)
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Simple body-centered
tetragonal
Simple cubic (isometric)
body-centered
face-centered
hexagonal
rhombohedral (trigonal)
Simple
body-centered
tetragonal
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Simple cubic (isometric)
body-centered
face-centered
5.3CRYSTAL HABIT:Crystal habit is the description of the outer appearance of a crystal. There are five types of crystal habit widely recognized: Platy: plates Tabular: moderate expansion of two parallel faces Prismatic: columns Acicular: needle-like Bladed: flat acicular These occur in all the six system.
5.3.1 METHODS OF MODIFICATIONS OF CRYSTAL HABIT:-
Excessive supersaturation. E.g. transform a prism or isodiametric crystals to needle shape. Cooling rate and agitation. E.g. naphthalene gives thin plates if rapidly cooled whereas slow evaporation yields prisms. The crystallizing solvent. E.g. resorcinol produces needles from benzene and squat prisms from butyl acetate. Similarly, iodoform crystallizes as hexagonal bipyramids from aniline and as prisms from cyclohexane.
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Addition of co-solvents or solutes. E.g. sodium chloride is cubic but urea produces octahedral habit.
Crystal habit can also be modified by adding impurities or poisons; for example, sulphonic acid dyes alter the crystal habit of ammonium, sodium and potassium nitrates. It can be quantitatively expressed in terms of aspect ratio (AR), defined as the ratio of length to width and values of AR approaching 1 (spherical or cube shape) are considered to be pharmaceutically good. It is preferable to keep the AR values below 5 so as to avoid problems with flow. AR in polar solvents was as high as 9.4 in comparisons with 5-6 in non-polar solvents.
6. CRYSTALLIZATION
6.1 DEFINITION Crystallization is the (natural or artificial) process of formation of solid crystals from a uniform solution. Crystallization is also a chemical solidliquid separation technique, in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs.
6.2 The crystallization process consists of two major events. Nucleation is the step where the solute molecules dispersed in the solvent start to gather into clusters, on the nanometer scale (elevating solute concentration in a small region), that becomes stable under the current operating conditions. These stable clusters constitute the nuclei. Nucleation can occur spontaneously or induce artificially by any foreign surface. These two cases are referred as homogenous and heterogenous nucleation respectively.
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The crystal growth is the subsequent growth of the nuclei that succeed in achieving the critical cluster size. Occur through 4 stagestransport through or from the bulk solution to an impingement site, which is not necessarily final site Adsorption at impingement site, where precursors may shed solvent molecules. Hence solvent must be transported back in soln. Diffusion of growth units of precursors from site of impingement to growth site. incorporation into lattice; for precursors, after desolvation. Thus, the growth site may also be a source of solvent that has possibility of, again, being adsorbed before escaping into soln.
Nucleation and growth continue to occur simultaneously while the supersaturation exists. Supersaturation is the driving force of the crystallization, hence the rate of nucleation and growth is driven by the existing supersaturation in the solution. Depending upon the conditions, crystals with different sizes and shapes are obtained. Once the supersaturation is exhausted, the solid-liquid system reaches equilibrium and the crystallization is complete, unless the operating conditions are modified from equilibrium so as to supersaturate the solution again. 6.2.1 Crystallization can be achieved by various methods, with 1) Solution cooling 2) Addition of a second solvent to reduce the solubility of the solute. (technique known as anti-solvent or drown-out) 3) Chemical reaction 4) Change in pH being the most common methods used in industrialpractice.
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Other methods, such as solvent evaporation, can also be used.
FIG.1 PROGRESSION OF CRYSTALLIZATION
Measurements of crystal growth rate:Divided into two groups, direct and indirect methods.
DIRECT MEASUREMENTS OF LINEAR CRYSTAL GROWTH RATESSingle crystals are always used for this purpose. Direct measurement of crystal dimensions under microscope (at beginning & after ending of experiment). Measurements using a travelling microscope, permitting continous observation of growth during experiment. Measurements using a screw micrometer.
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In addition, the growth rate can be calculated from measured increase in mass or volume. Gravimetric measurements with single crystals, in which increase in crystal mass is determined. The crystal is either weighed before or after ending experiment or weighed continously by suspending it on one arm of an analytical balance. The fluidised layer method, where the increase in mass of a greater number of crystals in fluidised bed in flowing soln is measured.
INDIRECT METHOD OF LINEAR CRYSTAL GROWTH RATEMeasurement in an agitated batch crystallizer, where the increase in crysta mass in crystalline suspension is measured. Measurement in a continous MSMPR crystallizer(mixed suspension,mixed product removal). Measurement of decrease in supersaturation in batch crystallizer.
7. ANALYTICAL METHODS FOR CHARACTERIZATION OF CRYSTAL FORMS :-
METHOD SAMPLE 1.Microscopy 2.Fusion methods (hot stage microscopy) 3. Differential scanning calorimetry (DSC/DTA) 4. Infrared spectroscopy 5. X-ray powder diffraction 6. Scanning electron microscopy 7. Thermogravimetric analysis 8. Dissolution/solubility analysis
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MATERIAL REQUIRED per
1mg 1mg 2-5mg 2-20mg 500mg 2mg 10mg mg to gm
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8. IMPORTANCE OF CRYSTALLINITY IN PREFORMULATION STUDIES:8.1 EFFECT ON SOLUBILITY & BIOAVAILABILITY The antibiotic, novobiocin is essentially inactive when administerd in crystalline form, but in amorphous form, absorption from g.i.t proceeds rapidly with good therapeutic response. Thus due to difference in solubility amorphous novobiocin is 10 times more bioavailable. The hormone insulin presents another striking e.g of different degree of activity that may result from use of different physical forms of it. Insulin is a protein that forms an extremely insoluble zinc-insulin complex when combined with zinc in presence of acetate buffer. Depending upon the pH of acetate buffer sol, the complex may be an amorphous ppt or crystalline material. NO. TYPE OF INSULIN 1 Prompt insulinzinc suspension (semilente) Extended insulinzinc suspension (ultralente) Insulin-zinc suspension (lente) FORM OF INSULIN ONSET OF ACTION Amorphous fast DURATION OF ACTION Short
Crystalline
slow
Long
30%amorphous+ 70%crystalline
fast
Intermediate
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The more soluble form of chloramphenicol palmitate, form B shows greater bioavailability after oral administration than least soluble form A. Similarly in chlortetracyclin hydrochloride form is more soluble and bioavailable than corresponding form. 8.2 CHEMICAL STABILITY In other instances, crystalline forms of drugs may be used because of greater stability than corresponding amorphous forms. e.g. crystalline forms of penicillin G as potassium or sodium salt are more stable. 8.3 SUSPENSION SYRINGEABILITY It is mostly a mechanical effect. A suspension of plate shaped crystals may be injected through a needle with a greater ease than one with needle shaped crystals of same dimensions. 8.4 EFFECT ON GRANULATION Sulphathiazole can exist in different crystalline forms out of which form III has water adsorption of 0.046mg/m2 while form I has water adsorption of 0.031mg/m2. so form III shows better wetting and so easy granulation. Use of amorphous form of calcium pentothenate in multi-vitamin tablets prepared by wet granulation process, is not desirable because polymorphic transformation makes the granulation mass sticky, making futher granulation virtually impossible. 8.5 HARDNESS OF TABLET Sulphamerazine is available in two different crystalline forms SMZ-I & SMZII. SMZ-II tablets show faster dissolution rate than SMZ-I due to difference in compressibility of both the forms. SMZ-I forms harder tablets than SMZ-II at same compression pressure and so it shows delayed release.
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Both these forms can be used in single tablet by compression coating in which the core is formed of SMZ-I and coat is made up of SMZ-II to get repeat action. 8.6 EFFECT ON CONSOLIDATION Substances possessing the cubic lattice arrangement were tabletted more satisfactorily than those with rhombohedral lattice. The isotropic nature of former group contribute to better tabletting because no alignment of particular lattice planes is required. In addition provide three equal planes for stress relief at right angles to each other. 8.7 DIRECTLY COMPRESSIBLE EXCIPIENTS The DC grade excipients are microgranulations, since they consist of masses of small crystallites randomly embedded in a matrix of glue-like (often amorphous) material. Such a combination imparts the desired overall qualities which results in strong tablet by providing a plastically deforming component (the matrix) to relieve internal stresses and strongly bonding surfaces (the faces of crystallites) to enhance consolidation. 8.8 POLYMORPHIC TRANSFORMATION Many drugs undergo polymorphic transformation during various processes. E.g. during grinding drugs like digoxin, estradiol, spironolactone, phenylbutazone undergo transformation. By granulation of theophylline with water converts into monohydrate from anhydrous form. Similarly by drying and compression also drugs undergo change in their form.
9. LATEST TECHNIQUES DEVELOMENTS IN CRYSTALLIZATION:9.1 SPHERICAL CRYSTALLIZATION-
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It has been developed by Yoshiaki and co-workers. It is a solvent exchange crystallization method in which crystal agglomeration is purposefully induced through the addition of third solvent termed as Bridging liquid which act as granulating agent. It is a novel technique to improve compressibility, good flowability and bioavailability of pharmaceuticals. In addition to this the tablets manufacture by this technique have greater mechanical strength and lower friability. Various drugs have been successfully undergone this process to acquire improved micromeritic properties like salicylic acid, mefenemic acid, aminophylline, tolbutamide and thus have shown increased dissolution rate.
METHODS OF SPHERICAL CRYSTALLIZATION
9.1.1 SIMPLE SPHERICAL CRYSTALLIZATION It is achieved by change of solvent or salting out. It causes formation of fine crystals and agglomeration. E.g. spherical crystallization of salicylic acid from ethanol by addition of water, using chloroform as bridging unit.
9.1.2 QUASI-EMULSION-SOLVENT-DIFFUSION METHOD Uniformly coated directly compressible agglomerates are obtained by using mixed system of two or three partially miscible solvents, i.e. bridging liquid-poor solvent system or good solvent-bridging liquid-poor solvent system. E.g. antirheumatic drug bucillamine was crystallized as spheres by this method using HPMC.
9.1.3 AMMONIA DIFFUSION METHOD

