Alpha1-antitrypsin Deficiency Figure 4: Effect of Lipids on Inactivation of Vesicular Stomatitis Virus by Solvent/Detergent Treatment
A. Unextracted Fr IV-1 10
2 4 6 8
0
0123 Time (hours)
45 Test material
Suspension only (no
added lipids)
B. Extracted Fr IV-1 10
2 4 6 8
0
0123 Time (hours)
TNBP PS20 0.00% 0.00% 0.02% 0.25% 0.03% 0.50%
45 Symbol Test material
Suspension with added lipids
C. Set-point PEG filtrate 10
2 4 6 8
0
0123 Time (hours)
TNBP PS20 0.00% 0.00% Not done 0.02% 0.25% 0.03% 0.50%
Symbol
VSV was spiked into unextracted fraction IV-1 (A), extracted fraction IV-1 (B) and set-point PEG filtrate (C) at 2AU and treated with 0.02%/0.25% or 0.03%/0.50% TNBP/PS-20. Suspensions were also spiked with extracted lipids before virus addition, followed by treatment with 0.02%/0.25% or 0.03%/0.50% TNBP/PS-20. Control suspensions containing no S/D and no additional lipids were spiked with virus and processed in parallel.
PEG = polyethylene glycol; S/D = solvent/detergent; TNBP = tri-n-butyl-phosphate; VSV = vesicular stomatitis virus.
rapid freezes and slow thaws, the high-protein PEG filtrates showed little or no reduction in absorbance, but transmittance dropped by ~40%. The unchanged A280 and decreased T580 are indicative of the formation of aggregates in the frozen and thawed suspensions. VSV was spiked into PEG filtrates with low aggregates (frozen and thawed never or once) and into highly aggregated PEG filtrates (frozen and thawed nine times). Each test material was treated with 0.02% TNBP/0.25% PS-20 or 0.01% TNBP/0.13% PS-20 at the lower limits for temperature and pH. Virus inactivation was essentially the same in all PEG filtrates. After one hour of treatment with 0.02% TNBP/0.25% PS-20 and three hours of treatment with 0.01% TNBP/0.13% PS-20, VSV was near or at the limit of detection, regardless of aggregate levels (see Figure 3).
Effect of Lipids on Virus Inactivation
The efficacy of S/D treatment in the presence of lipids was assessed using test materials generated at bench scale. Lipids were extracted from a suspension of fraction IV-1 paste and concentrated. Set-point PEG filtrate and the unextracted and extracted fraction IV-1 suspensions were diluted to 2AU and analyzed for lipid content.
The unextracted fraction IV-1 suspension contained 74µg/ml cholesterol, 45µg/ml triglycerides and no detectable fatty acids (see Table 4). Lipids were below detection in the set-point PEG filtrate and extracted fraction IV-1 suspension. After adding the lipid concentrate at 1:100 dilution, however, the average cholesterol, triglyceride, and fatty acid levels in the two suspensions were 120µg/ml, 110µg/ml, and 0.4mEq/l, respectively. VSV was spiked into the different suspensions, with or without added lipids, and treated with 0.03% TNBP/0.5% PS-20 (set-point S/D concentrations) or 0.02% TNBP/0.25% PS-20. Treatment of the lipid-containing unextracted fraction IV-1 suspension or the suspensions with no detectable lipids with set-point S/D concentrations resulted in inactivation of VSV to near or below detection level (see Figure 4). The addition of lipids to extracted fraction IV-1 suspension and to PEG filtrate
44
reduced the virucidal capacity of set-point concentrations, but S/D treatment was still effective since 5.0 log10 VSV was inactivated.
VSV inactivation during S/D treatment of PEG filtrate with 0.02% TNBP/0.25% PS-20 was no different from treatment with set-point concentrations, even after the addition of lipids. VSV inactivation during treatment of extracted fraction IV-1 suspension with the reduced S/D
concentrations was also 5.0 log10. Despite this, VSV inactivation was only 3.8 log10 in the lipid-containing unextracted fraction IV-1 suspension and in the extracted fraction IV-1 suspension after the addition of lipids.
Effect of Detergent on Virus Inactivation VSV inactivation by S/D treatment was compared using PS-20 or PS-80 (see Figure 5). Set-point PEG filtrate spiked with semi-pure VSV was incubated with a mixture of 0.02% TNBP and 0.25% PS-20 or PS-80. In addition, virus-spiked test solutions were treated with even lower S/D concentrations (0.01% TNBP/0.13% PS-20 or PS-80) or with only 0.25% detergent (PS-20 or PS-80). In the absence of S/D, VSV inactivation was minimal. Treatment with 0.02% TNBP/0.25% PS-20 or 0.01% TNBP/0.13% PS-20 inactivated VSV to below or near detection after one or three hours, respectively. Even incubation in 0.25% PS-20 only (no TNBP) inactivated 5.5 log10 VSV after five hours.
VSV inactivation was significantly less when PS-80 was used. After treatment with 0.25% PS-80 and with 0.01% TNBP/0.13% PS-80, VSV
inactivation was 1.7 and 2.1 log10, respectively. Virus inactivation was faster in the presence of 0.02% TNBP/0.25% PS-80, but after five hours only 3.3 log10 VSV reduction was achieved.
Discussion
Serpins, such as alpha1-PI, are susceptible to inactivation after traditional S/D treatments with 0.3% TNBP/1% detergent. In some cases, excipients
such as sucrose have been added to stabilize alpha1-PI as part of the US RESPIRATORY DISEASE 45
Log10VSV titer
Log10 VSV titer
Log10 VSV titer
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84