Tuesday, November 16, 2010

Cell Culturists….Are your human cells authenticated?

by Dr. Ray Nims

Until fairly recently, it has been common practice to authenticate human cell cultures using phenotypic status (e.g., receptor or protein expression) and animal species of origin testing. This level of authentication is better than none, but it is not sufficient to unambiguously identify a human cell culture. The result has been that we are still hearing about cases of misidentified human cells being used for biomedical research.

There are now methods available that are capable of rapidly and unambiguously identifying human cell lines, tissues, and cell preparations to the individual level. The recent demonstration of the potential utility of molecular technologies such as short tandem repeat (STR) and single nucleotide polymorphism (SNP) profiling for cell authentication has provided the impetus for development of a new standardized method for human cell authentication.

To this end, an ATCC Standards Development Organization workgroup with international representation has spent the past two years developing a consensus standard for the Authentication of Human Cell Lines through STR Profiling. The forthcoming Standard will provide guidance on the use of STR profiling for authenticating human cells, tissue, and cell lines. It will contain methodological detail on the preparation and extraction of the DNA, guidance on the appropriate numbers and types of loci to be evaluated and on interpretation and quality control of the results. Associated with the standard itself will be the establishment of a public STR profile database which will be administered and maintained by the National Center for Biotechnology Information (NCBI). The database primarily will contain STR profiles of commonly used cell lines.

                                            STR Profiling of Hela Cells

 An announcement that the Standard is now available for public 45-day review, comment, and vote was published in the October 22, 2010 issue of the ANSI newsletter Standards Action.

The benefits of the Standard will depend on the degree to which it is adopted and followed in the biomedical research and development and biopharmaceutical  communities. Taking a broader view, it is hoped that funding agencies and journals will begin to use such authentication standards as important considerations for funding or publishing research employing human cells. The quality and validity of funded and published research should benefit greatly as a result of the reduction in frequency of use of misidentified human cells.

The deadline for comments is December 6, 2010. There is still time to review the draft Standard and to voice your opinions and concerns.

Wednesday, November 3, 2010

Fry those mollicutes!

By Dr. Ray Nims

It is not only viruses that may be introduced into biologics manufactured in mammalian cells using bovine sera in upstream cell growth processes. The other real concern is the introduction of mollicutes (mycoplasmas and acholeplasmas). Mollicutes, like viruses, are able to pass through the filters (including 0.2 micron pore size) used to sterilize process solutions. Because of this, filter sterilization will not assure mitigation of the risk of introducing a mollicute through use of contaminated bovine or other animal sera in upstream manufacturing processes.

Does mycoplasma contamination of biologics occur as a result of use of contaminated sera? The answer is yes. Most episodes are not reported to the public domain, but occasionally we hear of such occurrences. Dehghani and coworkers reported the occurrence of a contamination with M. mycoides mycoides bovine group 7 that was proven to have originated in the specific bovine serum used in the upstream process (Case studies of mycoplasma contamination in CHO cell cultures. Proceedings from the PDA Workshop on Mycoplasma Contamination by Plant Peptones. Pharmaceutical Drug Association, Bethesda, MD. 2007, pp. 53-59). Contamination with M. arginini and Acholeplasma laidlawii attributed to use of specific contaminated lots of bovine serum have also occurred.

Fortunately, the risk of introducing an adventitious mollicute into a biologics manufacturing process utilizing a mammalian cell substrate may be mitigated by gamma-irradiating the animal serum prior to use. This may be done in the original containers while the serum is frozen. Unlike the case for viruses, in which the efficacy of irradiation for inactivation may depend upon the size of the virus, mollicute inactivation by gamma irradatiion has been found to be highly effective (essentially complete), regardless of the species of molicute. The radiation doses required for inactivation are relatively low compared to those required for viruses (e.g., 10 kGy or less, compared to 25-45 kGy for viruses). The gamma irradiation that is performed by serum vendors is typically in the range of 25-40 kGy. This level of radiation is more than adequate to assure complete inactivation of any mollicutes that may be present in the serum. For instance, irradiation of calf serum at 26-34 kGy resulted in ≥6 log10 inactivation of M. orale, M. pneumoniae, and M. hyorhinis. In the table below I have assembled the data available on inactivation of mollicutes in frozen serum by gamma-irradiation.

So, the good news is that gamma irradiation of animal serum that is performed to mitigate the risk of introducing a viral contaminant will also mitigate the risk of introducing a mollicute contaminant. If the upstream manufacturing process cannot be engineered to avoid use of animal serum, the next best option is to validate the use of gamma irradiated serum in the process.  In fact, the EMEA Note for guidance on the use of bovine serum in the manufacture of human biological medicinal products strongly recommends the inactivation of serum using a validated and efficacious treatment, and states that the use of non-inactivated serum must be justified.

References: Gauvin and Nims, 2010; Wyatt et al. BioPharm 1993;6(4):34-40; Purtle et al., 2006