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Useful for amphoteric drugs like enoxacin which is slightly soluble in water but soluble in acidic and alkaline solution. A mixture of three partially immiscible solvents, acetone-ammonia water-dichloromethane, was used. Here ammonia water acts as a bridging liquid and also good solvent for enoxacin. Acetone is water miscible but poor solvent. Thus enoxacin gets precipitated by solvent exchange without forming ammonium salt.
9.1.4 NEUTRALIZATION METHOD Tolbutamide dissolved in sodium hydroxide and HPEC aqueous solution was crystallized using this method. Hydrochloric acid was added to neutralize the solution and crystallize out fine crystals of drug.
9.2 CONTROLLED CRYSTALLIZATION-
Very useful method for getting microcrystals in very narrow size range for hydrophobic drugs. It is more effective than micronisation because it gives better bioavailability due to uniform sized particles. E.g. anti-inflammatory drug betamethasone dipropionate, triamcinolone acetonide, beclomethasone. Here the side effects caused by drug deposition in the throat is avoided and administered amount of drug can be lowered. This method was performed using solvent change method by instataneously mixing two liquids in presence of HPMC as stabilizing agent.
9.3 AMORPHOUS FORM STABILIZATION-
Amorphous forms have highest solubility but it is very unstable and on storage may get convert to crystalline form. So scientist developed a
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technique to stabilize the amorphous form. Drugs like ketoprofen, indomethacin, naproxen and progesterone were used for this purpose. NEUSILIN (amorphous magnesium alluminium silicate) was milled in the ball mill with the above drugs. The amorphous form thus formed was more stable than normal amorphous form and did not turned to crystalline form easily. Neusilin is an amphoteric compound so can be combined with both acidic and alkaline groups. Thus by using additives with high glass transition temperature or by selective hydrogen bonding with the stabilizing additives conversion of amorphous form to crystalline form can be prevented.
9.4 SUPER-CRITICAL FLUID CRYSTALLIZATION-
Useful method for selective production of polymorphs and pseudopolymorphs. In this technique precipitation of small organic molecules from aqueous solution was studied using a mixture of supercritical CO2 & ethanol as drying medium and as anti-solvent. Glycine has three polymorphs and can be selectively precipitated to either pure or form. When increase the ethanol concentration precipitation of metastable -glycine was preferred. Similarly increase ethanol concentration in extractant phase favoured precipitation of phenylalanine anhydrate over monohydrate form.
CONCLUSION
Thus, with all examples of the effects of habits, polymorphs, solvates and clathrates on optimising pharmaceutical formulations, the crystal chemistry has become a routine part of every pharmaceutical companys preformulation programme.
REFERENCES
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Pharm. Dosage forms and drug delivery system , ANSEL, 100, 151. Pharm. Dosage forms, LACHMANN and LIBERMANN, 1, 26-30. Modern pharmaceutics, BANKER,MARSHALL DEKKER INC. Pharm Encyclopedia, 3, 399. Pharm Encyclopedia, 12, 320-321. Advanced pharmaceutical solids, CARSTENSEN, 110, 6. Physical pharmacy, ALFRED MARTIN. Industrial pharmacy, LACHMANN and LIBERMANN. Physico-chemical principles of pharmacy, A.T.FLORENCE & D.ATTWOOD, 8-10. Pharmaceutics-the science of dosage form design, M.E.AULTON, 142-149. Pharmaceutical sciences, REMINGTON, 1358. Journ. Of Pharm. Sciences, (2007), 96, 990. Journ. Of Pharm. Sciences, (2006), 95, 26-30. Journ. Of Pharm. Sciences, (2006), 95, 446. Journ. Of Pharm. Sciences, (2006), 95, 1641. Journ. Of Pharm. Sciences, (2003), 92, 35-46. Journ. Of Pharm. Sciences, (1989), 78, 68-72. Journ. Of Pharm. Sciences, (1987), 76, 471-474. Journ. Of Pharm. Sciences, (1984), 73, 1407-1410. Journ. Of Pharm. Sciences, (1975), 64, 1264. Journ. Of Pharm. Sciences, (1963), 52, 781-791. Journ. Of Pharmaceutics and Pharmacology, (1975), 28, 94. Pharmaceutical research, (1994), 11. Int. Journ. Of Pharm., (2002), 241, 73-85. Advanced Drug Delivery Reviews, (2007), 59, 617-630. Chemical Abstract, (2007), 147(12), 1058.
1. Introduction of Particle size. a. Definition b. Classification of powder 2. Introduction of particle size distribution.
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a. Definition b. Methods for analysis c. Properties of drug that are affected by particle size d. Importance of Particle and particle size distribution. 3.Introduction of particle shape.
PARTCLE SIZE Definition:Particle size is a notion introduced for comparing dimensions of solid particles, liquid particles (called droplets), or gaseous particles (called bubbles) . The notion of particle size applies to: Colloidal particles Particles in ecology Particles present in particulate matter Particles that form a granular material The particle size of a spherical object can be unambiguously and quantitatively defined by its diameter. The above quantitative definition of particle size cannot be applied to non-spherical particles. GENERAL CLASSIFICATION OF PARTICLES BASED ON THEIR SIZE:Type of particle Coarse Powders Conventional Powders Fine Particles Very fine Particles Ultra-fine Particles Mesh opening size > 1000 m 50 m - 1000 m 1 m - 50 m 0.1 m - 1 m < 0.1 m
TYPE OF POWDER ACCORDING TO PRTICLE SIZE:Monodisperse powder:- all particles are of same size. Polydisperse powder:- particles of different size.
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Generally powder sample contains no. of irregular shape three dimensional particles so generally we consider Avg. Ps. Average particle size: Average size of the particles which are distributed in system. dmean = ( ndp+f / ndf)1/p p=1-particle length, p=2-surface, p=3-expression of volume, p=+ve arithmetic mean p= -ve harmonic mean, p= zero geometric mean PARTICLE SIZE DISTRIBUTION Definition:- The particle size distribution (PSD) of a powder, or granular material, or particles dispersed in fluid, is a list of values or a mathematical function that defines the relative amounts of particles present, sorted according to size. Systems for Collection of particle are practically always polydisperse. METHODS FOR PARTCLE SIZE ANALYSIS METHOD NOMINAL RANGE ( m ) <0 2-74 0.5-500 0.002-15 0.05-500 0.003-3.0 0.1-600 Martin s, Feret s, or equivalent circle diameter Volume weighted diameter Volume weighted mean diameter Volume weighted mean diameter Equivalent Spherical diameter [ ESD ] ESD ESD 0.05-50
SIZE DETERMINED
Sieving Dry Wet Microscopic Optical Electron Electrical Zone Sensing Photon Correlation Spectroscopy Laser Diffraction Elutriation Laminar flow Cyclone Centrifugal Classification Centrifugal Sedimentation
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3-75 8-50 0.5-50
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Mass accumulatiom Photo-extinction X-ray Gravity Sedimentation Pipettes & Hydrometers X-ray Hydrodynamic Chromatography Capillary Column Cascade Impactors Gas Permiability Gas Adsorption Nephelometry
0.05-100 0.01-5 ESD 1-100 0.2-65 ESD 0.1-6 0.05-30 0.01-40 0.005-50 >0.1 Aerodynamic diameter Mean surface weighted diameter Mean surface weighted diameter Total light scattering (size dependent)
01] MICROSCOPY:RANGE OF ANALYSIS:- By transmission electron microscope 0.001-0.1 micron. - By scanning electron microscope 0.01-1000 micron. - By light microscope 1-1000 micron. METHOD:- An emulsion or Suspension in diluted or Undiluted form is mounted on a slide Or ruled cell and placed on a mechanical stage. - The microscope eyepiece is fitted with a micrometer by which the size of the particles may be estimated. - Sometimes the field can be projected onto a screen where the particles are measured more easily, or a photograph can be taken from which a slide is prepared & projected on a screen for measurement. - The popular Measurements for Particle size are Martins diameter
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Ferets diameter Projected Area Diameter - A Particle Size Distribution Curve is plotted as seen in figure.
particle size distribution curve USES:- Scientist named Prasad & Wan used Video Recording equipment to observe, record, store & retrieve particle-size data from a microscopic examination of Tablet excipients including MCC , Na CMC , Na Starch Glycolate & Methyl Cellulose. ADVANTAGES - Easy and convenient - A size-frequency distribution curve can be plotted. - Can detect the presence of agglomerates. DISADVANTAGES - Diameter is obtained from only two dimensions - length and breadth - No estimation of the depth (thickness) of particle is available
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- The number of particles that must be counted to get a good estimate of the distribution makes the method slow and tedious. 02] SIEVING:RANGE OF ANALYSIS:- 5-12000 m. METHOD:-This method utilizes a series of standard sieves calibrated by the National Bureau of Standards. Methods may be simple shaking of the sample in sieves until the amount retained becomes more or less constant. Alternatively, the sample may be washed through with a non-reacting liquid (usually water) or blown through with an air current. ADVANTAGES - simplicity, cheapness, and ease of interpretation. DISADVANTAGES - The smallest practical sieve size is 20-40 m, and many PSDs are concerned with much smaller sizes than this. - A 20 m sieve is exceedingly fragile, and it is very difficult to get material to pass through it. - The amount of energy used to sieve the sample is arbitrarily determined. Overenergetic sieving causes attrition of the particles and thus changes the PSD, while insufficient energy fails to break down loose agglomerates. AIR JET SIEVING METHOD:Principle: A reverser air jet circulator beneath the sieve mesh, blowing oversize particles away from the mesh to blocking.
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USE:- Impinging Air jet can remove Micrometer-size particles from solid surface. [Chemical Abstracts, 149(3); August 2008:56449r] 03] SEDIMENTATION:- These are based upon study of the terminal velocity acquired by particles suspended in a viscous liquid. - Sedimentation time is longest for the finest particles. - Stokes given a theoritical description of the motion of falling under the influence of gravity. dst=[18 /(Pp-PL) g ] - A no. of Techniques based on Sedimentation Methods utilizing devices such as The Andereasen apparatus pipette or Recording Balances. - This technique is useful for sizes below 10 m. - For sub-micrometer particles which can't be reliably measured by above method due to the effects of Brownian motion. The Typical apparatus used which diperses the sample in liquid, then measures the optical density of successive layers using visible light or x-rays. 04] ELUTRITION:- Elutriation is a procedure in which the fluid moves in direction opposite to sedimentation movement. For example the particle will move in vertically down wards direction and fluid moves vertically upwards direction. If velocity of fluid is higher then the particle are carried upwards and vice versa. - Size of particle that will separate depends on their Viscosity & Density. - Separation in to several fractions may be affected by using no. of vessels of increasing diameter with suspension entering through bottom. 05] ELECTRONIC SCANNING ZONE (COULTER COUNTER):PAPER-910101 L.M. COLLEGE OF PHARMACY
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An example of this is the Coulter counter, which measures the momentary changes in the conductivity of a liquid passing through an orifice that take place when individual non-conducting particles pass through. The particle count is obtained by counting pulses, and the size is dependent on the size of each pulse. Advantages - Fastest counting. - 1000 Particle count at one second. - More reliable since no of particles are counted. - To study particle Growth & dissolution and the effect of antibacterial agent on the growth of micro-organism. 06] CAPILLARY HYDRODYNAMIC FRACTIONATION:Sample particles are fractionated according to size as they flow in a capillary tube. The particles are detected at the capillary outlet by an online detector, typically an ultraviolet (UV) detector. Particle size is given by the elution or transit time of the particles in the capillary. This elution time depends only on the particle hydrodynamic size and is independent of particle chemical composition and density. 07] LASER LIGHT SCATTERING METHODS:- In this method particle can be presented either in liquid or in air suspension. - Both the large particle and small particle analyzers are based on the interaction of laser light with particles. FRAUNHOFER DIFFRACTION:For particles that are much larger than the wave length of light, any interaction with particles causes light to be scattered in a forward direction with only a small change in angle. This phenomenon is known as Fraunhofer diffraction, and produces light intensity patterns that occur at regular angular intervals and are proportional to the particle diameter producing the scatter different diameter particle may be considered to be the sum of all the individual patterns produced by each particle in the size distribution. 08] X-RAY DIFFRACTION METHOD:Principle:PAPER-910101 L.M. COLLEGE OF PHARMACY
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- An x-ray irradiation produce a highly specific diffraction pattern from a crystal of material. An X-ray diffraction pattern from the crystal is formed a series of dots of varying intensity with fixed angular & recorded on photographic film. - It is powerful tool for particle size analysis. ADVATAGES:- very sensitive - use in identification of polimorphs DISADVANTAGES:- Very expensive 09] NEPHELOMETRY:Measurement of the scattered light intensity is the basis of nephelometry. Method:- A beam of light is directed through the test sample. Detectors are placed to measure the 90-degree scatter, the forward-scattered light, & the light transmitted through the sample. Excellent linearity and colour rejection are attained by electronically comparing the ratio of the output of the 90degree detector to the sum of the other two detectors. The design of the optical system makes the effect of stray light negligible. 10] CASCADE IMPACTION:Size Range:-0.05-30 m. Material:-Particle of all kind Method:- Air samples are withdrawn through device which consist of several stages on which particles are deposited on impaction plate. - Particles will impact on certain stage depending on their size. - A series of scanning microscopy that may be identified & related to a size range in which elevated counts were noted with a particle counter. Uses:- In combination with scanning microscopy use to identify small particles present in the air. -To determine the distribution of particles of respirable size. E.g Aerosol.
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[ Chemical abstracts, vol:149, No:3 (62143c), July 2008. ] Limitation:- particles bouncing off - over loading - fluctuation 11] ROTATING DRUM METHOD Material:-Dry powder, Granulates, Friable products. Size Range:-0.5-10000 microns This method is suitable to determine the distribution of particle of respirable or inhalable size. PROPERTIES OF DRUG THAT ARE AFFECTED BY PARTICLE SIZE AND PARTICLE SIZE DISTRIBUTION Surface area Density, Porosity and Compressibility Angle of repose and Flow property Bulkiness and Packaging Criteria Hygroscopicity Electrostatic charge SURFACE AREA:As particle size decrease the surface area of particle increases. Surface area is important for drug absorption, dissolution, solubility and bioavailability. DENSITY: The ratio of mass to volume is known as the density. As bulk density of pwd decrease, decrease PS more air absorbed on to surface. POROSITY: - Is define as ratio of void volume to bulk volume of Packing. Particle of very small PS increase porosity through decrease flowability due to absorbed moisture or Vander Wallas force. FLOW PROPERTY:Flow property can be increased by addition of small particles in to larger particle which fill the void space.
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Chapter-1
ANGLE OF REPOSE:As Particle size decreases angle of repose decreases and due to cohesive forces flow property increases. BULKINESS AND PACKAGING :As particle size increase bulkiness decreases. It is a reciprocal of bulk density. Uniformity of powder blend is imp. HYGROSCOPYCITY:Decrease in particles size give larger surface area that will give high susceptibility for moisture absorption.
ELECTROSTATIC CHARGES:Particle size, PSD, cohesion, adhesion and electrical double layer property is most affected by it. IMPORTANCE OF PS AND PSD:1. Particle size affects many physical properties of drug like surface area, density, porosity, compressibility, moisture absorption, surface properties like solubility, absorption, dissolution and bioavailability. 2. Tablet: - PS and PSD is important for selecting granulation process it also affect average tablet weight variation, granules properties like uniformity of color, size uniformity, also uniformity of dose, absorption, dissolution and finally bioavailability. [ Chemical abstracts, vol:149, No:4 (87139u), July 2008. ] 3. Suspension: - Sedimentation Rate, Suspendibility, redispersibility, coalescence and agglomeration. 4. Aerosol:- Particle Size of drug affects site of absorption in the bronchopulmonary tract. [ Chemical abstracts, vol:149, No:3 (62179), July 2008. ] 5. Bioavailability:- Drug whose BA is increase by PS reduction are Sulphadiazine,
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Chapter-1
Phenothiazen, Tolbutamide, Spironolactone, Aspirin, Nitrofurantoin. Greseoflavin: - If micronized than increases rate of absorption and finally the dissolution. Penicillin-G and Erythromycin if PS decreases, surface area increases if remain more time in contact with GIF so increases degradation. Poorly soluble hydrophobic drug:- If PS is decreases then increases chance of formation of agglomerates. In case of Nitrofurantoin increase in bioavaibility may resulted in increase in its side effects. PS & PSD also affects the porosity and bulkiness so affects packing.
REFERENCES 1. 2. 3. 4. 5. 6. Pharmaceutical particulates Matter by Thomas A. Barber ,page-177,269,300 to 345 Pharmaceutics by M. E. Aulton, 2nd edition Page 156 Lyklema, J. Fundamentals of Interface and Colloid Science, vol.2, page.3.208, 1995 Hunter, R.J. "Foundations of Colloid Science", Oxford University Press, 1989 Dukhin, S.S. & Derjaguin, B.V. "Electrokinetic Phenomena", J.Willey and Sons, 1974 ISO Standard 9276-5 "Representation of results of particle size analysis" (2004) Retrieved from "http://en.wikipedia.org/wiki/Particle_size_%28general%29"
7. Jillavenkatesa A, Dapkunas S J, Lin-Sien Lum, Particle Size Characterization, NIST Special Publication 960-1, 2001 8. Sivakugan N, Soil Classification, James Cook University Geoengineering lecture handout, 2000 9. James P M Syvitski (editor) (2007). Principles, Methods and Application of Particle Size Analysis. Cambridge University Press. ISBN-13: 9780521044615. Retrieved from "http://en.wikipedia.org/wiki/Particle_size_distribution" 10. The theory and Practice of industrial Pharmacy by Lechman, 3rd edition, Page 67 11. Encyclopedia of Pharmaceutical technology Dekker, vol-11; Page 237. 12. Cooper and Gunns Tutorial Pharmacy, 6th edition, Pages 174. 13. Ansels Pharmaceutical Dosage Forms and Drug Delivery Systems. 8th edition page-101. Chemical abstracts, vol:149, No:3 (62179u),July 2008. Chemical abstracts, vol:149, No:3 (62143c), July 2008. Chemical abstracts, vol:149, No:4 (87139u), July 2008. Chemical abstracts, vol:149, No:3 (62179), July 2008. Chemical abstracts, vol:147, No:11 (261206), August2007. Chemical abstracts, vol:147, No:10 (240676), sept2007.
PAPER-910101
Paper-910101
Chapter-1
STUDY QUESTIONS
1.Discuss types of powders according to particle size and enlist methods for method of particle size analysis which properties of drug are affected by particle size and particle size distribution? (1st test 5th April 2006). 2. Explain different phases of powder compaction and its evaluation. (1st test 30th March 2005). 3. Differentiate consolidation and compaction of powders. March 2005). (1st test 30th
PAPER-910101
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
(PREFORMULATION)
GUIDED BY:
Dr. R.K.PARIKH
PRESENTED BY:
SAURABH M.PHARM -1 YEAR-2009-10 ROLL NO-03
PHARMACEUTICS & PHARMACEUTICAL TECHNOLOGY
L.M.COLLEGE OF PHARMACY
AHMEDABAD-380009
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
CONTENTS
1) INTRODUCTION 2) ROLE IN PREFORMULATION 3) CLASSIFICATION OF THERMAL ANALYSIS. 4) DIFFERENT METHODS OF THERMAL ANALYSIS. 5) COMBINED TECHNIQUES 6) GENERAL PRINCIPLES INVOLVED IN THERMAL ANALYSIS. 7) INNOVATION IN THERMAL ANALYSIS 8) MAJOR APPLICATIONS OF TA IN PREFORMULATION A) CHARACTERIZATION OF HYDRATES & SOLVATES. B) STUDY OF POLYMER. C) DETECTION OF IMPURITY D) THERMAL ANALYSIS AS SCREENING TECHNIQUE. E) STUDY OF POLYMORPHISM. F) PREDICTION OF STABILITY OF DRUG. G) STUDY OF DEGREE OF CRYSTALLINITY. H) STUDY OF DRUG EXCIPIENT INCOMPATIBILITY. 9) MINOR APPLICATIONS- DSC IS A VALUABLE TOOL IN CHOICE OF SUPPOSITORY BASE. - IN STUDY OF POLYMER COMPOSITION, MISCIBILITY & INDIVISUAL CHARACTERIZATION. - STUDY OF TABLET COATING - DETERMINATIONS OF MELTING POINT. ETC - DETERMINATION OF MOISTURE CONTENT IN DRUG. - CHEKING TECHNOLOGICAL QUALITY GRADE OF DISINTEGRATE. - STUDY OF SOLID DRUG DISPERSION. - DETERMINATION OF DRYING TEMP. FOR DIFFERENT EXCIPIENTS. 10) 11) 12) 13) FT-IR SPECTROMETER X-RAY POWDER DIFFERACTION LIMITATIONS REFERENCES
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
Thermal analysis
INTRODUCTION Definition:
Thermal method of analysis are group of techniques in which changes in physical and /or chemical properties of a substance are measured as a function of temperature, while substance is subjected to controlled temp programme.
Thermal analytical methods can measure the following physical properties, 1) 2) 3) 4) 5) 6) 7) 8) 9) WEIGHT LOSS ON DRYING ENTHALPY TEMP GAS EVOLUTION ELECTRICAL CONDUCTIVITY OPTICAL CHARACTERISTIC MAGNETIC PROPERTIES CHANGES IN FORM IN DIMENSION VISCOELASTIC PROPERTIES OF SUBSTANCE
ROLE OF THERMAL ANALYSIS IN PREFORMULATION 1) They are unique methods in the field of polymer analysis & of high value for a solid state analysis. 2) They finds wide application in A) Detection of impurity B) Determination of moisture content in any drug substance or any excipient C) Study of polymorphism D) Characterization of hydrates & solvates E) Degree of Crystallinity F) Study of phase diagram G) Drug excipient compatibility study H) Study of complexation
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
Classification of Thermal Analytical Technique

Physical properties
1) MASS
Name of technique
A)THERMOGRAVIMETRY B)EVOLVED GAS ANALYSIS
Instrument used
THERMO BALANCE EVOLVED GAS DETECTOR DTA APPRATUS DIFFERENTIAL CALORIMETER TMA APPRATUS DILATOMETER
2) TEMP 3) ENTHALPY
4) MECHANICAL PROPERTIES (DIMENTION,VISCOELASTIC PROPERTIES) 5) ACOUSTIC CHARACTERISTIC 6) OPTICAL CHARACTERISTIC 7) MAGNETIC SUSCEPTIBILITY 8) ELECTRICAL RESISTANCE OR CONDUCTION
DIFFERENTIAL THERMAL ANALYSIS(DTA) DIFFERENTIAL SCANNING CALORIMETRY(DSC) A)THERMO MECHANICAL ANALYSIS B)THERMODILATOMETRY A)THERMO ACOUSTIMETRY B)THERMOSONIMETRY THERMOPTOMETRY THERMOMAGNETOMETRY THERMOELECTROMETRY
OTHER THERMAL ANALYSIS

1) Thermophotometry: measures light intensity 2 Thermoluminescences: measures light emitted by sample 3) Thermomicroscopy: visual examination of phase transformation 4) Microthermal analysis: measures thermal conductivity 5) Differential mechanical analysis: measures modulus, damping & viscoelastic behaviour 6) Emanation thermal analysis: measures release of radioactive emation from a substance as a function of temperature. 7) Thermoparticulate analysis: measures release of particulate matter from a substance.
Combined techniques:
A) COMBINATION WITH THERMOGRAVIMETRY
1)FOR BETTER INTERPRETATIONS A) TG-DTA B) TG-DSC 2)FOR IDENTIFICATION OF GAS INVOLVED IN THERMAL ANALYSIS A) TG-IR B) TG-GC-MS C) TG-MS B) COMBINATION WITH DSC A) DSC-THERMOMICROSCOPY B) DSC-FTIR C) DSC-TEM d)DSC-X-RD DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
GENERAL PRINCIPLE INVOLVED IN THERMAL TECHNIQUE

1) THERMOGRAVIMETRY
PRINCIPLE: Tg is a technique in which a change in the weight of a substance is recorded as a function of temperature or time.
Instrument: Instrument used for thermogravimetry is thermobalance

Major components of a Thermobalance
1) Sample container ,usually shallow platinum crucible. 2) Furnace Assembly 3) Automatic recording Balance(Micro balance)
Factors affecting Thermogravimetry analysis are
1) 2) 3) 4)
heating rate furnace atmosphere crucible geometry sample characteristic
Data recorded in form of curve known as Thermogram. Thermograms can be divided into two portions: 1) Horizontal portion: indicate region where there is no weight loss. 2) Curved portion: indicate regions of weight loss.
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
2)Differential Thermal analysis (DTA)

PRINCIPLE: A Technique in which the temperature difference between a substance & a reference material is measured as a function of temperature, while the substance & reference are subjected to a controlled temperature programme. the Difference in temperature is called as Differential temp(t) is plotted against temp. or a function of time. Physical changes usually result in Endothermic peak ,whereas chemical reactions those of an oxidative nature are exothermic. Endothermic reaction (absorption of energy) includes vaporization, sublimation, and absorption & gives downward peak. Exothermic reaction (liberation of energy) includes oxidation, polymerization, and catalytic reaction & gives upward peak.
3) Differential scanning calorimetry

