Biopharmaceutical

Technology BNN 40203

By Dr. Sity Aishah Mansur

1.1 Introduction to Pharmaceutical Products
1.2 Biopharmaceuticals and Pharmaceutical
Biotechnology
1.3 History of The Pharmaceutical Industry
1.4 The Age of Biopharmaceuticals
1.5 Biopharmaceuticals: Current Status and
Future Prospects
 Greek - Pharmacon (drug) & logos (discourse /
discussion)

 Agents affecting living processes :

• Hormones
• Drugs (pharmaceuticals)
• Growth factors
• Neurotransmitters

 The medicinal/organic chemists - create the candidate

compound
 The pharmacologist - responsible for testing the drug for
its pharmacological activity
 Ultimately will lead to the discovery of novel drugs for
therapeutic intervention
 French: Drogue – a dry herb
• A single active chemical entity present in a
medicine that is used for diagnosis, prevention
and treatment of diseases

 WHO (1966) – Drug is any substance or

product which is used or intended to be used
to modify or explore physiological systems or
pathological states for the benefit of the
recipient.
 Pharmacology studies the effects of drugs and
how they exert their effects:

• Paracetamol can reduce body temperature by

inhibiting an enzyme known as cyclooxygenase in
CNS, which is responsible for the synthesis of a
number of inflammatory mediators

• Penicillin (antibiotics) fights bacterial infections by

inhibiting a key enzyme (transpeptidase – forms
peptidoglycan cross-links) involves in the
synthesis of bacterial cell walls
chemistry medicine

Pharmacology

biochemistry
 Biologically active

 Contain biofunctional groups that attached to

it’s framework

 Able to induce biological response when

binds to its target
 Plant sources – Morphine, digoxin, quinine, atropine,
reserpine , vinca alkaloids and paclitaxel.
 Animal sources – Insulin, Thyroid extract, heparin,
gonadotrophins and antitoxic sera.
 Minerals – Liquid paraffin, magnesium sulfate,
magnesium trisilicate, ferrous sulfate and kaolin.
 Micro organisms – Bacteria and fungi – Penicillin,
Streptomycin
 Synthetic – Analgesics, hypnotics, anticancer drugs
and antimicrobials
 Genetic Engineering – Human insulin, growth
hormone genes
 Hybridoma technique – monoclonal antibodies origin
 1

Gibson M (2009) Pharmaceutical preformulation & formulation. 2nd edition. A Practical

Guide from Candidate Drug Selection to Commercial Dosage Form
1) Strategic research – based on company

Feasibility
Expertiseand competency
Therapeutic area
Market potential
2) Exploratory research

Mechanism of actions of drugs

Vital factors affecting the pathway

 EXAMPLES : drugs for diabetes treatment

•Insulin
•Metformin
•Glibenclamide / glyburide, Glipizide, Glimepiride,
Gliclazide
•Repaglinide, Nateglinide
•Pioglitazone, Rosiglitazone
•Sitagliptin, Vildagliptin
 Diabetes is a metabolic disorder where
production of insulin is insufficient or body
cannot effectively utilise insulin produced by
pancreas
 As a consequence, there will be high blood
glucose levels in the body
 Two classes of diabetes
• Type 1 diabetes mellitus (T1DM)
• Type 2 diabetes mellitus (T2DM)
 Aim of diabetic drugs – ultimately will reduce
blood glucose concentrations
Class Compound(s) Physiological action(s)
Biguanides Metformin Suppresses hepatic glucose production and
increases insulin sensitivity in muscle

Sulfonylureas Glibenclamide Binds to sulphonylurea receptors causes KATP

/ glyburide channels on β-cell plasma membranes to close,
Glipizide influx of Ca2+ and stimulation of insulin release
Glimepiride
Gliclazide

Meglitinides Repaglinide Binds to sulphonylurea receptors, but at different

Nateglinide binding sites to sulphonylurea causes KATP channels
on β-cell plasma membranes to close, influx of Ca2+
and stimulation of insulin release

