Sterility Assurance in

Pharmaceuticals

Apar Dholakia
Amneal Oncology Pvt. Ltd
Email: apard@amnealindia.com
 Agenda for today

• Background and History of sterility assurance

• Regulatory expectation
• Overview of SAL aspects
• Factors affecting SAL
• Aseptic process simulation
• Aseptic process design
• Recent advancement(s)
 Pharmaceutical Dosage Forms

Topical Oral Parenteral

Pharmaceutical Dosage
Forms Medical Device*

API*

Inhaler Ophthalmic Suppository

* For sterility assurance understanding
 Sterile, Sterility Assurance

• Sterility, “Absence of all Viable microorganism”

• Sterility Assurance and Sterility Assurance Level
• Basic rules of sterility Assurance,
– No processes/material shall add or increase microorganism
– Final process shall ensure removal of all microorganism
• Aseptic
– Drug product, container and closure are first subjected to
sterilization methods separately and brought together.
• Terminally Sterilized
– Filling and sealing of product container under high-quality
environment condition to have low bio-burden and then final
container is subjected to sterilization process.
 History Of Sterilization

1949: Guidance to industry

The Federal Food, Drug,
"Procedures for the 1963: First reference of
and Cosmetic (FDC) Act
Appraisal of the Toxicity of GMP came in FDA CFRs
of 1938
Chemicals in Food,"

1971/72 : Devonport case: 5% Dextrose infusion contaminated with

Klebsiella aerogenes & other gram negative bacteria in very high
concentration (excess of 106 bacteria/mL)

1972: Rocky mount incident: Contaminated with Enterobacter

agglomerans, Enterobacter cloacae and other Enterobacter spp.

Failed Test
Diagnostic error
4.00% Microbial contamination
6.00% 11.00%

1971: EU GMP “Guide to

Lack of Sterility Assurance 79.00% Pharmaceutical
Manufacturing Practice”
Sterile recall by type (2004 to 2011)
 Regulatory Expectation

• Good Manufacturing Practice (cGMP) regulations (CFR sections

CFR 210 and 211).
• Guidance for Industry Container and Closure System Integrity
Testing in Lieu of Sterility Testing as a Component of the
Stability Protocol for Sterile Products
• Microbiological Pharmaceutical Quality Control Labs;
• Sterile Drug Substance Manufacturers.

• EudraLex - Volume 4 - Good Manufacturing Practice (GMP)

guidelines
• Euradlex : Manufacture of sterile medicinal products, Annex 1.
 Regulatory Expectation

• PIC/S : Validation of Aseptic Processes PI 007-5

• Isolators Used for Aseptic Processing and Sterility Testing PI
014-3

• WHO GMP for sterile pharmaceutical products. Annex 6,

WHO Technical Report Series 961, 2011

• ISO 14644-1: Cleanrooms and associated controlled

environments; Part 1: Classification of air cleanliness
(2015);
• ISO 14698-1 and 2, Part 1 – Cleanrooms and associated
controlled environments – bio-contamination control –
General principles and methods; and Part 2 – Evaluation
and interpretation of bio- contamination data.

And many more….

 Overview of Sterility Assurance Aspects
• Training & qualification • Bio-burden • Physical: [Heat {Dry Heat; Moist Heat
• Oversight • Qualified etc}, Radiation sterilization, Filtration]
• Compatible
• Quality Culture • Chemical[Eto, Hydrogen Peroxide etc]

Man Material Method

Sterility
Assurance

Measurement Milieu Machine

HVAC Design & Qualification • Design

Calibration Program Qualification
EM program Water System D&Q •
Utilities D&Q • Preventive maintenance
 Factor(s) affecting SAL: Personnel
Limiting number of Aseptic Technique,
personnel in Train, train and
critical areas retrain

Frequent
Sanitization of Personal Qualification
hand gloves

Management Clean room garment

Oversight Design & Testing

Fact: Operators are the main source of internal particle generation

A typical clean room gowned operator would generate 10,000 Particles of 0.5µ or
larger/Sec/Ft2.
Assuming perfect mixing & High Air Change Rate, internal generation due to the operator
would be around 5000 Particles of 0.5µ or larger/Min/Ft 2 in a typical clean room.
 Factor(s) affecting SAL: Method
Method of sterilization, Moist Heat Sterilization
Area of implementation:
Terminal sterilization; SS parts and equipment not
used for single use; SIP of vessels, manufacturing
lines; closures; filters; heat stable devices.

