Enveloped Virus Inactivation by a Non-traditional Solvent/detergent Treatment Step inactivation procedure.8,9 The inclusion of excipients to S/D mixtures,


however, has the potential to stabilize viruses and adds complexity to the downstream purification process, which must be designed to remove the chemicals used in S/D treatment.


A variation of the standard S/D step for alpha1-PI was described that both effectively inactivates viruses and maintains the molecular function of the


protein without the addition of stabilizers. The Prolastin-C S/D treatment step uses 0.5% PS-20 as the detergent and TNBP concentrations of 0.03%.


At set-point conditions, 100% recovery of alpha1-PI activity was achieved after five hours of S/D treatment; after extended incubation for 48 hours, potency yields were 94%.18


In previous studies, inactivation of serpins


during S/D treatment of plasma was attributed primarily to the detergent Triton X-100 and not to TNBP.19


Our data show that when TNBP was increased five-fold from set-point concentrations to 0.15% and PS-20


concentrations were maintained at 0.5%, alpha1-PI recoveries decreased from 100% to 86%. Thus, high concentrations of TNBP may also contribute to the inactivation of serpins during S/D treatment.


Virus studies were performed to assess the effectiveness of these lower S/D concentrations to inactivate a variety of enveloped viruses. A step


capable of inactivating 4 log10 virus is generally considered to be an effective virus-inactivation step and this level of inactivation was achieved for WNV, VSV, PRV and BVDV after one hour at set-point conditions. Four


log10 inactivation could not be demonstrated for DHBV due to its low starting titers, but in all cases it was inactivated to below detection. WNV, PRV and BVDV were also inactivated to below detection levels. In contrast to the other viruses studied, the total input VSV was never consistently


inactivated to the limit of detection; nevertheless, 4 log10 was routinely achieved. The resistance of VSV to complete inactivation by S/D can most likely be attributed to the ability of the delipidated nucleocapsid-enclosed virion to infect cells at an efficiency approximately 10-5 to 10-6 times that of the lipid membrane-enclosed virion.20


This residual VSV infectivity could


be expected to remain even after a 4–5 log10 reduction in virus titer due to inactivation by any S/D treatment condition. This is further supported by


the fact that resistance of VSV to complete inactivation by standard S/D treatment has been reported elsewhere.21,22


As manufacturing processes are conducted within a range of process conditions, the virus reduction observed under set-point conditions may not be the same as the reduction achieved at the process limits. Robustness studies were performed to assure that virus inactivation was independent of variability in processing parameters. For these experiments, the effect of S/D concentration, temperature and pH on virus inactivation were evaluated separately (data not shown). The individual worst-case process conditions were then combined to test HIV-1 and VSV inactivation. HIV-1 and VSV were effectively inactivated to near or below detection after one to three hours. This period is well below the formal incubation time in production and provides a high level of assurance regarding pathogen safety. As the inactivation kinetics for the worst-case virus, VSV, were similar to the inactivation observed under set-point conditions, effective inactivation of all other viruses throughout the entire operating range for S/D treatment can be expected.


To gain a better understanding of virus inactivation by the non-traditional S/D treatment, experiments were also performed to evaluate virus


US RESPIRATORY DISEASE


B. PS80 10


2 4 6 8


0


0123 Time (hours)


TNBP 0.00% 0.00% 0.01% 0.02%


PS*


0.00% 0.25% 0.13% 0.25%


*PS = PS-20 or PS-80. VSV was treated using stocks of TNBP/PS-20 (A) or TNBP/PS-80 (B) at final concentrations of 0.01%/0.13% or 0.02%/0.25%. Control suspensions without S/D or with only 0.25% detergent were also spiked with virus and processed in parallel. PEG = polyethylene glycol; S/D = solvent/detergent; TNBP = tri-n-butyl-phosphate; VSV = vesicular stomatitis virus.


inactivation under conditions that were well outside the commercial manufacturing ranges. For these experiments, ‘outlier’ PEG filtrates were generated with protein and aggregate levels that exceeded those that would be encountered in manufacturing. VSV inactivation by 0.02% TNBP/0.25% PS-20 was essentially the same in the high-protein PEG filtrate and aggregated PEG filtrates as in the set-point PEG filtrates. When 0.01% TNBP/0.13% PS-20 was used, VSV inactivation was slightly delayed, but near or at detection by three hours in all test materials.


are a concern, so many product intermediates are filtered before S/D treatment. The data for Prolastin-C clearly show effective virus inactivation in the presence of high-protein and highly aggregated material, even at S/D concentrations below manufacturing ranges. Due to the effectiveness of the upstream precipitation and filtration steps to remove impurities, lipids cannot be detected in PEG filtrate. Nevertheless, the effect of lipids on the Prolastin-C S/D step was investigated because the researchers who originally developed the S/D method postulated that the concentration of lipids should have a greater impact on virus inactivation than protein concentration.23


The presence of aggregates that could shield and protect a virus from inactivation13


A concentrate of fraction IV-1


lipids was added to set-point PEG filtrate and extracted fraction IV-1 suspension to yield test materials with measurable concentrations of


45 Symbol 45


Figure 5: Effect of Detergent on Inactivation of VSV by S/D Treatment of PEG Filtrate


A. PS20 10


2 4 6 8


0 0


123 Time (hours)


45


Log10 VSV titler


Log10 VSV titer


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