Virus and Prion Safety of a New Preparation of Human Alpha1-proteinase Inhibitor, Prolastin®-C


single-parameter robustness evaluation were pH, temperature, and ethanol concentration. Since none of the parameters in the robustness evaluations had a significant effect on virus removal, a combined worst-case evaluation, where all parameters that affect virus reduction are adjusted simultaneously, was not performed. Under set-point


conditions, the PEG precipitation step removed 4.3 ± 0.2 log10 HIV-1 and approximately 3 log10 of the other viruses (BVDV, PRV, Reo3, HAV, and PPV) (see Table 3). Similarly, under set-point conditions, the


subsequent depth filtration step achieved 4 log10 or greater reduction of HIV-1, BVDV, PRV, Reo3, and PPV, all of which were reduced to their


respective limits of detection (see Table 4). HAV was removed to the


limit of detection across the depth filtration step (≥2.8 log10). Additional HAV reduction could likely have been achieved if a higher starting titer had been attainable.


The robustness of virus reduction across the PEG precipitation and depth filtration steps was evaluated using BVDV and PPV. Given their small size, these viruses tend to be more difficult to remove by either precipitation or filtration, thus representing a worst-case challenge for virus removal. Varying the process parameters did not result in significant differences in the LRVs compared with the LRVs observed with all parameters at set-point. Unlike other process steps that were evaluated, no significant impact on virus removal was observed during the single parameter robustness evaluations, therefore a combined worst-case evaluation was not warranted.


Virus removal by the 15nm nanofiltration step was evaluated using a panel of viruses that included both enveloped and non-enveloped


viruses. The nanofiltration step removed 4.2 ± 0.2 log10 PPV, ≥6.9 log10 HIV-1, ≥4.7 log10 BVDV, ≥5.2 log10 PRV, ≥5.1 log10 VSV, ≥4.3 log10 Reo3, and ≥5.5 log10 HAV under set-point conditions (see Table 5). Removal of PPV, the smallest virus studied (18–24nm) and therefore the worst-case


challenge to the 15nm nanofilter, was evaluated under robustness conditions with and without a post-nanofiltration rinse. In the absence


of a rinse, a minimum of 4.0 log10 removal of PPV was observed at all protein concentrations, filtration pressures, pHs, and filter loads evaluated (data not shown). When a post-nanofiltration rinse was


included in the process step, a minimum of 4.0 log10 removal of PPV was maintained for high protein concentration, high or low filtration pressure, high or low filter loads, or low pH. However, with a rinse,


PPV reduction was <4.0 log10 at very high pH and very low protein concentration. Therefore, the manufacturing limits for pH and


protein concentration ranges were narrowed until >4.0 log10 PPV removal was consistently observed utilizing a post-nanofiltration rinse (see Table 5). In a combined worst-case evaluation using the adjusted values for the pH and protein concentration, including a


post-nanofiltration rinse, 4.0 log10 PPV removal was demonstrated (see Table 5).


Virus Inactivation Studies


The Prolastin-C manufacturing process employs novel solvent/detergent treatment conditions.25


To ensure the virus inactivation by these novel


solvent/detergent treatment conditions is robust with respect to a variety of enveloped viral challenges, an expanded panel of viruses was evaluated. VSV was included within the panel and chosen as the worst-case virus for the robustness evaluation of this step because of its


US RESPIRATORY DISEASE


Table 7: Virus Reduction (Log10) of Individual Steps in the Prolastin-C Manufacturing Process


Log10 Virus Reduction Factor Enveloped Viruses Process


fractionation PEG


precipitation Depth


filtration S/D


treatment 15nm virus removal


Step Cold ethanol 3.4


4.3 3.5 2.8 ≥4.7 4.0 Non-enveloped Viruses HIV-1 BVDV PRV VSV Reo3 HAV PPV 3.9 ND ≥2.1 1.4 3.3 ND 3.3 3.0 ≥4.8 ND ≥4.0 ≥2.8 ≥6.2 ≥4.6 ≥4.3 ≥6.9 ≥4.7 ≥5.2 1.0 3.2 ≥4.4 5.1 NA* NA NA ≥5.1 ≥4.3 ≥5.5 4.2


nanofiltration** Global virus ≥25.5 ≥19.6 ≥21.5 ≥10.2† ≥13.7 ≥12.7 ≥12.8 reduction factor


*This step is only effective against enveloped viruses. **Includes a post-nanofiltration rinse.


†VSV reduction was only determined for the S/D treatment and 15nm virus removal nanofiltration steps.


BVDV = bovine viral diarrhea virus; HAV = hepatitis A virus; HIV-1 = human immunodeficiency virus type 1; NA = not applicable; ND = not determined; PEG = polyethylene glycol; PPV = porcine parvovirus; PRV = pseudorabies virus; Reo3 = reovirus type 3; S/D = solvent/detergent; VSV = vesicular stomatitis virus.


Table 8: Reduction (Log10) of TSE Infectivity and Prion Protein (PrPRES


) During the Prolastin-C


Manufacturing Process Process Step


Cold ethanol


TSE Infectivity Reduction PrPRES Removal by by Animal Bioassay


(log10 ID50) 6.0


fractionation (effluent I to effluent II + III) PEG precipitation plus ≥6.8 depth filtration


Western Blot (log10 PrPRES)


5.0


≥5.5 PEG = polyethylene glycol; TSE = transmissible spongiform encephalopathy. resistance to inactivation by solvent/detergent treatment.18,19 Under


set-point conditions (0.03% TNBP, 0.5% polysorbate 20), log10 reduction values of ≥4.6 log10 BVDV, ≥4.3 log10 PRV, 5.1 log10 VSV (see Table 6), and ≥5.4 log10 WNV were observed for this process step. The inactivation of VSV and HIV-1 was evaluated in combined worst-case


conditions of low solvent and detergent concentrations, low temperature, and low pH in combination with different Fraction IV-1 Paste suspension ratios. The kinetics of VSV inactivation were slightly slower under these conditions, but the overall reduction capacity was not significantly influenced (see Table 6 and Figure 3). Under combined


worst-case conditions, an LRV of ≥6.2 log10 was observed for HIV-1 with complete inactivation occurring within two hours (see Table 6 and Figure 3).


The reduction factors for all individual processing steps that achieved at least one log10 were combined to determine the global reduction factors.


37


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