Alpha1-antitrypsin Deficiency

Effective and Robust Enveloped Virus Inactivation by a Non-traditional Solvent/detergent Treatment Step
JoAnn Hotta, PhD,1 Shih-Fong Chao, PhD,1 Michelle Gall, PhD,1 Nathan J Roth, PhD,1 John Lang, BS2 and Douglas Lee, PhD1
1. Pathogen Safety, Talecris Biotherapeutics Inc., Research Triangle Park, North Carolina; 2. BioAnalytics, Talecris Biotherapeutics Inc., Clayton, North Carolina

Abstract
Solvent/detergent (S/D) treatment is a viral-inactivation procedure that has been used in most cases with a mixture of 0.3% tri-n-butyl-phosphate (TNBP) and 1% detergent, such as polysorbate 80, Triton X-100, or sodium cholate. The manufacturing process for Prolastin-C, a plasma-derived alpha1-proteinase inhibitor (alpha1-PI), includes S/D treatment but at non-traditional concentrations. Since the potency of alpha1-PI was negatively impacted at previously utilized concentrations of S/D, the manufacturing process for Prolastin-C was developed using 0.03% TNBP and 0.5% polysorbate 20. This report describes the robustness and efficacy of the step for enveloped virus inactivation at S/D concentrations that do not affect alpha1-PI activity. Bench-scale set-point experiments were conducted to evaluate virus inactivation by S/D treatment under standard manufacturing conditions and at broad ranges for temperature, pH, protein concentration, S/D concentration, and in the presence of lipids or highly aggregated suspensions. These studies present an alternative robust and effective method for S/D treatment using non-traditional low concentrations of TNBP and polysorbate-20.

Keywords
Alpha1-antitrypsin, alpha1-proteinase inhibitor, Prolastin, Prolastin-C, solvent, detergent, virus inactivation, TNBP, polysorbate 20
Disclosure: All authors are full-time employees of Talecris Biotherapeutics Inc. Acknowledgments: The authors would like to thank Jiagang Wang, Sharon Smith, Yavnika Patel, Brett Buno, and Heather Donohoe for their assistance in developing this step, and the viral validation and bioanalytical groups for their support in these studies. Received: October 5, 2010 Accepted: December 9, 2010 Citation: US Respiratory Disease, 2010;6:406 Correspondence: JoAnn Hotta, PhD, Pathogen Safety, Talecris Biotherapeutics Inc., 85 TW Alexander Drive, Research Triangle Park, NC 27709, US. E: joann.hotta@talecris.com

Support: This study was sponsored by Talecris Biotherapeutics Inc. (Research Triangle Park, NC 27709, USA). Editorial assistance was provided under the direction of the authors by Anne-Marie Manwaring, BSc, and Martin Kenig, DPhil, of PAREXEL and was supported by Talecris Biotherapeutics Inc.

Human alpha1-proteinase inhibitor (PI), also known as alpha1-antitrypsin, is a serine protease inhibitor (serpin) that is synthesized in the liver and travels to the lungs after secretion by hepatocytes into the blood. In the lungs, alpha1-PI inhibits neutrophil elastases and prevents them from degrading pulmonary connective tissue. Patients with inherited alpha1-PI deficiency have reduced levels of alpha1-PI in their serum and lungs, so they are at an increased risk for developing emphysema. Prolastin is a concentrate of human alpha1-PI that is administered to patients in an attempt to restore the elastaseserpin balance. Observational studies have shown that augmentation therapy may help to slow a decline in lung function.13 Modifications have recently been approved to the Prolastin manufacturing process and the resulting Prolastin-C process yields an alpha1-PI with improved functional activity and a high purity.4 Prolastin-C is derived from large pools of human plasma using a modified CohnOncley fractionation method.5,6 The Prolastin-C process incorporates two dedicated virus-reduction steps:

a solvent/detergent (S/D) treatment step located immediately downstream of the generation of polyethylene glycol (PEG) filtrate (see Figure 1); and a nanofiltration step. S/D treatment is a widely accepted method for enveloped virus inactivation and involves the incubation of a protein solution in a mixture consisting of an organic solvent, tri-n-butyl-phosphate (TNBP) and a non-ionic detergent. The exact composition of the S/D mixture varies depending on the manufacturing process and the target protein of interest. TNBP is generally used at concentrations of 0.10.3%, while the most commonly used detergents, polysorbate(PS)-80 (Tween 80), octoxynol (Triton X-100), and sodium cholate,7 are often added at concentrations of 0.11%. There are reports that indicate treatment with 0.3% TNBP/1% PS-80 or 0.3% TNBP/0.3% sodium cholate substantially lowers alpha1-PI activity unless stabilized by sucrose.8,9 To avoid the need for stabilizers, the Prolastin-C step uses the S/D combination 0.03% TNBP/0.5% polysorbate (PS)-20. This report summarizes the efficacy of the treatment in inactivating enveloped viruses.

