Alpha1-antitrypsin Deficiency


Table 3: Virus Inactivation by Solvent/Detergent Treatment of Polyethylene Glycol Filtrate Time (hours)


Log10 virus titer Set-point Crude


0 1 3 5


LRV


WNV 7.0


≤1.6 ≤1.6 ≤1.6 ≥5.4


VSV 6.7 2.6 2.2 2.2 4.5


VSV 8.2 2.4 2.3 2.3 5.9


PRV 6.4


≤2.1 ≤2.1 ≤2.1 ≥4.3


*Conditions for robustness experiments = 0.02% tri-n-butyl-phosphate/0.25% polysorbate-20. **Extended volume testing was employed to lower the assay detection limit for HIV-1.


BVDV = bovine viral diarrhea virus; DHBV = duck hepatitis B virus; HIV-1 = human immunodeficiency virus type 1; LRV = log10 reduction value; PEG = polyethylene glycol; PRV = pseudorabies virus; S/D = solvent/detergent; VSV = vesicular stomatitis virus; WNV = West Nile virus.


Figure 2: Effect of Protein Concentration on Inactivation of VSV by S/D Treatment of PEG Filtrate


10


2 4 6 8


0 01


PEG filtrate Set-point


2 345 Time (hours)


TNBP 0.00% 0.01% 0.02%


High protein


0.01% 0.02%


PS20 0.00% 0.13% 0.25%


0.13% 0.25%


VSV was spiked into set-point PEG filtrate (2AU) or high-protein PEG filtrate (6AU) and treated with 0.01%/0.13% or 0.02%/0.25% TNBP/polysorbate-20 or with no solvent/detergent (S/D). PEG = polyethylene glycol; TNBP = tri-n-butyl-phosphate; VSV = vesicular stomatitis virus.


PS-20 was used in robustness experiments to evaluate even lower concentrations. A third stock containing only PS-20 was used to test inactivation by PS-20 only (no TNBP). Deactivating stock solutions were also prepared with PS-80 and used in experiments to compare the virucidal capacity of different detergents.


S/D Treatment


For S/D treatment at production scale, a 100x stock of TNBP/PS-20 was added to PEG filtrate with final weight-based concentrations of 0.02–0.04% TNBP and 0.25–0.75% PS-20. The bench-scale S/D experiments were conducted by diluting a lipid concentrate and/or deactivating stock solutions into the product process intermediates, followed by the addition of virus.


The virus-spiked, TNBP and/or PS-containing suspensions were pH-adjusted and incubated at the desired temperature. Virus-spiked suspensions with no TNBP and no PS were also processed in parallel as


42 Virus Inactivation


During set-point experiments, different viruses and virus preparations were treated under manufacturing set-point conditions for pH, temperature, and S/D concentration (0.03% TNBP/0.5% PS-20). Aliquots for DHBV titration were removed at only two time-points: before and after S/D treatment. All other viruses were sampled at zero, one, three, and five hours. The data show that all viruses were inactivated to near or below the limit of detection and that the kinetics of virus inactivation were comparable regardless of virus preparation (see Table 3). The impact of variations in the S/D process was evaluated in robustness experiments.


US RESPIRATORY DISEASE Symbol Analytical Assays


TNBP was measured by gas chromatography using flame ionization detection as described elsewhere.9


Polyoxyethylene nonionic surfactants,


such as PS-20 or PS-80, were measured spectrophotometrically at 620nm using a cobalt complexation method.14


Total cholesterol was determined using an enzymatic assay that yielded a chromogenic quinine-imine dye with a maximum absorbance of 520nm.16


Alpha1-PI activity was determined


using a two-stage assay that measured the degree of inhibition of elastase activity.15


A peroxidase-coupled method was used to colorimetrically measure triglyceride concentrations,17


were assayed using the Wako NEFA C test kit (Richmond, VA). Results


Effect of Solvent/Detergent on Recovery of Alpha1-PI Activity


Aliquots for potency testing were removed from PEG filtrates after treatment with various concentrations of TNBP/PS-20. Product yields,


which were determined by comparing alpha1-PI activity before and after treatment, were lower, with increasing S/D concentrations (see Table 1). Incubation in traditional concentrations of S/D (0.3% TNBP/1% PS-20) yielded only 76% product recovery. Reducing S/D concentrations to 0.15%


TNBP/0.5% PS-20 increased alpha1-PI recovery to 86% and treatment with 0.03% TNBP/0.5% PS-20 achieved 100% recovery. The concentrations of TNBP/PS-20 and the conditions with the least impact on protein activity were used to determine the set-points and limits for all S/D processing parameters (see Table 2). Experiments were then conducted to evaluate the virucidal capacity of S/D treatment and to fine-tune manufacturing ranges.


while non-esterified fatty acids


untreated virus controls. Aliquots of virus titrations were removed from all suspensions immediately after spiking (t = zero hours), at the end of incubation and at various times in between.


Semi-pure


BVDV 6.2 1.7


≤1.6 ≤1.6 ≥4.6


DHBV 4.0 ND ND


≤0.5 ≥3.5


HIV-1 5.7 1.9


≤-0.5** ≤-0.5** ≥6.2


Robust* Semi-pure


VSV 8.3 2.4 2.4 2.4 5.9


Log10 VSV titer


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