Managing Your Environmental Isolates

Good Testing Practices

“Good practice includes the periodic

challenge of media with low levels of

organisms. This includes USP indicator

organisms as well as normal flora.”

-U.S. Food and Drug Administration: “Guide to Inspections of

Microbiological Pharmaceutical Quality Control Laboratories” (1993)

Detection & Identification

It is important to know:

– What is in the cleanroom environment

– The effectiveness of cleaning

and sanitization procedures

– The source of the contaminate

Sources of Contamination

Common culprits:

– Personnel

– Air

– Water

– Processes

Case Studies of Microbial Contamination in Biologic

Product Manufacturing” Suvama, et al., American

Pharmaceutical Review, 2011.

Raw Materials

Personnel

Facility

Utilities

Equipment

Processes

Tracking and Trending

Effective tracking and trending

analysis is essential

– An early warning signal for system

failures

– Provides clues to contamination source

Is It Objectionable?

21 CFR 211.84(d)(6) “Each lot of a component, drug product container,

or closure with potential for microbiological contamination that

is objectionable in view of its intended use shall be subjected to

microbiological tests before use.”

21 CFR 211.113(a) “Appropriate written procedures, designed to

prevent objectionable microorganisms in drug products not required to

be sterile, shall be established and followed.”

21 CFR 211.165(b) “There shall be appropriate laboratory testing, as

necessary, of each batch of drug product required to be free of

objectionable microorganisms.”

“What is an Objectionable Organism?” Sutton, Scott, Ph.D., American

Pharmaceutical Review, October 2012.

Is It Objectionable?

Ultimately, the laboratory must determine if

the organism is objectionable

– Sterile vs. non-sterile products

– Intended use

– Intended recipient

– Intended route of administration

Managing Risk with Testing

Disinfectant Challenge Test

Growth Promotion Test

Antimicrobial Effectiveness Test

Disinfectant Challenge Testing

“…the most frequently isolated microorganisms from an

environmental monitoring program may be periodically

subjected to use-dilution testing with the agents used in the

disinfection program to confirm their susceptibility, as there

are real differences among

different species in resistance

to the lethal effects of

different sanitizers.”

– USP 38 <1072>

Disinfectants and Antiseptics, 2015

Selecting the Right Isolates for Disinfectant

Testing

Provide scientific rationale for the organisms used for your

testing

Choose strains that represent common organisms isolates

from the facility

Choose organisms that are common contaminates or high

risk for the products manufactured at the facility

Avoid unnecessary audit findings

Growth Promotion Testing

The media used in the following tests should be

capable of growing recommended indicator

organisms and environmental isolates

– Environmental monitoring

– Media fills

– Sterility Test

Growth Promotion Testing

“The growth promotion test is a procedure used to

demonstrate the media in the microbiological

environmental monitoring program or in media-fill runs, are

capable of supporting growth of indicator microorganism

and of environmental isolates from samples obtained

through the monitoring program or their

corresponding ATCC strains.”

– USP 38 <1116> Aseptic Processing

Environments, 2015

Antimicrobial Effectiveness Testing

“The standard battery of challenge organisms… need not

prevent the inclusion of other species of microorganisms if

deemed useful to measure the biological activity of the

preservative system for a specific product.”– USP <51> Antimicrobial Effectiveness Testing, 2015

Product

Product

Antimicrobial Effectiveness Testing

Products of any category whose formulas establish more

extreme environmental conditions (pH, water activity, specific

formula conditions) may warrant addition of isolates capable of

survival if not growth under such extreme conditions allowing

measure of preservative efficacy versus intrinsic hostility.

– Antimicrobial Effectiveness Testing: Culture Selection and Inoculum Preparation,

Phil Geiss, 2013

Why Use Environmental Isolates?

Environmental isolates may be more resistant to

disinfectants because they adapt to the

environment and limited nutrients

The isolates may need special conditions to grow

Isolates can be harder to detect in

your product

Wild Strains versus Domesticated

Strains

“Wild microorganisms are able to modify their

properties according to their nutrient supply

(for example, phase variation in bacteria) and,

in particular, are able to adapt to limited nutrients.”– Multicellular microorganisms: laboratory versus nature, Zdena Palková, EMBO Reports, 2004

“There is a strong argument that environmental isolates are

the best challenge to media and for validation studies like

sterility test validation. They are the most sensitive micro-

organisms, having become recently exposed to

disinfectants, particular soils etc.”– The Use of Environmental Isolates, Tim Sandle, Pharmaceutical Microbiology blog, January 10,

2010

Preserving Isolate Options

Short-Term Preservation Methods

Long-Term Preservation Methods

Professional Preservation Services

Short-Term Preservation Methods

Preservation Method Pros Cons

Sub-culturing/

Refrigeration

+ Low tech - Highest risk of mutation

- Risk of contamination

- Must remain within 5 passages per

USP

Sub-zero Freezing

(-20°C)

+ Viability can be

maintained for 1-2 yrs.

- Ice crystals and electrolyte

fluctuations can damage organism

- Risk of contamination

- Freezer cost and maintenance

Long-Term Preservation Methods

Preservation

Method

Pros Cons

Ultra-low Freezing

Cryogenic Freezing

+ Reduces probability of

mutation

+ Longer survival rate

- Labor intensive

- Costly

- Must closely monitor temperature

- Vulnerable to power outages and

failures

Lyophilization + Reduced risk of intracellular

ice crystallization; halts all

enzymatic and non-enzymatic

reactions

+ Easy storage

- Requires specialized equipment

- Labor intensive

- Experimentation required; strains

react differently to process

Professional Preservation Services

Turn-key processes for isolate

management

– Isolate identification

– Isolate manufacturing

– Isolate storage

Characterization and seed preparation

Customized format

production

Shipping,

distribution and storage

Step 1: Characterization and Pre-

production

Strain characterization

Determine final isolate specifications for

testing

– Media and conditions should

be mimicked

– Packaging needs

– Distribution requirements

Establish a project timeline

Step 2: Production

Seed stock is established

3-phase quality control checks

– Before preservation

– After preservation

– After packaging

Seed stock for storage

and future useStock for immediate

production

Step 3: Certificates and Reports

Certificate of Analysis– Mean Assay Value and statistics

– Complete DNA sequencing report

– Organisms identification

– Biochemical testing results

Step 4: Shipping and Storage

Isolate is packaged,

shipped and distributed

Isolates are maintained

for future use

Cost Analysis for Outsourcing

Costs of Manual PrepLabor (hrs x wages)

- Handling isolates

- Quantitating samples

- Identification for mutation

- QC for contamination

- Record keeping for audits

Freezer

3rd party identification

Laboratory supplies

Costs of OutsourcingProject price

Identification (included)

Maintenance & storage

(included)

Vs.Higher Cost Lower Cost

Considerations for Outsourcing

Accreditation

Experience

Test-ready format options

Quality reputation

Finished goods storage

Technical support and service

Distribution options

Product warranty

Final Thoughts

All environmental isolates pose

risk in aseptic manufacturing

Current regulations require the

use of isolates in testing

Managing isolates can be difficult

and expensive, but there are

accredited providers to help

Contact Info

Kelly Hedlund

Midwest Regional Sales Manager Territory - IL, IN, MI, MN, ND, OH, WI

Cell: 320-249-4761

Email: khedlund@microbiologics.com

Website: www.microbiologics.com/custom-solutions

Thank you!