Virus and Prion Safety of a New Preparation of Human Alpha1-proteinase Inhibitor, Prolastin®-C


Figure 3: Kinetics of Virus Inactivation During the Solvent/Detergent Treatment Step of the Prolastin-C Process


WNV – set-point


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0


0.0 0 100 LRV = ≥5.4 200 Time (minutes) HBSS WNV LOD BVDV – set-point


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0


0.0 0 100 LRV = ≥4.6 200 Time (minutes) HBSS BVDV LOD HIV-1 combined worst case


2.0 3.0 4.0 5.0 6.0 7.0 8.0


1.0 -1.0


0.0 0 50 RPMI 100 LRV = ≥6.2 150 Time (minutes) HIV-1 LOD HBSS VSV BVDV = bovine viral diarrhea virus; HBSS = Hank’s balanced salt solution; HIV-1 = human


immunodeficiency virus type 1; LOD = limit of detection; LRV = log10 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 1/15,000 of the manufacturing scale. The subsequent process step, depth filtration of the PEG supernatant, was carried out at a scale of approximately 1/40,000 of the manufacturing scale. The nanofiltration experiments were performed using a bench-scale process that utilized a 15nm nanofilter with a filter surface area representing a 1/4,000-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 alpha1 potency, tri-n-butyl-phosphate (TNBP) concentration, and polysorbate 20 concentration.


US RESPIRATORY DISEASE LOD 200


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0


0.0 0 100 300


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0


0.0 0 100 HBSS 300


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0


0.0 0 100 HBSS PRV – set-point


Figure 4: Prion Reduction During the Polyethylene Glycol Precipitation/Depth Filtration Step of the Prolastin-C Process


1 2 3 4 5 6 7 8 9 101112131415


Not loaded LRV = ≥4.3 200


Time (minutes) PRV


VSV – set-point Not loaded Effluent 300 Not LOD loaded Paste


Fraction IV-1 NaCI suspension


LRV = 5.1 ± 0.2 200


Time (minutes) VSV


LOD VSV combined worst case Not loaded PEG filtrate


300


Not loaded


Pad extract


PEG = polyethylene glycol.


LRV = 4.4 + 0.4 200


Time (minutes) 300 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 1). Each experimental condition was evaluated in triplicate using independent lots of manufacturing intermediates. The


results are reported as the mean log10 reduction value (LRV) plus or minus one standard deviation. Up to five relevant and model enveloped viruses17


were tested for each process step evaluated. HIV-1 was selected as a clinically relevant blood-borne ribonucleic acid (RNA) virus, and is additionally a model for HIV-2 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 inactivation18,19


and because of its distinctive rod-like morphology (approximately 60nm by 200nm), which poses a unique challenge to a nanofiltration step.


Three relevant and model non-enveloped viruses were tested,17 all with


inherent resistance to physical and chemical inactivation. Reovirus type 3 (Reo3) is an RNA virus that serves as a general model for


33


Titer (log10TCID50/ml)


Titer (log10TCID50/ml)


Titer (log10TCID50/ml)


Titer (log10TCID50/ml)


Titer (log10TCID50/ml)


Titer (log10TCID50/ml)


Depth filtration


Centrifugation


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