Working Principle: • Approach from SAL perspective:

Saturated steam under pressure is • Probe temperature should remain between
applied. 121.1°C to 124°C during sterilization hold time.
Latent heat of condensation is • Exposed BIs should not show any growth after
transferred to load incubation.
• Minimum F0 value should be NLT 30 minutes in
all sensors.
• Recorded pressure shall be within limit during
the sterilization hold period (1.0 to 1.3 bars).
 Factor(s) affecting SAL: Method
Method of sterilization, Dry Heat Sterilization
Area of implementation:
Heat Stable, non-aqueous material
(Glassware, powder, metal objects)

Working Principle:

Application of superheated air to article. Val. Approach from SAL perspective:

Conduction, Convection and radiation
Also depyrogenation.
Coagulates protein of microorganism and
causes oxidative free radical damage.
Sometimes incinerates microbial cell
• The temperature in each Probe should
remain above 300°C for a minimum time of 3
minutes during total period of
Depyrogenation.
• The equivalent Depyrogenation time (FH)
achieved at all probes should be greater than
30 minutes considering a base temperature
of 250°C and a Z value of 46.4 minutes.
• Minimum 3 Log Endotoxin reductions should
meet in all the exposed endotoxin challenge
containers.
 Factor(s) affecting SAL: Method

Method of sterilization, Filtration

Area of implementation:
Fluids (Liquid and Gas)
Heat sensitive liquids

Working Principle: Approach from SAL perspective:

Mono-dispersed solution of
Sterilization by means of ‘removal’. Breviundimonas diminuta (ATCC 19146)
Porosity of 0.22µ having concentration of 107 CFU/cm2 is
‘Bubble point” for testing challenged; filtrate shall be sterile.

Product specific worst case process

conditions eg. Pressure differentials, flow
rate, time & temperature is determined .
 Factor(s) affecting SAL: Method
Method of sterilization, Gamma Radiation
Working Principle:
Gamma rays are a form of
electromagnetic radiation (Generated
generally from Cobalt 60), whereby
gamma radiation kills microorganisms by
destroying cellular nucleic acid.
Area of implementation:
• Plastic containers,
• Medical device,
• Final Packaging
Val. Approach from SAL perspective:
• Loading configuration
• Dose optimization (generally 25 kGy)
• Bacillus pumilus as biological indicator
• The aim of this validation step is to
determine the dose required to
achieve a sterility assurance of 1×10 −6
 Factor(s) affecting SAL: Method

• Other methods:
– Hydrogen Peroxide vapour sterilization
– Gaseous (Eto, Ozone, Chlorine gas etc)
– Ultraviolet light
– Microwaves
– Infrared radiation
– Ultrasonics
– Formaldehyde steam
– X- rays etc.
 Factor(s) affecting SAL: Method
Cleaning and disinfection of facility

• Suitability and efficacy of disinfectant against routine

environmental flora

• Sterile disinfectant in critical areas

• Hold time for disinfectant use

• Contact time of disinfectant
 Factor(s) affecting SAL: Environment (Milieu)

Air Classification HEPA filtered air,

based on process integrity, Air Changes,
requirement(s) Air Velocity

Viable and
nonviable particle
Drains
count monitoring

Clean area separation

Trending and
(Positive/negative
analysis
pressure)

Airlocks and Smooth, hard

interlocks surface, easily
cleanable
 Factor(s) affecting SAL: Machine

Minimize number
Pre-assembled /
and complexity of
SIPable
intervention

Atomization like
robotics Qualification

Sanitary fittings Preventive

and Valves maintenance

Design to ensure ‘First Air’

to product
 Factor(s) affecting SAL: Measurement

EM program:
Calibration Program
location, frequency

EM program: Microbial count of

Active, Passive, biological indicator/ D
surface, personal Value

EM Program:
Sterility Testing
Non-viable particle
 Factor(s) affecting SAL: Material

Water/Pure
Steam System Limit for bioburden
D&Q

Utilities (Nitrogen
/ Compressed air: Container Closure
Sterile filtered, design (CCIT..)
dry, Free from Oil
 Aseptic Process Simulation
• Process simulation using microbiological growth medium exposed
– Product contact surfaces of equipment train
– Container / closure system
– Critical environment
– Process interventions
• Study is designed considering
– Personnel, practices, number and type of interventions
– Manufacturing process complexities
– Intermittent process durations
– Container / Closure systems
– Line speeds & more...
• Failure investigation and Microbial identification
 Aseptic Process Design

• Risk Assessment

• Minimize exposure of sterile articles to potential hazard

• Limiting duration of exposure of sterile product elements

• Providing highest possible environment control,

optimizing process flow & Designing equipment to
prevent entrainment of lower quality of air
 What is Next?
• Advanced cleanroom technology
– Isolator Technology
– Robotics
• Rapid Microbiology
• Ready to use technology
• Disposable systems
• PAT, Google glass etc.
 Useful resources
• https://www.fda.gov/Drugs/default.htm

• https://
www.ema.europa.eu/en/human-regulatory/marketing-authorisation/mark
eting-authorisation-guidance-documents
• https://www.ich.org/home.html

• https://www.picscheme.org/

• https://www.pda.org/

• https://ispe.org/

• http://www.usp.org/

• https://www.iso.org/home.html