PRINCIPLE: It is a technique in which the energy necessary to establish a zero temp. difference between the sample & reference material is measured as a function of temp. Here, sample & reference material are heated by separate heaters in such a way that their temp are kept equal while these temp. are increased or decreased linearly. Endothermic reaction: if sample absorbs some amount of heat during phase transition then reaction is said to be endothermic. In endothermic reaction more energy needed to maintain zero temp difference between sample & reference. E.g. Melting, boiling, sublimation, vaporization, desolvation.
Exothermic reaction: if sample released some amount of heat during phase

transition, then reaction is said to be exothermic. In exothermic reaction, less energy needed to maintain zero temp difference between sample & reference. E.g crystallization, degradation, polymerization.
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
INSTRUMENT
Ideal DSC curve:
DSC Is widely used to measure glass transition temp & characterization of polymer. Glass Transition temp(Tg): Temp at which an amorphous polymer or an amorphous part of crystalline polymer goes from hard ,brittle state to soft, Rubbery state. 4) THERMO MECHANICAL ANALYSIS PRINCIPLE: A Technique in which changes in dimension of substance are measured as Function of temp. TMA is useful for the measurement of changes in shape(volume or dimension),penetration characteristic& viscoelastic properties of different material as a function of controlled temp elevation. DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
5) THERMOMICROSCOPY This Technique also known as Hot stage microscopy. This Technique essentially involves the observation of a sample through a
microscope fitted with a stage that can be heated or cooled at a controlled rate By This Method we can Determine a) investigation of the changes according to heat b) melt crystallisation c) detection of melting d) crystal transformation e) crystal pseudomorphs f) sublimation g) desolvation
6) MICROCALORIMETRY PRINCIPLE: Calorimetric technique deals with the measurement of heat evolved or
absorbed by chemical or physical process. Isothermal & adiabatic calorimetry are the diff ways of measuring the heat of samples maintained at constant temp. - The output of the instrument is measured by the rate of heat exchange (dq / dt) as a function of time.
INNOVATION IN THERMAL ANALYSIS a) Multielemental scanning thermal analysis(MESTA)

Method for the identification & characterization of solid substance. Simple,rapid,sensitive,alternative for routine examination of solid sample. Principle: Volatile components in the sample are carried to a high temp combustion tube where the C, N, S are oxidized to their respective oxides & detected by the detector. ADVANTAGE: SIMPLE Cost effectiveness of MESTA make it a promising tool for routine chemical analysis of solid substance. b) MICROTHERMAL ANALYSIS Microthermal analysis is a relatively new technique that combines the resolution of an Atomic forse microscope (AFM) with thermal conductivity measurement. MTA is useful for polymorph analysis. In this technique thermal conductivity Is measured as a function of temp. MTA used for Identification of components in compressed tablet Analysis of tablet coats. DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
c) Modulated DSC: Also called as oscillating DSC. Advantage of modulated DSC over traditional DSC method
Increased sensitivity Increased resolution Separation of complex transitions Measurement of crystallinity.
d) ROBOTIC SYSTEM
Most companies nowadays introduces to the robotic system with autosampling,
data manipulation. Also increased efficiency of analysis & increased accuracy.
e) DYNAMIC MECHANICAL ANALYSIS

Here the mechanical response of a sample is measured as it is deformed under oscillating
load against temp/time. Here, Dynamic Modulus and /or damping of a substance under oscillatery load is measured.
f) FAST SCAN DSC

FAST SCAN DSC distinguishes melting, melting degradation, sublimation,
& thermal stability of drug. Useful for stability study. Stability of drugs is based on a comparison of their thermal properties at various heating rates.
Application of thermal analysis in pre formulation
1. characterization of hydrates and solvates

Pre formulation studies is to identify the ability of drug to take up water and characterize the state of this water because this information is relevant in developing strategy for the process and storage of dosage form. TGA provides an important tool to characterize & quantify the moisture content in pharmaceutical material.
2. study of polymer
TGA: Thermogravimetric methods are largely limited to decomposition & oxidation
reaction & to such physical process like vaporization, sublimation, desorption. Qualitative analysis: Most important application of thermogravimetric methods are found in study of polymers.Thermogram provides information about decomposition mechanism for DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
various polymeric preparation. In addition, the decomposition patterns are characteristic for each kind of polymer& in some case can be used for identification purpose.
Quantitative information
Thermogram is also used for quantitative analysis of a polymeric material`. Example: polyethylene mixed with fine carbon black to inhibit degradation from exposure to sunlight. This analysis would be difficult by most other method.
3.
Detection of impurity
Thermal analytical system can be used for detection of impurities in pharmaceutical ingredient by recording TG Thermogram & DTA or DSC curves. This curves could be then be compared with the curve of reference standard . Any abnormal mass changes on TG curve & irregular endotherm or exotherm peaks on DTA or DSC curve would indicate the presence of impurity.
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
Basic of any calorimetric purity method A sharp melting endotherm indicates the relative purity where as broad asymmetric curve suggest impurity. The presence of minute amount of substance broadens its melting range & lowers its mp. Compare to other thermal methods, DSC is best method for detection of impurity. Eg. DSC of phenacetin.
4. study of polymorphism
Polymorphism is the tendency of a substance to crystallize in different crystalline modification. The all thermodynamic parameter in the polymorphism substance is different like melting, sublimation temperature, kinetics, stability, solubility, heat capacity, crystal hardness & shape which are extremely important for the dosages form. During preformulation, it is important to identify the polymorph that are stable & also imp. to determine whether polymorphic transition are possible within the temp. range used for stability studies, processing (drying, milling, mixing. granulation etc.) & storage. Eg. Mannitol occurs in four forms, all melting at the same temp. & they are non hygroscopic, only form B shows a small endotherm exotherm process.
Paper 910101 M.Pharm-(2009-10) 5.
THERMAL ANALYSIS
prediction of stability of drug

Stability test is one of the tests which req. the longest time in drug development. For a quick release of new drugs to the market, it is difficult to estimate stability speedy from exact information in preliminary stability tests. Thermal analysis is a ideal technique to solve this problem.
ADVANTAGES OF THERMAL ANALYSIS IN PREDICTION OF STABILITY
a) This method is very speedy. b) It only takes two weeks to predict stability of a drug substance in detail. c) The operations are simple d) The method required very small quantity of drug substance & total amount of sample being necessary to predict stability is very little so method is used even at an early developmental stage when production scale is small. e) Furthermore the accuracy & the precision of prediction using the method are equivalent to or better than those of preliminary tests. f) This method is very widely applicable to # Pharmaceutical intermediate # Pharmaceutical excipient # Agricultural chemical # Pesticides # Pharmaceutical raw material.
6) Degree of crystallinity
Partial crystallinity is also a type of polymorphism. The degree of crystallinity was determined by solution calorimetry. Principle of solution calorimetry
Basis on the fact that for many solids the amorphous form is higher in energy then the crystalline form. The heat of solution of the amorphous form is expected to be more exothermic then the crystalline form. The percentage of crystallinity (pc) may be determined in a partially crystalline sample according to the following equation.
100(H sample- H amorphous)

PC =
H crystal - H amorphous
where, sample is partially crystalline material amorphous is 100% amorphous standard & crystalline is 100% crystal standard.
Paper 910101 M.Pharm-(2009-10) 7)
THERMAL ANALYSIS
COMPATIBILITY STUDY OF DRUG WITH EXCIPIENT.
The early detection of Drug excipient Incompatibility is vital in pharmaceutical industry to avoid costly material wastage & time delays. DSC & TGA with the support of x-ray diffraction & infrared spectroscopy are used as screening technique for the compatibility testing of drug with excipient.
E.g. DRUG EXCIPIENT COMPATIBILITY STUDY a) DSC of sparfloxacin b) DSC of pvp C) DSC of 1:1 physical mixture of Drug:pvp
Conclusion incompatibility
Reason:
Absence of melting endotherm & exotherm is broad.
8) STUDY OF COMPLEXES & INCLUSION COMPOUNDS
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
The Disappearance of the DSC peak of the drug is the proof of Complexation in solid state. Figure shows, that no complexation was obtained for a drug with -cyclodextrin.
9) Study of Thermal behaviour Sugar ester.

They have wide range of HLB value so they can be used as surfactant or penetration enhancer. Aim of this study, to measure thermal properties of Sugar ester & to differentiate sugar ester with HLB. Thermal properties are measured with modulated DSC,& combined with HOT STAGE MICROSCOPY to visualized changes in sample during heating.
MINOR APPLICATIONS
- DSC is a valuable tool in choice of suppository base. - In study of polymer composition ,miscibility & individual characterization. - Study of tablet coating - Determinations of melting point. etc - Determination of moisture content in drug. - Checking technological quality grade of disintegrate. - Study of solid drug dispersion. - Determination of drying temp. for different excipients.
GENERAL PRINCIPLE & APPLICATION OF FTIR SPECTRUM & X-RAY DIFFRACTION METHOD.
Generally ,this technique are not consider as thermal technique, But if used in specific condition then this technique are classified as thermal technique. DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
A) Fourier transform infrared spectroscopy(FTIR)

Fourier transform infrared spectrometers can be single beam or Double beam. Commercial FTIR spectrometers are of single beam. A double beam instrument is designed to compensate for atmosphere. In most IR spectrometer ,the optical components are manufactured in sealed & desiccated compartment with a goal of reducing water & carbon dioxide interference.
ADVANTAGE:
- Simple - Accurate - Sensitive - Speedy
DISADVANTAGE:
1) Generally not used alone. 2) Gives peak at same wave number, Eg. so not differentiate polymorph.
B) X-RAY POWDER DIFFRACTION X-RAY powder Diffractometry is used to characterize spray dried & crystalline
material & the binary mixtures. PRINCIPLE: x-ray are Diffracted & order of this diffraction is measured in form of graph. Diffraction occurs as a result of the interaction of radiation with electron of atom.
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
Why only x-ray are used?

Because x-rays have wavelengths of about the same magnitude as the distance between the atoms or molecules of crystal. Theory Crystals to be examined are reduced to a fine powder and placed in a beam of monochromatic x-rays. -- Each tiny crystal is oriented at random with respect to the incident beam. - The crystals diffract x-ray similar to a diffraction grating. - related to and d by braggs equation:
n = 2 d sin
where, n = order of diffraction d = interatomic distance = angle of incidence - Diffraction occur as a result of the interaction of radiation with electron of atom. - when Braggs condition is fulfilled, a peak is detected.
Application: For structure determination: Identification Of Impurity: X-ray diffraction pattern of any specimens match with standard. Presence of Additional lines on the photograph of specimen, indicate the presence of impurity. e.g In cosmetic talc, the contaminant tremolite (a potentially carcinogen ) can be detected by x-ray diffraction technique. Characterization of polymorphism: Polymorphs having different lattice arrangement & give different x-ray Powder diffraction spectrum., so it is easily characterize by this technique. Characterize spray dried & crystalline material. For particle size analysis.
Limitations of Thermal analysis

1) low sensitivity for transitions involving small energies.
2) The interpretation of the curves & DSC studies should always Include several conditions & replicate. 3) Impurity consisting of molecules of same size, shape, & character as those of the major component are not detected by DSC, Because these impurity fit into the matrix of the major component without disruption of lattice forming solid solution or inclusion ,such impurity are not detected by DSC. 4) TGA used to studies hydrates & moisture study is not always reliable.
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
5) Thermal analysis are affected by number of factors like a) Sample weight b) Particle size c) Heating rate d) Atmospheric condition e) Instrumental factor f) Furnace atmosphere g) Crucible geometry
REFERENCES
The Science of Dosage form Design M.E Aulton Physical pharmacy, Alfred martin, fourth edition. The science & practice of Pharmacy,Remington,20th Edition. Instrumental methods of analysis ,Willard ,7th edition Journal of AOAC ,Jan/Feb 2007,volume-90,Number-1 Encyclopedia of pharmaceutical Technology ,Swarbrick & Boylan vol-15 Encyclopedia of pharmaceutical Technology ,Swarbrick & Boylan vol-06 Encyclopedia of pharmaceutical Technology ,Swarbrick & Boylan vol-02 Encyclopedia of pharmaceutical Technology ,Swarbrick & Boylan vol-07 Practical Pharmaceutical chemistry ,AH Beeckett & JB Stenlake,3 rd Edition vol02 Principle and Instrumental analysis, skoog-hollar-nieman, 5th edition. Journal of pharmaceutical sciences.vol-91,2002 Journal of pharmaceutical science,vol.-95, jan2006, page no.-159 Chemical Abstract,VOL-147,NO-6,125025 Chemical Abstract,VOL-147,NO-1,15935 Chemical Abstract,VOL-146,NO-25,134532 www.scci-inc.com/analytical www.springerlink.com www.ptli.com/test/opedia/tests www.wikipedia.com www.pubmed.com www.ijpsonline.com/article.asp www.thermal analysis.com www.doi.wiley.com/10.1002/jps.2600580529 www.ingentaconnect.com
Paper 910101 M.Pharm-(2009-10)
THERMAL ANALYSIS
Questions asked in exam 1) Explain the role of thermal analysis in preformulation (Guj. Uni. 2006) 2) Explain the application of any one technique based on thermal analysis? (Guj. Uni. 2005) 3) Enlist the modern technique and explain any one combined technique based on thermal analysis. (First internal 2006) 4) What do you mean by thermal analytical method? (first internal 2006) 5) Application of thermal analysis in Preformulation. (Guj uni.2007)
STUDY QUESTIONS 6) What are the limitations of thermal method? 7) What is thermal analysis ? classify thermal analysis? 8) write a note on general principle involved in different thermal methods ? 9) what are the innovations in thermal analysis? 10) Enlist the physical properties measured in different thermal methods & give detail of thermal method which is based on enthalpy measurement.
SEMINAR ON DRUG EXCIPIENT COMPATIBILTY STUDY

(As a part of preformulation study)
Guided By: Dr.R.K.Parikh
Prepared By: Krupa P.Mehta (M.Pharm-Sem-I)
DEPARTMENT OF PHARMACEUTICS & PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY,AHMEDABAD-9
POOL OF CONTENTS:
Incompatibility General aspects & Types Objectives Compatibility Tests A. Solid state reactions B. Liquid state reactions
Different Techniques used for compatibility testing Thermal methods of analysis Accelerated Stability Study FT-IR Spectroscopy DRS-Diffuse Reflectance Spectroscopy Chromatography Radiolabelled Techniques Vapour Pressure Osmometry Flourescence Spectroscopy
Incompatible impurities P-Glycoprotein inhibitor excipients Known incompatibilities Compatibility studies in different dosage forms
INCOMPATIBILITY:
DEFINITION:When we mix two or more API and / or excipient with each other & if they are antagonistic & affect adversely the safety, therapeutic efficacy, appearance or elegance then they are said to be incompatible. TYPES OF INCOMPATIBILITY: A. Physical incompatibility:- It involves the change in the physical form of the formulation which involves color changes, liquefaction, phase separation or immiscibility. B. Chemical incompatibility:- It involves undesirable change in formulation which is due to formation of new chemical comp. with undesirable activity or our formulation undergoes hydrolysis, oxidation, reduction, precipitation, decarboxylation, racemization. C. Therapeutic incompatibility:- It is type of in vivo compatibility. It involves change in therapeutic response of the formulation which is undesirable to patient as well as physician. OBJECTIVES:
-Why to screen excipients? 1.need to minimize no of model formulations 2.provide rational basis for selecting excipients 3.Formulation stability studies are time consuming. -Goal of the study( Identify the excipients that) 1.are compatible with API 2.do not have impact on the stability of API -Importance: 1.Stabity of formulation can be maximised. 2.Helps to avoid surprise problems. 3.Essential for IND submission. 4.Bridges drug discovery and drug development COMPATIBILITY TESTS: 2 Aspects of compatibility tests are: 1. Identification of compatible excipients for a formulation. 2. Identification of stable storage conditions
2 Types: Solid state reactions: - much slower and difficult to interpret. Liquid state reactions: - easier to detect - Acc. to Stability Guidelines by FDA following conditions should be evaluated for solutions or suspensions 1. Acidic or alkaline pH. 2. Presence of added substances 3. High oxygen and nitrogen atmospheres. 4. Effect of stress testing conditions.
STEPS IN COMPATIBILITY STUDY:
-There are THREE steps to consider. 1. Sample preparation 2. Storage 3. Method of analysis
SAMPLE PREPARATION: FOR SOLID STATE REACTIONS: SampleA: -mixture of drug and excipient SampleB: -SampleA+ 5% moisture SampleC: -Drug itself without excipients o All the samples of drug-excipient blends are kept for 1-3 weeks at specified storage conditions. o o o Then sample is physically observed . It is then assayed by TLC or HPLC or DSC. Whenever feasible, the degradation product are identified by MASS SPECTROSCOPY, NMR or other relevant analytical techniques. o
To determine Solid state stability profile of a new compound. of a new compound
weighed sample are placed in open screw cap vials and exposed directly to a variety of temp., humidity & light intensities for up to 12 weeks.
FOR LIQUID STATE REACTIONS: o o o Place the drug in the solution of additives. Both flint and amber vials are used. This will provide information about -Susceptibility to oxidation. -Susceptibility to light exposure. -Susceptibility to heavy metals. o In case of oral liquids, compatibility with ethanol, glycerin ,sucrose, preservatives and buffers are usually carried out. STORAGE CONDITION: The storage conditions used to examine compatibility can very widely in term of temp. & humidity, but a temp. of 50c for storage of compatibility sample is considered appropriate. Some compounds may require high temp. to make reaction proceed at a rate that can be measured over a convenient time period.
Analytical techniques used to detect Drug-Excipient Compatibility

1) Thermal methods of analysis I. DSC- Differential Scanning Calorimetry II. DTA- Differential Thermal Analysis 2) Accelerated Stability Study 3) FT-IR Spectroscopy 4) DRS-Diffuse Reflectance Spectroscopy 5) Chromatography I. SIC-Self Interactive Chromatography II. TLC-Thin Layer Chromatography III. HPLC-High Pressure Liquid Chromatography 6) Miscellaneous I. Radiolabelled Techniques II. Vapour Pressure Osmometry III. Flourescence Spectroscopy 5
DSC:- Differential Scanning Calorimetry.

