Enveloped Virus Inactivation by a Non-traditional Solvent/detergent Treatment Step


Figure 1: Process Flow Diagram for Purification of Alpha1-PI from Cohn Fraction IV-1 Paste


Fraction IV-1 paste


Suspend Add NaCI Add PEG Centrifuge


PEG effluent Depth filter


PEG filtrate


S/D treatment Chromatography Eluate


Chromatography Flow-through


Nanofiltration UF/DF Formulate/fill Freeze-dry


Prolastin-C PEG = polyethylene glycol; S/D = solvent/detergent; UF/DF = ultrafiltration/diafiltration.


Materials and Methods Product Process Intermediates


In order to represent typical product intermediates undergoing S/D treatment, PEG filtrates from the production facility were used as well as PEG filtrates that had been produced at bench-scale under manufacturing set-point conditions. Bioanalytical testing demonstrated that the ‘set-point’ bench-scale material was similar to the material produced at production scale with respect to impurity profile and specific activity. A ‘high-protein’ PEG filtrate was also generated by slightly modifying the purification procedure. These changes were introduced to generate material with a higher protein concentration and lower specific activity. Extracted fraction IV-1 suspension and a lipid concentrate were generated by adding chloroform to a suspension of fraction IV-1 paste and vortexing. After centrifugation, the lighter aqueous extracted fraction IV-1 suspension layer was removed and aerated to drive off the residual chloroform. The denser, lower, chloroform-containing lipid layer was re-extracted with chloroform and centrifuged again. The lipids were concentrated by evaporating the chloroform phase and the remaining material was solubilized in 1% PS-20.


Virus Preparation and Assay


West Nile Virus (WNV), bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), and their corresponding cells for propagation and assay were obtained from the American Type Culture Collection (Rockville, MD) or the Biological Research Faculty and Facility (Ijamsville, MD). The viruses were prepared as crude or semi-pure stocks.


Crude virus stocks were the clarified supernatants from infected cells that had been disrupted by freezing and thawing and/or sonicating and then centrifuged at low speed (e.g. 3000 x g). Semi-purified virus stocks were made by ultracentrifuging crude virus through a sucrose cushion, suspending the resulting virus pellet in a small volume of buffer, then


US RESPIRATORY DISEASE


passing the suspension through a 0.2μm filter. Semi-purified stocks of human immunodeficiency virus type 1 (HIV-1) were obtained from Cell Trends (Middletown, MD) and assayed on C8166 cells from the National Institutes of Health AIDS Research and Reference Reagent Program. All viruses were quantified by end-point dilution in 96-well microtiter plates as previously described.10,11


Table 2: Process Conditions for Solvent/Detergent Treatment


Parameter Manufacturing Scale: Target


Set-point TNBP % 0.03


(wt/wt) PS-20 (%) wt/wt


0.5 0.02–0.04 0.25–0.75 0.03 0.5 PS = Polysorbate; S/D = solvent/detergent; TNBP = tri-n-butyl-phosphate. 0.01, 0.02 0.13, 0.25 Virus Bench Scale: Range Set-point exp Robust exp


Table 1: Effect of Solvent/Detergent on Alpha1-PI Recovery


S/D Concentration Generate PEG filtrate


TNBP (%) 0.00 0.30 0.15 0.03


PS-20 (%) 0.00 1.00 0.50 0.50


Alpha1-PI Recovery (%)


100 76 86


100 PS = Polysorbate; S/D = solvent/detergent; TNBP = tri-n-butyl-phosphate.


Duck hepatitis B virus (DHBV), obtained from


Hepadnavirus Testing (Palo Alto, CA), consisted of serum from congenitally infected ducklings. Virus titers were relatively low, so the infected serum was concentrated with 10% PEG and 1M sodium chloride prior to use. DHBV experiments were performed at ATS Labs (Eagan, MN) and assayed as previously described.11


Virus titers were determined by a median tissue culture infectious dose 50 assay using the Spearman-Karber method.12


The limits of detection for


the assays were based on the results of cytotoxicity and/or viral interference experiments. Extended volume testing was employed to increase HIV-1 assay sensitivity when no virus could be detected. The theoretical titer was based on the probability of detecting virus with 95% confidence using the Poisson distribution as follows: c = -ln(0.05)/v = 3/v, where v was the total volume of sample tested.13


For test samples with no detectable virus, virus titers were expressed with ‘less than or equal to’ (≤)


symbols. Log10 virus reduction values were calculated by subtracting log10 virus titers after five hours of S/D treatment from log10 virus titers that had been determined for the untreated virus controls at t = 0 hour. Results


reported as ‘greater than or equal to’ (≥) indicate that the test samples had no detectable virus and that higher reduction values could have been demonstrated had the input virus spike been higher.


Solvent/Detergent Stock Solutions


Different S/D stock solutions were prepared for the bench-scale virus experiments. A 100x concentrate consisting of approximately 3% TNBP/50% PS-20 was used in experiments when targeting manufacturing set-point S/D concentrations. Another stock containing 2% TNBP and 25%


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