Biochemical, molecular characterization, and glycoproteomic analyses of alpha(1)-proteinase inhibitor products used for replacement therapy.

BACKGROUND

Isoelectric focusing (IEF) of alpha(1)-proteinase inhibitor (A1PI) shows that commercial products and plasma have different glycoisoform band patterns. Those in Aralast (Grifols Biologicals) reflect an anodal shift of glycoisoforms, which has caused concern. The protein, including glycoproteomic analyses, and structural features of A1PI products were investigated by state-of-the-art techniques.

STUDY DESIGN AND METHODS

Batches from Aralast, Prolastin (Bayer), and Zemaira (Aventis Behring LLC) were analyzed by high-resolution IEF and high-performance size-exclusion chromatography (HP-SEC). Preparative separated isoforms from IEF were further purified by chromatography and subjected to mass spectrometry for sequence analyses, peptide mapping, and glycosylation analysis. Deamidation was quantified by enzymatic isoaspartate detection. Multiple sequence alignments and structural bioinformatics analyses were performed.

RESULTS

In HP-SEC, Prolastin had the highest aggregate content at approximately 30 percent. Isoforms from all products purified by high-resolution IEF were sequenced with an amino acid coverage of more than 98 percent. Deamidation of Asn116 and Asn314 in A1PI was to found to some extent in all products and confirmed quantitatively by enzymatic analysis. There were no signs of methionine oxidation. Cys232 was found to be cysteinylated in A1PI in Prolastin and Aralast as in plasma, but not in Zemaira. All products showed truncation of the C-terminal lysine. Intact A1PI concentrates contained mainly diantennary, disialylated and smaller amounts of triantennary, trisialylated N-glycans. The percentage of fucosylation was similar in all products. Site-specific glycan analysis revealed bands M6 contained only diantennary glycans, whereas the more acidic bands M4 and M2 also carried triantennary structures. The most acidic isoforms, M2 in Prolastin and Zemaira and M0 in Aralast, additionally exhibited tetraantennary N-glycans.

CONCLUSION

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.