DSC is widely used to investigate and predict any physico-chemical interaction between drug and excipients involving thermal changes.. METHOD The preformulation screening of drug-excipient interaction requires 5 mg of drug, in 50% mixture (1 : 1) with excipient, to maximize the likehood of observing an interaction. Mixture should be examined under N2 to eliminate oxidative and pyrrolytic effects at heating rate ( 2, 5 or 100c / min) on DSC apparatus.
Drug: Ofloxacin Excipients: (1) Lactose (2) Starch (3) PVP (4) Talc
Trace 1 of figure 1-4 shows peak at 278.33 0C. (melting endothermic peak of Ofloxacin). Trace 3 (Physical mixture of Ofloxacin & Lactose) shows absence of peak at 278.33 0 C and slight pre shift in Lactose peaks. DSC RESULT-- INCOMPATIBLE
Trace 5 (Physical mixture of Ofloxacin & Starch) shows an early onset at 268.37 0C. DSC RESULTCOMPATIBLE
Trace 7 (Physical mixture of Ofloxacin & PVP) shows no change in position of endothermic peak for PVP but there is increase in peak area and size & shape of peak for Ofloxacin is also decreased. DSC RESULT-- INCOMPATIBLE
Trace 9 (Physical mixture of Ofloxacin & Talc) shows combine features of each component but there are evident changes in onset. DSC RESULTCOMPATIBLE
DSC Study of Ascorbic acid Pceutical formulations Excipients: Sod. Crosscarmellose, MCC, Lactose Thermal stability was performed on ascorbic acid std. samples, binary mix. Of ascorbic acid & excipients, under N2 & air atmospheres. IR & X-Ray Diffractometry: No chemical interaction However thermal stability of Pceutical formulations is different. Temp. of beginning of thermal degradation for Ascorbic acid is lowered of about 50C for MCC & 100C for Na-crosscarmellose & Lactose. Such facts must be considered for storage planning of tablets.
ADVANTAGES OF DSC OVER TRADITIONAL METHODS:1. fast (no long term storage of mixture is required prior to evaluation. 2. Reliable 3. Very less sample required (few mg.) LIMITATIONS:1. If thermal changes are very small, DSC cant be used. Therefore, it should always be supported by some non-thermal methods like TLC or FT-IR or XRPD. 2. DSC can not detect the incompatibilities which occur after long term storage. Eg. MCC / ASPIRIN.. DSC shows no incompatibilities between these two. But after long term storage MCC, being hygroscopic, absorb moisture and degradation of Aspirin occur due to moisture.
3. It is important to view results of such incompatibility testing with caution. 9
For Eg. Mg-stearate is incompatible with wide range of compounds when tested. Yet because it is used at low conc. (0.5-1%). Such low conc. rarely produces a problem in practice on long term storage & use. 3. Not applicable if test materials exhibit properties that make data interpretation difficult. Such as Eutectic formation, coincident melting & dissolution of one component in the melt of other.
DIFFERENTIAL THERMAL ANALYSIS(DTA)

Thermal Analysis is useful in the investigation of solid-state interactions. It is also useful in the detection of eutectics. Thermograms are generated for pure components and their physical mixtures with other components. In the absence of any interaction, the thermograms of mixtures show patterns corresponding to those of the individual components. In the event that interaction occurs, this is indicated in the thermogram of a mixture by the appearance of one or more new peaks or the disappearance of one or more peaks corresponding to those of the components. Drug : Enalepril maleate Excipients(Directly compressible diluent): (1) Avicel (2) Spray dried lactose (3) Emcompress (4) A-tab # In all the formulations excipients other than directly compressible vehicle are kept same. FORMULATION F1 (Avicel) F2 (Spray dried lactose) F3 (Emcompress) F4 (A-tab) RESULT OF DTA (interaction) + + + SHELF LIFE 3 month 15 MONTH 8 month 9 month INFERENCE Least suitable Ideal Not recommended Not recommended
ACCELERATED STABILITY STUDY

Different formulations of the same drug are prepared.(Eg. Enalepril maleate As above F1-F4) Samples are kept at 400C / 75 % RH. Chemical stability is assessed by analyzing the drug content at regular interval.
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Amt. of drug degraded is calculated. % Drug decomposed VS time (month) is plotted and determine with excipient combination in which drug attains maximum stability. DIFFUSE REFLECTANCE SPECTROSCOPY: Principle: Penetration of a portion of incident radiation flux into the interior of the solid sample, return of some portion of radiation to the surface of sample following partial absorption and multiple scattering at boundary of individual sample particles. Detects the decomposed products, along with physical and chemical adsorption of excipients on to A.P.I. and vice versa. Example: Ethanol mediated interaction between dextroamphatamine sulphate and spray dried lactose in solidsolid mixture:Discoloration of powdered mixture was accelerated by 2 amine and by storage at elevated temp. Two new absorption maxima were observed at 340 nm & 295 nm resply. A + L = AL AHMF The diffuse reflectance depends on the packing density of the solid, its particle size and its crystal form. Where these factors are adequately controlled, diffuse reflectance spectroscopy can be used to investigate physical and chemical changes occurring on solid surfaces. A shift in the diffuse reflectance spectrum of the drug due to the presence of the excipient indicates physical adsorption. whereas the appearance of a new peak indicates chemisorption or formation of a degradation product. DRS is more useful than HPLC assay to detect surface discoloration due to oxidation or reaction with excipients.
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CHROMATOGRAPHY
TLC: Thin Layer Chromatography HPTLC: High Performance Thin Layer Chromatography TLC is generally used as confirmative test of compatibility after performing DSC. Because if sample undergo negligible thermal changes, it will difficult to detect by thermal method. In TLC, Stationary phase consist of powder adhered onto glass, plastic or metal plate. Powder commonly used are Silica, Alumina, Polyamide, Cellulose & Ion exchange resin. Solution of Drug, Excipient & Drug: Excipient mixture are prepared & spotted on the same baseline at the ed of plate. The plate is then placed upright in a closed chamber containing the solvent which constitutes the Mobile phase. As the solvent moves up the plate, it carries with it the material. The materials that have stronger affinity for S.P. will move at slower rate. The material is identified by its Rf value. The position of the material on the plate is indicated by spraying the plate with certain reagents or exposing the plate to UV radiation. If there is no interaction between drug & excipient, the mixture will produce two spots. The Rf value of which are identical with those of individual drug & excipient. If there is interaction, the complex formed will produce a spot. The Rf value of which is different from those of the individual components. (2)C: SIC: Self Interactive Chromatography
# Applications:SIC is useful for proteinous drug products with excipients. EX: INF Tau A new anti- viral drug. Interactions of it with different types of buffers were studied by SIC. Here, buffer is used to prevent aggregations. # Method:SIC is a modified type of affinity chromatography. Here, drug is made immobilized as the SP and solution to be tested acts as MP. Measure, Rt (Retention time) and compare it with non- retained marker.
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# Principle:For different mobile phases (i.e. different excipients) the injected drug have different interactions (may be repulsive or attractive) with the SP of drug leads to shift in retention time. FIGURE(A) FIGURE(B) FIGURE(C)
When interaction is repulsive, a sharper peak is obtained at a shorter retention time.
When no net interaction between the immobilized drug, Rt =dead volume of column.
When attractive interactions, it will have longer retention time& wider peak.
HPLC (high pressure liquid chromatography) Characteristics: -The APIs and model compounds of diversified chemical structure was studied. -Elution rate: 7.5 ml/hr at ambient temp. -Allows the detection and quantification of impurities, which span a wide range of polarities, including nonpolar compounds. FLUORESCENT MEASUREMENT: -This technique is restricted to those compounds, which can generate florescence. As the no. of such compounds are restricted, this method is used in Analysis and not in preformulation. VAPOR PRESSURE OSMOMETRY & EQUILIBRIUM DIALYSIS Principle: samples of solutions and pure solvent are introduced into a temperaturecontrolled enclosure, which is saturated with solvent vapor.Since the vapor pressure of solution is lower than that of solvent, solvent vapor condenses on solution sample causing its temperature to rise. The temperature rise is predicted by Clausis Clapcyron equation. Characteristics: Either liquid or solid sample and must be soluble in organic solvent or in water Sample must not undego association in solution. Sample size is approx. 3 gms for multiple analysis.
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Measures a no. of avg. mole. Wt. of about 10,000 Daltons. This method measures interactions, & records the interaction caused by variation of particle no. RADIO LABELLED TECHNIQUES: It is important when the API is having radioactivity. Method is carried out by using either 3H or 13C. Highly sensitive method but the cost of carrying out the method & the availability of well established other techniques & methods, this method is generally not preferred.
INCOMPATIBLE IMPURITIES
# Chemical impurity profiles of the excipient can be very important in influencing the long term chemical stability performance of the formulated drug product. Eg. (1) DCP Sometimes, IRON may be present in DCP as impurities. & It is incompatible with MECLIZINE HCl . (Fe NMT 0.04%) (2) Evaluation of Hydroperoxides ( HPO) in common pharmaceutical excipients. POVIDONE PEG 400 HPC POLYSORBATE 80 Contains substantial conc. of HPOs with significant batch to batch OR manufacturer to manufacturer variations.
While MCC, Lactose, High M.wt PEG, Polyxamer contains less amt. of HPOs.
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# In solid dosage forms, PVP is commonly used as a bonder for wet granulation & often used at very low conc. However, the total HPO content is high enough in PVP to promote significant degradation when formulating oxidatively sensitive drugs. # 5% of PVP was shown to be responsible for N-oxide formation of Raloxifen HCl, due to high HPO content. # So for these excipients, active monitoring and control of HPOs by the supplier may be necessary. (3) Gelatin is also containing IRON as impurities, Dark spots may occur in the shell due to the migration of water soluble iron sensitive ingredients from fill material into the shell. P-GLYCOPROTEIN INHIBITOR EXCIPIENTS P-Glycoprotein is membrane associated transport protein. It is an efflux pump lies in tissue membranes. Research has shown that some excipients have p-Glycoprotein efflux-pump inhibiting properties. Thus they increase drug concentration in cells & hence enhance the effect of drug molecules. EXAMPLES: - 1) PEG-32 lauric glycerides. 3) Polysorbate-80 5) Polysorbate-85 7) PEG-35 castor oil Known Incompatibilities 2) PEG-50 Stearate 4) Polysorbate-20 6) PEG-40 hydrogenated castor oil
Functional group Primary amine
Incompatibility Type of reaction Mono & Di-saccharides Amine-Aldehyde & Amine-Acetal Ester, Lactone Basic component Ring opening, Ester base hydrolysis Aldehyde Amine, Carbohydrate Aldehyde-Amine, Schiff base Or Glycosylamine formation Carboxyl Base Salt formation Alcohol Oxygen Oxidation to Aldehyde & Ketones Sulfhydryl Oxygen Dimerization Phenol Metal Complexation Gelatin- Capsule Shell Cationic Surfactant Denaturation
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Excipient Parabens
Incompatibility Non ionic surfactants (Polysorbate 80) Plastic Containers
Type of reaction Micellization (Reduced antimicrobial activity) Absorption of Parabens Anti-microbial activity Reduced
Phenylmercuric # Anionic Emulsifying agents, Nitrate Suspending Agents, Talc, Nametabisulfite, Na-thiosulfate Halides
PEG
Penicillin & Bacitracin Phenol, Tannic acid & Salicylic acid Sulphonamide & Dithranol Film coating
Incompatible (forms less soluble halogen compds) Anti-bacterial activity reduced Softening & Liquifaction Discoloration Migration of PEG from tablet film coating, leading to interaction with core component
Compatibility studies in different dosage forms A) Drug-Excipient compatibility studies in solid dosage forms
Example 1:Millard reaction:- is a non-enzymatic bimolecular browning reaction between reducing sugar and an amine( 10/ 20) Mechanism:-
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Example 2: Effect of Excipients on Hydrate formation in wet masses containing Theophylline During wet granulation Theophylline Shows Pseudopolymorphic changes that may alter its dissolution rate. In the presence of moisture Theophylline monohydrate is formed which has slow dissolution rate. Diluents Used:
o - Lactose monohydrate
Minimum water absorbing capacity. So not able to prevent but enhanced Hydrate formation of Theophylline.
o Silicified MCC Highly water absorbing capacity. Able to inhibit the formation of Theophylline monohydrate at low moisture content. SILICIFIED MCC as a multifunctional pharmaceutical excipient Multifunctional excipient Characteristics offered by Prosolv are high compactibilty, high intrnsic flow, enhanced lubrication efficiency and improved blending properties. Provide tremendous advantages through out product life cycle. MCC is a dry binder- when comes in contact with water ,its compressibilty is decreased..but that is not the case with SMCC. (Ref:CA,Vol:151,No:6,August10,2009 ,131557w)
B) Drug excipient Compatibility Studies in Aerosols

Example 1:- Interaction of propellent-11 with aqueous drug products. Propellent 11 is trichloromonofluoromethane. Interaction of above with the aqueous drug is as follow.
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HCl corrodes the Al-container. Therefore, Propellent 11 is incompatible with aqueous products. Example 2: Beclomethasone- Hydroflouroalkane interactions: BDP is a Steroidal drug used in Asthma.
Manipulation of above interaction: BDP particles coated with amphiphilic macromolecular excipient by Spray drying.
Therefore, prevention of aggregation & production of physically stable suspension with excellent aerosolisation properties. o Anhydrous ethanol is corrisive to Al containers. -Hydrogen produced in the reaction increases the pressure of the container.So drugs containing polar solvents tend to be corrosive to bare Al. For containers which contain 2%Tin and 98% Lead -Lead reacts with the fatty acids(for product cont.soaps) to form Lead salts which cause valve clogging.
C) Drug Excipient Compatibility Studies in Parenteral products

Excipients are added to parenteral formulations to enhance or maintain active ingredient solubility and/or stability. Excipients also are important in parenteral formulations to assure safety, minimize pain and irritation upon injection, and control or prolong drug delivery. These are all examples of positive or synergistic interaction between excipient and drugs.
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(I) Anti-oxidants:A) Ascorbic acid: It is incompatible with those drugs which are acid- unstable. Eg. Penicillin-G, Phenylephrine HCl, Pyrilamine meleate, Salicylamide, Theobromine.
B) Na bisulfite: It is a strong Nucleophilic anti-oxidant. Epinephrine + Na bisulfite Sulphonic acid dvt. o It can be prevented by addition of Na-borate which produces complex with Epinephrine and prevent its interaction with Na-bisulfite. It is incompatible in Opthalmic solution containing Phenyl mercuric acetate especially when autoclaved.
C) Edetate salts: used in stabilization of drugs sensitive to metal catalyzed oxidation and / or photolysis. Edetate salts are incompatible with Zn Insulin, Thiomerosal, Amphotericin & Hydralazine HCl. (II)
Preservatives:
A) Phenolic Preservatives: Lente- Insulin + Phenolic preservative Break-down of Bi-sulphide Linkage in Insulin structure.
But when we formulate Protamine- Insulin formulation Phenol plays imp. role. It forms tetragonal oblong crystals which is responsible for prolong action of insulin.
(III) Surface active agents: A) Polysorbate 80: Solubilizing agent, Wetting agent, Emulsifying agent. One must concern about the residual peroxide present in Polysorbate. PS 80 Polyoxyethylene sorbitan ester of Oleic acid (Unsatd.F.A) PS 20 Polyoxyethylene sorbitan ester of lauric acid (Satd.F.A) So PS 20 is less prone to oxidation than PS 80. SURFACTANTS & CHELATING AGENTS DRUG Proteins EXCIPIENT Tween 80 and other nonionic polyether surfactants INTERACTION OBSERVED Surfactants undergo oxidation and the resultant alkyl hydroperoxides formed contribute to the degradation of protein.
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Protein formulations
Thiols such as Most effective in stabilizing protein cystiene, formulations containing peroxideglutathione and forming surfactants. thioglycerol Solubilize and stabilize drugs without apparent compatibility problems.
Dexamathasone, Modified Estradiol, cyclodextrins, Iterleukin-2 & Proteins and Peptides
( IV) Cosolvents: B) Sorbitol: Increase the degradation rate of Penicillin in Neutral and Aqueous solutions. C) Glycerol: Increase the mobility of freeze-dried formulation leading to peptide deamidation. COSOLVENTS Sr.No. INTERACTION DRUG EXCIPIENT OBSERVED 1. Nicotinamide & dimethylisosorbide Propylene-glycol Hemolysis (in vivo effect) Precipitation of Cremophor EL
2.
Paclitaxel, Diazepam, Cremophor EL Propaniddid and (polyoxyl 35 Alfaxalone castor oil)
OILS AND LIPIDS Sr. No. DRUG 1. Lidocaine Calcium chloride, phenytion sodium, tetracycline hydrochloride
EXCIPIENT Unpurified sesame oil Soybean oil
INTERACTION Degradation of lodocaine Incompatible with All.
2.
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BUFFERS,ANTIMICROBIALS & ANTIOXIDENTS DRUG N-nitrosourea 5-flurouracil EXCIPIENT Tris buffer Tris buffer INTERACTION Form stable complex with N-nitrosourea and retard the degradation of this agent. Tris buffer will degrade 5-flurouracil, causing the formation of two degradation products that can cause serious cardiotoxicities Incompatible
Chlorpromazine
Meta-cresol
Recombinant human interferon gamma Cisplatin
Benzyl alcohol Benzyl alcohol caused the aggregation of the protein
Sodium metabisulfite
Sodium metabisulfite inactivates cisplatin
REFERENCES: Pharmaceutical Dosage forms By Leon Lachman & Liberman Hand book of Pharmaceutical Excipients Remingtons Pharmaceutical Science,21st edition,2005. Modern Pharmaceutics by Banker & Rhodes,4th edition,2002. Theory and Practice of Industrial Pharmacy by Lachman & Lieberman. Int. J. Ph.Exci., Vol-1, Jan-2000, 153. Int. J. Ph.Exci., Nov-2002, 2283 Int. J. Ph.Exci.,jan-march,2003 J. Ph. Sci..,Vol-97, Jan-2007,106 J. Ph. Sci., Vol-95, May-2006, 976. J. Ph. Sci., Vol-95, May-2006, 1060. J. Ph. Sci., Vol-95, June-2006, 1342. J. Ph. Sci., Vol-93, Jan-2004,132 J. Ph. Sci., Vol-93, Nov-2004, 2755. J. Ph. Sci., Vol-92, May-2003, 516. JPS 2002, Vol. 91, No. 9-12, page 2283-2296 C.A. vol:146, No:25,June 18 :2007,507180t C.A. vol:147, No:4, July 23 :2007,79121 CA,Vol:151,No:6, August10,2009 ,131557w
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Paper 910101 M.Pharm-(2009-10)
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ORGANIC VOLATILE IMPURITIES AND THEIR REGULATORY LIMITS