Thiazolidinedio Pioglitazone Agonise and activate nuclear transcription factor

nes Rosiglitazone peroxisome-proliferator-activated-receptor-γ
(PPAR-γ) in adipose tissue, increase adipogenesis,
reduce release of fatty acids and increase insulin
sensitivity in muscle and liver
Class Compound(s) Physiological action(s)
Dipeptidyl Sitagliptin Inhibits DPP-4 action causes
peptidase-4 (DPP- Vildagliptin an increase in concentrations
4) inhibitor of native GLP-1 and GIP

Glucagon-like Exendin-4 Bind to and agonise GLP-1

peptide-1 (GLP-1) Liraglutide receptor, causing increase in
receptor agonists Exenatide-long- glucose-dependent insulin
acting release (LAR) secretion, glucagon
Lixisenatide suppression, slow gastric
Albiglutide emptying and reduce appetite

SGLT2 inhibitors Dapagliflozin Inhibits SGLT2 action causes

Canagliflozin reduction in renal glucose
reabsorption

Inzucchi SE 2002 Oral antihyperglycaemic therapy for type 2 diabetes scientific review. The Journal of the American
Medical Association 287 360-372
Tahrani AA, Bailey CJ, Del Prato S & Barnett AH 2011 Management of type 2 diabetes: new and future developments in
treatment. The Lancet 378 182-197.
3) Potential drug selection

 Optimisation by testing in vitro and in vivo.

 Most desired characteristics are selected:

 Potency
 Selectivity
 Duration of action
 Safety / toxicology assessment
  Glucagon-like peptide-1 or GLP-1 (7-37)
 Incretin hormone
 Oral administration of glucose or food intake, GLP-1 is
secreted by L-cells in intestine – hence the term
incretin
 Primary target  pancreas
 Stimulate insulin release by pancreatic beta cells
 Activity of GLP-1 is limited by short half-life (3-5 min)
 Degraded by dipeptidyl peptidase-4 which cleaves
Alanine residue at position 2 of N-terminal –
dipeptides and inactivated GLP-1(9-37) released
 GLP-1 agonists – modified to protect the compounds
from DPP-4 activity and renal clearance
Baggio & Drucker 2007 Biology of incretins : GLP-1 and GIP. Gastroenterology 132 2131-2157
 Based on this article
Knudsen LB, Nielsen PF, Huusfeldt PO, Johansen NL,
Madsen K, Pedersen FZ, Thøgersen H, Wilken M &
Agersø H 2000 Potent derivatives of glucagon-like
peptide-1 with pharmacokinetics properties suitable
for once daily administration. Journal of Medicinal
Chemistry 43 1664-1669.

 How are the GLP-1 derivatives synthesised?

 Receptor experiments (in vitro)?
 Pharmacokinetics experiments (in vivo)?
Functional receptor assay

•Activation of GLP-1R by
native GLP-1stimulates
adenylate cyclase activity
and production of cAMP
•Potential GLP-1-based
drugs have to induce
similar or higher cAMP
levels – similar potency or
more potent!

Knudsen LB, Nielsen PF, Huusfeldt PO, Johansen NL, Madsen K, Pedersen FZ, Thøgersen H, Wilken M & Agersø H
2000 Potent derivatives of glucagon-like peptide-1 with pharmacokinetics properties suitable for once daily
administration. Journal of Medicinal Chemistry 43 1664-1669.
 Pharmacokinetic
experiment

• GLP-1 analogues were

administered at a dose of
0.5 nmol/kg bw
• Native GLP-1 at a higher
dose 15 nmol/kg bw
• Blood samples collected
at 0, 2, 4, 6, 8, 24, 36,
48, 72 h after dosing
• GLP-1
radioimmunoassay was
performed.

