FRAGMENTATION OF THERAPEUTIC HUMAN-IMMUNOGLOBULIN PREPARATIONS

Some commercial batches of human therapeutic immunoglobulins (Ig) have been found to show evidence of molecular fragmentation when examined by molecular sizing methodologies including sodium dodecyl sulphate po lyacrylamide gel electrophoresis [SDS-PAGE] and size exclusion high pe rformance liquid chromatography (SE-HPLC). These batches all demonstra ted impaired immunobiological activity (efficacy) as assessed by Fc fu nction measured using a rubella haemolytic assay and as such are likel y to be subpotent for therapeutic use. Fragmented Igs were characteriz ed by the presence of at least three protein bands and peaks additiona l to monomeric IgG. Incubation of Igs with blood enzymes (plasmin and kallikrein) reproduced the fragmentation patterns observed for intrins ically degraded batches, suggesting that fragmentation occurred by con tamination with these proteases from the source material (human blood) during manufacture. Intravenous Igs (IVIG) were found to be more susc eptible to proteolysis than intramuscular Igs, probably as a consequen ce of the post-fractionation processing that some IVIGs receive which may induce molecular alterations, allowing enzyme access and fragmenta tion. Two of the products examined were found to be relatively resista nt to proteolysis and both were formulated by processes that limit enz yme activity. These processes were inclusion of an enzyme inhibitor, a lpha(2)-macroglobulin, and formulation at acidic pH. Enzyme carry-over into the final product is a likely cause of Ig fragmentation, and red uction in levels of such contamination should lead to improvements in product stability and efficacy.