( AS A PART OF PREFORMULATION)
GUIDED BY: DR.R.K.PARIKH
PRESENTED BY: KALPESH G. VYAS M.PHARM PART-I ROLL.NO :-10 YEAR:2009-10
PHARMACEUTICS & PHARMACEUTICAL TECHNOLOGY
L.M.COLLEGE OF PHARMACY AHMEDABAD-380009
Paper 910101 M.Pharm-(2009-10)
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TABLE OF CONTENTS :
(1)INTRODUCTION (2)TYPES OF IMPURITIES (3)SOURCES OF IMPURITIES (4)ICH HARMONISED TRIPARTITE GUIDELINE
(4.1)INTRODUCTION (4.2)SCOPE OF THE GUIDELINE (4.3)GENERAL PRINCIPLES (4.3.1)Classification of residual solvents by risk assessment (4.3.2)Methods for establishing exposure limits (4.3.3)Options for describing limits of class 2 solvents (4.3.4)Analytical procedures (4.3.5)Reporting levels of residual solvents (4.4)LIMITS OF RESIDUAL SOLVENTS (4.4.1)Solvents to be avoided (4.4.2)Solvents to be limited (4.4.3)Solvents with low toxic potential (4.4.4)Solvents for which no adequate toxicological data was found
(5)AS PER U.S. PHARMACOPOEIAL REGULATORY LIMITS (6)DECISION TREE FOR IDENTIFICATION & QUALIFICATION
Paper 910101 M.Pharm-(2009-10)
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(1) INTRODUCTION
Impurities in pharmaceuticals are the unwanted chemicals that remain with active pharmaceutical ingredients (APIs),or develop during formulation, or upon aging of APIs and formulated APIs to medicines. Websters dictionary defines impurity are something that is impure or makes something else impure. Presence of this unwanted impurities may influence the efficacy & safety the pharmaceutical products. But sometimes the presence of some impurities may not deleteriously impact on drug quality if therapeutic efficacy that similar to or greater than the drug substance itself. Neverthless, a drug substance can considered as compromised with respect to purity even if contains an impurity with superior pharmacological or toxicological properties. So identification & isolation of impurities are more important. It is identifyed &isolated by using following analytical methodMass spectroscopy Fourier transform ion cyclotron resonance mass spectroscopy(FTCR-MS) Nuclear magnetic resonance(NMR) High performance liquid chromatography(HPLC) Also different pharmacopoeias, such as the British pharmacopoeia(BP),united state pharmacopoeia(USP),are slowly incorporating limits to allowable levels of impurities present in the APIs or formulation. The international conference on harmonization-(ICH) has published guidelines on impurities in new drug substance.
(2)TYPES OF IMPURITY
Two types of impurities in medicines(a)impurities associated in with APIs & (b)impurities that forms are created during formulation & or with aging or that are related to formulated forms. According to ICH guidelines impurities associated with APIs are classified in to following categories(a)Organic impurities (b)Inorganic impurities (c)Residual solvents (d)Other materials
(a)Organic impurities
Starting materials Process related impurities Intermediates Degradation products
Paper 910101 M.Pharm-(2009-10)
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(b)Inorganic impurities
Salts Catalysts Ligands Heavy metals
(c)Residual solvents (d)Other materials

Filter aids Charcoal According to United State Pharmacopoeias(USP) (a)Impurities in official articles (b)Ordinary impurities (c)Organic volatile impurities Also the following terms have used by the USP to describe impuritiesForeign substances The materials that are introduced by contamination or adulteration, not as a consequence of synthesis or preparation, are labeled foreign subs.. e.g., pesticides in oral analgesic. Toxic impurities These impurities have significant undesirable biological activity. Concomitant components Bulk pharmaceuticals chemicals may contain concomitant components. e.g. Antibiotics that are mixtures and are geometric and optical isomer. Signal impurities These impurities include some process-related impurities or degradation products that provide key information about the process. Ordinary impurities The species of impurities in bulk pharmaceutical chemicals that are innocuous by virtue of having no significant undesirable biological activity in the amounts present are called ordinary impurities. Organic volatile impurities OVIs are generally solvents used in the synthesis or during the formulation of the drug product.
Paper 910101 M.Pharm-(2009-10)
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(3)SOURCES OF IMPURITIES Starting materials

These are the materials that are used to begin the synthesis of an APIs. e.g. paracetamol bulk, there is a limit test for p-amino phenol, which could starting materials for some one manufactures or be intermediate for others. Intermediates The compounds during synthesis of the desired materials are called intermediates. Penultimate intermediate(Final intermediates) As the name suggests ,this is the last compound in the synthesis chain prior to the production of the desired compound. By-products The unplanned compound produced in the reaction are generally called byproducts. e.g. In paracetamol bulk diacetylated paracetamol may form as a By-products.
Interaction products
This is a slightly more comprehensive term than the two described above(byproducts and transformation products);however ,it is more difficult to evaluate in that it considers interactions that could occur between various chemicals-intentionally or unintentionally.Two types of interaction products that can be commonly encountered are drug substance-excipient interactions and drug substance container /closer interactions. Related products This term tends to suggest that the impurity is similar to the drug substance. e.g. Hydrates in Isoniazide which produce hepatotoxicity. Degradation products These are the compound produced because of decomposition of material of interest or APIs. e.g. The degradation of penicillins and cephalosporins is well known example.The presence of -lactam ring as well as that of an amino group in the C6/C7 chain plays critical role in there degradation.
Paper 910101 M.Pharm-(2009-10)
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Route1
Route2
Route3
Route4
Route5
SM1
solvent1 catalyst
SM2 (SM2)
INT (INT)
solvent2 reagent
(BP1,BP2..) DS (DS) (D1,D2.)
Figure1:General reaction scheme for a drug substance synthetic process.
SM=Starting material INT=Intermediate DS=Drug substance BP=By-product D=Degradation product SM=Starting material impurity with potential to form INT
Paper 910101 M.Pharm-(2009-10)
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(4)ICH HARMONISED TRIPARTITE GUIDELINE

INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENT FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE
ICH HARMONISED TRIPARTITE GUIDELINE
IMPURITIES: GUIDELINE FOR RESIDUAL SOLVENTS

Q3C(R3)
Impurities: Guideline for Residual Solvents Impurities: Guideline for Residual Solvents (Maintenance) PDE for Tetrahydrofuran (in Q3C(R3)) Q3C Q3C(M)
Recommended for Adoption at Step 4 of the ICH Process on 17 July 1997 by the ICH Steering Committee. Birth of ICH at April 1990. This Guideline has been developed by the appropriate ICH Expert Working Group and has been subject to consultation by the regulatory parties, in accordance with the ICH Process. At Step 4 of the Process the final draft is recommended for adoption to the regulatory bodies of the European Union, Japan and USA
(4.1)INTRODUCTION
The objective of this guideline is to recommend acceptable amounts for residual solvents in pharmaceuticals for the safety of the patient. The guideline recommends use of less toxic solvents and describes levels considered to be toxicologically acceptable for some residual solvents. Residual solvents in pharmaceuticals are defined here as organic volatile chemicals that are used or produced or remains after the manufacture of drug substances or excipients, or in the preparation of drug products. The solvents are not completely removed by practical manufacturing techniques. Appropriate selection of the solvent for the synthesis of drug substance may enhance the yield, or determine characteristics such as crystal form, purity, and solubility. Therefore, the solvent may sometimes be a critical parameter in the synthetic process. This guideline does not address solvents deliberately used as excipients nor does it address solvates. However, the content of solvents in such products should be evaluated and justified. Since there is no therapeutic benefit from residual solvents, all residual solvents should be removed to the extent possible to meet product specifications, good manufacturing practices, or other quality-based requirements.
(4.2) SCOPE OF THE GUIDELINE

Residual solvents in drug substances, excipients, and in drug products are within the scope of this guideline. Therefore, testing should be performed for residual solvents when
Paper 910101 Chapter 1 OVIS M.Pharm-(2009-10) Preformulation production or purification processes. It is only necessary to test for solvents that are used or produced in the manufacture or purification of drug substances, excipients, or drug product. Although manufacturers may choose to test the drug product, a cumulative method may be used to calculate the residual solvent levels in the drug product from the levels in the ingredients used to produce the drug product. If the calculation results in a level equal to or below that recommended in this guideline, no testing of the drug product for residual solvents need be considered. If, however, the calculated level is above the recommended level, the drug product should be tested to ascertain whether the formulation process has reduced the relevant solvent level to within the acceptable amount. Drug product should also be tested if a solvent is used during its manufacture. This guideline does not apply to potential new drug substances, excipients, or drug products used during the clinical research stages of development, nor does it apply to existing marketed drug products. The guideline applies to all dosage forms and routes of administration. Higher levels of residual solvents may be acceptable in certain cases such as short term (30 days or less) or topical application. Justification for these levels should be made on a case by case basis.
(4.3) GENERAL PRINCIPLES (4.3.1) Classification of Residual Solvents by Risk Assessment

The term "tolerable daily intake" (TDI) is used by the International Program on Chemical Safety (IPCS) to describe exposure limits of toxic chemicals and "acceptable daily intake"(ADI) is used by the World Health Organization (WHO) and other national and international health authorities and institutes. The new term "permitted daily exposure" (PDE) is defined in the present guideline as a pharmaceutically acceptable intake of residual solvents to avoid confusion of differing values for ADI's of the same substance. Residual solvents assessed in this guideline are listed in Appendix 1 by common names and structures. They were evaluated for their possible risk to human health and placed into one of three classes as follows:
Class 1 solvents: Solvents to be avoided

Known human carcinogens, strongly suspected human carcinogens, and environmental hazards. e.g.,Benzene, Carben tetrachloride, 1,1-Dichlorobenzene, etc
Class 2 solvents: Solvents to be limited

Non-genotoxic animal carcinogens or possible causative agents of other irreversible toxicity such as neurotoxicity or teratogenicity. Solvents suspected of other significant but reversible toxicities. e.g.,Acetonitrile,Chlorobenzene,Chloroform,Cyclohexane,1,4-Dioxane,etc.
Class 3 solvents: Solvents with low toxic potential

Solvents with low toxic potential to man; no health-based exposure limit is needed. Class 3 solvents have PDEs of 50 mg or more per day. e.g.,Acetone, Anisol, 1-Butanol, Cumene, Ethyl ether,etc.
Class4 solvents: Solvents for which no toxicological data have been found
Paper 910101 Chapter 1 M.Pharm-(2009-10) Preformulation Sovents for which no adequate toxicological have been found. e.g.,1,1-Dietoxypropane,Trifluroacetic acid,Ethyl acetate,etc.
OVIS
(4.3.2)Methods for Establishing Exposure Limits:The Gaylor-Kodell method of risk assessment, is appropriate for Class 1 carcinogenic solvents. Only in cases where reliable carcinogenicity data are available should extrapolation by the use of mathematical models be applied to setting exposure limits. Exposure limits for Class 1 solvents could be determined with the use of a large safety factor (i.e., 10,000 to 100,000) with respect to the no-observed-effect level (NOEL). Detection and quantitation of these solvents should be by state-of-the-art analytical techniques. Acceptable exposure levels in this guideline for Class 2 solvents were established by calculation of PDE values. PDE is derived from the no-observed-effect level (NOEL), or the lowest-observed effect level (LOEL) in the most relevant animal study as follows: PDE = NOEL x Weight Adjustment F1x F2 x F3 x F4 x F5 The PDE is derived preferably from a NOEL. If no NOEL is obtained, the LOEL may be used. The modifying factors are as follows: F1 = A factor to account for extrapolation between species F1 = 5 for extrapolation from rats to humans F1 = 12 for extrapolation from mice to humans F1 = 2 for extrapolation from dogs to humans F1 = 2.5 for extrapolation from rabbits to humans F1 = 3 for extrapolation from monkeys to humans F1 = 10 for extrapolation from other animals to humans F1 takes into account the comparative surface area: body weight ratios for the species concerned and for man. Surface area (S) is calculated as: S = kM0.67 in which M = body mass, and the constant k has been taken to be 10. F2 = A factor of 10 to account for variability between individuals. A factor of 10 is generally given for all organic solvents, and 10 is used consistently in this guideline. F3 = A variable factor to account for toxicity studies of short-term exposure F3 = 1 for studies that last at least one half lifetime (1 year for rodents or rabbits; 7 years for cats, dogs and monkeys). F3 = 1 for reproductive studies in which the whole period of organogenesis is covered. F3 = 2 for a 6-month study in rodents, or a 3.5-year study in non-rodents. F3 = 5 for a 3-month study in rodents, or a 2-year study in non-rodents. F3 = 10 for studies of a shorter duration.
Paper 910101 Chapter 1 OVIS M.Pharm-(2009-10) Preformulation In all cases, the higher factor has been used for study durations between the time points, e.g a factor of 2 for a 9-month rodent study.
F4 = A factor that may be applied in cases of severe toxicity, e.g. non-genotoxic carcinogenicity, neurotoxicity or teratogenicity. In studies of reproductive toxicity, the following factors are used: F4 = 1 for fetal toxicity associated with maternal toxicity F4 = 5 for fetal toxicity without maternal toxicity F4 = 5 for a teratogenic effect with maternal toxicity F4 = 10 for a teratogenic effect without maternal toxicity F5 = A variable factor that may be applied if the no-effect level was not established When only an LOEL is available, a factor of up to 10 could be used depending on the severity of the toxicity. The weight adjustment assumes an arbitrary adult human body weight for either sex of 50 kg. This relatively low weight provides an additional safety factor against the standard weights of 60 kg or 70 kg that are often used in this type of calculation. It is recognized that some adult patients weigh less than 50 kg; these patients are considered to be accommodated by the built in safety factors used to determine a PDE. If the solvent was present in a formulation specifically intended for pediatric use, an adjustment for a lower body weight would be appropriate.
(4.3.3)Options for Describing Limits of Class 2 Solvents:Two options are available when setting limits for Class 2 solvents. Option 1: The concentration limits in ppm stated in Table 2 can be used. They were calculated using equation given below by assuming a product mass of 10 g administered daily.
Concentration (ppm) = 1000 x PDE Dose

