Genetic Engineering & Biotechnology News

JUL 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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GENengnews.com | Genetic Engineering & Biotechnology News | JULY 2018 | 19 what the cell line is, you could potentially apply the wrong testing," says Henry Chiou, Ph.D., associate director, cell biology, Life Science Solutions, Thermo Fisher Scientific. This could lead to a significant safety risk for patients, although Dr. Chiou admits that it's an "extreme scenario." Adventitious viruses are not the only in- visible contaminants that scientists working with cell cultures need to be wary of—myco- plasma bacteria, which even high-magnifica- tion, bright-field, or phase microscopy can- not detect, have become common contami- nants. Mycoplasma often outcompete cells for essential nutrients without altering the turbidity or pH of the culture. Not only can contamination cause decreased cell growth and protein production, but some species of mycoplasma are pathogenic. "GMP sites can be closed down for mul- tiple months to clean out bioreactors and all surfaces purely because of mycoplasma detection. It's a huge deal from a business perspective as well as a quality perspective," says Jeff Hou, Ph.D., manager of cell culture development, BioProcess Sciences, Pharma Services Group, Patheon (part of Thermo Fisher Scientific). Multiple methods exist for detecting my- coplasma contamination, including direct culture, indirect culture, and PCR assays. Considered the gold standard for myco- plasma detection, the direct-culture method uses a nutritionally enriched broth and care- fully controlled environmental conditions to cultivate mycoplasma on agar plates. Both the characteristic "fried egg" appearance of colonies on the agar and a distinct color change in the broth indicate mycoplasma contamination. The direct-culture method requires lengthy incubation periods, however, and it takes a minimum of 28 days to complete. In addition, while sensitive, it cannot detect mycoplasma species or strains that are noto- riously difficult to grow in culture. Instead, scientists use the indirect culture test to detect noncultivable mycoplasma using fluores- cent DNA stains, which bind to mycoplasma DNA and reveal contamination through ex- tranuclear fluorescence. PCR-based assays have become a popular technique for detecting both cultivable and noncultivable mycoplasma, and organiza- tions such as the ATCC offer PCR-based mycoplasma detection services and kits. The ATCC's Universal Mycoplasma Detection Kit can detect over 60 species of the tiny bacteria. "There are a couple of testing labo- ratories that have demonstrated the com- parability between qPCR tests and the gold standard," notes Dr. Hou. Functionality Testing Although cell line identity, purity, and sterility must be validated during the manu- facture of protein-based therapeutics, the endgame is all about the product, and even whole-genome sequencing can't guarantee the cell substrate will yield large quantities of functional, high-quality protein. Thus, the final step in validation assesses the cell line's ability to consistently produce high-quality protein. "After you produce the protein, you need to test the mRNA level by qPCR, and you need to check the protein by Western blot using a specific antibody to make sure the protein is what you want," explains Leon Song, M.D., director, GenScript. The next step, he says, is to check protein activ- ity using a bioassay designed to test protein function. Biopharmaceutical manufacturers play a different game than preclinical researchers— with different rules and referees. Validating cell line identity, purity, sterility, and func- tionality using methods that meet regulatory requirements ensures product success and patient safety. "No one assay will provide all of the answers that a manufacturer needs—a multimodal approach provides greater con- fidence that a particular cell line is authentic and will provide the functionality needed for efficiency, productivity, and safety," says Dr. Hantman. Bioprocessing

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