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


Figure 3: Effect of Aggregates on Inactivation of VSV by S/D Treatment of PEG Filtrate


A. Set-point PEG filtrate aggregation 3


2 1 0 0–1x FT number A280


B. Set-point PEG filtrate virus reduction 10


FT


2 4 6 8


0


012 345 Time (hours)


C. High-protein PEG filtrate aggregation 6


4 2 0 0–1x FT number A280


D. High-protein PEG filtrate virus reduction 10


FT


2 4 6 8


0


012 345 Time (hours)


Aggregate levels in set-point PEG filtrate (A) and high-protein PEG filtrate (C), which had been frozen and thawed zero to one or nine times, were ascertained by measuring absorbance at 280nm and transmittance at 580nm. VSV was spiked into set-point (B) or high-protein (D) PEG filtrates that had been frozen and thawed zero to one times, and then treated with 0.01%/0.13% or 0.02%/0.25% TNBP/PS-20. Suspensions that had been frozen and thawed nine times were spiked with virus and treated with 0.01%/0.13% or 0.02%/0.25% TNBP/PS-20. Control suspensions without S/D were also spiked with virus and processed in parallel. FT = freeze thaw; PEG = polyethylene glycol; S/D = solvent/detergent; TNBP = tri-n-butyl-phosphate; VSV = vesicular stomatitis virus.


US RESPIRATORY DISEASE


0– 1x


9x


TNBP PS20 0.00% 0.00% 0.01% 0.13% 0.02% 0.25% 0.01% 0.13% 0.02% 0.25%


Symbol T580 9x 100


25 50 75


0


0– 1x


9x


TNBP PS20 0.00% 0.00% 0.01% 0.13% 0.02% 0.25% 0.01% 0.13% 0.02% 0.25%


Symbol T580 9x 100


25 50 75


0


Table 4: Lipid Content of Test Suspensions Test Suspension


Unextracted fr IV-1


suspension Extracted fr IV-1


suspension Extracted fr IV-1


suspension + added lipids


Set-point PEG filtrate Below detection Below detection Below detection Set-point PEG filtrate 130 + added lipids


120 0.4


CHOL = cholesterol; FA = fatty acids; fr = fraction; PEG = polyethylene glycol; TG = triglyceride.


During single-parameter robustness experiments, one process variable was set at the manufacturing limit while all other process parameters were maintained at their targeted set-points. Data from these experiments demonstrated that the VSV inactivation rate was slightly slower at the lower limits indicated for S/D concentration (0.02% TNBP/ 0.25% PS-20), temperature and pH. The individual worst-case conditions were then combined and used to test HIV-1 and VSV (see Table 3).


Under combined worst-case conditions, HIV-1 was inactivated to the limit


of detection (≥6.2 log10) within three hours of S/D treatment. Inactivation of VSV was biphasic, as 5.9 log10 virus was rapidly inactivated by one hour, followed by low levels of infectivity where one or two positive wells were


occasionally observed. As VSV was the only virus that was not consistently inactivated to below detection, and since crude virus preparations had significantly lower titers and were inactivated at essentially the same rate as semi-purified virus, semi-purified VSV was deemed the most S/D-resistant virus. For this reason, semi-purified VSV was used in all subsequent robustness studies.


Effect of High Protein on Virus Inactivation The effect of higher-than-anticipated protein concentrations on the ability of S/D treatment to inactivate virus was tested. For the studies, total protein was determined as the absorbance of suspensions at 280nm and results were reported as absorbance units (AU). The average concentration for bench-scale set-point PEG filtrates was 2AU, while the high-protein PEG filtrates averaged 6AU. VSV was spiked into set-point and high-protein PEG filtrates and treated with 0.02% TNBP/0.25% PS-20 or 0.01% TNBP/0.13% PS-20 at the worst-case lower conditions for temperature and pH. Virus inactivation was essentially the same for both PEG filtrates (see Figure 2). VSV was near or at the limit of detection after one hour of treatment with 0.02% TNBP/0.25% PS-20 and after three hours of treatment with 0.01% TNBP/0.13% PS-20, regardless of protein concentration.


Effect of Aggregates on Virus Inactivation To assess the effectiveness of S/D to inactivate virus in the presence of aggregates, set-point and high-protein PEG filtrates were rapidly frozen and slowly thawed multiple times to induce aggregation. The extent of protein aggregation was ascertained by measuring absorbance at 280nm (A280) and transmittance at 580nm (T580). After nine cycles of freezing and thawing, the A280 of the set-point PEG filtrates remained the same but the T580 decreased from 98% to 72% (see Figure 3). Similarly, after nine


43


CHOL (µg/ml) TG (µg/ml) 74


45


FA (mEq/l) Below detection Below detection Below detection Below detection 110 100 0.4


% Transmittance


% Transmittance


Log10 VSV titer


Protein concentration


Log10 VSV titer


Protein concentration


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