Here, PDE is given in terms of mg/day and dose is given in g/day. These limits are considered acceptable for all substances, excipients, or products. Therefore this option may be applied if the daily dose is not known or fixed. If all excipients and drug substances in a formulation meet the limits given in Option 1, then these components may be used in any proportion. No further calculation is necessary provided the daily dose does not exceed 10 g. Products that are administered in doses greater than 10 g per day should be considered under Option 2. Option 2: It is not necessary for each component of the drug product to comply with the limits given in Option 1. Option 2 may be applied by adding the amounts of a residual solvent present
Paper 910101 Chapter 1 OVIS M.Pharm-(2009-10) Preformulation in each of the components of the drug product. The sum of the amounts of solvent per day should be less than that given by the PDE.
Consider an example of the use of Option 1 and Option 2 applied to acetonitrile in a drug product. The permitted daily exposure to acetonitrile is 4.1 mg per day; thus, the Option 1 limit is 410 ppm. The maximum administered daily mass of a drug product is 5.0 g, and the drug product contains two excipients. The composition of the drug product and the calculated maximum content of residual acetonitrile are given in the following table. Component Amount in formulation (gm) 0.3 0.9 3.8 5.0 Acetonitrile content (ppm) 800 400 800 728 Daily exposure (mg) 0.24 0.36 3.04 3.64
Drug substance Excipient 1 Excipient 2 Drug product
Excipient 1 meets the Option 1 limit, but the drug substance, excipient 2, and drug product do not meet the Option 1 limit. Nevertheless, the product meets the Option 2 limit of 4.1 mg per day and thus conforms to the recommendations in this guideline. Consider another example using acetonitrile as residual solvent. The maximum administered daily mass of a drug product is 5.0 g, and the drug product contains two excipients. The composition of the drug product and the calculated maximum content of residual acetonitrile is given in the following table. Component Amount in formulation (gm) 0.3 0.9 3.8 5.0 Acetonitrile content (ppm) 800 2000 800 1016 Daily exposure (mg) 0.24 1.80 3.04 5.08 In this
Drug substance Excipient 1 Excipient 2 Drug product
example, the product meets neither the Option 1 nor the Option 2 limit according to this summation. The manufacturer could test the drug product to determine if the formulation process reduced the level of acetonitrile. If the level of acetonitrile was not reduced during formulation to the allowed limit, then the manufacturer of the drug product should take other steps to reduce the amount of acetonitrile in the drug product. If all of these steps fail to reduce the level of residual solvent, in exceptional cases the manufacturer could provide a summary of efforts made to reduce the solvent level to meet the guideline value, and provide a risk benefit analysis to support allowing the product to be utilised with residual solvent at a higher level.
(4.3.4) Analytical Procedures:Residual solvents are typically determined using chromatographic techniques such as gas chromatography. Other methods are LC-MS, HPLC, Mass Spectroscopy. Any harmonised procedures for determining levels of residual solvents as described in the pharmacopoeias should
Paper 910101 Chapter 1 OVIS M.Pharm-(2009-10) Preformulation be used, if feasible. Otherwise, manufacturers would be free to select the most appropriate validated analytical procedure for a particular application.
If only Class 3 solvents are present, a nonspecific method such as loss on drying may be used. Solvents are used frequently to dissolve film-coating material to facilitate application onto compressed tablets. These tablets are subjected to air drying to remove all organic solvents from coat of finished product. The residual levels of these organic solvents in tablet core and film coats are critical, because they cause undesirable side effects.
Determination of Methylene chloride organic volatile impurity in marketed formulations of Ciprofloxacin, Norfloxacin, Pefloxacin, and Ofloxacin: The most sensitive among the methods for monitoring the amount of residual solvents in the marketed solid dosage formulations is the Gas chromatographic method. First, the Standard Analytical Grade is taken and diluted with particular solvent for ex. Methylene chloride as impurity solvent diluted with dimethyl sulfoxide. The retention time is recorded in Gas chromatogram. Then, tablets which are coated or formulated with this solvent are taken and crushed and extracted with diluting solvent. So whatever methylene chloride present that will extract out and that filtered. Retention time of this filtrate taken. As per retention time of standard methylene chloride, peak intensity or area, amount of test methylene calculated. (4.3.5) Reporting levels of residual solvents:Quantitative results should be presented numerically, and not in general terms such as complies or meets limits. Below 1.0 %, the results should be reported to two decimal places (e.g. 0.06%, 0.31%);at and above 1%, the results should be reported to one decimal place (e.g. 1.3%). Impurities should be designated by code number or by an appropriate descriptor e.g. retention time
(4.4) LIMITS OF RESIDUAL SOLVENTS:(4.4.1) Solvents to Be Avoided:Solvents in Class 1 should not be employed in the manufacture of drug substances, excipients, and drug products because of their unacceptable toxicity or their deleterious environmental effect. However, if their use is unavoidable in order to produce a drug product with a significant therapeutic advance, then their levels should be restricted as shown in Table 1, unless otherwise justified. 1,1,1- Trichloroethane is included in Table 1 because it is an environmental hazard. The stated limit of 1500 ppm is based on a review of the safety data.
Solvent Concentration Limit (ppm) 2 4 5 8 Concern
Benzene Carbon tetrachloride 1,2-Dichloroethane 1,1-Dichloroethene
Carcinogen Toxic and environmental Hazard Toxic Toxic
Paper 910101 M.Pharm-(2009-10) 1,1,1-Trichloroethane
Chapter 1 Preformulation 1500
OVIS
Environmental hazard
(4.4.2) Solvents to Be Limited:Solvents in given table should be limited in pharmaceutical products because of their inherent toxicity. PDEs are given to the nearest 0.1 mg/day, and concentrations are given to the nearest 10 ppm. The stated values do not reflect the necessary analytical precision of determination. Precision should be determined as part of the validation of the method.
TABLE 2. Class 2 solvents in pharmaceutical products.
SOLVENT Acetonitrile Chlorobenzene Chloroform Cyclohexane 1,2-Dichloroethene Dichloromethane N,N-Dimethylacetamide N,N-Dimethylformamide 1,4-Dioxane 2-Ethoxyethanol Ethylene glycol 6.2 620 Ethylene glycol Formamide Hexane Methanol 2-Methoxyethanol Methylbutylketone
PDE (MG/DAY) 4.1 3.6 0.6 38.8 18.7 6.0 10.9 8.8 3.8 1.6 6.2 6.2 2.2 2.9 30.0 0.5 0.5
CONCENTRATIO N LIMIT (PPM) 410 360 60 3880 1870 600 1090 880 380 160 620 620 220 290 3000 50 50
Paper 910101 M.Pharm-(2009-10) Methylcyclohexane

N-Methylpyrrolidone Nitromethane Pyridine Sulfolane Tetralin Toluene 1,1,2-Trichloroethene Xylene*
Chapter 1 Preformulation 11.8

48.4 0.5 2.0 1.6 1.0 8.9 0.8 21.7
OVIS
1180 4840 50 200 160 100 890 80 2170
* usually 60% m-xylene, 14% p-xylene, 9% o-xylene with 17% ethyl benzene.
(4.4.3) Solvents with Low Toxic Potential:Solvents in Class 3 (shown in Table 3) may be regarded as less toxic and of lower risk to human health. Class 3 includes no solvent known as a human health hazard at levels normally accepted in pharmaceuticals. However, there no long-term toxicity or carcinogenicity studies for many of the solvents in Class 3. Available data indicate that they are less toxic in acute or short-term studies and negative in genotoxicity studies. It is considered that amounts of these residual solvents of 50 mg per day or less (corresponding to 5000 ppm or 0.5% under Option 1) would be acceptable without justification. Higher amounts may also be acceptable provided they are realistic in relation to manufacturing capability and good manufacturing practice. Table 3. Class 3 solvents which should be limited by GMP or other quality based requirements. Acetic acid, Isobutyl acetate, 1-Butanol, 3-Methyl-1-butanol, Heptane, Anisole, Methyl acetate, Butyl acetate, Acetone, Isopropyl acetate, 2-Butanol, Methylethyl ketone,
Paper 910101 M.Pharm-(2009-10) tert-Butylmethyl ether, 2-Methyl-1-propanol, Ethanol, 1-Propanol, Ethyl formate, Tetrahydrofuran
Chapter 1 Preformulation Methylisobutyl ketone, Dimethylsulfoxide, 1-Pentanol, Ethyl ether, Propyl acetate
OVIS
Cumene, Pentane, Ethyl acetate, 2-Propanol, Formic acid,
(4.4.4) Solvents for which No Adequate Toxicological Data was Found The following solvents (Table 4) may also be of interest to manufacturers of excipients, drug substances, or drug products. However, no adequate toxicological data on which to base a PDE was found. Manufacturers should supply justification for residual levels of these solvents in pharmaceutical products. Table 4. Solvents for which no adequate toxicological data was found are given below. 1,1-Diethoxypropane, Methyltetrahydrofuran Isooctane, acid Methylisopropyl ketone, 2,2-Dimethoxypropane, Trichloroacetic acid, 1,1-Dimethoxymethane, Petroleum ether, Isopropyl ether Trifluoroacetic
(5)As per U.S. Pharmacopoeia regulatory limits

Organic volatile impurities Benzene Chloroform 1,4-Dioxane Methylene chloride Trichloroethylene USP/NF limits before 2003 (ppm) 100 50 100 500 100 USP/NF 2003 limits (ppm) Not specific 60 380 600 80
FIGURE2: DECISION TREE FOR IDENTIFICATION AND QUALIFICATION

Is impurity greater than identification threshold?
DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY PHARMACY-09 Yes L.M.COLLEGE OFNo

No action
Paper 910101 Chapter 1 M.Pharm-(2009-10) Preformulation C.A. vol:150, No:26,june 29:2009,570442c
OVIS
Polymer-metal nanocomposites with two dimensional gold nanoparticle for sensoric application of organic volatile compounds
Increase in distance
Polymer
Gold nanoparticle
Swell polymer
Questions:
Paper 910101 M.Pharm-(2009-10)
OVIS
(1) Classify impurities present in APIs? Sept-2004 (2) How will you calculate PDE in OVIs? Apr-2005 (3) OVIs Sept-2006 (4) Classify impurities and how they are addressed in new drug substance? Apr-2006
Study Questions:
What What
are impurities? Write a note on sources of impurities are residual solvents? Why we take care in selection of appropriate solvents? What is PDE? Classify residual solvents by risk assessment. How will you calculate PDE of class-1 and class-2 solvents? Describe a limits of residual solvents Draw a decision tree for identification & qualification
References: (1) www.ICH.org (2) USP Vol -II P.No.-1746 (3) IJPS vol-68, may-June 2006 P.No.-368 (4) IJPER vol-40 Apr- June2006.
(5) Advance drug delivery review vol-59, issue-1 (6) Indian Drugs May 2008 vol-45 (7) C.A. vol:150, No:26,june 29:2009,570442c
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
PREFORMULATION STUDY OF BIOTECHNOLOGICAL PRODUCTS AND REFERENCE GUIDELINES
GUIDED BY:
DR.R.K PARIKH
PREPARED BY:
Vijay makvana M.Pharm. -1 ROLL NO:1
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
Pre-formulation studies as an essential guide to formulation development and manufacturing of pharmaceuticals

CONTENTS
Introduction to BIOTECHNOLOGY PHYSICO - CHEMICAL parameters of biotechnological products BIOLOGICAL characterization EXCIPIENT COMPATIBILITY studies STRESS STUDIES of Biopharmaceuticals STABILITY aspects of Biopharmaceuticals METHODS to IMPROVE the stability FORMULATION APPROACHES to protein stabilization FORMULATION DEVELOPMENT consideration GUIDELINES on the stability of Biopharmaceuticals Interpretation through CASE STUDY
INTRODUCTION TO BIOTECHNOLOGY
"Biotechnology means any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use." Karl Ereky coined the word biotechnology from Hungary during 1917 The Dawn of Biotech is considered from the first production of insulin around 1970 to present. Biotechnological drugs official in IP96: Vaccines Hepatitis B virus Japanese encephalitis, Rabies, Human rabies vaccine (neural tissue) Diphtheria, tetanus and pertuses (adsorbed), Tetanus vaccine (adsorbed), Cholera, Live measles, Live yellow fever, Oral polio virus, Plague, Small pox (freeze dried), Tetanus, DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10) Typhoid, Typhus, Rickettsial
BIOTECHNOLOGY
Others Hyaluronidase inj., Insulin, Dried human antihaemophilic fraction, Human euglobulin, Bovine euglobulin, Freeze dried human coagulation factor, Chorionic gonadotrophin, Human serum globulin, Human plasma protein fraction, Human normal serum albumin, Human normal immunoglobulin, Immune human serum globulin, Levothyroxin, Human albumin solution.
PHYSICO-CHEMICAL PROPERTIES
1. Purity, Impurities and Contaminants 2. Structural Characterization 3. Conformation Study 4. Physical Characterization 4.1 SOLID STATE PROPERTIES. 4.1.1 Crystallinity 4.1.2 Hygroscopicity 4.1.3 Molecular weight 4.1.4 Thermal Denaturation temperature 4.1.5 Extinction coefficient. 4.2 SOLUTION PHASE PROPERTIES 4.2.1 Ionic Strength 4.2.2 pH 4.2.3 Adsorption 4.2.4 Solubility 4.2.5 Surface interaction
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
1. PURITY, IMPURITIES AND CONTAMINANTS The impurities in the products are: Process related and Product related Process related impurities in the drug substance may include cell-culture media, host cell proteins, DNA, monoclonal antibodies or chromatographic media used in purification, solvents & buffer components and should be minimized by the appropriate well controlled manufacturing process. Product related impurities in the drug substance are molecular variants with properties different from those from the desired product during the manufacture &/or storage. The methods used for determination includes Peptide mapping, HPLC (mainly RPHPLC),Mass spectrometry, Amino acid analysis, N-terminal sequencing. CONTAMINANTS 1. Protein based product 2. Pyrogen detection 3. DNA 4. Microbial DETERMINATION TECHNIQUE SDS-PAGE ,HPLC ,Immunological approaches like immunoassay Amino acid analysis ,N-terminal sequencing ,peptide mapping Rabbit pyrogen test (exogenous pyrogen) and LAL Test(endotoxins) DNA Hybridization studies(dot-blot assay)utilizes radiolabel led DNA probes virus specific DNA probes , immunoassays.
2.STRUCTURAL CHARACTERIZATION Protein architecture are typically described in terms of primary ,secondary ,tertiary & quaternary & the order of stability is from primary>>>quaternary. Protein structure is determined by following techniques, SDS-gel electrophoresis ,HPLC (mainly RP-HPLC) ,Amino acid analysis ,N-terminal sequencing etc 3. CONFORMATION A proteins conformation is the most elusive of its physical properties in that it may be the most difficult not only to ascertain but to maintain. One must establish the other conformational forms present have no undesirable chemical, physical and biological effects. It can be determined by the following technique ,X-ray diffraction ( to study 3Dprotein conformation ,Circular dichrosim ,Peptide mapping ,N-terminal sequencing or amino acid to determine amino acid sequence. ,Spectroscopic techniques ( UV,IR, Raman, Fluorescence) This technique provides a valuable clues as to the proteins physical and chemical stability and may yield important information on structure function relationships as well.
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
4. PHYSICAL CHARACTERIZATION 4.1 SOLID STATE PROPERTIES
4.1.1 Crystallinity:
Many peptides exhibit in an amorphous state than crystalline one.(as the method of isolation involved are lyophilisation and precipitation one) Lack of crystallinity is characterized by the low intensity peaks or broad bands in the POWDER X-ray pattern. Other methods used are X-ray Diffraction pattern, Solid state NMR (SSNMR),Differential scanning calorimetry ( DSC), Thermo gravimetric analysis (TGA).
4.1.2 Hygroscopicity:
The changing environmental conditions can affect the stability of the hygroscopic products The estimation of water content done by the HPLC method
4.1.3 Molecular weight:

The molecular size of peptides and proteins are 1 to 3 orders of magnitude larger than that of conventional drug molecules, is a major factor limiting their diffusion across the biological membranes. Biopharmaceuticals molecular weight ranges from 600 to more than 10,000daltons in their non-aggregated state Larger the size ,lower the permeation across biological membranes g.i.t, ocular epithelium ,stratum corneum considered as impermeable barriers to the permeation to the latter. Molecular size also restricts diffusion of the biopharmaceutical across the polymeric barriers, used in the fabrication of proteins and peptides delivery systems.
4.1.4 Extinction coefficient (molar absorptivity):

For quantification purpose, it is desirable to measure at a particular UV-VIS wavelength for the measurement of the protein content.
4.1.5 Thermal denaturation temperature:

Most proteins can be reversibly denatured with an increase in temperature and continued increase can result in irreversible denaturation (seen in enzymes). Also in some case, protein unfolds and refolds into new structure different then original native one. FREEZE DRYING of proteins in presence of appropriate buffer excipients can minimize the problem
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
Thus, careful analysis of physical properties and biological activity as a function of storage temperature is extremely important. Thus it is necessary to determine a particular limit i.e. thermal denaturation temperature (temperature at which proteins denaturation initiates) to set the acceptable storage criteria for biopharmaceuticals 4.2 SOLUTION PHASE PROPERTIES
4.2.1 Adsorption:
Loss of peptides and proteins from solution by adsorption to various surfaces is a common phenomenon and should be addressed early in the preformulation activities. In the case of where adsorption is due to ionic interaction of the peptide with the silanol groups on the glass surface, can be prevented by silylating the glass (flasks, syringes,etc) with organosilanes such as Prosil 28. Other surfaces include tubing , syringes, membrane fiber filter,etc
4.2.2 Solubility and charge:

The solubility of a protein is a useful indicator of changes that may occur in the protein conformation If a protein denatures, more hydrophobic groups may be exposed to the aqueous phase from the interior of the protein. Crystallization and polarity are major physico-chemical parameters determining the drug solubility 4 commonly used solvent approaches are pH control, Co-solvency ,Micellization, complexation Solubilisation approaches includes 1) Alteration of the crystal structure of a solute (decrease in particle size) 2) Modification of a solvent by adding solubilising agent 3) Most globular proteins are soluble in water, dilute acid and dilute salt. Examples of peptide solubility: Peptide Pectin Human gastrin Growth hormone releasing peptides Substance P,angiotensin I,II,or III Analogs chemical modifications -endorphin ,somatostatin,TRH LHRH soluble in DMSO ammonium acetate buffer DMSO,Methanol,Ethanol Acetic acid Water or acetic acid
4.2.3 pH:
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
pH has profound effect on protein stability ,folding and rates of reaction which chemically alter the amino acid residues In some cases it may be possible to formulate the protein under conditions where chemical modifications and proteolysis are minimized as much as possible while maintaining the protein in misfolded conformation which is readily and rapidly refolded to native form upon administration.
4.2.4 Ionic strength:

The concentration of counter ions is known to be an important property in mediating electrostatic interactions in molecule, and have large effect on stability of a protein as well as its solubility. The DebyeHuckel theory suggest an important parameter for assessing the concentration of the counter ions ,ionic strength :
I = Ci zi2
Where Ci: molar concentration of its counter ions and with charge zi2 Therefore, in the development of a formulation buffer for proteins, it is especially crucial that attention be paid to the types of counter ions used as wall as ionic strength. For example: Guanidium HCl is a strong naturant of protein where as Guanidium sulphate at equivalent concentration can stabilize proteins.
4.2.5 Surface interactions:

The potential of interaction is of great concern with regard to maintaining native protein structure The type and degree of interaction depend not only on physical and chemical properties of both protein and surface but also on the stresses placed on the system. It results in protein conformational changes, the degree and the reversibility of which are dependent on time temperature, agitation and type of surface. The adsorption was irreversible as surface bound protein could not be completely eluted with detergent, organic solvents or denaturants under varying conditions. The mechanisms involved in the interaction are Formation of multiple bonds and Interfacial forces In order to minimize or prevent the adsorption with the use of nondenaturing surfactant in the protein solution.
BIOLOGICAL CHARACTERIZATION
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
Validated biological assays should be performed at the preformulation stage to assess the
biological activity. For the complex molecules, the physio-chemical information may be extensive but unable to confirm the higher order structures, a biologic activity with a specific quantitative measure. Radio receptor assays (RIA) may be used to relate binding of a protein hormone to a cellular receptor and may be used to asses activity protein fragments, produced by the enzymatic degradation Immunogenicity is ability to induce the formation of antibodies when combined with antigenicity is the ability to react with specific antibodies can potentially result in the development of hypersensitivity reactions. Techniques for minimizing the immunogenicity (considered at the preformulation stage ) 1. Cloned peptides and proteins 2. Conjugation of peptides and proteins to low molecular weight compounds 3. Conjugation of peptides and proteins to various polymers such as dextran, albumin, dlamino acids, poly vinyl pyrrolidone, polyethylene glycol,etc E.G. ALBUMIN BASED PACLITAXEL FOR METASTATIC CANCER* Solvent based formulation of paclitaxel can be administered i.v. in case of breast cancer but it produce hypersensitivity ,neutropenia and neuropathy. Nanoparticles technology using human albumin protein exploits natural pathway to selectively deliver the drug to tumor while avoiding some of toxicities of solvent based formulations. (*ref; C.A, .vol.148, no.26, 2008, 592550x) This conjugation causes increase in water solubility, reduce in immunogenicity, extended circulation half-life in vivo, Retention of biological activity. Provide the route of administration E.G. ORAL DELIVERY OF INSULIN BY CONJUGATION WITH VITAMIN B12 * Limitation of oral delivery of insulin: 1. Proteolysis in GIT 2. Absorption at intestinal entrocyte To overcome this problem, we decided to use the dietary uptake pathway of vitamin B12. Mammals have an active transport mechanism in the GIT for the absorption and the cellular uptake of relatively large vitamin B12. (*ref: C.A., vol.148, No.26, 2008, 592657m)
EXCIPIENT COMPATIBILITY STUDIES

Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
The development of a suitable pharmaceutical formulation involves the screening of a number of physiological acceptable buffers,,salts,Chelators,antioxidants,surfactants,solvents co-solvents, preservatives ,dispersants, bulking agents etc For example the presence of polyethylene glycol may stabilize protein structure by mechanism of decreasing the protein surface area in contact with the solvent., this decrease is obtained by folding the polypeptide chain into compact structure Or by protein self association There lie preferential interaction between proteins and salts, sugars and other compounds to their effectiveness as stabilizers against thermal denaturation. Case study: 1. The objective of this study was to investigate the influence of formulation excipients on the physical characteristics and aerosolization performance of insulin dry powders for inhalation . The insulin dry powders were prepared by a spray drying technique using excipients such as sugars (trehalose, lactose and dextran), mannitol and amino acids (L-leucine, glycine and threonine) HPLC and mouse blood glucose method were used for determination of insulin content. The powder properties were determined and compared by scanning electron microscopy, thermogravimetric analysis and size-distribution analysis by a time-of- flight technique. Powders with good physical properties were achieved by combination of insulin and trehalose This study suggests that L-leucine could be used to enhance the aerolization behavior of the insulin dry powders for inhalation and trehalose could potentially be used as an excipient in the formulation. 2. Inhibition of aggregation of aluminum hydroxide adjuvant during freezing and drying.* Aluminum salts are widely used to increase immunogenicity of recombinant protein vaccines. However, when vaccine formulated with these adjuvants are frozen or lyophilized, loss of efficacy often reported. this loss of potency is usually attributed to aggregation of adjuvant particles during processing. By cooling alhydrogelTM formulation as faster rates or by addition of sufficient amount of glass forming excipient such as trehalose, aggregation of alhydrogel can be prevented. (*ref: JPS 97:2049-2061, 2008)
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
BIOPHA EXCIP PROBLEMS RMACEU IENTS TICAL DPT Toxoids Formal dehyde Induce aggregation due to cross-linking that produces inter-molecular cross links. Macromolecular associative diminishes the immunogenicity and complicate the release of vaccine from controlled release vehicles such as PLG Alumin Increase in Crystallinity with time at room um temperature resulting in the desorption of salts antigen. Also catalyze chemical degradation of PRP antigen in HB Vaccines Succina Crystallization during freezing and unfolding te of protein buffer
REMEDIES
Process optimization
Vaccines
Process optimization
Lyophiliz ed IF-
Acetate Lyophilize buffer d products Mannit GCSF ol
Sublimes during manufacturing (manufacturing incompatibility)
Glycolate buffer replacement (remains in amorphous state).Add glycine and lysine acts as a buffer and lyoprotectant. Use sorbitol which remains in amorphous state. Add glycine which renders the product amorphous.
Crystallizes out during manufacturing ,loss of activity
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
STRESS STUDIES
During such preformulation study, the protein/peptide will be exposed to various stresses, key degradation products will be identified, and appropriate stability-indicating assays will be developed. The protein/peptide products will be exposed to the following stresses: 1. Elevated temperatures, e.g., 37 and 45 degree C 2. pH's between 3.0 and 8.0 3. Ultraviolet and fluorescent light 4. Agitation and shear 5. Oxidation 6. Freeze-thawing From this, the following stress-induced changes will be investigated: 1. Aggregation and/or precipitation 2. Deamidation or cyclic imides formation (if relevant amino acid residues are present in the sequence) 3. Oxidation (if relevant amino acid residues are present in the sequence) The following analytical methods will be used to determine the degradation product From amongst these, stability-indicating methods will be identified. Size-exclusion HPLC Reversed-phase HPLC Ion-exchange HPLC Electrophoresis Turbidity assay Protein concentration assay
Characte rizations Accelerated stability studies Stress Studies Key Degradation products
Examples Heat, Freezing,pH,Light, Agitation, Oxidation, Dehydration, Surfaces, Shear, etc. Aggregation, Oxidation, Deamidation, Cleavage, Surface adsorption, surface denaturation, etc. HPLC, Electrophoresis, Spectrometry, article count, turbidity, etc.
Stabilityindicating assays
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
DEGRADATION PROCESSES OF PROTEIN PHARMACEUTICALS
1. PHYSICAL INSTABILITY
1.1 Denaturation 1.2 Surface adsorption 1.3 Aggregation 1.4 Precipitation. 2. CHEMICAL INSTABILITY 2.1 Hydrolysis 2.1.1 Deamidation 2.1.2 Intramolecular aminolysis 2.1.3 Transpeptidation 2.2 Oxidation 2.3 Racemization 2.4 -elimination 2.5 Disulphide exchange
1. PHYSICAL INSTABILITY:
Physical instability refers to physical transformation in the higher order structure of the protein Macromolecules (proteins) undergo a variety of structural changes independent of chemical molecular modifications Physical instability of proteins result in changes in 3-D protein structure due to unfolding and refolding ,changes in hydrogen bonding and changes in hydrophobic interactions. When protein unfold, they loose their globular structure and the unfolded molecule can undergo further inactivation by adsorption to surfaces, aggregation with other protein molecules or by chemical reaction. The loss of the globular structure of proteins is referred to as denaturation.
1.1 Denaturation / conformational changes :

It is alteration of global fold of a native molecule i.e. disruption of tertiary and frequently secondary structure. It essentially involves the physical change of protein rather than its chemical composition.
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
Factors affecting denaturation are: Temperature, pH, Ionic strength, addition of organic solutes (e.g. urea ,guanidine salts,acetamide) or ,organic solvents ( e.g. alcohol, acetone). It can be reversible or irreversible. Reversible denaturation can be defined as unfolding of the polypeptide which can be reversed by removal of denaturant. Irreversible denaturation defined as unfolding process where the original structures cannot be recovered. Solubility of proteins in aqueous solution is physical property and occurs when protein to solvent interaction are more favorable than protein to protein interactions Denaturation ,thus results in ,decreasing solubility., loss of biological activity ,loss of crystallizing ability ,alteration in reactivity of the constituent groups, changes in molecular shape and susceptible to enzymatic hydrolysis In order to obtain active proteins from natural sources, it is necessary to solubilizes it in the denaturants and to allow the protein to refold by removal of the denaturant.
1.2 surface adsorption:

Peptides and proteins are amphiphilic i.e. they possess both polar ,hydrophilic and non-polar hydrophobic amino acids residues, hence have tendency to be adsorbed at interfaces such as air-water and air-solid Hydrophobic, non-polar amino acids prefer hydrophobic environment such as air, surfaces of glass or plastic Thus, they rearrange and denature on adsorption at an interfaces On adsorption ,they form van der walls ,hydrophobic and ion pair bonds which may result in further denaturation of the molecules Biological activity is changed or lost due to adsorption This also causes a reduction in the concentration of drug available. The loss of polypeptide and protein as a result of interfacial adsorption may occur during purification, formulation, storage or delivery. Example INSULIN is adsorbed to the surfaces of delivery pumps, glass and plastic containers, insides of intravenous bags and tubing etc.
1.3 Aggregation /non-covalent aggregation (self- association) & precipitation :

Denatured ,unfolded proteins may rearrange to form aggregates in which the hydrophobic amino acid residues of different molecules associate together Aggregation on a macroscopic scale resulting in precipitation Both the phenomenon can follow interfacial adsorption
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
The extent of it is dependent on the relative hydrophilicity or hydrophobicity of the surfaces and accelerated by hydrophobic surfaces. The types of vials and stoppers used are therefore of important. E.g.-insulin frosting on the walls of the container. Precipitation is accelerated in presence of large air-water interface, indicating that adsorbed protein at the air-water interface is denaturing, aggregating and finally precipitating. Agitation increases aggregation and precipitation The formation of aggregates or ppts in the insulin reservoir of implanted insulin pumps has been a major problem associated with this system
Chemical stability involves molecular modification of the protein molecule via the making
or breaking of covalent bonds, resulting in the formation of a new chemical entity 2.1 Hydrolysis:
2. CHEMICAL INSTABILITY:
2.1.1 Deamidation:
Deamidation refers to the hydrolysis of the side chain amide linkage of an amino acids residues of protein pharmaceutical to form carboxylic acid especially asparagines and/or glutamine ,yielding respectively Mechanism involves the formation of a succinimide intermediate, depending on the bonds cleaved during hydrolysis, can also result in Transpeptidation or racemization. Proteins that undergo deamidation in vitro include Human growth hormone, growth hormone, Porcine and Bovine Insulin formuation,Adrenocorticotropic hormone (ACTH),Prolactin,Lysozyme,Secretin etc Phosphate buffers are most prone to increase the deamidation rates in protein. The rate of deamidation is favored by extremes of pH, elevated temperature and ionic strength and is more influenced by tertiary and quaternary interactions than by primary sequence and slowest rates of deamidation occurring near pH 6.
2.1.2 Proteolytic degradation /proteolysis:

It is characterized by hydrolysis of 1 or more peptide (amide) bonds in the protein backbone, resulting in loss of biological activity. And susceptibility to cleavage is dependent on residues involved. Asparagines residues are more unstable than other residues and asp-pro bond is particularly labile. It occurs when the proteins are exposed to extremes of pH or high temperatures or when proteins are exposed to proteolytic enzymes. In general at neutral pH and room temperature, peptide bonds are relatively stable in relation to hydrolysis DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
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For example, lysozymes is inactivated irreversibly by heating at 90-100C The most obvious source is through bacterial contamination, also introduced during isolation and recovery of recombinant proteins, avoided by storing the protein in the cold under sterile conditions. Resistance to proteolysis seems to be dependent upon higher levels of protein structure. Denaturation renders protein susceptible to proteolytic degradation Strategies may be adopted to minimize the likelihood of proteolytic degradation of the protein product: Minimizing processing times limits the duration of contact between protein and proteases Processing at low temperature (4C) reduce the rate of proteolytic activity. Use of the specific protease inhibitor, in homogenization buffer, using cocktail of such inhibitor is thus more effective. Often such inhibitors are avoided due to their toxicity. Inhibitors PMSF ( Phenyl methylsulphonyl fluoride) Benzamidine Pepstatin A EDTA Protease class inhibited Serine proteases and cysteine proteases Serine proteases Aspartic proteases Metalloproteases
2.2 Oxidation:
The side-chain of histidine, methionine, lysine, tyrosine & tryptophan residues in the proteins are susceptible to oxidation. It is common during the synthesis, isolation & storage. The thioether group of Methionine is particularly susceptible to oxidation especially atmospheric oxygen and acidic pH and thiol group of Cysteine can be oxidized to sulfonic acid. Factors such as pH, temperature, trace amount of metal ions (catalysts), buffer content used and O2 tension can affect the rates of oxidative reactions. Oxidation can best minimized by replacing the air in the headspace of the final product container with an inert \gas such as nitrogen and /or the addition of antioxidants to the final product. Peptide hormones for which biological activity reported to be lost by Methionine oxidation include :Corticotrophin, Human growth hormone ( contain 3 methionine) Parathyroid hormone ,Corticotrophin releasing factor ,Gastrin ,Calcitonin
2.3 Racemization:
Paper 910101 M.Pharm-(2009-10)
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All amino acids with the exception of Gly. are chiral at the carbon bearing the side chain and are subject to base catalyzed racemization It renders the protein NON-METABOLIZABLE forms of amino acids and reduces the biological activity. The rate of racemization depends on particular amino acids and are influenced by temperature ,pH.,ionic strength and meta ion chelation Aspartic acid and serine residues are prone to racemization
2.4 Disulfide bond exchange:

Disulphide bonds provide co-valent structural stabilization in proteins ,may break and reform incorrectly ,altering the 3-D structure ,thereby affecting biological activity Exchange is Catalyzed by thiols , arises as a result of hydrolytic cleavage of disulphide ,in alkaline and neutral media and can be controlled by thiol scavengers like N-ethyl maleimide and p-mercuribenzoate.
METHODS TO IMPROVE PHYSICAL STABILITY:

Additives such as metal ions ,polyalcohol ,surfactants ,reducing agents ,chelating agents and other proteins ,amino acids ,fatty acids and phospholipids Specific ion binding sites can be introduced on protein molecules in relation to the aggregation and precipitation.eg salts Both ionic and non-ionic surfactants can stabilize proteins by preventing adsorption of proteins at interfaces At low concentration in aqueous solutions ,polyhydric alcohol such as glycerol by selective salvation .(excluding the water molecules)
METHODS TO IMPROVE CHEMICAL STABILITY:

Different methods used are: 1. Chemical modification of protein molecules: Synthetic polymers like PEG and Polyoxyethylene can be coupled to protein molecules chemically to enhance the chemical stability. Comparatively, lipids coupled with Insulin can increase the absorption of Insulin and hence can increase the activity. 2. Site directed mutagenesis: This technique creates specific mutations that alter the amino acid sequence of proteins and create new proteins.
Paper 910101 M.Pharm-(2009-10) 3. Appropriate choice of conditions:
BIOTECHNOLOGY
Chemical stability can be obtained by the appropriate choice of conditions such as physical state, pH, temperature, preservative and ionic strength. Proteins can be stabilized if they are in a dry state. Lower temperatures can also enhance the stability. Dosage forms can be formulated in a way that they are solid and freeze dried products.
METHOD TO IMPROVE STABILITY

Problems Deamidation, cyclic imides formation Aggregation, precipitation Truncation Oxidation pH optimization pH optimization; addition of sugars, salts, amino acids, and/or surfactants pH optimization, protease removal Excipient purity analysis, addition of freeradical scavengers (mannitol, sorbitol), use of a competitive inhibitor (methionine) Addition of surfactants or excipient proteins Addition of sugars or amino acids Solutions
Surface denaturation, adsorption Dehydration
Stabilizer excipient
Conce.
Mechanism of stabilization Biopharmaceutical Protein stabilized , and interferons , globulin preparation,IL2,urokinase,erythro poietin Various interferon preparations,erythro poietin,factorVIII,u rokinase Polypeptide stabilization.
1.Human Decrease the surface adsorption 0.1serum albumin 0.2% stabilize the native (HSA) conformation Effective cryoprotectant Minimizes detrimental effects of freeze drying process. 2 Glycine, 0.5-5% Reducing surface lysine or adsorption arginine Inhibits aggregate formation stabilizes the conformation 3.Several Stabilizes the protein in polyols solution. glycerol, Carbohydrates are added mannitol, prior to the freeze drying sorbitol PEG to provide the physical bulk to the freeze dried cake
Paper 910101 M.Pharm-(2009-10) 4.surfactants: Tween series
Chapter 1 Preformulation LIMITE Reduce the protein D aggregation by reducing the surface tension and increase the solubility of the protein
BIOTECHNOLOGY
Vaccines. -globulin preparations. Sodium do-decyl sulphate for nonglycolated IL-2
FORMULATION APPROACHES TO PROTEIN STABILIZATION 1. Protein stabilization in solution using additives:

Concentration of salts has profound effect on protein solution and aggregation. E.g. for human growth hormone, a Sodium phosphate buffer concentration of about 5 mM produced less aggregation of protein.
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
For tissue plasminogen activator, solubility of protein at optimally stable pH was insufficient for therapeutic application. So, positively charged amino acid, arginine was included in formulation to increase protein solution at desired pH range. Non ionic surfactants like, polysorbate 80 inhibits protein aggregation because of greater tendency of surfactant molecule to align themselves at the liquid-air interface, so, excluding the proteins from interface and inhibiting surface denaturation. Use of cyclodextrin or 2-Hydroxy propyl- -cyclodextrin solubilizes ovine growth hormone and prevents shaking induced precipitation.
2. Protein stabilization in the dried solid state:

Lyophilization and spray drying used to dehydrate heat sensitive molecules and thereby inhibit the degradative reactions that may be observed when proteins are formulated in solution
Mechanism for Stabilization of Dried Proteins:

Binding of additives to the dried protein to act as a water substitute after removal of hydration shell. As water substitutes, sugars such as lactose can serve to partially satisfy Hydrogen bonding requirements of polar groups in dried proteins. So, bulking agents are used to enhance the appearance of the lyophilized product.E.g. Mannitol is widely used as bulking agent which produces an elegant lyophilized cake. It also exerts a cryoprotective effect and its concentration in a formulation can be adjusted to achieve an isosmotic solution on reconstitution. Other polyhydric alcohols such as sorbitol and sugars, such as sucrose, dextrose and dextran are also used as bulking agents.. Thermal properties of importance for Lyophilization cycle development include freezing temperature of formulation, temperature at which frozen formulation will melt or collapse and the temperature above which products will rapidly degrade. The moisture level and physical character of the lyophilized cake (amorphous or crystalline) compatible with long term stability also needs to be considered during development of the Lyophilization cycle. Formulation of crystalline cake is affected by temperature cycling during Lyophilization and the nature of the excipient in the formulation. Final moisture content of an amorphous lyophilized cake is higher owing to decrease transport of water vapour from the cake during drying. Increasing moisture decreases the physical stability of the lyophilized cake and may leads to collapse of the cake with storage. In addition to affecting physical stability of the lyophilized product, the moisture level may affect the Physico chemical stability of the protein itself.
2.1 Lyophilization and protein formulation development:

