title: Low pH inactivation for xenotropic gamma retrovirus in recombinant human TNF-α receptor immunoglobulin G and mechanism of inactivation.
abstract: CHO-derived recombinant proteins for human therapeutic are used commonly. There are noninfectious endogenous retroviruses in CHO cells. Validation study for inactivation process is required. Murine xenotropic gamma retrovirus (X-MulV) is a model virus in validation study. In our previous study, optimum conditions for X-MulV inactivation were sifted. In this study, we performed a further research on low pH inactivation for evaluation of X-MulV clearance in manufacturing of recombinant human TNF-α receptor immunoglobulin G fusion proteins (rhTNF-α) for injection. Cell-based infectivity assay was used for the evaluation of X-MulV clearance. RhTNF-α were spiked with X-MulV and were inactivated at pH 3.60 ∼ 3.90, 25 ± 2 °C, and 0 ∼ 240 min, respectively. Samples incubated at the conditions for 15 ∼ 180 min were not inactivated effectively. For 4 h incubation, log10 reductions were achieved 5.0 log10. Biological activity of rhTNF-α incubated at pH 3.60, 25 °C for 4 h, which was assayed on murine L929 fibroblasts 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 pH 3.60, and it may be the mechanism of low pH inactivation.
authors.affiliation: Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. Electronic address: haijiaosky@126.com.
authors.forename: Rong
authors.initials: R
authors.lastname: Ma
authors.affiliation: Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. Electronic address: cuixl2812@sina.com.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Cui
mesh_terms: D000818:Animals; D001483:Base Sequence; D002415:Cats; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017452:Receptors, IgG / Q000276:immunology*; D011995:Recombination, Genetic; D012190:Retroviridae / Q000502:physiology*; D012689:Sequence Homology, Nucleic Acid; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
chemical_list: D004279:DNA, Viral; D017452:Receptors, IgG; D014409:Tumor Necrosis Factor-alpha
keywords: CHO; CHO cells; Chinese hamster ovary; Env; Envelope; Envelope gene; L929 cells; Low pH; Murine xenotropic gamma retrovirus; PCR; PG-4 cells S + L-; Polymerase chain reaction; Recombinant human TNF receptor immunoglobulin G fusion proteins; X-MulV; rhTNF-α