September 15, 2016 (Vol. 36, No. 16)

Joaquina Mascarenhas Ph.D. Senior R&D Scientist/Team Lead MilliporeSigma
Nikolay Korokhov Ph.D. Principal Scientist MilliporeSigma
Ademola Kassim Scientist MilliporeSigma
Trissa Borgschulte Ph.D. Manager MilliporeSigma
Henry George Manager, R&D Operations MilliporeSigma
Audrey Chang Ph.D. Director of Global Development Services MilliporeSigma
David Onions Ph.D. Independent Consultant MilliporeSigma
Kevin J. Kayser Ph.D. Director R&D Upstream and Systems MilliporeSigma

Special Cell Lines Developed for Resistance to the Minute Virus of Mouse

Chinese hamster ovary (CHO)-based animal component-free (ACF) systems are currently responsible for producing more than $50 billion of therapeutic products a year. 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 Minute Virus of Mice (MVM), also known as Mouse Minute Virus (MMV), remains a continuing challenge. On average, there is one major episode of MVM contamination every five years. For example, MVM was reported by Genentech in CHO cell lines in 1993 and 1994. Amgen reported contamination in 2006, followed by Merrimack in 2009.

Parvoviruses are among the most treatment-resistant viruses known. Their small size poses a challenge to risk mitigation strategies (e.g., nanofiltration systems). Although infrequent, infection of a fermenter can be catastrophic for a manufacturer, resulting in the loss of product, temporary withdrawal from the market, and extensive clean-down costs, which can reach a total in the tens of millions of dollars.

In addition to the loss of associated revenue, a contamination event can also have a potential impact on drug supply and patient safety as well as have regulatory implications.

New Approach to Viral Safety

Adding MVM resistance to a CHO bioproduction cell line would be significant insurance against the main cause of virus-induced catastrophic failure in ACF fermentation systems. With this in mind MilliporeSigma has developed a new approach to viral safety.

As experts in biopharma cell-line engineering, we asked ourselves if it would be possible to engineer a host CHO cell line to actually be resistant to infection by MVM. In our work, we evaluated engineering the CHOZN® GS/ parental cell line to create a new host cell line that would be resistant to MVM infection through elimination of the major receptors used by the virus to enter cells. The goal was to engineer a host cell line resistant to MVM infection, while maintaining desired productivity and product quality profiles.

Data generated in our group demonstrates that resistance against MVM virus contamination can be incorporated into CHO production cell lines, adding another level of “defense” against the devastating financial consequences of this virus infection, without compromising monoclonal antibody yield or quality.

While the exact functional receptor for MVM binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated. The CMP-sialic acid transporter (solute carrier family 35A1 or SLC35A1) is responsible for transporting sialic acid into the Golgi apparatus. Knocking out function of this gene in a cell results in asialylated glycan structures, which block MVM’s ability to bind to and enter the cell.

Zinc finger nuclease (ZFN) technology was used to knock out SLC35A1 in the CHOZN GS/ host cell line. Limited dilution was used to generate single cell clones and Sanger sequencing was used to confirm modifications in the target gene.

While the wild-type cells displayed immediate loss of viability and inhibition in cell growth upon MVM infection, the absence of sialic acid on the SLC35A1 knock-out cell line led to complete resistance to MVM infection (Figure 1).

Figure 1. Viable cell density of SLC35A1 knockout and wild-type CHO cells with and without exposure to MVM at a multiplicity of infection of 1.0.

Demonstrated Resistance

To test for a lack of viral replication in the knockout cells, wildtype and Slc35A1 knockout cells were incubated with MVM for 3 hours and then washed to remove non-specifically bound virus. In an extended culture assay samples were collected at time 0 hours and every 24 hours up to 96 hours and analyzed by qPCR using a probe against the MVM NS2 gene. Viral genome copies increase with extended time in the wildtype cells. In the SLC35A1 knockout cells, the viral genome copy number does not rise above inoculum levels indicating complete resistance to MVM infection (Figure 2).

Multiple SLC35A1-knockout clones were evaluated for growth compared to wild-type cells. Viable cell density was measured over a 13-day assay. The SLC35A1-knockout clones showed normal clonal variability in growth patterns compared to the wild-type cells. The top four clones were chosen based on growth profiles. These clones, along with the wild-type cells, were transfected with a vector expressing a model IgG1 molecule.

Figure 2. Wild-type and SLC35A1-knockout cells exposed to MVM. MVM replication is abrogated in SLC35A1-knockout cell line.

The transfected populations were plated into pools in 96-well plates and 7-day static productivity was measured. The four clones demonstrated a range in titers, that when compared to the wild-type titer, indicated the genetic modification did not have a detrimental effect on growth or productivity (Figure 3).

The culmination of this work has led us to the first commercially available gene-editing approach to modify CHO cells to be resistant to MVM: CentinelTM Intelligent Virus Defense. Using this platform, MilliporeSigma can modify customers’ CHO cell lines to provide viral resistance to MVM. Alternatively, customers can purchase the ZFN pairs to engineer cell lines directly.

The company’s BioReliance® testing services can validate MVM resistance and demonstrate the virus is not propagated in the cell line. A patent application has been submitted for this approach to viral resistance.

This technique could be applied to different CHO host cell lines, as well as to therapeutic protein-producing clonal cell lines. Incorporation of viral resistance to the MVM virus in the CHO host and subsequent production cell lines adds yet another level of defense to the current risk-mitigation strategies used, providing even greater ensurance of production of safely delivered cell-derived products.

Figure 3. Titer obtained from 15 pools isolated from each of the transfected populations. Pools from each population are ordered from highest titer to lowest.

Joaquina Mascarenhas, Ph.D., is senior R&D scientist/team lead, Nikolay Korokhov, Ph.D., principal scientist, Ademola Kassim, scientist, Trissa Borgschulte, Ph.D., manager, Henry George, manager, R&D operations, Audrey Chang, Ph.D., director of global development services, David Onions, Ph.D., independent consultant, Kevin Kayser, Ph.D., director R&D upstream and systems. All authors are at MilliporeSigma. For more information contact Erika Holroyd ([email protected]).

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