Alpha1-antitrypsin Deficiency


Table 1: Relevant and Model Viruses Used to Evaluate the Virus Reduction Capacity of the Prolastin-C Manufacturing Process


Attribute


Relevant or Model for


Family* Strain


HIV-1 HIV-1 HIV-2 HTLV


Retroviridae RF


Nucleic acid* RNA Enveloped* Size (nm)


Yes 80–100 Physicochemical Low


Resistance Propagation System


Propagation Medium


Assay system H9 cells


BVDV HCV


Flaviviridae Ky22


RNA Yes


50–70 Low


MDBK cells (ATCC CCL-22)


RPMI 1640 + 10% DMEM + 5% FBS + 1X P/S C8166-45 cells


HS + NHG BT cells


Assay medium RPMI 1640 + 10% DMEM + 2% FBS + 1X P/S


HS + NHG


PRV HHV CMV EBV


Herpesviridae dl tk


VSV Reo3 HAV


Enveloped virus Non-enveloped HAV virus


Rhabdoviridae Indiana


RNA Yes


Reoviridae Abney


(ATCC VR-2074) (ATCC VR-1238) (ATCC VR-232) DNA Yes


RNA No


120–200 Medium


Mv1Lu cells


(ATCC CCL-64) DMEM + 2% FBS + NHG Mv1Lu cells


(NIH AIDS RRRP (ATCC CRL-1390) (ATCC CCL-64) Cat#404)


DMEM + 2% FBS + NHG


100–430 High


BHK-21


(ATCC CCL-10) DMEM + 2% FBS + NHG BHK-21 cells (ATCC CCL-10)


DMEM + 2% FBS + NHG


60–80 Medium BS-C-1 cells


(ATCC CCL-26) DMEM + 2% FBS + NHG BS-C-1 cells


(ATCC CCL-26)


DMEM + 2% FBS + NHG


Picornaviridae 175/18f


PPV B19V


Parvoviridae NADL-2


(ATCC VR-1402) (ATCC VR-742) RNA No


DNA No


25–30 High


BS-C-1 cells


(ATCC CCL-26) DMEM + 2% FBS + NHG FRhk cells


DMEM + 2% FBS + NHG


18–24 Very high MPK cells


(ATCC CCL-166) DMEM + 6% FBS + NHG MPK cells


(ATCC CRL-1688) (ATCC CCL-166)


DMEM + 10% FBS + NHG


*International Committee on Taxonomy of Viruses Database (ICTVdB) [http://www.ncbi.nlm.nih.gov/ICTVdb/index.htm]. ATCC = American Type Culture Collection; B19V = parvovirus B19; 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-1 = human immunodeficiency virus type 1; HIV-2 = human immunodeficiency virus type 2; HS = horse serum; HTLV = human T-lymphotropic virus; MDBK = Madin–Darby bovine kidney; MPK = mini pig kidney; Mv1Lu = 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; Reo3 = reovirus type 3; RNA = ribonucleic acid; RPMI 1640 = Roswell Park Memorial Institute (cell culture medium); VSV = vesicular stomatitis virus.


Table 2: Summary of Virus Reduction (Log10) for the Cold Ethanol Fractionation Step of the Prolastin-C Manufacturing Process


Process Step Parameter pH EtOH Cold ethanol fractionation Low


Set-point High Low


Set-point High


Temperature Low


Set-point High


3.4 3.4 3.4 HIV-1 BVDV


3.5 ± 0.1 3.5 ± 0.2 3.2 ± 0.2 3.5 ± 0.1 3.5 ± 0.2 3.9 ± 0.1 3.5 ± 0.0 3.5 ± 0.2 3.1 ± 0.5


PRV 3.9 3.9 3.9


Log10 Virus Reduction ± SD Reo3 ≥1.9 ≥2.1 ≥1.8 ≥1.8 ≥2.1 ≥1.8 ≥2.6 ≥2.1 ≥2.6


HAV


1.4 ± 0.1 1.4 ± 0.3 1.1 ± 0.3 1.1 ± 0.1 1.4 ± 0.3 2.0 ± 0.1 1.5 ± 0.7 1.4 ± 0.3 1.1 ± 0.3


BVDV = bovine viral diarrhea virus; EtOH = ethanol; HAV = hepatitis A virus; HIV-1 = human immunodeficiency virus type 1; PEG = polyethylene glycol; PPV = porcine parvovirus; PRV = pseudorabies virus; Reo3 = reovirus type 3; SD = standard deviation.


non-enveloped viruses, the DNA virus porcine parvovirus (PPV) is a surrogate for human parvovirus B19 (a very small virus known for its very high resistance to inactivation), and hepatitis A virus (HAV) is a relevant bloodborne RNA virus of 27–30nm diameter. PRV stocks were propagated in mink lung (Mv 1 Lu) cells. PPV stocks were propagated in mini pig kidney (MPK) cells. Reo3 and HAV stocks were propagated in African Green Monkey kidney cells (BS-C-1). BVDV stocks were propagated in BVDV-free Madin-Darby bovine kidney (MDBK) cells. VSV stocks were propagated in baby hamster kidney (BHK-21) cells. Virus-infected cells and supernatants were disrupted by freeze–thawing to release virus, and the cell lysates were stored at not more than –68°C until used. The virus spike for each experiment was prepared by thawing


34


the virus-infected cell lysates, clarifying by low-speed centrifugation (e.g. 4,100 x g, 15 minutes at 5°C) to remove the cell debris, and collecting the clarified supernatants. Preparations of HIV-1 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 ~4000 x g for 15 minutes at 5°C) were utilized to evaluate virus reduction for the upstream manufacturing steps whereas semi-pure virus preparations (crude virus preparations ultracentrifuged (~80,000 x g for four to 24 hours) through a 20% sucrose cushion) were used to


US RESPIRATORY DISEASE PPV


1.3 ± 0.6 1.0 ± 0.3 1.0 ± 0.1 0.6 ± 0.1 1.0 ± 0.3 1.1 ± 0.1 1.0 ± 0.4 1.0 ± 0.3 0.1 ± 0.1


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