Alpha 1 -antitrypsin Deficiency

Transcription

1 Alpha -antitrypsin Deficiency Virus and Prion Safety of a New Preparation of Human Alpha -proteinase Inhibitor, Prolastin -C Nathan J Roth, PhD, Michael D Burdick, PhD, Kang Cai, PhD, Hilton Renfrow, DVM, Greg Buczynski, PhD, Wendy P Osheroff, PhD, JoAnn Hotta, PhD and Douglas C Lee, PhD Pathogen Safety, Talecris Biotherapeutics Inc., Research Triangle Park, North Carolina Abstract The manufacture of Prolastin -C (alpha -proteinase inhibitor [human]) uses a modified process for Prolastin that incorporates two new dedicated virus reduction steps: a unique solvent/detergent treatment and a small pore nanofiltration. The Prolastin-C manufacturing process was investigated for its capacity to remove or inactivate relevant viruses or models of relevant viruses by evaluating individual process steps across production ranges for key operating parameters. To address the theoretical risk of prion transmission, the Prolastin-C process was also investigated for its capacity to decrease the infectivity of an experimental agent of transmissible spongiform encephalopathy, considered as a model for the variant Creutzfeldt Jakob disease and Creutzfeldt Jakob disease agents. These studies demonstrate that the manufacturing process for Prolastin-C maintains a high safety margin from the risk of transmission of infectious viruses with a wide range of physicochemical properties and maintains the capacity to effectively remove prions, if present in the starting material. Keywords Alpha -antitrypsin, alpha -proteinase inhibitor, Prolastin, Prolastin-C, pathogen reduction, prion, protein, purity, virus, transmissible spongiform encephalopathy Disclosure: All authors are full-time employees of Talecris Biotherapeutics Inc. Acknowledgments: The following reagents were obtained through the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: C8- cell line from Dr Robert Gallo; H9 cell line from Dr Robert Gallo. Received: October, Accepted: December 9, Citation: US Respiratory Disease, ;: 9 Correspondence: Nathan J Roth, PhD, Pathogen Safety, Talecris Biotherapeutics Inc., PO Box, 8 TW Alexander Drive, Research Triangle Park, NC 779. E: nathan.roth@talecris.com Support: This study was sponsored by Talecris Biotherapeutics, Inc. (Research Triangle Park, NC 779, US). 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. Prolastin (alpha -proteinase inhibitor [human]) has been used since 988 in patients with alpha -antitrypsin deficiency. Observational and placebo-controlled studies have shown that Prolastin attenuates the decline in lung function. Prolastin also has an excellent tolerability profile 9 and record of pathogen safety. A new preparation of purified human alpha -proteinase inhibitor (tradename Prolastin -C, alpha -proteinase inhibitor [human]) is produced from large pools of human plasma using a modification of the Prolastin manufacturing process. Additional purification steps incorporated into the modified manufacturing process result in a more concentrated final product with a higher functional activity relative to Prolastin and also excellent purity. As with all plasma-derived products, there is a potential risk for pathogen transmission if the plasma manufacturing pool contains plasma units from infected donors. Multiple complementary pathogen reduction strategies are employed during manufacture to minimize this risk (see Figure ). Overall, the pathogen safety of plasma-derived protein therapies relies on an integrated approach that includes careful screening and management of donors; testing of plasma donations and manufacturing pools for bloodborne pathogens using both serological and/or nucleic acid amplification technology assays; and integrating process steps into the manufacturing process that have the capability to reduce potential pathogen burdens using independent mechanisms of action. Virus reduction for biological therapies encompasses both virus removal and virus inactivation strategies. Virus removal is the physical separation of the virus from the product, while virus inactivation is designed to render the virus (by physical or chemical means) incapable of causing an infection. The manufacturing process for Prolastin-C incorporates two new dedicated virus reduction steps: a novel solvent/detergent treatment step for enveloped virus inactivation and small pore nanofiltration for removal of viruses as small as approximately nm. These steps replace the pasteurization step used TOUCH BRIEFINGS

