Process Development and Spiking Studies for Virus Filtration of r-hFSH - This study on a recombinant human follicle stimulating hormone demonstrates the use of virus filters to reduce the risk of cont


Process Development and Spiking Studies for Virus Filtration of r-hFSH
This study on a recombinant human follicle stimulating hormone demonstrates the use of virus filters to reduce the risk of contamination.

BioPharm International
pp. 24-30

Description of tests for hydraulic performance

Prior to an experimental run, the VPro Virus filters were wetted with water for injection (WFI) and then conditioned by filtering the appropriate buffer for 5-7 m at a pressure drop of 30-50 psi. At the end of the buffer conditioning step, the normalized buffer permeability (NBP) was recorded and compared to the typical Normalized Water Permeability (NWP) values for the devices before proceeding with the filtration run.

The protein solution, used for the study, was a recombinant human Follicle Stimulating Hormone (r-hFSH, protein concentration 0.1 gm/L) solution from Bharat Serums & Vaccines Ltd. All the buffers and protein solutions were 0.2 μm filtered. After wetting of Vpro filter, the protein feedstock was placed into the pressure vessel and the operating pressure of 30 psi was set accordingly on the pressure regulator. Once drop-wise flow was observed from the Vpro micro (3.1 filtration area) device, the cumulative volume being filtered (V) as a function of time (t) was recorded. Approximately 200 ml of feed volume was used in each trial. Each trial was carried out for at least 60 m unless gross plugging was observed.

Time/volume (t/V) in the y-axis as a function of time (t) in the x-axis was plotted to draw a curve, the slope of this curve was used to calculate the capacity (Vmax) and the reciprocal of the y-axis intercept was used to calculate initial volumetric flow (Qi) (see Equation 1) of the filter based on Vmax gradual pore plugging model (3, 4).

The process filter area requirement (Amin) (under unspiked condition) can be estimated from Equation 1:

Virus spiking study

The virus spiking study was performed at Charles River Biopharmaceutical Services GmbH, Cologne. The virus stock solutions were ultracentrifuged, the virus pellet suspended in phosphate buffered saline (PBS), and subsequently prefiltered: the Murine Leukemia Virus (MuLV) and Pseudo Rabies Virus (PRV) stocks were 0.45μm filtered, the Reo-3 stock 0.22 μm filtered, and the Minute Mice Virus (MVM) 0.1 μm filtered. After prefiltration, samples from the four virus solutions were withdrawn and analyzed for the virus titer.

The virus spiked test item was prepared as follows: 200 mL of separate four aliquots of the r-hFSH protein (protein concentration 0.1 gm/L) was spiked individually in duplicate with 2 mL of each of ultracentrifuged and prefiltered virus stock solutions (spike ratio 1%) of MVM, Reo-3, MuLV, and PRV. The solution was mixed and samples withdrawn from the spiked pool and analyzed for virus titer (spiked test item). The spiked protein solution was additionally prefiltered: MuLV and PRV using 0.45μm, Reo-3 0.22 μm, and the MVM 0.1 μm filter. A sample was withdrawn and analyzed for virus titers (load, prefiltered). An aliquot of the prefiltered spiked protein solution was kept at room temperature for the entire process time to determine the stability of the virus load during virus retentive filtration (hold, prefiltered). The Viresolve Pro test system was set up (Figure 1) and the NWP and NBP were determined to ensure proper wetting of the Viresolve Pro filters. All the Vpro virus filters that were used for the study were chosen from the Vpro validation kit (VPro virus filters of 'Vpro Validation kit' that is available preintegrity tested by Merck Millipore). The filtration was performed at room temperature at 30 psi test pressure, with nitrogen as the pressure source. The operating conditions and feed used were representative of worst-case manufacturing conditions. The individual virus spiked feed solution was loaded into separate test systems and the filtration process started. Each of the filtrate was collected in separate containers that were continuously being weighed on a balance. The filtration was terminated when the desired volumes of at least 120 mL for each of the MuLV, PRV, and Reo-3 (fraction A) spiked feed streams were obtained. No process interruption/flushing during the spiking studies were done, nor it is practiced in manufacturing. For MVM, the prefixed success criteria for filtration was at least 60 mL for first fraction (filtrate fraction A), at least 40 mL for the second fraction (filtrate fraction B), and at least 20 mL for the third fraction (filtrate fraction C). The total mass of each filtrate was determined. After mixing, a sample was withdrawn from each of the filtrate and analyzed for viral titer by endpoint titration and by Large Volume Plating (Fraction A to C) as follows.

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