Demonstration of viral clearance is crucial for the recombinant proteins produced from mammalian cell culture. Size exclusion-based filtration is one of the methods for viral clearance valid for different types of mammalian viruses. In this study, the initial hydraulic performance of the virus filtration has enabled the development of a validation protocol and high-log reduction values (LRVs) for an appropriate panel of mammalian viruses (during spiking studies) with the desired throughput. This is essential to minimize the filter area, the cost of this unit operation, and to achieve a robust process. All of the four panel viruses (i.e., MuLV, PRV, Reo-3, and MVM) were evaluated during spiking studies. A high throughput of 1250 L/m 2 was obtained with a high flux because of low protein concentration. This was validated for a predefined throughput of 387 L/m 2 with high values of LRVs (>5) achieved resulting in the relatively small area of virus filter (0.04m 2 ) for a 50-L batch volume to be processed in 2 h. This study on a recombinant human follicle stimulating hormone (rhFSH) demonstrates the use of virus filters to reduce the risk of contamination and provide a robust process of virus filtration.
Recombinant proteins and monoclonal antibodies (mAbs) produced using mammalian cell culture-based expression systems are required to demonstrate robust virus clearance during downstream purification to meet regulatory guidelines (1). The guidelines also indicate that the process steps identified to provide and/or claim virus clearance should be validated for their ability to clear virus.Membrane filtration, which is based on the principle of size exclusion, is often employed as one of the techniques for virus clearance in the downstream processing of cell-culture derived biotherapeutics. To determine the appropriate virus filter (type and size) for a particular feed stream, a hydraulic performance study is generally performed using a scaled down model. Such studies are typically performed with the relevant drug-feed material to determine the expected flow rate and throughput (2). After determination of the hydraulic performance, a virus validation study is carried out using a scaled down model. The virus validation study entails spiking an appropriate panel of mammalian viruses (typically four different viruses of different sizes, structure, and type) into the feed material and determining the virus clearance ability (expressed as log reduction value) of the filter for each virus. The validated throughput (litres/square meter), under the spiked condition, also stipulates the maximum design throughput for the virus filter in a manufacturing process. The work embodied in this paper represents the collaborative effort between Bharat Serums and Vaccines Ltd., India (R&D group) and Merck Millipore, India (Biomanufacturing Sciences Network Group) to develop and optimize the hydraulic performance of a virus filtration step. After initial development, efforts were carried out to establish the optimum flux and process throughput, and a good laboratory practice (GLP) virus validation (spiking) study was carried out at Charles River Biopharmaceutical Services, GmbH, Cologne, Germany.
MATERIALS AND METHODS
Equipment setup for hydraulic performance