Regulatory bodies around the world expect downstream purification processes to demonstrate robust clearance of model adventitious viruses in time for execution of phase 3 clinical trials and product licensure (1,2,3). Model viruses selected for these studies should represent a diversity of viral physicochemical properties, and the clearance methods applied should include orthogonal mechanisms such as clearance based on size alongside chemical inactivation. Virus filtration is a critical unit operation used in numerous purification processes of monoclonal antibodies (MAbs), recombinant proteins, and plasma-derived biopharmaceuticals.

PRODUCT FOCUS: ANTIBODIES AND OTHER RECOMBINANT PROTEINS



PROCESS FOCUS: DOWNSTREAM PROCESSING



WHO SHOULD READ: MANUFACTURING, PROCESS DEVELOPMENT, AND ANALYTICAL PERSONNEL



KEYWORDS: VIRAL SAFETY, MMV/MVM, XMULV, FILTRATION, SCALE-UP



LEVEL: INTERMEDIATE

Virus filtration unit operations have been shown to be scalable, robust, and reproducible (4, 5). Initial sizing of membranes usually involves a V_max study on a small-scale filter followed by linear scale-up maintaining a constant ratio of process volume per square meter area. However, the ability of a virus filter to clear viruses is ultimately determined by validation studies involving virus-spike and clearance experiments. A representative feedstock is spiked with a preparation of model virus, this feedstock is passed through the filter, and viral clearance is determined after measuring recovered levels in filtrate using qualified/validated scale-down systems.

Because the level of impurities in a viral spike is much higher than in a nonspiked feed, significant degradation in volumetric capacity of a virus filter is sometimes observed when processing a spiked load (4). It has been noted that the ability of virus filters to clear small parvoviruses may be degraded when their membranes are fouled, as shown by accelerated decays in flow when using spiked loads (6, 7). This raises a question of whether virus filter sizing for routine processing should be based on capacity as determined using nonspiked feed or the more limited capacity obtained with a spiked feed during process validation (which, in most cases, is significantly smaller).

Here we present a novel procedure for approaching the validation of adventitious virus removal while successfully resolving those issues. It must be pointed out that this method is tested only in certain virus filters from Millipore Corporation (www.millipore.com) — but these particular filters are widely used in the industry, so the method should have broad application.

Materials and Methods

Optiscale-25 devices with Viresolve NFP membranes (3.5 cm²) were purchased from Millipore. Such filters are designed to be used in normal flow mode (dead-end mode) and can be operated at constant pressure or constant flow. Optiscale-40 devices with Viresolve prefilters (5.0 cm²) were also purchased. They combine charged-modified depth-filtration media with a 0.1-µm nominally rated cellulose membrane layer at the bottom.

Load Material and Virus Assays for the Study: Load material used for this study came from clinical manufacturing runs using an intermediate product pool, with a concentration of 4–8 mg/mL at pH 7.5 and conductivity of 8–10 mS/cm.

Viruses used for this study are listed in Table 1 along with their physical properties. We assayed our load samples for cytotoxicity and interference of xenotropic murine leukemia virus (XMuLV) and murine minute virus (MMV) infectivity in PG-4 (feline S⁺L⁻) cells and 324 K cells, respectively. Cytotoxicity, interference, and infectivity assays were performed by BioReliance Corporation (www.bioreliance.com). For validation spiking studies, virus titers were 1.0 × 10^7.7 particles/mL for XMuLV and 1.0 × 10^7.9 particles/mL for MMV. For the development study, virus titer was 1.6 × 10⁷ particles/mL. A viral spike volume of 0.05% (v/v) relative to the entire filtrate volume (v/v) was used in all spiking studies. LRV data at a specific percent of flow delay came from instantaneous samples (2 mL) collected from the permeate. We determined flow decay using this equation:

Table 1: Vi ruses used in this study (Click to enlarge)

Experimental Method

Vmax Test Procedure: We used Optiscale devices with an effective membrane area of 3.5 cm² and carried out our trials under a constant feed pressure of 30 psi. After a filter was connected to the set-up, buffer was added to the feed vessel, which was then pressurized to the test pressure. Then the buffer was allowed to flow through the membrane. We calculated buffer permeability values to ensure that the entire filter area was used. Next, the buffer was drained from the pressure vessel, and our protein solution was then added to the pressure vessel. The feed vessel was pressurized to the test pressure, and we recorded the weight of filtrate collected over time.

For some experiments, a Viresolve prefilter (VPF) was used in line with the Viresolve NFP filters in a similar ratio of membrane areas as used in offline experiments. For offline experiments the feed was prefiltered first through VPF, then loaded onto the NFP filter. For the latter, we maintained residence time through the VPF the same as for the downstream NFP filtration.