Alpha1-antitrypsin Deficiency


Characterization of a New Alpha1-proteinase Inhibitor from Human Plasma—Prolastin®-C


Todd Willis, PhD,1 Kevin Wee, PhD,2 Koen van der Drift, PhD2 and Gerold Mohn, PhD2 1. BioAnalytics, Talecris Biotherapeutics Inc., Clayton, North Carolina; 2. BioAnalytics, Talecris Biotherapeutics Inc., Raleigh, North Carolina


Abstract


Scientific advances have enabled modifications of the manufacturing process of Prolastin® (human alpha1-proteinase inhibitor) to significantly increase active content and functional activity. The objective of this study was to determine the biochemical characteristics of the new product,


Prolastin®


-C, including potency, purity, and glycan profile. Prolastin-C was characterized using a panel of analytical methods consisting of anti-neutrophil elastase capacity, immunonephelometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and capillary gel electrophoresis (SDS-CGE), capillary zone electrophoresis, Western blotting, isoelectric focusing, size exclusion high-performance liquid chromatography, and mass spectrometry. Six lots of Prolastin-C were characterized. These had a mean functional activity of 54.3mg/ml (approximately twice that of Prolastin). The average purity of Prolastin-C was 97% and the monomeric content was 89%. The glycan profile was comparable to that found in normal human plasma, while mass spectrometry confirmed the expected mass and presence of the single cysteine in Prolastin-C. Five plasma protein impurities could be quantified, accounting for approximately 4% of the total protein content.


Keywords Alpha1-antitrypsin, alpha1-proteinase inhibitor, Prolastin, Prolastin-C, functional activity, purity, potency, glycan profile


Disclosure: All authors are full-time employees of Talecris Biotherapeutics Inc. Received: October 5, 2010 Accepted: December 9, 2010 Citation: US Respiratory Disease, 2010;6:27–30 Correspondence: Todd Willis, BioAnalytics, Talecris Biotherapeutics Inc., 8368 US 70 West, Clayton, NC 27520. E: todd.willis@talecris.com


Support: This study was sponsored by Talecris Biotherapeutics Inc. (Research Triangle Park, NC 27709). 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.


Alpha1-proteinase inhibitor (alpha1-PI) is a serine protease inhibitor (serpin) with a molecular weight of approximately 52kDa. This


single-chain acidic glycoprotein consists of 394 amino acids and is approximately 12% carbohydrate, with three glycosylation sites and two primary glycoforms (designated M4 and M6).1–3


By inhibiting the


proteolytic enzymes released by inflammatory cells, alpha1-PI protects tissues from damage. In the lung, it inhibits neutrophil elastase,


providing a regulatory mechanism that helps to prevent the degradation of elastin, thus protecting the elasticity of the lungs.


Deficiency of alpha1-PI (alpha-1 antitrypsin deficiency) is an autosomal, co-dominant, hereditary disorder characterized by low serum and lung


levels of alpha1-PI.4–7 Severe forms of the deficiency are frequently


associated with progressive, moderate-to-severe emphysema that may become apparent in adults at any age, regardless of smoking history, potentially resulting in a lower life expectancy.1,4,5,7


Prolastin® is a plasma-derived human alpha1-PI that is administered as an intravenous infusion. It has been in clinical use since 1988 as


augmentation therapy for patients with alpha1-PI deficiency and has got an excellent safety profile.8–11


© TOUCH BRIEFINGS 2010 Since 1988, scientific advances have enabled Materials and Methods


The following analytical methods were used to evaluate six final container lots of Prolastin-C manufactured at commercial scale.


Potency


The functional activity of alpha1-PI was determined by measuring antineutrophil elastase capacity. The rate of the elastase activity


reaction, measured with a chromogenic substrate (Suc-(Ala)3-pNA), is 27


modifications to improve the Prolastin manufacturing process. These modifications include the incorporation of additional ion-exchange chromatography (a further purification step) as well as solvent detergent treatment and nanofiltration (pathogen reduction steps).12 improvements in the manufacturing process have significantly increased


The


the concentration of alpha1-PI while maintaining excellent purity. The new alpha1-PI product, prepared using the modified Prolastin manufacturing process, has been designated Prolastin®-C.


The aim of this article is to describe the biochemical characteristics of Prolastin-C, including functional activity, purity, protein composition, molecular mass distribution, and glycan profile.


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