Production and Post-Translational Modification of Tissue Plasminogen Activator (tPA) in Namalwa Cells

N-linked glycosylation is a major post-translational event having profound influence on the quality of recombinant proteins destined for therapy and hence it is extensively studied [1]. Glycosylation is affected by the type of host cell, environmental stimuli, and protein structure [2]. This study will investigate the first two factors by characterising a model protein: tissue plasminogen activator (tPA) expressed in Chinese hamster ovary (CHO) and a human lymphoblastoid cell line (Namalwa). tPA is a fibrin-specific serine protease of plasminogen involved in the dissolution of vascular fibrin clots in vivo. It exists as a mixture of single-chain and two-chain (A and B) glycoproteins (66-72kDa) with four potential N-linked glycosylation sites at Asn117 (invariably high mannose type) Asn184 (variably occupied complex type) Asn218 (never glycosylated) and Asn448 (invariably complex type) and an O-linked fucose occurs at Thr61. Type I tPA has three sites occupied with N-linked oligosaccharides and type II has only two sites occupied [3]

Effect of Lipid Supplements on the Production and Glycosylation of Recombiant Interferon-Gamma Expressed in CHO Cells

The effects of lipids on the glycosylation of recombinant human interferon-gamma expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-grycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon-gamma deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon-gamma glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process