Alpha1-antitrypsin Deficiency


molecular weight associated with loss of the additional free cysteine linked by cysteinylation to the single cysteine in alpha1-PI.


Measurement of specific plasma protein impurities in Prolastin-C by immunonephelometry detected only low levels of albumin (0.5%), alpha-1 acid glycoprotein (0.38%), apolipoprotein A-1 (0.035%), haptoglobin (0.92%), and IgA (2.4%). On average, these five protein impurities accounted for approximately 4% of the total protein content,


with alpha1-PI comprising 96% of the total protein content. Eleven other potential plasma protein impurities evaluated by immunonephelometry were below their limits of detection.


Discussion Recent improvements to the Prolastin manufacturing process have led


to the production of Prolastin-C, a human alpha1-PI product with a functional activity approximately twice that of Prolastin. The increase in functional activity of Prolastin-C was accomplished through process improvements that decreased plasma protein impurities and increased


alpha1-PI-specific activity. Ultimately, the higher functional activity of Prolastin-C improves patient convenience by significantly reducing the time required to administer the product. Infusion times with Prolastin-C are approximately 15 minutes.


Protein purification processes can have subtle effects on the integrity of any protein extracted. This is evident from the differences previously identified between the various alpha1-PI products.3


In normal human


plasma (and Prolastin), Cys232 of alpha1-PI is cysteinylated (the cysteine is linked to an additional cysteine amino acid).2


interaction of the protein with its physiological ligands.3


This may influence the It is also


important to retain this cysteinylation in the product because it minimizes the formation of a protein dimer linked through a disulfide bond. In Prolastin-C, this cysteinylated cysteine was shown to be


1. American Thoracic Society/European Respiratory Society Statement, Standards for the diagnosis and management of individuals with alpha-1 antitrypsin deficiency, Am J Respir Crit Care Med, 2003;168:818–900.


2.


Kolarich D, Weber A, Turecek PL, et al., Comprehensive glyco-proteomic analysis of human alpha1-antitrypsin and its charge isoforms, Proteomics, 2006;6:3369–80.


3.


Kolarich D, Tureckek PL, Weber A, et al., Biochemical, molecular characterization, and glycoproteomic analyses of alpha(1)-proteinase inhibitor products used for replacement therapy, Transfusion, 2006;46:1959–77.


4. 5.


Brantly M, Nukiwa T, Crystal RG, Molecular basis of alpha-1-antitrypsin deficiency, Am J Med, 1988;84:13–31.


Crystal RG, Brantly ML, Hubbard LC, et al., The alpha-1-antitrypsin gene and its mutations. Clinical consequences and strategies for therapy, Chest, 1989;95:196–208.


6.


Hutchison DC, Natural history of alpha-1-protease inhibitor deficiency, Am J Med, 1988;84:3–12.


7. 8. 9.


Hubbard RC, Crystal RG, Alpha-1-antitrypsin augmentation therapy for alpha-1-antitrypsin deficiency, Am J Med, 1988;84:52–62.


Bernhardt T, Cortes R, Post license safety of Prolastin, Eur Respir J, 2006;28(Suppl. 50):518S.


Wencker M, Banik N, Buhl R, et al., Long-term treatment of alpha1-antitrypsin deficiency-related pulmonary emphysema with human alpha1-antitrypsin. Wissenschaftliche Arbeitsgemeinschaft zur Therapie von Lungenerkrankungen (WATL)-alpha1-AT-study group, Eur Respir J, 1998;11:428–33.


10. Stoller JK, Fallat R, Schluchter MD, et al., Augmentation therapy with alpha1-antitrypsin: patterns of use and adverse events, Chest, 2003;123:1425–34.


11. Petrache I, Hajjar J, Campos M, Safety and efficacy of alpha- 1-antitrypsin augmentation therapy in the treatment of patients with alpha-1-antitrypsin deficiency, Biologics, 2009;3:193–204.


12. Lebing W, Michelle SP, Ndarathi, C, et al., Modification of the manufacturing process for the plasma-derived human


present by mass spectrometry following RP-HPLC (see Figure 4), as is the case in normal plasma.


The glycan profile of Prolastin-C was comparable to naturally-occurring


alpha1-PI in normal human plasma, which exists as an acidic glycoprotein. In plasma, alpha1-PI has some heterogeneity due to post-translational modifications, principally in the three complex


These N-glycans have different numbers of sialic acid residues, conferring slightly different levels of negative charge that can be detected using IEF.2


N-glycans that are covalently linked to three different asparagine residues.3


There are two primary glycoforms (M4 and M6)


and other minor glycoforms (which include M2). Correct glycosylation of glycoproteins is important, as it can affect protein folding,13 activity,14


specific interactions with receptors15,16 and half-life.17


biologic In this


study, the IEF profile of Prolastin-C was comparable to that of normal human plasma, indicating that the modified purification process does not appreciably alter the normal glycoform composition.


Conclusion


In conclusion, this study shows that the alpha1-PI in Prolastin-C is of a higher potency compared with Prolastin, while maintaining excellent


purity and the same physical and chemical characteristics. n


Todd Willis, PhD, is Director of BioAnalytical Operations at Talecris Biotherapeutics Inc. He provides analytical support to work teams within technical operations, process development, pathogen safety, and quality operations. He was a key member of the Prolastin-C project development team and was responsible for characterizing the purification process and final drug product, and preparing regulatory submission documents for the licensure of Prolastin-C. Todd obtained his PhD in biochemistry from Colorado State University.


alpha1-proteinase inhibitor leading to Prolastin®-C, US Resp Dis, 2010;6:22–6.


13. Morais VA, Costa MT, Costa J, N-glycosylation of recombinant human fucosyltransferase III is required for its in vivo folding in mammalian and insect cells, Biochim Biophys Acta, 2003;1619:133–8.


14. Moll M, Kaufmann A, Maisner A, Influence of N-glycans on processing and biological activity of the nipah virus fusion protein, J Virol, 2004;78:7274–8.


15. Ma BY, Mikolajczak SA, Yoshida T, et al., CD28 T cell costimulatory receptor function is negatively regulated by N-linked carbohydrates, Biochem Biophys Res Commun, 2004;317:60–7.


16. Takahashi M, Tsuda T, Ikeda Y, et al., Role of N-glycans in growth factor signaling, Glycoconj J, 2004;20:207–12.


17. Manzella SM, Dharmesh SM, Beranek MC, et al., Evolutionary conservation of the sulfated oligosaccharides on vertebrate glycoprotein hormones that control circulatory half-life, J Biol Chem, 1995;270: 21665–71.


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