Biologic Stress Tests
Viral induction studies: A useful but underutilized tool in cell bank characterization
The urgency to provide new and safe vaccines—witness the recent Ebola crisis—highlights the need for effective and innovative safety testing practices to avoid contaminations, especially for vaccines produced from live cells. The viral contaminant in the polio vaccine (produced from 1955-1963) originally came from the monkey kidney cells used to produce the vaccine. More recently, viral contaminations originating from the cells or cell growth products have been found in poultry, feline and canine vaccines. Safety testing procedures must encompass all materials and processes in the vaccine production including source materials, such as cell banks, intermediate process harvests and the final products.
Characterization of the cells in the cell bank used to produce a vaccine is the first step in assuring the safety of pharmaceutical biologics. Cell bank characterization is a series of analyses that confirm the identity, quality, genetic stability, and viral safety of cells used in the processing of vaccines, monoclonal antibodies, gene therapies, and other biopharmaceuticals.
Typical cell bank characterization safety tests, such as In Vitro Adventitious Virus Assays (AVA), detect infectious or replicating viral contaminants. However, in any particular sample, and at any stage of the manufacturing process, a virus may be present but not actively replicating and, therefore, not detected by conventional infectivity assays. These hidden endogenous or latent viruses require innovative tests to find them.
One such test is an induction study, which employs specific chemical compounds to induce virus expression or replication in cells. Following induction, latent and endogenous viruses can then be detected through subsequent assays which include Transmission Electron Microscopy (TEM) testing for visible virus particles, and Quantitative Fluorescent PCR (QF-PCR) testing for either retroviral reverse transcriptase activity or for viral DNA sequences.
Recently, the virology team at Charles River-Malvern designed and executed an induction study to test a pharmaceutical client's cell bank. Based on previous publications, we established two strategies to induce either DNA viruses or retroviruses (see Biologics, 37:196-201, 2009).
The initial studies aimed to balance toxicity with successful induction. Our studies tested four compounds (singly and in combination) on the client's cell bank, a Mardi-Darby Canine Kidney (MDCK) adherent cell line. Two compounds were used to induce DNA viruses, and two compounds to induce retroviruses. A series of decreasing compound concentrations and multiple exposure times were used to optimize the induction conditions. The confluence and resulting toxicity was monitored for up to five days. A 24-hour exposure time was selected for each compound concentration. Two positive control cell lines containing endogenous viruses were selected to monitor the effectiveness of the induction study and the optimal detection time of the induced viruses.
Once the induction study was performed on both client cells and the corresponding positive and negative (uninduced) controls, the samples were given to the molecular biology group for further evaluation.
QF-PCR assays were developed and qualified to detect four families of DNA viruses. Canine sequences were targeted to identify viruses in the sample MDCK cell line, while human viruses were targeted to identify human viruses that may grow in the canine cell line and potentially be present in the final pharmaceutical product. The second PCR assay, a PCR-based Reverse Transcriptase Assay (PBRT, PERT), was performed to detect broad-range retrovirus activity in the induced cell supernatant samples. Additionally, cell pellets were tested following induction by TEM to detect virus particles within cells.
The final results of this particular induction study did not detect any human or canine viruses in either uninduced or chemically induced MDCK cells by QF-PCR or TEM. In addition, no detectable retroviruses were observed in either uninduced or chemically induced MDCK cells by the PBRT assay or TEM. As expected, viruses were detected in the positive control cells using TEM, as well as multiplexed, broad-range DNA QF-PCR and PBRT assays.
Characterization of this MDCK cell bank included routine safety testing for infectious viruses, as well as custom induction studies to detect latent or endogenous viruses. These results show that multiple tests are crucial to provide an increased level of confidence that each cell bank and pharmaceutical product is safe and free of viral contaminants.