STRUCTURAL CHARACTERIZATION OF THE N-GLYCANS OF A HUMANIZED ANTI-CD18MURINE IMMUNOGLOBULIN-G

This study characterized the N-glycans of a humanized immunoglobulin G (4) (IgG(4)) expressed in NS/O mouse myeloma cells and directed agains t the CD18 family of adhesion-promoting receptors on leukocytes. The N -glycans were released from the purified recombinant IgG by N-glycanas e treatment, purified by Sephadex G-50 chromatography, and fractionate d by Bio-Gel P-4 chromatography into three oligosaccharide pools. Each pool was analyzed individually by glycosyl composition analysis, high -pH anion-exchange chromatography with pulsed amperometric detection ( HPAEC-PAD), 600-MHz H-1-NMR spectroscopy, and electrospray-ionization mass spectrometry. In addition, each of the three pools was subfractio nated by HPAEC and the isolated subfractions that contained sufficient material were hydrolyzed and analyzed for glycosyl composition by HPA EC-PAD. The overall results indicate the presence of five oligomannosi de-type structures (containing 5 to 8 Man residues) which are not usua lly found in IgG, and the presence of eight diantennary (mostly trunca ted) N-acetyllactosamine-type structures which are typical of mouse an d human IgGs. The N-acetyllactosamine-type structures were heterogeneo us with regard to alpha(1 --> 6) fucosylation of the linkage GlcNAc, a nd the presence or absence of GlcNAc and/or Gal beta(1 --> 4)GlcNAc ex tending the core pentasaccharide (Man(3)GlcNAc(2)). No evidence was fo und for the presence of sialic acid or bisecting GlcNAc residues on th e N-acetyllactosamine-type chains. The latter finding suggests that th e N-glycans of this humanized IgG are of the mouse type. (C) 1994 Acad emic Press, Inc.