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

Figure 1: Process Flow Diagram for Purification of Alpha 1 -PI from Cohn Fraction IV-1 Paste
Fraction IV-1 paste Suspend Add NaCI Add PEG Centrifuge PEG effluent Depth filter PEG filtrate S/D treatment Chromatography Eluate Chromatography Flow-through Nanofiltration UF/DF Formulate/fill Freeze-dry Prolastin-C
PEG = polyethylene glycol; S/D = solvent/detergent; UF/DF = ultrafiltration/diafiltration.

Table 1: Effect of Solvent/Detergent on Alpha1-PI Recovery

S/D Concentration TNBP (%) 0.00 0.30 PS-20 (%) 0.00 1.00 0.50 0.50 100 76 86 100 Alpha1-PI Recovery (%)

Generate PEG filtrate

0.15 0.03

PS = Polysorbate; S/D = solvent/detergent; TNBP = tri-n-butyl-phosphate.

Table 2: Process Conditions for Solvent/Detergent Treatment

Parameter Manufacturing Scale: Target Set-point TNBP % (wt/wt) PS-20 (%) wt/wt
PS = Polysorbate; S/D = solvent/detergent; TNBP = tri-n-butyl-phosphate.

Virus Bench Scale: Set-point exp 0.03 0.5 Robust exp 0.01, 0.02 0.13, 0.25

Range 0.020.04 0.250.75

0.03 0.5

Materials and Methods

Product Process Intermediates
In order to represent typical product intermediates undergoing S/D treatment, PEG filtrates from the production facility were used as well as PEG filtrates that had been produced at bench-scale under manufacturing set-point conditions. Bioanalytical testing demonstrated that the set-point bench-scale material was similar to the material produced at production scale with respect to impurity profile and specific activity. A high-protein PEG filtrate was also generated by slightly modifying the purification procedure. These changes were introduced to generate material with a higher protein concentration and lower specific activity. Extracted fraction IV-1 suspension and a lipid concentrate were generated by adding chloroform to a suspension of fraction IV-1 paste and vortexing. After centrifugation, the lighter aqueous extracted fraction IV-1 suspension layer was removed and aerated to drive off the residual chloroform. The denser, lower, chloroform-containing lipid layer was re-extracted with chloroform and centrifuged again. The lipids were concentrated by evaporating the chloroform phase and the remaining material was solubilized in 1% PS-20.

passing the suspension through a 0.2m filter. Semi-purified stocks of human immunodeficiency virus type 1 (HIV-1) were obtained from Cell Trends (Middletown, MD) and assayed on C8166 cells from the National Institutes of Health AIDS Research and Reference Reagent Program. All viruses were quantified by end-point dilution in 96-well microtiter plates as previously described.10,11 Duck hepatitis B virus (DHBV), obtained from Hepadnavirus Testing (Palo Alto, CA), consisted of serum from congenitally infected ducklings. Virus titers were relatively low, so the infected serum was concentrated with 10% PEG and 1M sodium chloride prior to use. DHBV experiments were performed at ATS Labs (Eagan, MN) and assayed as previously described.11 Virus titers were determined by a median tissue culture infectious dose 50 assay using the Spearman-Karber method.12 The limits of detection for the assays were based on the results of cytotoxicity and/or viral interference experiments. Extended volume testing was employed to increase HIV-1 assay sensitivity when no virus could be detected. The theoretical titer was based on the probability of detecting virus with 95% confidence using the Poisson distribution as follows: c = -ln(0.05)/v = 3/v, where v was the total volume of sample tested.13 For test samples with no detectable virus, virus titers were expressed with less than or equal to () symbols. Log10 virus reduction values were calculated by subtracting log10 virus titers after five hours of S/D treatment from log10 virus titers that had been determined for the untreated virus controls at t = 0 hour. Results reported as greater than or equal to () indicate that the test samples had no detectable virus and that higher reduction values could have been demonstrated had the input virus spike been higher.