Knudsen LB, Nielsen PF, Huusfeldt PO, Johansen NL, Madsen K, Pedersen FZ, Thøgersen H, Wilken M & Agersø H
2000 Potent derivatives of glucagon-like peptide-1 with pharmacokinetics properties suitable for once daily
administration. Journal of Medicinal Chemistry 43 1664-1669.
 You are provided with these articles
Neumiller JJ & Campbell RK 2009 Liraglutide: a once-daily incretin
mimetic for the treatment of type 2 diabetes mellitus. The
Annals of Pharmacotherapy 43 1433-1444.
Rolin et al. 2002 The long-acting GLP-1 derivative NN2211
ameliorates glycemia and increases -cell mass in diabetic mice.
Am J Physiol Endocrinol Metab 283 E745–E752

 What is Liraglutide? Provide the structure.

 How does it differ from native GLP-1?
 How does Liraglutide improve glycaemic control?
 In vivo studies that have been done?
 Class : Glucagon-like peptide-1 (GLP-1)
 Name of drug : Liraglutide
 Liraglutide (NN2211, Victoza) is an effective GLP-1
analogue and has high sequence homology (approx.
97%) to native GLP-1
 Liraglutide differs from native GLP-1 sequence at
lysine 26 and 34, where lysine 26 has an addition of
C-16 free fatty acid via a γ-glutamic acid spacer
while at position 34, lysine is substituted by an
arginine residue
 The additional acyl chain in Liraglutide promotes
non-covalent binding of the GLP-1 mimetic to serum
protein albumin and prevents renal clearance, thus
prolonging its action ~ 13 hours
 Like native GLP-1, liraglutide binds and
activates the GLP1 receptor, leading to :
• insulin release in the presence of elevated
glucose concentrations
• decrease in glucagon secretion
• delay in gastric emptying
• increase satiety
 Native GLP-1 – AA 26 – no FA chain
AA 34 – lysine residue

Neumiller JJ & Campbell RK 2009 Liraglutide: a once-daily incretin mimetic

for the treatment of type 2 diabetes mellitus. The Annals of
Pharmacotherapy 43 1433-1444.
Rolin et al. 2002 The long-acting GLP-1 derivative NN2211 ameliorates glycemia and increases -cell mass in
diabetic mice. Am J Physiol Endocrinol Metab 283 E745–E752
Rolin et al. 2002 The long-acting GLP-1 derivative NN2211 ameliorates glycemia and increases -cell mass
in diabetic mice. Am J Physiol Endocrinol Metab 283 E745–E752
 • Histological exams on
pancreas revealed :
- Increased proliferation
rate
- Increased beta cell mass

• Previous study – GLP-1

and its analogues inhibit
beta cells apoptosis

• Exendin-4 is still
subjected to renal
clearance – shorter half-
life than NN2211

Rolin et al. 2002 The long-acting GLP-1 derivative NN2211 ameliorates glycemia and increases -cell mass
in diabetic mice. Am J Physiol Endocrinol Metab 283 E745–E752
Props :

•High potency
pharmacology
•High selectivity

•noncarcinogenic
safety
•nontoxic
4) Exploratory development

Testing potential drugs in healthy human –

absorption, metabolism, then in human with
disease

Phase I clinical studies (Proof of concept)

•20-80 healthy volunteers
•Receive simple formulation potential drugs
 Safety, tolerability, pharmacokinetics and
pharmacodynamics tested

 Often include dose ranging or dose escalating

 Food effect – fasted & nonfasted

 Unwanted side effects seen – programme stopped!

 Dosage regimen
Absorption
Metabolism Pharmacokinetics
Distribution What body does to the drug

Elimination
Concentration in plasma and site of action

Pharmacodynamics
What drug does to the body

PHARMACOLOGIC
EFFECT

toxicity CLINICAL RESPONSE effectiveness

 Following preclinical studies, clinical trials were
done.
Agerso H, Jensen LB, Elbrond B, Rolan P & Zdravkovic M
2002 The pharmacokinetics, pharmacodynamics, safety
and tolerability of NN2211, a new long-acting GLP-1
derivative, in healthy men. Diabetologia 45 195-202.