2 Alpha -antitrypsin Deficiency Figure : Overview of Steps in the Prolastin-C Manufacturing Process That Contribute to Overall Virus and Prion Safety Figure : Characterization Data for the Polyethylene Glycol Precipitation and Depth Filtration Steps Donor screening Donation testing Manufacturing pool testing Plasma receiving Plasma pooling Cryoprecipitate separation Fraction I separation Fraction II + III separation Virus/TSE removal Alpha -PI potency (mg/ml) Fraction IV- NaCI suspension (n=) Lot A Lot B Lot C Alpha -PI potency (mg/ml) Unspiked PEG filtrate (n=) % HS % FBS Fraction IV- separation Suspend fraction IV- paste PEG precipitation Depth filtration Virus removal Virus removal TSE removal Alpha -PI (mg/ml) Alpha -PI (mg/ml) S/D treatment Enveloped virus inactivation Lot A Lot B Lot C Unspiked % HS % FBS Chromatography Chromatography -nm nanofiltration Prolastin-C Virus removal Albumin (mg/ml) Albumin (mg/ml) PEG = polyethylene glycol; TSE = transmissible spongiform encephalopathy. Lot A Lot B Lot C Unspiked % HS % FBS in the Prolastin process. In contrast to viruses, prions (PrP) are misfolded pathogenic proteins that are associated with a number of transmissible spongiform encephalopathies (TSEs), including variant Creutzfeldt Jakob disease (vcjd). There are currently no diagnostic assays with the necessary specificity and sensitivity to test plasma for the presence of low levels of prions. Therefore, the risk for transmitting TSEs is minimized by the exclusion of donors who are determined to have elevated risk for harboring prion diseases, the use of low-risk raw materials, and including steps in the manufacturing process that demonstrate the capability to remove prions. The Prolastin-C manufacturing process includes prion removal steps common to both the Prolastin and Prolastin-C processes that have demonstrated significant prion removal. We report on the results from studies that evaluated the capacity of the individual steps in the Prolastin-C manufacturing process to inactivate or remove viruses and to reduce the infectivity of an experimental agent of TSE, considered to be a model for the vcjd agent. Materials and Methods Characterization of Bench-scale Models of Each A bench-scale model was developed for each of the manufacturing process steps evaluated. The bench-scale processes modeled the full-scale manufacturing processes with respect to critical process parameters such as ph, temperature, pressure, flow rate, and protein concentration. The accuracy of the manufacturing simulation was ascertained by measuring several marker proteins and other parameters in characterization experiments, which were performed in lga (mg/ml) Lot A Lot B Lot C Unspiked % HS % FBS Gray boxes define the upper and lower limits of target ranges defined by the mean ± three standard deviations derived from testing of intermediate samples from manufacturing-scale processing. FBS = fetal bovine serum; HS = horse serum; IgA = immunoglobulin A; PEG = polyethylene glycol. the absence of a pathogen spike. The validity of the bench-scale models to represent the corresponding manufacturing steps was evaluated by comparing the analytical characterization data for the resultant process intermediates produced at bench scale and manufacturing scale. To control for the potential impact of adding a virus or TSE spike on the performance of the bench-scale processes, experiments were performed where cell culture medium (to mimic a virus spike) or normal hamster brain homogenate (to mimic a TSE spike) were added to the starting intermediate. The effect of the mock spike on the bench-scale model was evaluated by comparing the analytical characterization data for the resultant process intermediates with analytical characterization data for the process intermediates from unspiked experiments. The bench-scale studies of the cold ethanol fractionation (effluent I to effluent II plus III) process step were carried out at a scale approximately /, of the commercial manufacturing scale. The lga (mg/ml) US RESPIRATORY DISEASE

3 Virus and Prion Safety of a New Preparation of Human Alpha -proteinase Inhibitor, Prolastin -C Figure : Kinetics of Virus Inactivation During the Solvent/Detergent Treatment Step of the Prolastin-C Process Figure : Prion Reduction During the Polyethylene Glycol Precipitation/Depth Filtration Step of the Prolastin-C Process Titer (log TCID /ml) Titer (log TCID /ml) WNV set-point Titer (log TCID /ml) PRV set-point LRV =. LRV =.. HBSS WNV LOD HBSS PRV LOD BVDV set-point Titer (log TCID /ml) VSV set-point LRV =. LRV =. ±.. HBSS BVDV LOD HBSS VSV LOD Depth filtration Centrifugation Fraction IV- NaCI suspension Paste Effluent Pad extract Titer (log TCID /ml) HIV- combined worst case VSV combined worst case LRV =... LRV = RPMI HIV- LOD HBSS VSV LOD BVDV = bovine viral diarrhea virus; HBSS = Hank s balanced salt solution; HIV- = human immunodeficiency virus type ; LOD = limit of detection; LRV = log reduction value; PRV = pseudorabies virus; RPMI = Roswell Park Memorial Institute (cell culture medium); TCID = tissue culture infectious dose; VSV = vesicular stomatitis virus; WNV = West Nile virus. bench-scale model of the polyethylene glycol (PEG) precipitation step used to evaluate virus reduction was approximately /, of the manufacturing scale. The subsequent process step, depth filtration of the PEG supernatant, was