It is characterized by two primary concerns: 1 Excipient selection to optimize long term stability of protein in the dried state. 2. Effect of excipient on processing parameters of Lyophilization cycle such as maximum and minimum drying temperature and cycle length and on the appearance of the lyophilized powder cake. DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
Freezing and drying during Lyophilization is a source of inactivation for proteins. Freezing causes gradual concentration of solutes surrounding the protein molecule s water is removed by the phase change. The high concentration of salts causes the potential to shift in pH due to crystallization of buffer component, limiting solubility of protein itself as it concentration can leads to protein inactivation. For selection of a buffer for a lyophilized formulation, the effect of temperature on pH and buffer solubility needs to be considered. The effects of pH changes of freezing due to greater solubility of Potassium phosphate. Recovery of protein activity after freezing can be influenced by addition of certain protective additives, such as sugars, polyols, amino acids and salts.
2.2 Spray drying of protein pharmaceuticals:

It is alternative to freeze drying proteins as a process capable of drying thermally labile materials. The particles are dried in the air stream in seconds, owing to high surface area contact with drying gas. Its major advantage over Lyophilization is particle size and shape of final dried powder. It can produce spherical particles that have good flow properties and the process can be adopted to produce particles of a range of sizes dependent on the application. Disaccharide molecules protect proteins during drying. So, they may be effective stabilizers during the spray drying process. E.g. sucrose has a protective effect on both the spray drying and Lyophilization of oxhemoglobin.
GUIDELINES ON THE STABILITY OF THE BIOTECHNOLOGICAL PRODUCT 1. Preamble

The applicant should develop the proper supporting stability data for biotechnological product and consider many external conditions that can affect the product's potency, purity, and quality. The purpose of this document is to give guidance to applicants regarding the type of stability studies that should be provided in support of marketing applications.
2. Scope of the annex
Stability Testing of New Drug Substances and Products'' applies to well-characterized proteins and polypeptides, their derivatives and products of which they are components, and which are isolated from tissues, body fluids, cell cultures, or produced using recombinant deoxyribonucleic acid (r-DNA) technology. DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
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The document does not cover antibiotics, allergenic extracts, heparins, vitamins, whole blood, or cellular blood components.
3. Selection of Batches
Where bulk material is to be stored after manufacture, but before formulation and final manufacturing, stability data should be provided on at least three batches for which manufacture and storage are representative of the manufacturing scale of production. A minimum of 6 months stability data at the time of submission should be submitted in cases where storage periods greater than 6 months are requested and for those less than 6 months determined on case by case basis.
3.1 Drug Substance (Bulk Material)
3.2 Sample Selection
Where one product is distributed in batches differing in fill volume (e.g., 1 milliliter (mL), 2 mL, or 10 mL), unit age (e.g., 10 units, 20 units, or 50 units), or mass (e.g., 1 milligram (mg), 2mg, or 5 mg), samples to be entered into the stability program may be selected on the basis of a matrix system and/or by bracketing. The differences in the samples for the same drug product should be identified as, for example, covering different batches, different strengths, different sizes of the same closure, and, possibly, in some cases, different container/closure systems. Where the same strength and exact container/closure system is used for three or more fill contents, the manufacturer may elect to place only the smallest and largest container size into the stability program, i.e., bracketing.
4. Stability-Indicating Profile
Consequently, the manufacturer should propose a stability- indicating profile that provides assurance that changes in the identity, purity, and potency of the product will be detected. At the time of submission, applicants should have validated the methods that comprise the stability-indicating profile, and the data should be available for review. The determination of which tests should be included will be product-specific.
4.1 Protocol
The protocol should include all necessary information that demonstrates the stability of the biotechnological product throughout the proposed expiration dating period including, for example, well-defined specifications and test intervals.
4.2 Potency
For the purpose of stability testing of the products described in this guideline, potency is the specific ability or capacity of a product to achieve its intended effect and is determined by a suitable in vivo or in vitro quantitative method. For that purpose, a reference material calibrated directly or indirectly against the corresponding national or international reference material should be included in the assay. Potency studies should be performed at appropriate intervals as defined in the stability protocol and the results should be reported in units of biological activity calibrated, whenever possible, against nationally or internationally recognized standards. Where no national or DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
international reference standards exist, the assay results may be reported in in-house derived units using a characterized reference material.
4.3 Purity and Molecular Characterization
For the purpose of stability testing, tests for purity should focus on methods for determination of degradation products. The degree of purity, as well as the individual and total amounts of degradation products of the biotechnological product entered into the stability studies, should be reported and documented whenever possible. The use of relevant physicochemical, biochemical, and immunochemical analytical methodologies should permit a comprehensive characterization of the drug substance and/or drug product (e.g., molecular size, charge, hydrophobicity) and the accurate detection of degradation changes that may result from Deamidation, oxidation, sulfoxidation, aggregation, or fragmentation during storage. Methods that may contribute to this include electrophoresis (SDS-PAGE, immunoelectrophoresis, Western blot, isoelectro focusing), high-resolution chromatography (e.g., reversed-phase chromatography, gel filtration, ion exchange, affinity chromatography), and peptide mapping. Wherever significant qualitative or quantitative changes indicative of degradation product formation are detected during long-term, accelerated, and/or stress stability studies, consideration should be given to potential hazards and to the need for characterization and quantification of degradation products within the long-term stability program.
4.4 Other Product Characteristics
Visual appearance of the product (color and opacity for solutions/suspensions; color, texture, and dissolution time for powders), visible particulates in solutions or after the reconstitution of powders or lyophilized cakes, pH, and moisture level of powders and lyophilized products.
5. Storage Conditions 5.1 Temperature

As most finished biotechnological products need precisely defined storage temperatures, the storage conditions for the real-time/real-temperature stability studies may be confined to the proposed storage temperature.
5.2 Humidity
Biotechnological products are generally distributed in containers protecting them against humidity. Therefore, where it can be demonstrated that the proposed containers (and conditions of storage) afford sufficient protection against high and low humidity, stability tests at different relative humidity can usually be omitted.
5.3 Accelerated and Stress Conditions

Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
The expiration dating should be based on real-time/real-temperature data. However it is strongly suggested that studies be conducted on the drug substance under accelerated and stress conditions. Studies under accelerated conditions may provide useful support data for establishing the expiration date, provide product stability information or future product development (e.g., preliminary assessment of proposed manufacturing changes such as change in formulation, scale-up), assist in validation of analytical methods for the stability program, or generate information that may help elucidate the degradation profile of the drug substance or drug product. Also useful in determining whether accidental exposures to conditions other than those proposed (e.g., during transportation) are deleterious to the product and also for evaluating which specific test parameters may be the best indicators of product stability. It help to reveal patterns of degradation, if so, such changes should be monitored under proposed storage conditions.
5.4 Light 5.5 Container/Closure

Stability studies should include samples maintained in the inverted or horizontal position (i.e., in contact with the closure), as well as in the upright position, to determine the effects of the closure on product quality. The applicant should demonstrate that the closure used with a multiple-dose vial is capable of withstanding the conditions of repeated insertions and withdrawals so that the product retains its full potency, purity, and quality for the maximum period specified in the instructions-foruse on containers, packages, and/or package inserts.
5.6 Stability after Reconstitution of Freeze-Dried Product
The stability of freeze-dried products after their reconstitution should be demonstrated for the conditions and the maximum storage period specified on containers, packages, and/or package inserts.
6. Testing Frequency
The shelf lives of biotechnological products may vary from days to several years. With only a few exceptions, however, the shelf lives for existing products and potential future products will be within the range of 0.5 to 5 years. When shelf lives of 1 year or less are proposed, the real-time stability studies should be conducted monthly for the first 3 months and at 3 month intervals thereafter. For products with proposed shelf lives of greater than 1 year, the studies should be conducted every 3 months during the first year of storage, every 6 months during the second year, and year of storage, every 6 months during the second year, and annually thereafter.
7. Specification:
Although biotechnological products may be subject to significant losses of activity, physicochemical changes, or degradation during storage, international and national
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
regulations have provided little guidance with respect to distinct release and end of shelf life specifications. Recommendations for maximum acceptable losses of activity, limits for physicochemical changes, or degradation during the proposed shelf life have not been developed for individual types or groups of biotechnological products but are considered on a case-by-case basis. Each product should retain its specifications within established limits for safety, purity, and potency throughout its proposed shelf life
8. Labeling
For most biotechnological drug substances and drug products, precisely defined storage temperatures are recommended. Specific recommendations should be stated, particularly for drug substances and drug products that cannot tolerate freezing. These conditions, and where appropriate, recommendations for protection against light and/or humidity, should appear on containers, packages, and/or package inserts.
CASE STUDY : 1) PRELIMINARY INVESTIGATION OF INTERLEUKIN 2 PROLIPOSOMES

The IL-2 proliposomes prepared by vacuum freeze-drying system. The IL-2 activity measured using MTT colorimeter and particle diameter determined by laser diffraction system.Results showed that IL-2 proliposomes was non-finite white powder with particle diameter of 0.10-1 m and the activity of 5 x 10 6 units /g. The encapsulation rate of IL-2 proliposomes was 95 % . IN CONCLUSION , IL-2 Proliposomes with higher encapsulation rate and stability was obtained
1) METHODS FOR INCREASING THERMAL STABILITY OF LIVE VIRUS VACCINES BY USING HEAVY WATER :
Deuterating purified hepatitis A vaccines with heavy water of high concentration The concentration of heavy water is in the range from 36 % to 85 %.Stability ( including nucleic acid stability of the hepatitis A vaccine is greatly increased ,particularly hepatitis A vaccine which is stored at 4-8 C. The quality of hepatitis A vaccines is improved . It is proven that heavy water is effective in protecting thermostable hepatitis A virus strain H2 (HAV-H2)Accordingly ,method enables live attenuated hepatitis A vaccines to retain potencyduring storage and transport
2) PHYSICO-CHEMICAL CHARACTERISTICS OF MAGNETIC MICROSPHERES CONTAINING TISSUE PLASMINOGEN ACTIVATOR

Magnetic carrier system developed to target tissue plasminogen activator ( t PA) to a thrombosis.The microsphere were superparamagnetic with a magnetization of 6.9-8.7emu/g .The author encapsulated 5% tPA by mass which eluted from microspheres to produced a solution concentration of 5.3-19.6 g/ml in tPA which exceeds the theoretical thrombolysis concentration
2 RECOMBINANT HUMAN INTERLEUKIN-2

The aim was to prevent the adsorption of the IL-2 at the glass-vial interface and for these effects of various excipients are studied through RP-HPLC, SDS-PAGE & biological assay.It was found DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
Paper 910101 M.Pharm-(2009-10)
BIOTECHNOLOGY
that Poly sorbate 80 at higher concentration decreases the stability of IL-2 in solution. Antioxidants such as methionine and EDTA-Na2 diminished the oxidation rate of API.Amino-acids such as glutamine, glycine & histidine stabilized IL-2 in different grades & glycine at 5 mg/ml allowed for the best stability behavior
3 ANTHRAX POWDER FOR NASAL MUCOSAL DELIVERY:

Recombinant proactive antigen is most thermally stable within pH range 6-8.Spray dried formulations displayed substantial improvement in storage stability over liquid formulations.These studies were carried out by circular dichroism and UV - visible absorption and fluorescent spectroscopies.
4 RECOMBINANT RICIN TOXIN A-CHAIN VACCINE:

The method first uses spectroscopic techniques to evaluate the stability of rRTA as a function of temperature and pH. Following identification of optimal pH conditions, light scattering and fluorescence assays are employed to screen a wide variety of compounds for their abilities to stabilize rRTA. Once stabilizers were identified, the ability of rRTA to adsorb to aluminum salt adjuvants was evaluated. Desorption of the protein from the adjuvant was also analyzed. Using this approach, the optimal formulation conditions for rRTA were determined to be pH 6.0 utilizing glycerol as a stabilizer and Alhydrogel as an adjuvant.A rapid 3 step process was employed for its preformulation study
STUDY QUESTIONS
1) Describe the solid-state properties in brief as a part of the physical characterization in preformulation studies 2) Elucidate the importance of solution-phase properties in the biotechnological products. 3) Discuss in detail the excipient compatibility studies of the biotechnological products 4) Importance of stress studies in preformulation and how it is carried out ? 5) Discuss the methods to improve the physical instability of biopharmaceuticals 6) SITE DIRECTED MUTAGENESIS way to solve the chemical instability of biopharmaceuticals. justify this statement 7) Discuss guidelines considering the stability of the biotechnological products in brief. 8) Describe the formulation approaches to protein pharmaceuticals 9) Write about the stability indicating profile of protein pharmaceuticals QUESTIONS ASKED IN EXAMINATIONS: 1) Which factors are to be considered for preformulation studies of biotechnological products? 2) How the stability of proteins affected in presence of excipients
REFERENCES :
1. Modern Pharmaceutics.- Drugs and the Pharmaceutical Science by G S. Banker and C T. Rhodes. pg. 843-867. 2. Biotechnology and Pharmacy by J.M.Pizzuto, H.R.Manasse , pg 118 - 124 3. Pharmaceutical Biotechnology by S W. Zito pg : 83-90 DEPT. OF PHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY L.M.COLLEGE OF PHARMACY-09
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4. Biotechnology & Pharmacy by J M. Johnson & M. E. Johnson pg : 116 137 5. Pharmaceutical Biotechnology by D. J. A. Crommelin and R D. Sindelar 6. Biopharmaceuticals: Biochemistry and Biotechnology by G Walsch ,pg 115- 124,131 7. Peptide and Protein Drug Delivery by V H.L.Lee , pg.769 - 783 8. Biotechnology & Its Application in Pharmacy by G T.Kulkarni , pg. 124 -126 9. J Pharm Sci. 2007 Jan; 96(1):44-60 10. J Pharm Sci. 2004 Jul; 93(7):1912-23 11. J Pharm Pharmacol. 2005 Jan; 57(1):31 12. Chemical Abstract: Vol: 147, no: 1, 2007, p.no 1860 13. Chemical Abstract: Vol: 145, no: 18, 2006, 363311r 14. Chemical Abstract: Vol: 147, no: 1, 2007, p.no1850) 15. JPS 97:2049-2061, 2008 16. C.A., vol.148, No.26, 2008, 592657m 17. C.A, .vol.148, no.26, 2008, 592550x

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DT-23/09/2009

SEMINAR ON PHYSICOCHEMICAL FACTORS UNDER PREFORMULATION STUDY

GUIDED BY: DR.R.K.PARIKH

PRESENTED BY: NITU-K- CHANGOIWALA M.PHARM - I ROLL NO - 04 YEAR-2009-10

DEPT. OF PHARMACEUTICS & PCEUTICAL TECHNOLOGY L.M. COLLEGE OF PHARMACY, ABAD-09.

L.M. COLLEGE OF PHARMACY

CONTENTS:I. PHYSICAL CHARACTERISTICS

L.M. COLLEGE OF PHARMACY

L.M. COLLEGE OF PHARMACY

FLOW CHART FOR PREFORMULATION STUDY:

Receive drug substance

Determine their solubility, partition coefficient, pKa, dissolution rate.

Stability testing at normal and exaggerated condition.

[B] SOLUBILITY ANALYSIS

L.M. COLLEGE OF PHARMACY

Method to determine solubility

L.M. COLLEGE OF PHARMACY

Uses of these equations:1) 2) 3) 4)

L.M. COLLEGE OF PHARMACY

L.M. COLLEGE OF PHARMACY

P > 1 Lipophilic drug. P < 1 Hydrophilic drug.

L.M. COLLEGE OF PHARMACY

Dissolution Solid drugs in GI fluid Solution of drug in GI fluid

Absorption Drugs in systemic circulation

L.M. COLLEGE OF PHARMACY

[2] Particulate dissolution

[1] Solid state stability

Techniques for solid state stability studies:[JPS April 2007;

[2] Solution State Stability

L.M. COLLEGE OF PHARMACY

Free radical chain process:a) INITIATION

Activation RH Light, Heat R + H

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RO2 + X RO2 + RO2

Inactive product Inactive product

a) Reducing agent. b) Chain inhibitors of radical induced decomposition.

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L.M. COLLEGE OF PHARMACY

Convert oxygen from its ground state to singlet excited state.

Eg. Diphenhydramine, Chloroquine, Methotrexate.

Eg. Chlorpropamide, Furosemide.

(4) Decarboxylation in anti-inflammatory agents.

Eg. Naproxen, Flurbiprofen, Benzoxaprofen.

Eg. Noradrenaline, Doxepine.

Eg. Metronidazole Oxidiazine Yellow color.

L.M. COLLEGE OF PHARMACY

Prevention of Photodecomposition:1) Suitable packing.

[Eg. Nifedipine is manufactured under Na light].

Colorant - Curcumine, Azorubine. Pigments - Iron oxide, Titanium dioxide.

VIII. ENZYME DECOMPOSITION

L.M. COLLEGE OF PHARMACY

SUMMARY: The preformulation evaluation of new drug substances has become

L.M. COLLEGE OF PHARMACY

L.M. COLLEGE OF PHARMACY

SEMINAR ON Hygroscopicity , Powder Rheology & Compaction Properties

GUIDED BY : Dr. R. K. PARIKH

PRESENTED BY:SANJAY C. MODI M-PHARM- I YEAR-2009-10 ROLL NO. -7

DEPT. OF PHARMACEUTICS AND TECHNOLOGY L.M.COLLEGE OF PHARMACY AHMEDABAD-09.

(B) METHOD OF DETERMINATION :

(I)-Non hygroscopic (II)- Slightly hygroscopic (III)-Moderately hygroscopic (IV)-Very hygroscopic

(C) IMPORTANCE OF MEASUREMENT :

(D) METHODS OF IMPROVEMENT :

New smart excipient: