No abstract

N-Glycans at Asn(297) in the Fc domain of IgG molecules are required for Fc receptor-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In this study we have specifically remodeled the Fc N-glycans of intact recombinant IgG(1) therapeutic monoclonal antibody (Mab) products, Rituxan and Herceptin, with a soluble recombinant rat beta-1,4-N-acetylglucosaminyltransferase III (rGnTIII) produced by baculovirus-infected insect cells. N-Glycan remodeling in vitro permitted a controlled and selective transfer of a bisecting beta1,4-linked GlcNAc to the core beta-linked mannose of degalactosylated Mab N-glycans to yield Mabs varying in bisecting GlcNAc content from 31% to 85%. This was confirmed by analysis of N-glycans by both normal phase HPLC and MALDI-MS, the latter yielding the expected mass increase of 203.2 Da with no other oligosaccharide modifications evident. ADCC of remodeled Rituxan and Herceptin Mabs was determined using peripheral blood mononuclear cells as effectors and either CD20(+) (SKW6.4 and SU-DHL-4) or Her2(+) (SKBR-3) target cells, respectively. A conserved 10-fold increase in ADCC was observed for both remodeled therapeutic Mabs with high (>80%) bisecting GlcNAc content. In contrast, although the presence of a bisecting GlcNAc had minimal effect on CDC, degalactosylation of Rituxan reduced CDC by approximately half, relative to unmodified (variably galactosylated) control Mab. In summary, our data suggests that in vitro remodeling of therapeutic Mab Fc N-glycans may be utilized to control the therapeutic efficacy of Mabs in vivo and to offer a more "humanized" glycoform profile for recombinant Mab products.

Glycosylation is the most extensive of all the posttranslational modifications, and has important functions in the secretion, antigenicity and clearance of glycoproteins. In recent years major advances have been made in the cloning of glycosyltransferase enzymes, in understanding the varied biological functions of carbohydrates, and in the accurate analysis of glycoprotein heterogeneity. In this review we discuss the impact of these advances on the choice of a recombinant host cell line, in optimizing cell culture processes, and in choosing the appropriate level of glycosylation analysis for each stage of product development.

One of the most significant problems in industrial bioprocessing of recombinant proteins using engineered mammalian cells is the phenomenon of cell line instability, where a production cell line suffers a loss of specific productivity (qP). This phenomenon occurs with unpredictable kinetics and has been widely observed in Chinese hamster ovary (CHO) cell lines and with all commonly used gene expression systems. The underlying causes (both genetic and physiological) and the precise molecular mechanisms underpinning cell line instability have yet to be fully elucidated, although recombinant gene silencing and loss of recombinant gene copies have been shown to cause qP loss. In this work we have investigated the molecular mechanisms underpinning qP instability over long-term sub-culture in CHO cell lines producing recombinant IgG1 and IgG2 monoclonal antibodies (Mab's). We demonstrate that production instability derives from two primary mechanisms: (i) epigenetic--methylation-induced transcriptional silencing of the CMV promoter driving Mab gene transcription and (ii) genetic--progressive loss of recombinant Mab gene copies in a proliferating CHO cell population. We suggest that qP decline resulting from loss of recombinant genes is a consequence of the inherent genetic instability of recombinant CHO cell lines.