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
was obtained with a high flux because of low protein concentration. This was validated for a predefined throughput of 387
with high values of LRVs (>5) achieved resulting in the relatively small area of virus filter (0.04m
) 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
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,
MATERIALS AND METHODS
Equipment setup for hydraulic performance
The experimental setup is shown in Figure 1. The apparatus consisted of the pressure vessel to hold the protein solution, along with necessary valves, the Viresolve
Virus filter (Vpro, Merck Millipore) device (3.1 cm2, parvovirus reduction filter), digital electronic balance, and a collection vessel for the filtrate. Air pressure regulators
were used to control filtration pressure during the tests.
Figure 1: Experimental setup for hydraulic performance and spiking study. (ALL FIGURES ARE COURTESY OF THE AUTHORS)