carried out at a scale of approximately /, of the manufacturing scale. The nanofiltration experiments were performed using a bench-scale process that utilized a nm nanofilter with a filter surface area representing a /,-fold scale-down from the nanofilter used in manufacturing. Solvent/detergent treatment is scale independent, requiring only a water bath for holding the sample at the proper temperature. During the evaluation of virus inactivation, critical parameters such as solvent concentration, detergent concentration, protein concentration, ph, and temperature were measured and controlled. Mock-spiked and unspiked controls were incubated side-by-side with the virus-spiked samples and analyzed for alpha potency, tri-n-butyl-phosphate (TNBP) concentration, and polysorbate concentration. Titer (log TCID /ml) PEG = polyethylene glycol. PEG filtrate Evaluation of Virus Reduction The virus reduction capacity of each manufacturing process step was evaluated by spiking process intermediates with relevant or model viruses (see Table ). Each experimental condition was evaluated in triplicate using independent lots of manufacturing intermediates. The results are reported as the mean log reduction value (LRV) plus or minus one standard deviation. Up to five relevant and model enveloped viruses 7 were tested for each process step evaluated. HIV- was selected as a clinically relevant blood-borne ribonucleic acid (RNA) virus, and is additionally a model for HIV- and human T-lymphotropic virus. Bovine viral diarrhea virus (BVDV) is an RNA virus that is commonly used to model hepatitis C virus. Pseudorabies virus (PRV) is a model for the human herpes viruses and other large enveloped DNA viruses, including cytomegalovirus and Epstein Barr virus. West Nile virus (WNV) is a relevant bloodborne virus that was evaluated only in the solvent/detergent treatment studies. Vesicular stomatitis virus (VSV) is a rabies virus that is not considered to be a bloodborne pathogen, but was evaluated because of its relative resistance to solvent/detergent inactivation 8,9 and because of its distinctive rod-like morphology (approximately nm by nm), which poses a unique challenge to a nanofiltration step. Three relevant and model non-enveloped viruses were tested, 7 all with inherent resistance to physical and chemical inactivation. Reovirus type (Reo) is an RNA virus that serves as a general model for US RESPIRATORY DISEASE

4 Alpha -antitrypsin Deficiency Table : Relevant and Model Viruses Used to Evaluate the Virus Reduction Capacity of the Attribute HIV- BVDV PRV VSV Reo HAV PPV Relevant or HIV- HCV HHV Enveloped virus Non-enveloped HAV B9V Model for HIV- CMV virus HTLV EBV Family* Retroviridae Flaviviridae Herpesviridae Rhabdoviridae Reoviridae Picornaviridae Parvoviridae Strain RF Ky dl tk Indiana Abney 7/8f NADL- (ATCC VR-7) (ATCC VR-8) (ATCC VR-) (ATCC VR-) (ATCC VR-7) Nucleic acid* RNA RNA DNA RNA RNA RNA DNA Enveloped* Yes Yes Yes Yes No No No Size (nm) Physicochemical Low Low Medium High Medium High Very high Resistance Propagation H9 cells MDBK cells MvLu cells BHK- BS-C- cells BS-C- cells MPK cells System (ATCC CCL-) (ATCC CCL-) (ATCC CCL-) (ATCC CCL-) (ATCC CCL-) (ATCC CCL-) Propagation RPMI + % DMEM + % DMEM + % DMEM + % DMEM + % DMEM + % DMEM + % Medium FBS + X P/S HS + NHG FBS + NHG FBS + NHG FBS + NHG FBS + NHG FBS + NHG Assay system C8- cells BT cells MvLu cells BHK- cells BS-C- cells FRhk cells MPK cells (NIH AIDS RRRP (ATCC CRL-9) (ATCC CCL-) (ATCC CCL-) (ATCC CCL-) (ATCC CRL-88) (ATCC CCL-) Cat#) Assay medium RPMI + % DMEM + % DMEM + % DMEM + % DMEM + % DMEM + % DMEM + % FBS + X P/S HS + NHG FBS + NHG FBS + NHG FBS + NHG FBS + NHG FBS + NHG *International Committee on Taxonomy of Viruses Database (ICTVdB) [ ATCC = American Type Culture Collection; B9V = parvovirus B9; BT = bovine turbinate; BVDV = bovine viral diarrhea virus; CMV = cytomegalovirus; DMEM = Dulbecco s Modified Eagle medium; DNA = deoxyribonucleic acid; EBV = Epstein Barr virus; FBS = fetal bovine serum; H9 = single cell clone derived from the specific HUT 78 cell line, HT; HAV = hepatitis A virus; HCV = hepatitis C virus; HHV = human herpesvirus; HIV- = human immunodeficiency virus type ; HIV- = human immunodeficiency virus type ; HS = horse serum; HTLV = human T-lymphotropic virus; MDBK = Madin Darby bovine kidney; MPK = mini pig kidney; MvLu = mink lung epithelial cell; NHG = non-essential amino acids; NIH AIDS RRRP = National Institutes of Health AIDS Research and Reference Reagent Program; PPV = porcine parvovirus; PRV = pseudorabies virus; Reo = reovirus type ; RNA = ribonucleic acid; RPMI = Roswell Park Memorial Institute (cell culture medium); VSV = vesicular stomatitis virus. Table : Summary of Virus Reduction (Log ) for the Cold Ethanol Fractionation Step of the Log Virus Reduction ± SD Parameter HIV- BVDV PRV Reo HAV PPV ph Low. ±..9. ±.. ±. Set-point.. ±..9.. ±.. ±. High. ±..8. ±.. ±. EtOH