Virus Preparation and Assay

West Nile Virus (WNV), bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), and their corresponding cells for propagation and assay were obtained from the American Type Culture Collection (Rockville, MD) or the Biological Research Faculty and Facility (Ijamsville, MD). The viruses were prepared as crude or semi-pure stocks. Crude virus stocks were the clarified supernatants from infected cells that had been disrupted by freezing and thawing and/or sonicating and then centrifuged at low speed (e.g. 3000 x g). Semi-purified virus stocks were made by ultracentrifuging crude virus through a sucrose cushion, suspending the resulting virus pellet in a small volume of buffer, then

Solvent/Detergent Stock Solutions

Different S/D stock solutions were prepared for the bench-scale virus experiments. A 100x concentrate consisting of approximately 3% TNBP/50% PS-20 was used in experiments when targeting manufacturing set-point S/D concentrations. Another stock containing 2% TNBP and 25%

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Alpha1-antitrypsin Deficiency
Table 3: Virus Inactivation by Solvent/Detergent Treatment of Polyethylene Glycol Filtrate
Time (hours) Set-point Crude WNV 0 1 3 5 LRV 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 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 Log10 virus titer Robust* Semi-pure VSV 8.3 2.4 2.4 2.4 5.9

*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 8
Log10 VSV titer

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.

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 Alpha1-PI activity was determined using a two-stage assay that measured the degree of inhibition of elastase activity.15 Total cholesterol was determined using an enzymatic assay that yielded a chromogenic quinine-imine dye with a maximum absorbance of 520nm.16 A peroxidase-coupled method was used to colorimetrically measure triglyceride concentrations,17 while non-esterified fatty acids were assayed using the Wako NEFA C test kit (Richmond, VA).

6 4 2 0 0 1 2 3 4 5 Time (hours)
PEG filtrate Set-point TNBP 0.00% 0.01% 0.02% High protein 0.01% 0.02% PS20 0.00% 0.13% 0.25% 0.13% 0.25% Symbol

Results
Effect of Solvent/Detergent on Recovery of Alpha 1 -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.

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 0.020.04% TNBP and 0.250.75% 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

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.

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

A. Set-point PEG filtrate aggregation
3 100

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 Set-point PEG filtrate + added lipids
CHOL = cholesterol; FA = fatty acids; fr = fraction; PEG = polyethylene glycol; TG = triglyceride.

CHOL (g/ml) 74 Below detection 110

TG (g/ml) 45 Below detection 100

FA (mEq/l) Below detection Below detection 0.4

Protein concentration

% Transmittance

75 50

1 25 0 01x A280 FT number 9x T580 0

Below detection 130

Below detection 120

Below detection 0.4

B. Set-point PEG filtrate virus reduction

10 FT 8 TNBP PS20 Symbol

Log10 VSV titer

0.00% 0.00% 6 4 2 0 0 1 2 3 4 5 Time (hours) 9x 0 0.01% 0.13% 1x 0.02% 0.25% 0.01% 0.13% 0.02% 0.25%

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.

C. High-protein PEG filtrate aggregation

6 100

Protein concentration

% Transmittance

75 50

2 25 0 01x A280 FT number 9x T580 0

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.

D. High-protein PEG filtrate virus reduction

10 FT 8 TNBP PS20 Symbol

Log10 VSV titer

0.00% 0.00% 6 4 2 0 0 1 2 3 4 5 Time (hours) 9x 0 0.01% 0.13% 1x 0.02% 0.25% 0.01% 0.13% 0.02% 0.25%

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

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.

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Alpha1-antitrypsin Deficiency
Figure 4: Effect of Lipids on Inactivation of Vesicular Stomatitis Virus by Solvent/Detergent Treatment A. Unextracted Fr IV-1
10 8 Log10 VSV titer Log10VSV titer 6 4 2 0 0 1 2 3 4 5 Time (hours) Test material Suspension only (no added lipids) TNBP 0.00% 0.02% 0.03% PS20 0.00% 0.25% 0.50%

B. Extracted Fr IV-1
10 8

C. Set-point PEG filtrate

10 8 Log10 VSV titer 6 4 2 0

6 4 2 0 0 1 2 3 4 5 Time (hours) Symbol Test material Suspension with added lipids TNBP 0.00% 0.02% 0.03%

Time (hours) PS20 0.00% 0.25% 0.50% Symbol Not done

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).

