Adventitious agent contamination risk mitigation: engineering MMV virus resistance into CHO cells

The introduction of animal origin free (AOF) media has significantly reduced the incidence of adventitious virus contamination in biological production systems. Nevertheless, contamination by the parvovirus Mouse Minute Virus (MMV) remains a continuing challenge. Although infrequent, infection of a fermenter can be catastrophic for a manufacturer, and can also have a potential impact on drug supply, patient safety and have regulatory implications. In this work, we evaluated engineering Chinese Hamster Ovary (CHO) cell lines to create a new host cell line that would be resistant to MMV infection by modifying the major receptors used by the virus to enter cells. The goal was to engineer a host cell line resistant to MMV infection, while maintaining productivity and product quality profiles. Our strategy is outlined below. Attachment to a cell surface receptor is a key first step in the infection cycle for viruses. While the exact functional receptor for MMV binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated [1]. Moreover, MMV has been shown to preferentially bind to α-2,3 sialylated glycans with a type-2 Galβ1-4GlcNAc motif. Our approach was to systematically knock out genes affecting sialylation and then challenge each cell line for their ability to resist viral entry. Figure 1 lists the genes that were knocked out by ZFN mediated cell line engineering, and the corresponding typical glycosylation phenotypes expected on N-linked and O-linked glycans. https://static-content.springer.com/image/art%3A10.1186%2F1753-6561-9-S9-O2/MediaObjects/12919_2015_Article_3185_Fig1_HTML.jpg Figure 1 ZFN mediated gene knock outs affecting terminal sialic acid on glycosylation structures tested for susceptibility to MMV infection. MMV infectivity studies were conducted on the knock out clones identified in Figure 1 at various multiplicities of infection e.g. 0.1 viruses per cell, far higher than would be encountered in a fermenter infection. Infection of the cells was analyzed by PCR for the presence of viral genomic sequences as described in the schematic in Figure 2. https://static-content.springer.com/image/art%3A10.1186%2F1753-6561-9-S9-O2/MediaObjects/12919_2015_Article_3185_Fig2_HTML.jpg Figure 2 (a) Schematic of the qPCR based assay for MMV virus attachment, internalization, and single cycle replication. In brief the virus is incubated with the cells at an MOI of 0.1 for 3 hours. Samples are washed 3 times to remove excess unbou