Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.

Chinese hamster ovary and murine myeloma NS0 cells are currently favored host cell types for the production of therapeutic recombinant proteins. In this study, we compared N-glycan processing in GS-NS0 and GS-CHO cells producing the same model recombinant glycoprotein, tissue inhibitor of metalloproteinases 1. By manipulation of intracellular nucleotide-sugar content, we examined the feasibility of implementing metabolic control strategies aimed at reducing the occurrence of murine-specific glycan motifs on NS0-derived recombinant proteins, such as Galalpha1,3Galbeta1,4GlcNAc. Although both CHO and NS0-derived oligosaccharides were predominantly of the standard complex type with variable sialylation, 30% of N-glycan antennae associated with NS0-derived TIMP-1 terminated in alpha1,3-linked galactose residues. Furthermore, NS0 cells conferred a greater proportion of terminal N-glycolylneuraminic (sialic) acid residues as compared with the N-acetylneuraminic acid variant. Inclusion of the nucleotide-sugar precursors, glucosamine (10 mM, plus 2 mM uridine) and N-acetylmannosamine (20 mM), in culture media were shown to significantly increase the intracellular pools of UDP-N-acetylhexosamine and CMP-sialic acid, respectively, in both NS0 and CHO cells. The elevated UDP-N-acetylhexosamine content induced by the glucosamine/uridine treatment was associated with an increase in the antennarity of N-glycans associated with TIMP-1 produced in CHO cells but not N-glycans associated with TIMP-1 from NS0 cells. In addition, elevated UDP-N-acetylhexosamine content was associated with a slight decrease in sialylation in both cell lines. The elevated CMP-sialic acid content induced by N-acetylmannosamine had no effect on the overall level of sialylation of TIMP-1 produced by both CHO and NS0 cells, although the ratio of N-glycolylneuraminic acid:N-acetylneuraminic acid associated with NS0-derived TIMP-1 changed from 1:1 to 1:2. These data suggest that manipulation of nucleotide-sugar metabolism can promote changes in N-glycan processing that are either conserved between NS0 and CHO cells or specific to either NS0 cells or CHO cells.

Catalase is an antioxidant enzyme that has been shown to inhibit apoptotic or necrotic neuronal death induced by hydrogen peroxide. We report the purification of a contaminating antiapoptotic activity from a commercial bovine liver catalase preparation by following its ability to inhibit apoptosis when applied extracellularly in multiple death paradigms. The antiapoptotic activity was identified by protein microsequencing as arginase, a urea cycle and nitric oxide synthase-regulating enzyme, and confirmed by demonstrating the presence of antiapoptotic activity in a >97% pure preparation of recombinant arginase. The pluripotency of recombinant arginase was demonstrated by its ability to inhibit apoptosis in multiple paradigms including rat cortical neurons induced to die by glutathione depletion and oxidative stress, by 100 nM staurosporine treatment, or by Sindbis virus infection. The protective effects of arginase in these apoptotic paradigms, in contrast to previous studies on excitotoxic neuronal necrosis, are independent of nitric oxide synthase inhibition. Rather, arginase-induced depletion of arginine leads to inhibition of protein synthesis, resulting in cell survival. Because inhibitors of nitric oxide synthesis and of protein synthesis have been shown to decrease necrotic and apoptotic death, respectively, in animal models of stroke and spinal cord injury, arginine-depleting enzymes, capable of simultaneously inhibiting protein synthesis and nitric oxide generation, may be propitious therapeutic agents for acute neurological diseases. Furthermore, our results suggest caution in attributing the cytoprotective effects of some catalase preparations to catalase.

The biological activity of therapeutic proteins is strongly dependent on the stability of their folded state, which can easily be compromised by degradation. Oxidation is one of the most common causes of degradation and is typically associated with impairment of the native protein structure. Methionine residues stand out as particularly susceptible to oxidation by reactive oxygen intermediates even under mild conditions. Consequently, methionine oxidation has profound effects on protein activity up to the point of adverse biological responses. Of immediate importance therefore is finding affordable approaches for rapid detection of methionine oxidation before any substantial structural changes can ensue. Herein we report that vibrational bands at 1,044 and 1,113 cm⁻¹ in the mid-infrared region can serve as characteristic markers of methionine oxidation in oxidatively stressed protein therapeutics, monoclonal antibodies (IgG1 and its antigen-binding fragment). Such Fourier-transform infrared (FTIR) markers underpin rapid detection assays and hold particular promise for correlation of methionine oxidation with protein structure and function.