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


Table 3: Summary of Virus Reduction (Log10) for the PEG Precipitation Step of the Prolastin-C Manufacturing Process Process Step


Parameter pH PEG


PEG precipitation of fraction IV-1 Suspension


Low


Set-point High Low


concentration Set-point High


Buffer volume Low to paste


Set-point


suspension High ratio


4.3 ± 0.2 4.3 ± 0.2 4.3 ± 0.2 HIV-1 BVDV


2.9 ± 0.1 2.8 ± 0.3 2.9 ± 0.1 3.4 ± 0.1 2.8 ± 0.3 2.8 ± 0.4 3.2 ± 0.1 2.8 ± 0.3 2.6 ± 0.3


PRV 3.3 ± 0.2 3.3 ± 0.2 3.3 ± 0.2


Log10 Virus Reduction ± SD Reo3


HAV 3.3 ± 0.1 3.3 ± 0.1 3.3 ± 0.1 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 PPV


3.2 ± 0.2 3.2 ± 0.3 3.3 ± 0.6 3.2 ± 0.1 3.2 ± 0.3 3.3 ± 0.4 3.5 ± 0.4 3.2 ± 0.3 2.8 ± 0.6


BVDV = bovine viral diarrhea virus; HAV = hepatitis A virus; HIV-1 = human immunodeficiency virus type 1; PEG = polyethylene glycol; PPV = porcine parvovirus; PRV = pseudorabies virus; Reo3 = reovirus type 3.


Table 4: Summary of Virus Reduction (Log10) for the Depth Filtration Step of the Prolastin-C Manufacturing Process Process Step


Parameter pH Depth filtration of PEG effluent Low


Set-point High


Buffer volume Low to paste


Set-point


suspension High ratio


≥4.7 ≥4.7 HIV-1


BVDV ≥4.0


4.0 ± 0.2 3.7 ± 0.1 4.1 ± 0.1 4.0 ± 0.2 4.4 ± 0.4


PRV ≥4.8 ≥4.8


Log10 Virus Reduction ± SD Reo3


HAV ≥4.0 ≥4.0 ≥2.8 ≥2.8


PPV ≥4.4 ≥4.4 ≥4.4 ≥4.1 ≥4.4


5.4 ± 0.4


BVDV = bovine viral diarrhea virus; HAV = hepatitis A virus; HIV-1 = human immunodeficiency virus type 1; PEG = polyethylene glycol; PPV = porcine parvovirus; PRV = pseudorabies virus; Reo3 = reovirus type 3.


evaluate virus removal by the nanofiltration step. All virus preparations were filtered through a sequential series of filters with decreasing pore size to remove any remaining cell debris and disperse virus aggregates prior to spiking into manufacturing intermediates. To ensure that the virus preparations were monodispersed for the nanofiltration studies, PPV preparations were filtered through a 35nm nanofilter and HAV preparations were filtered through a 75nm nanofilter.


Process intermediates, which were generally obtained from clinical- or commercial-scale manufacturing, were spiked with virus at a ratio of 1:20 to 1:1,000 (virus to intermediate). The spiked intermediates were initially processed using the bench-scale models with all parameters set at the optimal manufacturing ranges (set-point). During the process step, samples were removed to determine virus inactivation kinetics or virus partitioning. The toxicity of sample matrix for each cell line was determined in the absence of virus to differentiate matrix effects from actual virus cytopathology. Experiments were performed to determine whether the manufacturing intermediates interfered with virus detection. The results of the cytotoxicity assay, combined with the results of the viral interference assay, determined the detection limit for the assay system. The detection limit was based on the lowest dilution of sample for which no cytotoxicity or viral interference was observed.


Virus titers were determined using serial half-log10 dilutions of samples in cell-based assays and were quantitated as tissue culture infectious


dose at 50% infectivity (TCID50) using the method of Spearman–Kärber.20 Those samples that did not have any positive wells were treated based on the probability of detecting infectious virus at 95% confidence using


US RESPIRATORY DISEASE the Poisson distribution.21 solvent/detergent treatment, expanded volume testing19


For evaluation of HIV-1 inactivation during was used


to increase the probability of detecting virus. The ability of each step to inactivate or remove virus was determined by comparing the total virus in the initial spiked material (input) to the total virus in the same material after processing (output).


The overall virus reduction capacity of the Prolastin-C manufacturing process (global virus reduction factor) was calculated by summing virus reduction factors from the individual process steps. Only process steps


achieving at least one log10 reduction were included in the calculation of the global virus reduction factor. Where reduction to the limit of detection was achieved, results are reported as greater than or equal to the reduction factor; a greater reduction could potentially be achieved if a higher titer virus spike was used.


As production parameters (such as pH and temperature) are subject to some variability, the robustness of each virus removal or inactivation step was investigated by determining virus reduction at or just outside the standard Prolastin-C manufacturing ranges. For the robustness studies, critical and key parameters were evaluated individually and in combined worst-case experiments. A parameter evaluated individually was adjusted to a value at or just outside the extremes of the allowable manufacturing range while all other parameters were maintained within the manufacturing ranges. The parameters that were found to significantly impact virus reduction were further evaluated simultaneously in combined worst-case experiments.


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