A much improved Ph. Eur. Chapter 5.3.2

By Dr. Ray Nims

Vaccine manufacturers intending to market in the EU should be aware of a recent change in the European Pharmacopoeia (Ph. Eur.) chapter 5.2.3 Cell substrates for production of vaccines for human use. This chapter addresses the characterization of vaccine cell substrates. The section on Test Methods for Cell Cultures within the chapter includes an instruction to perform a co-cultivation study. The language previously was as follows: “Co-cultivation. Co-cultivate intact and disrupted cells separately with other cell systems including human cells and simian cells. Carry out examinations to detect possible morphological changes. Carry out tests on the cell culture fluids to detect haemagglutinating viruses. The cells comply with the test if no evidence of any extraneous agent is found.”

This section has been changed, as of Ph. Eur. version 7.2 effective in January of 2011, to the following: “Co-cultivation. For mammalian and avian cell lines, co-cultivate intact and/or disrupted cells separately with other cell systems including human cells and simian cells. For insect cell lines, extracts of disrupted cells are incubated with other cell systems, including human, simian, and at least 1 cell line that is different from that used in production, is permissible to insect viruses and allows detection of human arboviruses (for example BHK-21). Carry out examinations to detect possible morphological changes. Carry out tests on the cell culture fluids to detect haemagglutinating viruses, or on cells to detect haemadsorbing viruses. The test for haemagglutinating viruses does not apply for arboviruses to be detected in insect cells. The cells comply with the test if no evidence of any extraneous agent is found.”

So what is the big deal? Co-cultivation is a commonly employed technique for detecting infectious retrovirus in a cell bank. It is effective for this purpose because the chances for spread of infectious virus from test cell to indicator (host) cell are optimized by the cultivation of live cells of each kind in close proximity. The endpoint of the retrovirus assay, be it reverse transcriptase enzyme induction or rescue of an S+L- virus, is not interfered with by the presence of two cell types in one culture. The same is not always true for a co-cultivation of a test cell with an indicator (host) cell for detection of infectious virus when morphological changes (viral cytopathic effects) are one of the assay endpoints. The reason is that the diploid human cells (e.g., MRC-5 or WI-38) used as one of the indicator cells in such assays are rapidly displaced during co-cultivation with intact continuous cell lines used to produce vaccines, such as the simian cell Vero. The result of this is that within a short period of time in co-cultivation, the test culture is no longer predominated by the diploid cell but rather by the test cells and observation of the culture for cytopathic effects becomes problematic. Changing the language of this section to read “…co-cultivate intact and/or disrupted cells separately with other cell systems…” allows the user to eliminate the inoculation of intact test cells onto a diploid indicator cell.

The other useful modification to the language of this section is the following addition: “For insect cell lines, extracts of disrupted cells are incubated with other cell systems, including human, simian, and at least 1 cell line that is different from that used in production, is permissible to insect viruses and allows detection of human arboviruses (for example BHK-21).” Testing of insect cells for extraneous virus is only marginally effective when it is conducted per the usual method of inoculating another insect cell. Why? The insect cells that are available are most commonly suspension cultures, making observation for cytopathic effect problematic. The extraneous viruses that are of most concern for an insect production cell are the arboviruses (viruses transmitted via insect vectors). It has been known for some time that the Syrian hamster cell line BHK-1 is an excellent host cell for detecting arboviruses. The new language in this section of Ph. Eur. chapter 5.2.3 now clears the way for the use of the monolayer BHK-1 cell line to be used for the testing of insect cells for extraneous virus. In this regard the Ph. Eur. chapter is now more closely aligned with the World Health Organization’s 2009 Evaluation of cell substrates for the production of biologicals: revision of WHO recommendations. The latter has the following passage: "For instance, in the case of insect cell substrates, certain insect cell lines may be used for detection of insect viruses, and BHK cells may serve for the detection of arboviruses."

   

Taken together, the recent changes to Ph. Eur. Chapter 5.2.3 greatly improve the chapter and the viral safety testing of vaccine production cell banks specifically proscribed within it.