FLUOROPHORE-LABELED CARBOHYDRATE ANALYSIS OF IMMUNOGLOBULIN FUSION PROTEINS - CORRELATION OF OLIGOSACCHARIDE CONTENT WITH IN-VIVO CLEARANCEPROFILE

CTLA4 is a membrane receptor on cytotoxic T cells whose interaction wi th the B7 counterreceptor on B cells is important in alloantigen respo nses. Soluble recombinant human and murine CTLA4 were produced using e ither Chinese hamster ovary or NS-0 cell lines. Expression vectors wer e constructed containing the gene coding for the extracellular domain of CTLA4 fused to either human IgG1 hinge, CH2, and CH3 domains or mur ine IgG2a hinge, CH2, and CH3 domain genes. These glycoproteins were p roduced in hollow-fiber or packed-bed-type bioreactors and purified fr om conditioned media by protein A affinity chromatography. Batches of purified CTLA4lg were analyzed for size, composition, and isoelectric point (pl) patterns by standard protein methods; oligosaccharide and m onosaccharide profiles using several carbohydrate specific techniques; and in vivo clearance profiles using a murine model. Significant diff erences were observed between lots in their pl, clearance, and carbohy drate profiles. Higher overall pl values correlated with accelerated a lpha-phase clearance and changes in oligosaccharide composition as det ermined by lectin binding analysis and electrophoresis of fluorophore- conjugated carbohydrates. Preparations exhibiting slower clearance pro files had oligosaccharides with higher quantities of N-acetylneuramini c acid and were predominantly of an N-linked biantennary complex-type. Conversely, batches with accelerated clearance profiles had less dete ctable N-acetylneuraminic acid. Oligosaccharides from murine CTLA4lg p roduced in NS-0 cells had terminal N-glycolylneuraminic acid but no de tectable N-acetylneuraminic acid and had concomitant accelerated clear ance. These data suggest that the presence and quantity of N-acetylneu raminic acid is an important component in predicting CTLA4lg plasma cl earance rates and that production lots can be analyzed for oligosaccha ride heterogeneity and sialic acid content by electrophoresis of fluor ophore-conjugated carbohydrates. (C) 1995 John Wiley & Sons, Inc.