| CHO-derived recombinant proteins for human therapeutic are used commonly. There are noninfectious endogenous retroviruses in Chinese hamster ovary (CHO) cells. Inactivation process is required in manufacturing for the safety of human use. In this study, we established a low pH method for evaluation of murine xenotropic gamma retrovirus (X-MulV) clearance in manufacturing of recombinant human TNF-a receptor immunoglobin G fusion proteins (rhTNF-a) for injection. X-MulV is used as a model virus in evaluation of viral inactivation in biological products derived from CHO cells. Mus. dunni cells are used to support the replication of X-MulV particles at present. In consideration of the characters of X-MulV, we tried to propagate the viruses on CHO cells, a non-murine cell line. The viruses were harvested from CHO cells from day2to day7post-infection, respectively. Reverse transcription-SYBR green fluorescence quantitative polymerase chain reaction (RT-qPCR) was performed to quantify the X-MulV particles on different days. The development of X-MulV on CHO cells was a typical single-step growth curve. The titers of viral stock harvested on day7were8.78±0.25log10PFU per mL, which were assayed on PG-4cells. Based on these data, we conclude that CHO cells could be a host organism for X-MulV particles. X-MulV produced on CHO cells could be used in the evaluation at a lab-scale of viral clearance in pharmaceutical proteins derived from CHO cells.Relevant parameters were analyzed and screened by orthogonal analysis and multiple linear regression analysis. Parameters including pH, temperatures, time and protein content were investigated for their impact on X-MulV inactivation. Cell-based infectivity assay was used for the evaluation of X-MulV clearance. RhTNF-a were spiked with X-MulV and were inactivated by the sifted conditions. Reverse transcriptase (RT) activities in inactivated samples were detected by real-time SYBR green product-enhanced reverse transcriptase assay (PERT). The pH and temperatures were critical factors in X-MulV inactivation. Simultaneously, there was an interaction between temperature and time on inactivation. Protein concentration and time had a less impact on inactivation. Inactivation conditions of pH3.50,4℃or25℃and4hours were chosen to achieve greater than4.0log10inactivation of X-MulV. RT activities were decreased significantly in samples that were inactivated with low pH and there were no difference between4℃or25℃.We performed a further research on low pH inactivation for evaluation of X-MulV clearance in manufacturing of rhTNF-a. RhTNF-a were spiked with X-MulV and were inactivated at pH3.60-3.90,25±2℃, and0-240min, respectively. Samples incubated at the conditions for15～180min were not inactivated effectively. For4h incubation, log10reductions were achieved5.0log10. Biological activity of rhTNF-a incubated at pH3.60,25℃for4h, which was assayed on murine L929fibroblasts cells, was not affected by low pH. Env gene of X-MulV, which was detected by conventional PCR method for the first time, was not detected after incubation at pH3.60, and it may be the mechanism of low pH inactivation. This method was used for the evaluation for the viral clearance in CHO-derived recombinant TNF-a protein, which was provided by a manufacturer in Guangzhou.The optimal conditions of inactivation were pH3.60,25℃and4hours. This method could be used at the lab scale for evaluation of viral clearance in pharmaceutical proteins derived from CHO cells in manufacturing process. |
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