US Respiratory Disease Volume 6 reg

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 John Lang, BS2

Michelle Gall, PhD,1 and Douglas Lee, PhD1 1. Pathogen Safety, Talecris Biotherapeutics Inc., Research Triangle Park, North Carolina; 2. BioAnalytics, Talecris Biotherapeutics Inc., Clayton, North Carolina Nathan J Roth, PhD,1

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&#xAE;

-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;6:40&#x2013;6 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&#xAE; is a concentrate of human alpha1-PI that is administered to patients in an attempt to restore the elastase&#x2013;serpin balance.

Observational studies have shown that augmentation therapy may help to slow a decline in lung function.1&#x2013;3

Modifications have recently been approved to the Prolastin manufacturing

process and the resulting Prolastin&#xAE;-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 Cohn&#x2013;Oncley fractionation method.5,6

dedicated virus-reduction steps: 40 The Prolastin-C process incorporates two 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.

&#xA9; TOUCH BRIEFINGS 2010

&#x2022;

a solvent/detergent (S/D) treatment step located immediately downstream of the generation of polyethylene glycol (PEG) filtrate (see Figure 1); and

&#x2022; 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 0.1&#x2013;0.3%, 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 0.1&#x2013;1%.

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