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MICROBIOLOGICAL QC 101

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Page 3Ensuring Safety in Biologics Production

Page 4Unwanted Guests: A Focus on Mycoplasma

Page 5Infographic: From Problems to Resolutions

Page 6Ins-and-Outs of the Microbial Limits Test

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The Scientist 2020 the-scientist.com3

MICROBIOLOGICAL QC 101

Ensuring Safety in Biologics Production

Quality control (QC) scientists working in biomanufacturing facilities must assure the quality of the final products by testing raw materials,

supplies, packaging, production laboratories, and equipment throughout the manufacturing lifecycle. QC scientists follow Good Manufacturing Practice (GMP) regulations during this process to minimize contamination risks at each step of biologic production and to avoid an unusable final product. With the emergence of drug-resistant strains of microorganisms, QC testing is more important than ever.

Airborne Microbes

Airborne microbes are almost exclusively bacteria dispersed into the air around us from the surfaces of our skin cells1, but they also include fungi, spores, and even viruses. These microbes may negatively impact biologic production. Specialized cleanroom clothing helps to eliminate the main source of contamination—human particulate matter—because it is made from lint-free material that acts as a filter against particles, but it does not eliminate contaminants entirely. Air sampling with active devices is therefore imperative for QC scientists for evaluating the microbes in any biopharmaceutical production environment. These scientists use two different physical methods for air monitoring: impaction or filtration.

Impaction involves removing particles from an air stream by forcing the air stream into a sharp bend; particles above a certain size cannot follow the air stream around the bend and instead strike a collection surface at the bend. Scientists collect the particles then assess their mass and composition. Numerous airborne microbial impaction-testing products and accessories are commercially available; the majority of these effectively collect airborne microorganisms at low impaction speeds.

Filtration involves intake and filtration of air to capture microbes and particles over a fixed time period. Many fixed and portable air filtration devices are available through several different companies, including those that use gelatin membrane filters that can capture even the smallest viruses.

Sterility Testing

A large part of a QC scientist’s job is to test the raw materials and intermediate products in closed, sterile biopharmaceutical production systems to ensure that they are free from viable

microorganisms. Microorganisms may enter sterile systems in contaminated raw materials, through faulty instruments that lead to a formerly closed system becoming “open,” or from poor sampling techniques.

There are numerous ways to perform sterility testing. According to the United States Pharmacopeia (USP) <71> Sterility Test guidelines, membrane filtration sterility testing requires filtering pharmaceutical products through a 0.45 or 0.2 µM membrane filter2. Scientists rinse the filters with an appropriate fluid and transfer them to a specific growth media for 14 days, during which they check daily for growth. Growth media is usually fluid thioglycollate medium (FTM), which promotes the growth of anaerobic microorganisms, or soybean casein digest medium (SCDM), which promotes the growth of aerobic microorganisms.

Non-filterable pharmaceutical products, such as powders and creams, benefit from direct transfer sterility testing. Scientists transfer products directly to FTM or SCDM, incubate for 14 days, and check every day for growth.

Closed system membrane filtration devices help maximize safety and convenience for QC scientists performing sterility testing. Furthermore, products that act as a sterile barrier for fast, aseptic transfer from closed, sterile systems also help QC scientists to take samples whenever needed. These samples can be transferred to rapid sterility testing devices, which help QC scientists quickly confirm bacterial or fungal contamination. This is particularly useful for testing advanced therapy medicinal products (ATMPs), medicines for human use that are based on genes, tissues or cells, before patient administration. Various fast sterility testing products are available, including those based on adenosine triphosphate (ATP) bioluminescence for results within five days, or real-time PCR for results within three hours.

References

1. T. Sandle, “16 - Cleanrooms and environmental monitoring,” in Pharmaceutical Microbiology, Woodhead Publishing, pp. 199-217, 2016.

2. USP <71> Sterility Tests, https://www.usp.org, accessed 14th January 2020

The Scientist 2020 the-scientist.com4

MICROBIOLOGICAL QC 101

Unwanted Guests: A Focus on Mycoplasma

Mycoplasma are often considered the trickiest of all microorganisms to detect and prevent, due to their tiny size and inherent antibiotic resistance.

