Characterization of operating parameters for XMuLV inactivation by low pH treatment.

To ensure the viral safety of protein therapeutics made in mammalian cells, purification processes include dedicated viral clearance steps to remove or inactivate adventitious and endogenous viruses. One such dedicated step is low pH treatment, a robust and effective method commonly used in monoclonal antibody production to inactivate enveloped viruses. To characterize the operating space for low pH viral inactivation, we performed a statistically designed experiment evaluating the effect of pH, temperature, hold duration, acid type, and buffer concentration on inactivation of the retrovirus model, XMuLV. An additional single factor experiment was performed to study the effect of protein concentration. These data were used to generate predictive models of inactivation at each time point studied, which can be used to identify conditions for robust and effective XMuLV inactivation. At pH 3.6, XMuLV inactivation was rapid, robust, and relatively unaffected by the other factors studied, providing support for this as a generic viral inactivation condition for products that can tolerate this low pH. At pH 3.7 and 3.8, other factors besides pH affected XMuLV inactivation. By understanding the impact of each factor on inactivation, the factors can be manipulated within the operating space to ensure effective inactivation while achieving desired product quality goals.

Clearance of the rodent retrovirus, XMuLV, by protein A chromatography.

Protein A chromatography is the most common unit operation used in the manufacture of therapeutic monoclonal antibodies (mAbs) due to its high affinity and specificity for the IgG Fc domain. However, protein A chromatography is often not effective for viral clearance. Typical log reduction values (LRV) for the model retrovirus XMuLV range between 1 and 4 logs, while effective steps such as viral filtration can achieve 5-7 logs of clearance. XMuLV LRVs obtained on protein A are reproducible for a given mAb, but can vary widely for different mAbs, even with the same operating conditions. In order to understand the mechanism of XMuLV clearance on protein A, we have investigated its partitioning on Mabselect SuRe protein A resin and explored how the virus interacts with resin, product, and impurities. The results show that XMuLV has some interaction with the resin backbone and ligand, but also appears to bind to and coelute with the mAb. The interaction with product was further examined by evaluating the effect of feed conditions, loading, and different washes on XMuLV partitioning on the column. Understanding the mechanism of XMuLV removal on a protein A, resin provides insight into the variability and low viral clearance of this step and suggests ways in which the removal of virus by this step can be improved.

Using high throughput screening to define virus clearance by chromatography resins.

High throughput screening (HTS) of chromatography resins can accelerate downstream process development by rapidly providing information on product and impurity partitioning over a wide range of experimental conditions. In addition to the removal of typical product and process-related impurities, chromatography steps are also used to remove potential adventitious viral contaminants and non-infectious retrovirus-like particles expressed by rodent cell lines used for production. This article evaluates the feasibility of using HTS in a 96-well batch-binding format to study removal of the model retrovirus xenotropic murine leukemia virus (xMuLV) from product streams. Two resins were examined: the anion exchange resin Q Sepharose Fast Flow™ (QSFF) and Capto adhere™, a mixed mode resin. QSFF batch-binding HTS data was generated using two mAbs at various pHs, NaCl concentrations, and levels of impurities. Comparison of HTS data to that generated using the column format showed good agreement with respect to virus retentation at different pHs, NaCl concentrations and impurity levels. Results indicate that NaCl concentration and impurity level, but not pH, are key parameters that can impact xMuLV binding to both resins. Binding of xMuLV to Capto adhere appeared to tolerate higher levels of NaCl and impurity than QSFF, and showed some product-specific impact on binding that was not observed with QSFF. Overall, the results demonstrate that the 96-well batch-binding HTS technique can be an effective tool for rapidly defining conditions for robust virus clearance on chromatographic resins.

Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes.

One measure taken to ensure safety of biotherapeutics produced in mammalian cells is to demonstrate the clearance of potential viral contaminants by downstream purification processes. This paper provides evidence that cation exchange chromatography (CEX), a widely used polishing step for monoclonal antibody (mAb) production, can effectively and reproducibly remove xMuLV, a retrovirus used as a model of non-infectious retrovirus-like particles found in Chinese hamster ovary cells. The dominant mechanism for xMuLV clearance by the strong cation exchanger, Fractogel SO 3⁻, is by retention of the virus via adsorption instead of inactivation. Experimental data defining the design space for effective xMuLV removal by Fractogel SO 3⁻ with respect to operational pH, elution ionic strength, loading, and load/equilibration buffer ionic strength are provided. Additionally, xMuLV is able to bind to other CEX resins, such as Fractogel COO⁻ and SP Sepharose Fast Flow, suggesting that this phenomenon is not restricted to one type of CEX resin. Taken together, the data indicate that CEX chromatography can be a robust and reproducible removal step for the model retrovirus xMuLV.