Biochemical, molecular characterization, and glycoproteomic analyses of α₁‐proteinase inhibitor products used for replacement therapy*

Abstract

Protein chemical characterization of A1PI showed that all A1PI products to some extent differ from A1PI circulating in human plasma. Bioinformatic analysis indicated that removal of C-terminal Lys394 and cysteinylation of Cys232 are unlikely to affect structure and/or function of A1PI but cysteinylation may influence interaction between A1PI and its physiologic ligands. Aralast, Prolastin, and Zemaira contain the same set of N-glycans in the same ratios as those in normal human plasma A1PI. Tri- and tetraantennary structures are responsible for the partitioning into IEF isoforms, with the migration shift of Aralast not being due to any difference in the N-glycosylation, but to the partial loss of the C-terminal lysine.

Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes

“…The glycans predicted by the MS analysis of unmodified proteins are consistent with published data on the structure of the glycans (21,25). Together, these results indicate that Cst-II is able to modify a variety of glycan structures including bi-, tri-, and tetraantennary glycans with either α2,3-or α2,6-linked terminal sialic acids.…”

“…A1AT contains three N-linked glycan sites, of which 74% are biantennary and possess a terminal α2,6-linked sialic acid. An additional 14% of the glycans are triantennary with a mixture of α2,3/6-linked terminal sialic acid residues (21). Factor IX is a coagulation factor used to treat hemophilia B, a congenital disease caused by a factor IX deficiency (22,23).…”

The Changing Fate of a Secretory Glycoprotein in Developing Maize Endosperm

“…Phytase was isolated from the extracts by sequential ammonium sulfate fractionation as described by Arcalis et al (2004) and concentrated by ultrafiltration before separation by 12% (w/v) SDS-PAGE under reducing conditions. The bands corresponding to phytase were excised, destained, carbamidomethylated, digested with trypsin, and extracted from gel pieces as described (Kolarich and Altmann, 2000;Kolarich et al, 2006). Peptide fractionation and analysis using a Q-TOF Ultima Global (Waters Micromass) mass spectrometer was performed as described previously (Kolarich and Altmann, 2000;Van Droogenbroeck et al, 2007).…”

Recent Advances in the Research and Development of Alpha-1 Proteinase Inhibitor for Therapeutic Use

“…In addition to all that, multistep manufacturing procedures are known to induce various protein alterations, such as aggregation and chemical modifications (e.g., deamidation, cysteinylation, and C-terminal truncation). Some modifications can be observed by IEF and other techniques (Cowden et al, 2005;Kolarich et al, 2006aKolarich et al, , 2006b) and reflected in the product specifications. Currently there are no data that would demonstrate whether these alterations affect the in vivo activity, safety, efficacy or immunogenicity of 1 -PI therapeutic preparations.…”