Managing Your Environmental Isolates

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 Personnel Utilities Raw Materials Facility Equipment Common culprits: Personnel Air Water Processes Case Studies of Microbial Contamination in Biologic Product Manufacturing Suvama, et al., American Pharmaceutical Review, 2011. 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 microorganisms, 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 Professional Preservation Services Long-Term Preservation Methods Short-Term Preservation Methods

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 Ultra-low Freezing Cryogenic Freezing Pros + Reduces probability of mutation + Longer survival rate Cons - 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 Characterizatio n and seed preparation Customized format production Shipping, distribution and storage

Step 1: Characterization and Preproduction 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 use Stock 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 Prep Labor (hrs x wages) - Handling isolates - Quantitating samples - Identification for mutation - QC for contamination - Record keeping for audits Freezer 3 rd party identification Laboratory supplies Higher Cost Vs. Costs of Outsourcing Project price Identification (included) Maintenance & storage (included) 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!