Low. ±..8. ±.. ±. Cold ethanol fractionation Set-point.. ±..9.. ±.. ±. High.9 ±..8. ±.. ±. Temperature Low. ±... ±.7. ±. Set-point.. ±..9.. ±.. ±. High. ±... ±.. ±. BVDV = bovine viral diarrhea virus; EtOH = ethanol; HAV = hepatitis A virus; HIV- = human immunodeficiency virus type ; PEG = polyethylene glycol; PPV = porcine parvovirus; PRV = pseudorabies virus; Reo = reovirus type ; SD = standard deviation. non-enveloped viruses, the DNA virus porcine parvovirus (PPV) is a surrogate for human parvovirus B9 (a very small virus known for its very high resistance to inactivation), and hepatitis A virus (HAV) is a relevant bloodborne RNA virus of 7 nm diameter. PRV stocks were propagated in mink lung (Mv Lu) cells. PPV stocks were propagated in mini pig kidney (MPK) cells. Reo and HAV stocks were propagated in African Green Monkey kidney cells (BS-C-). BVDV stocks were propagated in BVDV-free Madin-Darby bovine kidney (MDBK) cells. VSV stocks were propagated in baby hamster kidney (BHK-) cells. Virus-infected cells and supernatants were disrupted by freeze thawing to release virus, and the cell lysates were stored at not more than 8 C until used. The virus spike for each experiment was prepared by thawing the virus-infected cell lysates, clarifying by low-speed centrifugation (e.g., x g, minutes at C) to remove the cell debris, and collecting the clarified supernatants. Preparations of HIV- used to evaluate the solvent/detergent treatment and nanofiltration steps were propagated using H9 cells. The supernatants were concentrated using tangential flow filtration and the virus was pelleted by ultracentrifugation. In general, clarified, crude virus preparations (cell lysate centrifuged at ~ x g for minutes at C) were utilized to evaluate virus reduction for the upstream manufacturing steps whereas semi-pure virus preparations (crude virus preparations ultracentrifuged (~8, x g for four to hours) through a % sucrose cushion) were used to US RESPIRATORY DISEASE

5 Virus and Prion Safety of a New Preparation of Human Alpha -proteinase Inhibitor, Prolastin -C Table : Summary of Virus Reduction (Log ) for the PEG Precipitation Step of the Log Virus Reduction ± SD Parameter HIV- BVDV PRV Reo HAV PPV ph Low.9 ±.. ±. Set-point. ±..8 ±.. ±.. ±.. ±.. ±. High.9 ±.. ±. PEG Low. ±.. ±. PEG precipitation of fraction concentration Set-point. ±..8 ±.. ±.. ±.. ±.. ±. IV- Suspension High.8 ±.. ±. Buffer volume Low. ±.. ±. to paste Set-point. ±..8 ±.. ±.. ±.. ±.. ±. suspension High. ±..8 ±. ratio BVDV = bovine viral diarrhea virus; HAV = hepatitis A virus; HIV- = human immunodeficiency virus type ; PEG = polyethylene glycol; PPV = porcine parvovirus; PRV = pseudorabies virus; Reo = reovirus type. Table : Summary of Virus Reduction (Log ) for the Depth Filtration Step of the Log Virus Reduction ± SD Parameter HIV- BVDV PRV Reo HAV PPV ph Low.. Set-point.7. ± High.7 ±.. Depth filtration of PEG effluent Buffer volume Low. ±.. to paste Set-point.7. ± suspension High. ±.. ±. ratio BVDV = bovine viral diarrhea virus; HAV = hepatitis A virus; HIV- = human immunodeficiency virus type ; PEG = polyethylene glycol; PPV = porcine parvovirus; PRV = pseudorabies virus; Reo = reovirus type. evaluate virus removal by the nanofiltration step. All virus preparations were filtered through a sequential series of filters with decreasing pore size to remove any remaining cell debris and disperse virus aggregates prior to spiking into manufacturing intermediates. To ensure that the virus preparations were monodispersed for the nanofiltration studies, PPV preparations were filtered through a nm nanofilter and HAV preparations were filtered through a 7nm nanofilter. Process intermediates, which were generally obtained from clinical- or commercial-scale manufacturing, were spiked with virus at a ratio of : to :, (virus to intermediate). The spiked intermediates were initially processed using the bench-scale models with all parameters set at the optimal manufacturing ranges (set-point). During the process step, samples were removed to determine virus inactivation kinetics or virus partitioning. The toxicity of sample matrix for each cell line was determined in the absence of virus to differentiate matrix effects from actual virus cytopathology. Experiments were performed to determine whether the manufacturing intermediates interfered with virus detection. The results of the cytotoxicity assay, combined with the results of the viral interference assay, determined the detection limit for the assay system. The detection limit was based on the lowest dilution of sample for which no cytotoxicity or viral interference was observed. Virus titers were determined using serial half-log dilutions of samples in cell-based assays and were quantitated as tissue culture infectious dose at % infectivity (TCID ) using the method of Spearman Kärber. Those samples that did not have any positive wells were treated based on the probability of detecting infectious virus at 9% confidence using the Poisson distribution. For evaluation of HIV- inactivation during solvent/detergent treatment, expanded volume testing 9 was used to increase the probability of detecting virus. The ability of each step to inactivate or remove virus was determined by comparing the total virus in the initial spiked material (input) to the total virus in the same material after processing (output). The overall virus reduction capacity of the Prolastin-C manufacturing process (global virus reduction factor) was calculated by summing virus reduction factors from the individual process steps. Only process steps achieving at least one log reduction were included in the calculation of the global virus reduction factor. Where reduction to the limit of detection was achieved, results are reported as greater than or equal to the reduction factor; a greater reduction could potentially be achieved if a higher titer virus spike was used. As production parameters (such as ph and temperature) are subject to some variability, the robustness of each virus removal or inactivation step was investigated by determining virus reduction at or just outside the standard Prolastin-C manufacturing ranges. For the robustness studies, critical and key parameters were evaluated individually and in combined worst-case experiments. A parameter evaluated individually was adjusted to a value at or just outside the extremes of the allowable manufacturing range while all other parameters were maintained within the manufacturing ranges. The parameters that were found to significantly impact virus reduction were further evaluated simultaneously in combined worst-case experiments. US RESPIRATORY DISEASE

6 Alpha -antitrypsin Deficiency Table : Robustness of Virus Reduction (Log ) During the nm Virus Removal Nanofiltration Step of the Log Virus Reduction ± SD Parameter HIV- BVDV PRV VSV Reo HAV PPV Protein Low.. concentration Set-point High.8. Pressure Low.8. Set-point High.7. ph Low.. nm nanofiltration Set-point High.7. Filter load, Low.. kg/m Set-point High.. Process interruptions () NT. NT NT NT NT. Combined low protein and NT NT NT NT NT NT. high ph BVDV = bovine viral diarrhea virus; HAV = hepatitis A virus; HIV- = human immunodeficiency virus type ; NT = not tested; PPV = porcine parvovirus; PRV = pseudorabies virus; Reo = reovirus type ; VSV = vesicular stomatitis virus. Table : Summary of Virus Reduction (Log ) for the Solvent/Detergent Treatment Step of the Parameter Log Virus Reduction ± SD S/D treatment HIV- BVDV PRV VSV Set-point ND... ±. Combined worst-case (low S/D, low temperature, and low ph) ND ND ND. ±. with low buffer volume to paste suspension ratio Combined worst-case (low S/D, low temperature, and low ph). ND ND.9 ±. with set-point buffer volume to paste suspension ratio * determined. BVDV = bovine viral diarrhea virus; HIV- = human immunodeficiency virus type ; ND = not determined; PRV = pseudorabies virus; S/D = solvent/detergent; VSV = vesicular stomatitis virus. Evaluation of Prion Reduction The capacity of the Prolastin-C manufacturing process to reduce TSE infectivity was evaluated by spiking process intermediates with a TSE agent preparation containing the pathogenic form of the prion protein (PrP Sc ). The spike material was prepared as previously described. Spike volumes were less than or equal to one th the volume of the intermediate spiked. The levels of TSE agent in the intermediates and resulting fractions were analyzed using two methodologies. A Western blot method was used to detect a marker for TSE infectivity (the protease-resistant form of the prion protein, PrP RES ) and TSE infectivity was directly assessed by end-point dilution in Syrian hamsters. In the Western blot assay, samples were prepared in a serial half-log dilution series and then assayed as previously described. For the infectivity assay, samples were prepared in a serial whole log dilution series. Groups of six animals were inoculated for each dilution tested. The number of animals with clinical signs of scrapie in each group of injected animals was converted to a titer (log infectious dose % per ml, ID /ml) using the method of Spearman and Kärber. The titers were used to calculate prion loads of the process intermediates, which were in turn used to calculate the TSE infectivity reduction factor for the process step. Results Characterization of Bench-scale Models of Each Intermediates generated using the bench-scale models were tested for potency and content of alpha -proteinase inhibitor, content of other proteins, critical reagent concentrations (such as ethanol concentration in the effluent I to effluent II plus III step), and other biochemical attributes. The bench-scale models were representative of the manufacturing processes as the data from analytical testing were within three standard deviations of the mean from testing of intermediates generated at both clinical and manufacturing scales (data not shown). As an example, the results from the alpha potency and protein content testing performed on intermediates generated at bench scale for the PEG precipitation and depth filtration steps are shown in Figure. Virus Removal Results The effluent I to effluent II plus III step was evaluated for its capacity to remove a variety of enveloped and non-enveloped viruses. The effluent I to effluent II plus III step removed. log HIV-,. log BVDV,.9 log PRV,. log Reo,. log HAV, and. log PPV at operating set-points (see Table ). The parameters selected for the US RESPIRATORY DISEASE

7 Virus and Prion Safety of a New Preparation of Human Alpha -proteinase Inhibitor, Prolastin -C single-parameter robustness evaluation were ph, temperature, and ethanol concentration. Since none of the parameters in the robustness evaluations had a significant effect on virus removal, a combined worst-case evaluation, where all parameters that affect virus reduction are adjusted simultaneously, was not performed. Under set-point conditions, the PEG precipitation step removed. ±. log HIV- and approximately log of the other viruses (BVDV, PRV, Reo, HAV, and PPV) (see Table ). Similarly, under set-point conditions, the subsequent depth filtration step achieved log or greater reduction of HIV-, BVDV, PRV, Reo, and PPV, all of which were reduced to their respective limits of detection (see Table ). HAV was removed to the limit of detection across the depth filtration step (.8 log ). Additional HAV reduction could likely have been achieved if a higher starting titer had been attainable. The robustness of virus reduction across the PEG precipitation and depth filtration steps was evaluated using BVDV and PPV. Given their small size, these viruses tend to be more difficult to remove by either precipitation or filtration, thus representing a worst-case challenge for virus removal. Varying the process parameters did not result in significant differences in the LRVs compared with the LRVs observed with all parameters at set-point. Unlike other process steps that were evaluated, no significant impact on virus removal was observed during the single parameter robustness evaluations, therefore a combined worst-case evaluation was not warranted. Virus removal by the nm nanofiltration step was evaluated using a panel of viruses that included both enveloped and non-enveloped viruses. The nanofiltration step removed. ±. log PPV,.9 log HIV-,.7 log BVDV,. log PRV,. log VSV,. log Reo, and. log HAV under set-point conditions (see Table ). Removal of PPV, the smallest virus studied (8 nm) and therefore the worst-case challenge to the nm nanofilter, was evaluated under robustness conditions with and without a post-nanofiltration rinse. In the absence of a rinse, a minimum of. log removal of PPV was observed at all protein concentrations, filtration pressures, phs, and filter loads evaluated (data not shown). When a post-nanofiltration rinse was included in the process step, a minimum of. log removal of PPV was maintained for high protein concentration, high or low filtration pressure, high or low filter loads, or low ph. However, with a rinse, PPV reduction was <. log at very high ph and very low protein concentration. Therefore, the manufacturing limits for ph and protein concentration ranges were narrowed until >. log PPV removal was consistently observed utilizing a post-nanofiltration rinse (see Table ). In a combined worst-case evaluation using the adjusted values for the ph and protein concentration, including a post-nanofiltration rinse,. log PPV removal was demonstrated (see Table ). Virus Inactivation Studies The Prolastin-C manufacturing process employs novel solvent/detergent treatment conditions. To ensure the virus inactivation by these novel solvent/detergent treatment conditions is robust with respect to a variety of enveloped viral challenges, an expanded panel of viruses was evaluated. VSV was included within the panel and chosen as the worst-case virus for the robustness evaluation of this step because of its Table 7: Virus Reduction (Log ) of Individual Steps in the Log Virus Reduction Factor Enveloped Viruses Non-enveloped Viruses Process HIV- BVDV PRV VSV Reo HAV PPV Step Cold ethanol...9 ND... fractionation PEG..8. ND... precipitation Depth.7..8 ND..8. filtration S/D.... NA* NA NA treatment nm virus removal nanofiltration** Global virus reduction factor *This step is only effective against enveloped viruses. **Includes a post-nanofiltration rinse. VSV reduction was only determined for the S/D treatment and nm virus removal nanofiltration steps. BVDV = bovine viral diarrhea virus; HAV = hepatitis A virus; HIV- = human immunodeficiency virus type ; NA = not applicable; ND = not determined; PEG = polyethylene glycol; PPV = porcine parvovirus; PRV = pseudorabies virus; Reo = reovirus type ; S/D = solvent/detergent; VSV = vesicular stomatitis virus. Table 8: Reduction (Log ) of TSE Infectivity and Prion Protein (PrP RES ) During the Prolastin-C Manufacturing Process TSE Infectivity Reduction PrP RES Removal by by Animal Bioassay Western Blot (log ID ) (log PrP RES ) Cold ethanol.. fractionation (effluent I to effluent II + III) PEG precipitation plus.8. depth filtration PEG = polyethylene glycol; TSE = transmissible spongiform encephalopathy. resistance to inactivation by solvent/detergent treatment. 