They are also one of the most prevalent microorganisms within biopharmaceutical manufacturing facilities. Lucille B. Robinson et al., from the Arthritis Research Unit at the Veterans Administration Hospital in Washington, DC, and Department of Microbiology at Johns Hopkins University first recognized the potential for cell culture system contamination by mycoplasma in 19561,2. Now, researchers estimate contamination rates as high as 35% in established cell cultures2. Mycoplasma lead to chromosomal aberrations, gene-expression pattern changes, membrane antigenicity alterations, cell growth inhibition, DNA fragmentation, compromised protein or virus production, cell metabolism inhibition, and increased cell death3. Viable but non-culturable (VBNC) mycoplasma are of particular concern in QC testing; it can go undetected by traditional mycoplasma-culturing methods, risking false-negative results.

Where do Mycoplasma Come From?

Three major sources lead to mycoplasma contamination of cell cultures in the laboratory: infected cells sent from another lab; contaminated cell culture medium reagents such as serum and trypsin; and laboratory personnel infected with Mycoplasma orale or M. fermentans. Other contaminants include M. hyorhinis originating from swine sources, or M. arginine and M. hominis from bovine sources3. These may come from contaminated media, sera, or reagents, improper sterilization of supplies, media, and solutions (e.g., uneven heating in an autoclave), closing and opening of incubators equipped with fans and air currents, airborne particles generated during culture manipulations, other mycoplasma-contaminated cell cultures, and even liquid nitrogen3; although mycoplasma do not proliferate in liquid nitrogen, they survive and can infect cultures stored in the liquid. Overuse of antibiotics, which results in resistant bacteria, and improper sealing of culture dishes can also allow mycoplasma into cultures; when the top, bottom, or sidewalls of dishes or flasks and their caps become wet, mycoplasma transfer by capillary action to the wet surfaces3.

Eliminating Mycoplasma with Antibiotics

Although the first course of action when discovering mycoplasma-infected cultures should be to discard them and start from scratch with fresh, uncontaminated stock, this is not always feasible; slow-growing cultures, rare cell types, or cell lines producing

unique proteins might warrant treatment. In such cases, antibiotic treatment remains the gold standard for treating mycoplasma-contaminated cell cultures. While many antibiotics are impotent against mycoplasma, some antibiotics do show promise, such as those from the quinolone, tetracycline, and macrolide families. Generally, researchers require two or more antibiotics to completely eliminate mycoplasma contamination. These treatments may take weeks or months to complete.

Preventing Mycoplasma Contamination

Because mycoplasma are hard to detect and difficult to get rid of, preventing contamination is essential. Researchers can do so by improving aseptic techniques, following Good Manufacturing Practices (GMP, or Good Laboratory Practices; GLP), obtaining sera and media from reputable manufacturers, using antibiotics sparingly and responsibly, discarding mycoplasma-contaminated cells, quarantining new cells of any origin, reducing aerosol generation, and routinely testing cultures for contamination2.

Mycoplasma Testing

Numerous tests are available to test for mycoplasma contamination; direct growth on agar or in broth, DNA staining, PCR, ELISA, enzymatic procedures, and RNA labeling are all options. Direct growth methods are lengthy, costly, and unreliable for VBNC species. DNA staining is quick to perform, but interpreting the results can be difficult. In microbiological QC situations where time and quality are of the essence, qPCR kits offer early detection; however, stringent assay designs are required to avoid DNA contamination or Taq polymerase inhibition4.

Mycoplasma contamination comes from many sources, and it spreads quickly. The key to prevention in pharmaceutical laboratories is frequent testing. Simple, fast mycoplasma detection kits enable frequent testing and help reduce the risk of contamination.

References

1. L.B. Robinson and R.H. Wichelhausen, “Contamination of human cell cultures by pleuropneumonialike organisms,” Science, 124(3232):1127-48, 1956.

2. S.E. Armstrong et al., “The scope of mycoplasma contamination within the biopharmaceutical industry,” Biologicals, 38(2): 211-3, 2010.

3. L. Nikfarjam and P. Farzaneh, “Prevention and detection of mycoplasma contamination in cell culture,” Cell J, 13(4): 203-12, 2012.

4. C.C. Uphoff et al., “Detection of mycoplasma contamination in cell cultures,” Curr Protoc Mol Biol, 106:28.4.1-14, 2014.

