Fluorophore‐labeled carbohydrate analysis of immunoglobulin fusion proteins: Correlation of oligosaccharide content with in vivo clearance profile

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Abstract
CTLA4 is a membrane receptor on cytotoxic T cells whose interaction with the B7 counterreceptor on B cells is important in alloantigen responses. Soluble recombinant human and murine CTLA4 were produced using either Chinese hamster ovary or NS‐0 cell lines. Expression vectors were constructed containing the gene coding for the extracellular domain of CTLA4 fused to either human lgG1 hinge, CH2, and CH3 domains or murine lgG2a hinge, CH2, and CH3 domain genes. These glycoproteins were produced in hollow‐fiber or packed‐bed‐type bioreactors and purified from 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 monosaccharide profiles using several carbohydrate specific techniques; and in vivo clearance profiles using a murine model. Significant differences were observed between lots in their pl, clearance, and crbohydrate profiles. Higher overall pl values correlated with accelerated α‐phase clearance and changes in oligosaccharide composition as determined by lectin binding analysis and electrophoresis of fluorophore‐conjugated carbohydrates. Preparations exhibiting slower clearance profiles had oligosaccharides with higher quantities of N‐acetylneuraminic acid and were predominantly of an N‐linked biantennary complex‐type. Conversely, batches with accelerated clearance profiles had less detectable N‐acetylneuraminic acid. Oligosaccharides from murine CTLA4lg produced in NS‐0 cells had terminal N‐glycolylneuraminic acid but no detectable N‐acetylneuraminic acid and had concomitant accelerated clearance. These data suggest that the presence and quantity of N‐acetylneuraminic acid is an important component in predicting CTLA4lg plasma clearance rates and that production lots can be analyzed for oligosaccharide heterogeneity and sialic acid content by electrophoresis of fluorophore‐conjugated carbohydrates. © 1995 John Wiley & Sons, Inc.