8,9 Under set-point conditions (.% TNBP,.% polysorbate ), log reduction values of. log BVDV,. log PRV,. log VSV (see Table ), and. log WNV were observed for this process step. The inactivation of VSV and HIV- was evaluated in combined worst-case conditions of low solvent and detergent concentrations, low temperature, and low ph in combination with different Fraction IV- Paste suspension ratios. The kinetics of VSV inactivation were slightly slower under these conditions, but the overall reduction capacity was not significantly influenced (see Table and Figure ). Under combined worst-case conditions, an LRV of. log was observed for HIV- with complete inactivation occurring within two hours (see Table and Figure ). The reduction factors for all individual processing steps that achieved at least one log were combined to determine the global reduction factors. US RESPIRATORY DISEASE 7

8 Alpha -antitrypsin Deficiency Global reduction factors for the Prolastin-C manufacturing process are. log for HIV-, 9. log for BVDV,. log for PRV,. log for VSV,.7 log for Reo,.7 log for HAV, and.8 log for PPV (see Table 7). Greater reduction could potentially have been achieved if virus with higher titers was available for the input spike. Prion Reduction Studies The TSE reduction capacity of the cold ethanol fractionation step (effluent I to effluent II plus III) was evaluated using both Western blot and animal infectivity assay. The PrP RES reduction factor as assessed by Western blot was. log (see Table 8). The TSE infectivity reduction determined using the animal bioassay was. log ID (see Table 8). The results obtained from the two assays are consistent given that the Western blot assay has a sensitivity that is approximately log lower than that of the infectivity assay. The TSE reduction capacity of the coupled PEG precipitation and depth filtration steps as assessed by Western blot was. log PrP RES (see Table 8 and Figure ). The results from an animal infectivity assay of the same samples yielded a reduction of.8 log ID (see Table 8). Higher reduction may have been demonstrated if a higher titer spike had been available. The results obtained with the two assays are consistent given the differences in sensitivity between the methodologies. Discussion Prolastin-C is a next-generation alpha -proteinase inhibitor produced using a modification of the existing Prolastin process. Both the Prolastin and Prolastin-C processes incorporate manufacturing steps that minimize the residual risk for virus and prion transmission. The Prolastin-C process incorporates two new dedicated virus reduction steps: solvent/detergent treatment to inactivate enveloped viruses and small pore nanofiltration to remove viruses down to less than nm in size. The incorporation of these process modifications had no impact on the purity or potency of the product. Other studies presented here demonstrate that steps common to the Prolastin and Prolastin-C processes provide reduction of prion infectivity. Thus, both the Prolastin and Prolastin-C processes yield products with excellent pathogen safety profiles. The virus safety studies employed a panel of viruses encompassing a wide range of physicochemical properties,7 that presents a broad spectrum of challenges to the manufacturing process with respect to virus reduction (see Table ). This strategy provides assurance that the process is robust with respect to viral challenges of both known, unknown, and emerging viruses should they be present. The efficacy of virus reduction was also robust, being largely unaffected by fluctuations in the relevant processing parameters for the different manufacturing steps. For the cold ethanol fractionation step, there was no significant difference in virus removal at varying values of ph, ethanol concentration, and temperature set at or outside the manufacturing limits. The PEG precipitation step removed both enveloped and non-enveloped viruses when ph, PEG concentration, and paste suspension ratio were varied, with no difference in virus removal observed at operating extremes compared with production set-points. The depth filtration step also contributed to the overall virus reduction for the manufacturing process, providing robust removal of both enveloped and non-enveloped viruses that was not influenced by variations at the extremes of the operating ranges for ph, PEG concentration, or paste suspension ratio. The virus reduction capacity of the solvent/detergent treatment step was robust when evaluated with HIV- and VSV at worst-case conditions (a combination of low solvent concentration, low detergent concentration, low temperature, and low ph), although VSV inactivation was not complete. It should be noted that the resistance of VSV to complete inactivation by solvent/detergent treatment can most likely be attributed to the ability of the delipidated nucleocapsid-enclosed virion to infect cells at an efficiency approximately - to - of that of the lipid membrane-enclosed virion. 