Differential affects of low glucose on the macroheterogeneity and microheterogeneity of glycosylation in CHO-EG2 camelid monoclonal antibodies

Abstract

Background: The demand for high yield recombinant protein production systems has focused industry on culture media and feed strategies that optimize productivity, yet maintain product quality attributes such as glycosylation. Minimizing media components such as glucose, reduces the production of lactate, but may also affect glycosylation. The first steps in the glycosylation pathway involve the synthesis of lipid-linked oligosaccharides (LLOs). Glycan macroheterogeneity is introduced by variation in site-specific glycosylation with the transfer of the oligo-saccharide to the protein. Further modification of the oligosaccharide can occur through processing reactions, where some sugars are removed and additional sugars added. This produces microheterogeneity of the glycan pool. Both macroheterogeneity and microheterogeneity may be affected by fermentation conditions. The objective of this study has been to investigate the effect of variable concentrations of glucose on the glycosylation patterns of a camelid monoclonal antibody produced in Chinese hamster ovary (CHO) cells and to further evaluate their effect on components of the N-glycosylation pathway. Material(s) and Method(s): A CHO cell line recombinantly expressing chimeric antibodies EG2 with a camelid single domain fused to human Fc regions was used in this study. Cells were inoculated at 2.6 x 106 cells/ml into 7 shake flasks (250 ml) each containing 80 ml of media with a different initial glucose concentration varying from 0 to 25 mM. The cultures were maintained and monitored under standard shaking conditions in an incubator over a 24 hr period. Cells were harvested and quenched to stop any subsequent metabolic activities [1]. LLOs were extracted from the cells using a previously established method [2]. Mild acid cleaved glycans were labeled with 2- aminobenzamide and analyzed by high performance liquid chromatography (HPLC) using the technique of hydrophilic interaction liquid chromatography (HILIC). The structures were assigned using standard GU values from the GlycoBase database (NIBRT.ie) [3] and confirmed by Mass spectrometric analysis. Antibodies were purified from culture supernatants with a Protein A affinity column and run under denaturing conditions on 8-16% SDS-PAGE gels and stained with Coomassie Brilliant Blue (CBB). The density ratio between upper and lower bands was determined by densitometry. The protein bands were removed by scalpel, washed, and treated with Peptide-NGlycosidase F for 18 h to remove the attached glycans. MS analysis was carried out on the MALDI-TOF/TOF mass spectrometer to confirm aglycosylated Mabs in the lower band, and glycosylated proteins present in the upper band. The isolated N-linked glycans were labeled with 2-AB [4]. Glycan structures were assigned using standard GU values from HILIC analysis in GlycoBase. Structures were confirmed by exoglycosidase enzymatic digestion arrays according to method of Royle et al (2010). Result(s): Peaks corresponding to the LLOs from each of the previously described cultures with varying glucose concentration cultures were compared (Figure 1.A.). Samples from cultures containing 25mM glucose displayed a prominent large peak with a GU value of 11.7 representing 63% of the total LLOs and designated as the Glc3Man9GlcNAc2a structure (Figure 1.A.). Small peaks were designated as Glc2Man9GlcNAc2, Glc1Man9GlcNAc2, Man9GlcNAc2, Man5GlcNAc2 and Man2GlcNAc2 structures. For cells grown at an initial glucose concentration of less than 15 mM the predominant peak was Man2GlcNAc2 with a significant level of the Man5GlcNAc2 structure but the percentage of the Glc3Man9GlcNAc2 structure was reduced significantly to 2.9% of the overall LLOs. It is important to note that these cultures (