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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Page 19 of 33

18 | JULY 2018 | Genetic Engineering & Biotechnology News | sequence that allows differentiation between animal species, and an extensive database exists for species identification. However, similar to isoenzyme analysis, DNA barcod- ing cannot distinguish cell lines at the sub- species level. At the opposite end of the spectrum, some companies have begun to use next- generation sequencing (NGS) to perform whole-genome or whole-exome sequencing on cell lines. The method is increasing in popularity as sequencing costs decrease be- cause it provides a massive amount of data on the cell line. In contrast to DNA barcod- ing, whole-genome sequencing can differen- tiate between cell lines originating from the same species and closely related cell lines, but it requires a reference to perform a compara- tive analysis. For identification at the subspecies level, "the industry is really hot for cell line au- thentication by short tandem repeat (STR) analysis," says Dr. Hantman. Originally developed by forensics labs to aid in crimi- nal investigations, STR analysis uses short, repeated DNA sequences for identification. The number of repeated units often varies between individuals, and each location on a chromosome where these repeats occur rep- resents a different contour in an individual's genetic fingerprint. STR analysis has become the gold stan- dard for authenticating human cell lines, and the availability of do-it-yourself kits from companies such as Thermo Fisher Scientific has increased accessibility. Thermo Fisher Scientific's AuthentiFiler™, Identifiler™Plus, Identifiler™ Direct, and GlobalFiler™ PCR amplification kits contain primers for 9, 15, 15, and 21 different autosomal STR mark- ers, respectively—as well as the sex marker, amelogenin. These, and similar kits, use fluo- rescently labeled primers optimized to allow co-amplification of multiple STR loci in a single PCR reaction. Following amplification, researchers can perform size- and color-based separation and detection of STR loci using capillary electrophoresis and compare the results to reference profiles in databases to verify cell- line identity. Scientists can also create inter- nal references using GeneMapper ® ID-X and GeneMapper 5 software (Thermo Fisher Scientific). The software also allows scientists to monitor longitudinal changes in STR profiles, which in combination with a broad range of STR markers, can detect genetic drift. With every division, cells can pick up genetic muta- tions, and these mutations can cause discrep- ancies between STR profiles. While regulato- ry agencies realize that genetic drift can cause an imperfect match, these discrepancies can cause uncertainty and complicate the inter- pretation of results. A more substantial barrier to widespread use of STR analysis, however, is the current lack of kits, guidelines, and databases for nonhuman cells. While human cell lines have become popular among manufacturers, Chinese hamster ovary (CHO) and mouse cell lines are also commonly used in protein production. Unfortunately for biopharma, the FBI didn't have much incentive for chas- ing down Chinese hamsters during the devel- opment of DNA fingerprinting. The National Institute of Standards and Technology (NIST) has teamed up with the ATCC to form a consortium of 13 labora- tories to expand the use of STR analysis to mouse cell lines. The Mouse Cell Line Au- thentication Consortium (MCLAC) selected murine cell lines because of their widespread use in both biomanufacturing and preclinical research. MCLAC members have tested 50 of the most commonly used mouse cell lines using a multiplexed PCR assay developed by NIST. The results of this work should be published later this year, says Jamie Almeida, bioassay methods group, NIST. "The consor- tium's data will lead to validated STR pro- files for the mouse cell lines tested," she adds. "These profiles will be available on the NCBI BioSample database." Testing for Contamination In cell line validation, identity must be confirmed as well as purity and steril- ity. Cross-contamination can compromise product yield, quality, and safety. One of the reasons it's important to identify the cells used to manufacture therapeutic proteins has to do with a practice that is meant to protect against cross-contamination. Manufacturers select panels for adventitious virus testing based on the cell line species. Human lines will undergo testing for HIV and other virus- es that infect human cells, whereas murine lines undergo a different battery of tests. "If you don't have clear identification of Cell Line Validation Continued from page 16 Bioprocessing ThermoFisher's GeneMapper® 5 Software allows users to process and compare short tandem repeat (STR) profiles between samples after co- amplification and fragment analysis of STR loci using an STR analysis kit and capillary electrophoresis, respectively. Having the capability to monitor changes in STR profiles over time can help researchers spot both contamination and genetic drift in their cell cultures. A scientist pipettes samples into a gel, performing one of the most hands-on operations in isoenzyme analysis, a relatively rapid and inexpensive technique for identifying cell lines at the species level and detecting cross-contamination. The technique uses electrophoretic banding to reveal small differences in the structure and motility of different intracellular enzyme variants. Charles River Laboratories

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