 How do we determine the pharmacokinetics &

pharmacodynamics of a drug?
 Possible adverse effects?
Agerso H, Jensen LB, Elbrond B, Rolan P & Zdravkovic M 2002 The pharmacokinetics, pharmacodynamics, safety
and tolerability of NN2211, a new long-acting GLP-1 derivative, in healthy men. Diabetologia 45 195-202.
5) Full development

 Long-term safety & clinical studies completed

in patients with the disease

 Phase II
• dose-ranging studies, tolerability in patients
(hundreds)
• Intended commercial formulation developed
 Phase III

• Candidate drug must be in its intended

commercial formulation
• Tested in hundreds to thousands patients
(single-blind or double-blind trials) – potential
drug, placebo, first line drug
• High efficacy and safety
• Approved medical drug into the market

 Phase IV

• Watch drug’s long term effects

Further reading : https://www.cancer.org/treatment/treatments-and-
side-effects/clinical-trials/what-you-need-to-know/phases-of-clinical-
trials.html
Gibson M (2009)
  Biopharmaceuticals
• produced by genetic
engineering, protein
engineering or cell
engineering
technologies
• Further reading
Carter PJ 2011 Introduction to current and
future protein therapeutics: A protein
engineering perspective. Experimental
Cell Research 317 1261-1269.
Kim et al. 2005 Antibody engineering for
the development of therapeutic
antibodies. Molecules & Cells 20 17-29.
 Pharmaceuticals
• Chemically/synthetical
ly produced
• Further reading
Knudsen LB, Nielsen PF, Huusfeldt PO,
Johansen NL, Madsen K, Pedersen
FZ, Thøgersen H, Wilken M & Agersø
H 2000 Potent derivatives of
glucagon-like peptide-1 with
pharmacokinetics properties
suitable for once daily
administration. Journal of Medicinal
Chemistry 43 1664-1669.
GENETIC ENGINEERING
•1953 – the discovery of the structure of DNA
•1960s-1970s – isolation of restriction enzymes and their use
to analyse DNA structure
•1972-73 – cloning & recombinant DNA technique developed.
First bacterial gene cloned
•1974 – first expression in a bacterium of a gene from a
different species
•1977 – first complete genetic code of an organism (a phage-
5375 bases)
•1978 – bacteria produce human somatostatin & insulin from a
synthetic gene
•1982 – recombinant DNA of human insulin approved for use
in GB and the USA by FDA
Taylor DJ et al. Chapter 22, 3rd edition Biological Science
Terms & definition :
•Cloning – making many identical copies of a molecule
•Plasmid – a small circular pieces of DNA found in
certain bacteria
•Bacteriophage / phage – virus which can inject their
DNA into bacteria for replication
•Recombinant DNA – DNA formed after a piece from
one organism is joined to a piece from another
organism
•Vector / cloning vector - acts as a carrier for the DNA
to be cloned (normally the phage or plasmid)
 Genetic engineering in bacteria has 5 stages :

STAGE 1 obtain a copy of the required gene from the

donor organism
STAGE 2 insert the gene in a vector
STAGE 3 vector is introduced into the host (bacteria)
STAGE 4 select the cells which have taken up DNA of the
donor
STAGE 5 clone the gene

https://www.youtube.com/watch?v=5ffl-0OYVQU
STAGE 1 – obtaining a copy of the desired gene
3 methods used to get a copy of a gene :

1)Gene is copied from mRNA, using reverse

transcriptase
2)Gene is synthesised artificially
3)Shotgun approach – chopping up the DNA with
restriction enzymes and searching for the piece with
the required gene
• Cells which express the
required gene is identified ( e.g
pancreatic beta cells for
insulin)
• Isolate mRNA (red)
• Reverse transcriptase will
produce a complementary DNA
(cDNA blue)
• Degradation of mRNA – single-
stranded cDNA is made
• Complementary DNA strand is
synthesised by DNA
polymerase
• Double-stranded cDNA formed
 • Sequence of bases of a gene is known
• 4 bases : Adenine (A), Guanine (G), Thymine
(T), Cytosine (C)
• A…..T, G…..C
• Gene is constructed using nucleotides (each
base is part of one nucleotide)
• The nucleotides are joined accordingly
• Possible for short gene – somatostatin 14 AAs
only