8 The residual VSV infectivity observed after a log to log inactivation by the novel solvent/detergent treatment is consistent with previous observations of incomplete VSV inactivation by standard solvent/detergent treatments. 8,9 The virus reduction capacity observed for this step was not influenced by the buffer to fraction IV- paste suspension ratio and high protein concentration had no effect on virus inactivation even when evaluated under the combined worst-case conditions. Under set-point operating conditions with a post-nanofiltration rinse, the nm nanofiltration step provided effective and robust removal of viruses as small as 8 nm (PPV). Using PPV as a worst-case model, the nanofiltration step was shown to be robust throughout the operating ranges of the Prolastin-C manufacturing process. In the absence of a post-nanofiltration rinse, no critical parameters were identified. Without a rinse, the nanofiltration step provided robust virus removal across the pressure, ph, filter load, number of process interruptions, and total protein concentration ranges evaluated. When evaluated with a post-nanofiltration rinse to maximize product recovery, high ph and low total protein concentration were identified as critical parameters that affected virus reduction. As a result, the operating ranges for ph and protein concentration were adjusted to assure that at least. log PPV was removed. The robustness of virus removal by nanofiltration under worst-case conditions (high ph, low total protein concentration, and a % rinse simultaneously) was confirmed (see Table ). These results support the operation of the process step without a rinse or with a rinse, if necessary, to maximize product recovery. The capacity of the Prolastin-C manufacturing process to remove an experimental agent of TSE was also evaluated. We used hamster-adapted scrapie strain K agent in this study as a model for human TSE agents because its partitioning trend in the presence of PEG is similar to that of vcjd, sporadic CJD, and Gerstmann Sträussler Sheinker syndrome. 9 Cold ethanol fractionation and the coupled PEG precipitation and depth filtration steps are common to the manufacturing process for both Prolastin and Prolastin-C and are effective prion removal steps. These steps, which rely on different conditions for precipitating and partitioning proteins, were each shown to remove a minimum of log ( %) of TSE infectivity. Taken together, the removal of prion infectivity demonstrated for these steps provides assurance that low levels of TSE agent infectivity, if present in the plasma manufacturing pool, would be removed. 8 US RESPIRATORY DISEASE

9 Virus and Prion Safety of a New Preparation of Human Alpha -proteinase Inhibitor, Prolastin -C These data demonstrate that the Prolastin-C manufacturing process has a high capacity to inactivate and/or remove a diverse variety of enveloped and non-enveloped virus challenges as well as infectious prions. The novel solvent/detergent treatment step provides robust inactivation of enveloped viruses while maintaining alpha -proteinase inhibitor activity. The nm nanofiltration step introduces a robust, dedicated orthogonal step for removal of viruses. Three steps common to the Prolastin and Prolastin-C manufacturing processes, cold ethanol fractionation, PEG precipitation, and depth filtration, also contribute to the reduction of viruses as well as TSE infectivity. Therefore, the Prolastin-C manufacturing process provides the product with a high demonstrated margin of safety from the risk for transmission of infectious agents. n Nathan J Roth, PhD, is the Director of Pathogen Safety, overseeing the Viral Validation, TSE, and Support and Compliance sections within the Talecris Pathogen Safety department. He is a biochemist/microbiologist by training, having earned a BS degree in cellular, molecular, and microbial biology and a PhD in biochemistry from the University of Calgary. Prior to joining Talecris, Dr Roth was part of a team of scientists at VI Technologies involved in the development of novel methods for inactivation of viruses and protozoa and removal of transmissible spongiform encephalopathy (TSE) pathogens from blood components. He also gained relevant experience in biopharmaceutical process development at Centeon (now CSL Behring), where he co-invented a patented method for preparing a diafiltered stabilized blood product. 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