Manufacturing of Sterile Products

Session 1

Historical Background & Environmental Microbiology

Presentation Focus

1. Microbiology

2. Gram Staining

3. Microbiology in Sterile Operations

4. Sterility & Statistical Capability

Disclaimer

The views expressed in this

presentation are not necessarily

of the Drug Regulatory Authority

of Pakistan

It is an informal communication

that represents the best current

judgment and does not constitute

an advisory opinion or binding or

otherwise obligate or commit the

DRAP, to the views expressed.

This presentation is designed by

the speaker after consulting

international reference

documents

Karachi University

National Seminars,

Conferences

Experience of Hospital Pharmacy

Experience of

Regulatory Services

International Meetings &

Trainings

Manufacturing of Sterile Products

Microbiology

Microbiology

Tiny living things

Microorganisms that exist as single cells capable of growth, respiration, reproduction independent of other cells of the same or different kind.

Microbe Size

Microbial Growth

Manufacturing of Sterile Products

Gram Staining

Gram Staining

“I have therefore published

the method, although I am

aware that as yet it is very

defective and imperfect; but

it is hoped that also in the

hands of other investigators

it will turn out to be useful”

Hans Christian Gram, 1884

Gram Staining

Gram Staining

Gram Staining

Manufacturing of Sterile Products

Microbiology in Sterile Operations

Environmental Requirements

Nutrients Respiration Temperature

Humidity pH

Water also carry the microorganisms and supports growth too

Respiration

Obligate Aerobes

• Must have oxygen to grow

Facultative Aerobes

• Grow with or without oxygen

Obligate Anaerobes

• Cannot grow if oxygen is present

Temperature

Psychrophiles

• Low temperature 0-20°C

Mesophiles

• Mid temperature 15-45°C

Thermophiles

• High temperature 40-70°C

Extremes

Volcanic Vents

• High temperature

Antarctica

• Low temperature

• Low nutrients

Mount Everest

• O2, low temperature, low nutrients

Sahara Desert

• low moisture, high temperature, low nutrients

WFI Systems

• Low nutrients

Microorganisms

Fungi

Eukaryotes

More animal than plant

No Photosynthesis Use carbon fixed by other organisms

Microscopic Fungi

Yeast

• Single cell

Mold

• Multi-cellular filaments

Microorganisms

Bacteria

• Prokaryotes

• Bacteria

• Most abundant of all microorganisms

Virus

• Small

• Genetic material with a protective coat

• Must infect host cell to reproduce

Endospores

Dormant, tough, non-reproductive structure

Forms in response to environmental stress

Can survive for extended periods of time

Produced by Gram Positive (+) Bacillus (rod)

Microbe Movement

Microbes do not …

• Crawl

• Swim

• Fly

Microbes must be carried by …

• Air flow

• Water flow

• Contact and transfer

Putting it all to use

Human Operation/ skin/ hands & hair

1Small/

medium colony

White, beige to yellow

Circular and

convexButtery

Staphylococcus species

Cocci, Gram +ve

Putting it all to use

Environment such as soil (seasonal factor)Through shoe, air etc.

2 Medium/ large colony

Off white to beige

Circular/ irregular

and convex/ raised

Mucoid, dry to grainy

Clostridium species

Rods, Gram +ve

Putting it all to use

Generally found in water (equipment piping )

3Small to medium colony

White, off white to beige

Circular/ irregular

and convex

Mucoidto

buttery

Pseudomonades, E. coli species

Rods, Gram -ve

Compendial Organisms (USP … ICH)

Staphylococcus aureus

• Aerobic

• Gram +ve

• Coccus

Pseudomonas aeruginosa

• Aerobic

• Gram -ve

• Rod

Bacillus subtilis

• Aerobic

• Gram +ve

• Rod

• Spore forming

Clostridium sporogenese

• Anaerobic

• Gram +ve

• Rod

• Spore forming

Candida albicans

• Yeast

Aspergillusbrasiliensis

• Mold

Sterility Test-Growth Promotion (support the growth of wide range of organisms)

Manufacturing of Sterile Products

Sterility & Statistical Capability

Sterility

A strict definition of sterility is the complete absence of life or inability to reproduce

• Cont’d

However, sterility as defined in the pharmaceutical industry has a more complex technical definition, and is expressed in quantitative terms

Sterility

The probability definition for sterility derives from the fact that microbial death follows a geometric progression .i.e. the cells do not all die at once when exposed to lethal conditions

• Cont’d

The US FDA defines sterilization as the

“reduction of a microbial population to 10° (or one cell) plus an additional 6 log-cycles”

Sterility (Statistical Capability-1 in a million)

Sterility is defined as the “probability of a non-sterile unit (PNSU) in a lot or batch of product”

PNSU is also referred to as the “Sterility Assurance Level” or SAL

A product is considered sterile when the probability of a non-sterile unit is < 1 in 1, 000, 000 units; or a SAL of 10-6

Exponential Death

Microorganisms die at a constant

rate over a period of time

For e.g. one million bacteria

with a 90% death rate/ min

1st min

900, 000 dead/ 100, 000 survivors

2nd min

90, 000 dead/ 10, 000 survivors

3rd min

9, 000 dead/ 1, 000 survivors

4th min

900 dead/ 100 survivors

5th min

90 dead/ 10 survivors

6th min

9 dead/ 1 survivor

7th min

• ?

Constant set of lethal conditions develop resistance