Contaminated culture detected—needs fast identification

Use mass spectrometry-based biotypers to rapidly identify an organism based its unique proteomic fingerprint

PROBLEM

RESOLUTION

Long QC test times for Advanced Therapy Medicinal Products (ATMPs)

Use real-time PCR methods to detect bacterial and fungal contamination within a short timeframe

PROBLEM

RESOLUTION

Transfer a sample out of a closed, sterile system

Use a liquid transfer system that does not require filter manipulation and acts as a sterile barrier

PROBLEM

RESOLUTION

Suspected mycoplasma contamination

Use PCR testing kit for fast mycoplasma detection

PROBLEM

RESOLUTION

Fast sterility testing is required before ATMPs can be administered to patients

Use real-time PCR for results within three hours

PROBLEM

RESOLUTION

Airborne microorganisms have entered a cleanroom

Ensure that all sta� are outfitted with correct cleanroom attire, and perform airborne particle testing

PROBLEM

RESOLUTION

The Scientist 2020 the-scientist.com6

MICROBIOLOGICAL QC 101

Ins-and-Outs of the Microbial Limits Test

To assure the quality of non-sterile pharmaceutical, healthcare, or cosmetic products, QC microbiologists commonly perform the microbial limits test (MLT) for

raw materials, in-process samples, and final products throughout the manufacturing process. The MLT assesses the number and type of viable microorganisms present in non-sterile products and determines if microorganisms exceed quantitative limits. The MLT covers testing for common microbial contaminants, including bile tolerant gram-negative bacteria.

Harmonizing Microbial Limits Testing Worldwide

Historically, the United States Pharmacopeia (USP) <61> included instructions for performing Microbial Limits Testing (MLT). The chapter provided directions on how to estimate numbers of viable aerobic microorganisms present in a manufactured nonsterile product and included information about how to perform a Total Plate Count (TPC) for aerobic organisms and yeasts or molds, and how to detect Specified Microorganisms. USP <61> was equivalent to the Japanese Pharmacopoeia Chapter 35, “Microbial Limit Test (MLT),” and similar to the British Pharmacopoeia (BP) chapter B1 and sections of the European Pharmacopeia (Ph Eur).

Because there was so much overlap between each Pharmacopeia, the USP standardized the MLT worldwide in 2009. They separated the previous version of the MLT, USP <61>, into 2 chapters: USP <61> Microbial Enumeration Tests (MET)1, which describes a quantitative test that determines the total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) present in a sample, and USP <62> Tests for Specified Microorganisms2, which details a test for the presence of Staphylococcus aureus, Escherichia coli, Salmonella, Pseudomonas aeruginosa, total gram negative bacteria, Clostridium and Candida albicans in a product. These two USP chapters are now commonly referred to as the “harmonized MLT.”

Performing the Microbial Limits Test

Researchers prepare samples by pooling random, multiple portions of the products to be tested. Researchers first neutralize, inactivate, dilute or filter out any antimicrobial

substances present in test samples. They take great care to prevent microbial contamination during the testing procedure.

QC scientists enumerate microbes according to USP <61> using plate count test methods, including the pour-plate method or surface-spread method, or membrane filtration test methods. Various filtration units, filtration systems, membrane filters, and culture media products are commercially available.

To test for specified microorganisms according to USP <62>, QC scientists prepare samples using approaches similar to those described in USP <61>, including neutralizing the product, enriching the sample in an appropriate neutralizer broth, and streaking the enriched sample on selective media. Before or during specified microorganism testing, scientists must perform a one-time suitability of recovery test; this demonstrates the ability of the methods to detect specific microorganisms, and is designed to show that any antimicrobial properties of the product do not inhibit possible growth recovery.

Interpreting Results

Generally, scientists report two results for microbial enumeration tests for USP <61>: The Total Aerobic Microbial Count (TAMC), which is equal to the number of colony-forming units (cfu) found using soybean-casein digest agar, and the Total Combined Yeasts and Molds Count (TYMC), which is equal to the number of cfu found using Sabouraud dextrose agar. For USP <62>, scientists report the presence or absence of the specific organisms mentioned above. They perform suitability testing by inoculating the product with <100 cfu of specific organisms.

References

1. USP <61> Microbial Enumeration Tests, https://www.usp.org, accessed 14th January 2020

2. USP <62> Tests for Specified Microorganisms, https://www.usp.org, accessed 14th January 2020

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