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Yield improvement for manufacture of α 1 ‐proteinase inhibitor
Author(s) -
Zimmerman T. P.
Publication year - 2006
Publication title -
vox sanguinis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.68
H-Index - 83
eISSN - 1423-0410
pISSN - 0042-9007
DOI - 10.1111/j.1423-0410.2006.00841.x
Subject(s) - yield (engineering) , leupeptin , dissolution , chemistry , protease , pi , chromatography , tris , protease inhibitor (pharmacology) , nuclear chemistry , biochemistry , enzyme , materials science , organic chemistry , biology , metallurgy , human immunodeficiency virus (hiv) , antiretroviral therapy , viral load , immunology
Background and Objectives The aim of this study was to increase the yield of active α 1 ‐proteinase inhibitor (α 1 ‐PI) from Cohn fraction IV‐1 paste during the manufacture of this therapeutic protein and to investigate the molecular mechanism for this yield increase. Materials and Methods Dissolution experiments with IV‐1 paste investigated the impact of different variables on the yield of α 1 ‐PI activity. Solutions of IV‐1 paste prepared under different conditions were assayed for evidence of protease activity by Western blots of α 1 ‐PI following SDS‐PAGE, by azocaseinolytic and amidolytic (S‐2288) assays, and by zymography, and for the extent of α 1 ‐PI oligomerization by Western blots following nondenaturing PAGE. Results Minor modification of the manufacturing process by combining dissolution of IV‐1 paste with the subsequent pH adjustment (to 9·25–9·50 with NaOH), achieved by addition of a standard concentration of NaOH to the 10‐m m Tris base dissolvent for IV‐1 paste, was found to give a highly reproducible 9·4 ± 0·9% increase in yield of active α 1 ‐PI. Solutions of IV‐1 paste prepared with this combined dissolvent contained reduced amounts of low molecular weight fragments of α 1 ‐PI, reduced protease activity, and reduced amounts of oligomers of α 1 ‐PI. Addition of the protease inhibitor leupeptin to the 10‐m m Tris base dissolvent for IV‐1 paste also caused an increase in the yield of α 1 ‐PI activity. Conclusions Dissolution of IV‐1 paste in a more alkaline medium gave a significant increase in the yield of active α 1 ‐PI. This yield increase was attributed to a reduction both in protease activity and in the extent of oligomerization of α 1 ‐PI.