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.

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 74g/ml cholesterol, 45g/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 120g/ml, 110g/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

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

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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 45 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

Figure 5: Effect of Detergent on Inactivation of VSV by S/D Treatment of PEG Filtrate A. PS20
10
VSV titer Log
10

8 6 4 2 0 0 1 2 3 Time (hours) 4 5

B. PS80
10
VSV titler Log
10

8 6 4 2 0 0 1 2 3 Time (hours) PS* 0.00% 0.25% 0.13% 0.25% Symbol 4 5

TNBP 0.00% 0.00% 0.01% 0.02%

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

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Alpha1-antitrypsin Deficiency
cholesterol, triglycerides and fatty acids. VSV inactivation by S/D treatment was then compared in the two suspensions before and after lipid addition, as well as in a lipid-containing unextracted fraction IV-1 suspension. Treatment with set-point concentrations of S/D (0.03% TNBP/0.5% PS-20) was very robust and inactivated 5.05.8 log10 VSV in all suspensions in the presence or absence of lipids. Treatment with lower concentrations of S/D (0.02% TNBP/0.25% PS-20) was slightly different. For set-point PEG filtrate, virus inactivation (5.0 log10) was the same before and after the addition of lipids. However, for extracted fraction IV-1 suspension virus inactivation was significantly lower after the addition of lipids and similar to the 3.8 log10 reduction achieved in the lipid-containing unextracted fraction IV-1 suspension. To the best of our knowledge, these are the first studies to formally evaluate the impact of lipids on S/D treatment. The data indicate that the presence of lipids may directly interfere with virus inactivation, but the extent of the interference may be dependent on the inactivation matrix. The final set of experiments evaluated the effect of detergents on S/D treatment and compared VSV inactivation by S/D mixtures containing PS-20 or PS-80. In the complete absence of S/D, little or no inactivation occurred; but when 0.02% TNBP/0.25% PS-20 was used, the virus was inactivated to near or below detection by one hour. Treatment with 0.25% PS-20 in the absence of TNBP also effectively inactivated >5 log10 virus, but only after five hours. Thus, rapid and robust inactivation of enveloped virus for this step requires the presence of both TNBP and PS-20. Replacing PS-20 with PS-80 significantly reduced the virucidal capacity of the step, as effective 4 log10 virus inactivation was not achieved even after treatment for five hours. The superiority of PS-20 over PS-80 has previously been reported in FVIII/vWF studies using traditional concentrations of S/D.24 Our data extend these findings and show that the effectiveness of low concentrations of S/D for virus inactivation in the Prolastin-C process is dependent on the use of PS-20. S/D treatment removes infectivity by disrupting lipid membranes. TNBP acts as an organic solvent that removes lipids from the membranes, while non-ionic detergents stabilize TNBP and disrupt lipid layers for easier extraction of lipids.25 Differences in TNBP stabilization by PS-20 or PS-80 are not known. Explanations for the discrepancies in virus inactivation by TNBP in combination with PS-20 or PS-80 will require additional studies.

Conclusion
In summary, these studies present a method for S/D treatment that involves the use of very low concentrations of TNBP and PS-20 while maintaining the potency of the protein. Treatment of alpha1-PI PEG filtrate with 0.04% TNBP/0.75% PS-20the upper manufacturing limit for S/Dmaintained product potency without the need for stabilizers. Treatment with 0.01% TNBP/0.13% PS-20concentrations that are below theset-point manufacturing ranges for S/Deffectively inactivated the worst-case enveloped test virus. Inactivation was not influenced by variations in protein concentration, temperature, pH, or aggregates. Effective virus inactivation was even achieved when lipids, not normally detected in PEG filtrate, were added. The data demonstrate the robustness of this S/D regimen for enveloped virus inactivation in the purification process for Prolastin-C. n

JoAnn Hotta, PhD, is Section Head of the Viral Methods group in the Pathogen Safety Department at Talecris Biotherapeutics Inc. In this role she is responsible for the evaluation and development of new technologies and methods to inactivate and/or remove viruses in manufacturing processes for plasma-derived proteins. She helped develop many of the processes at Talecris, including those for Gamunex and Prolastin.

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