Nucleotide – monomer of nucleic acid; composed of a nitrogenous base,

a five-carbon sugar (ribose or deoxyribose), and at least one phosphate
group.
• Using restriction enzymes – endonucleases
• Endo – internal; nuclease – nucleic acid
• Endonucleases recognise particular sequence of
bases and cut these points – restriction sites.
• Different enzymes attack different sites –
produce DNA pieces of different size (restriction
fragments)
• These fragments were separated and identified
by gel electrophoresis and autoradiography -
obtain the desired gene
• Disadvantage – not specific & relies on chopping
up all the DNA
- Separate
fragments of
DNA of
different
length
- Fragments
produced by
chopping up
with
restriction
enzymes
- Largest
fragments
have slow
movement –
more
difficult to
pass
through the
gel
STAGE 2 – putting genes into a vector

•Vector used is plasmid DNA or phage

DNA

•PLASMID VECTOR
-Much smaller than cromosomal DNA
-Easily separated based on the size
-Bacterial cells broken down &
centrifuged – separation of
chromosomal DNA (in pallet) & plasmid
(in supernatant)
-Plasmids are purified and cut with
restriction enzyme
-Restriction fragments (from donor) mix
with plasmids and joined by sticky ends
by DNA ligase.
• Larger pieces of DNA that can reliably
be carried by plasmids
• Part of phage DNA is replaced with the
desired DNA
• Part replaced doesn’t involve in
replication, hence cloning unaffected.
• Sticky ends –
staggered cut
formed by
restriction
enzymes; EcoR1,
HindIII & BamHI
• Blunt ends –
produced by
HindII & HpaI
STAGE 3 – introducing vector DNA into the host
(transformation)

•Commonly used host – E.coli

•Normal inhabitant of human gut & grows rapidly with
doubling time of 30 min.
•For the use in laboratory, mutant E.coli was developed
– cannot infect human if it escapes with the foreign
genes
•Plasmid vector added to a flask containing a culture of
E.coli
•Calcium ions (CaCl2) added, followed by a brief heat
shock – make cell membrane permeable to DNA and
allowing the plasmids to enter
•Transformation occurs – process of adding new DNA
to bacterial cell
• Phage vector introduced by infection of a bacterial
lawn growing on an agar plate
STAGE 4 – selecting the transformed bacteria
•When plasmids are mixed with bacteria, 2 events could occur.
1) Not all bacteria will take up the plasmids
2) Not all plasmids will have taken up the donor DNA

Overcome by using plasmids which have these features :

•Resistance for a particular antibiotic
-When bacteria are grown in a medium containing that antibiotic,
only the transformed bacterial cells will survive and multiply

AND

Blue-white screening :
•Contain a gene for β-galactosidase (lacZ – a reporter gene) which
has a group of restriction sites added
-When DNA inserted at the restriction site, the gene will NOT work –
no hydrolysis of X-gal to produce insoluble blue compound
-Therefore, colonies lack of donor DNA grown on medium containing
X-gal will appear blue
-Colorless colonies contain the donor DNA are isolated for cloning
STAGE 5 – cloning the DNA

•E.coli containing desired gene are plated out onto

nutrient agar in petri dishes
•They grow and divide once every 30 min
•Eventually colonies formed.
•For phage, one recombinant DNA molecule can
produce > 1012
identical copies of itself (together with the required
gene) in less than one day
•The combination of both techniques will result in
billions of clones produced in a very short time
Rader RA 2013 FDA biopharmaceutical product approval and trends in 2012. Bioprocess
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biopharmaceuticals production: a Finnish perspective. European Journal of Pharmaceutics and
Biopharmaceutics 59 (2005) 397–405