The first steps in the glycosylation pathway involve the synthesis of lipid-linked oligosaccharides (LLOs). collection recombinantly expressing chimeric antibodies EG2 with a camelid single domain name 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 Obatoclax mesylate (GX15-070) ml of media with a different initial glucose concentration varying from 0 to 25 mM. The cultures were managed 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 your 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 conversation liquid chromatography (HILIC). The structures were assigned using standard GU values from your GlycoBase database (NIBRT.ie) [3] and confirmed by Mass spectrometric analysis. Antibodies were purified from culture supernatants with a Protein A Obatoclax mesylate (GX15-070) affinity column and run under denaturing conditions on 8-16% SDS-PAGE gels and stained with Coomassie Amazing 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-N-Glycosidase F for 18 h to remove the attached glycans. MS analysis was carried out around the MALDI-TOF/TOF mass spectrometer to confirm aglycosylated Mabs in the lower band, and glycosylated proteins present Rabbit Polyclonal to GNA14 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). Results Peaks corresponding to the LLOs from each of the previously explained cultures with varying glucose concentration cultures were compared (Physique 1.A.). Samples from cultures made up of 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 (Determine 1.A.). Small peaks were designated as Glc2Man9GlcNAc2, Glc1Man9GlcNAc2, Man9GlcNAc2, Man5GlcNAc2 and Man2GlcNAc2 structures. For cells produced 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 Obatoclax mesylate (GX15-070) Glc3Man9GlcNAc2 structure was reduced significantly to 2.9% of the overall LLOs. It is important to note that these cultures (15mM glucose) were under conditions of glucose depletion for at least 4 h prior to harvest. Open in a separate window Physique 1 The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody.A. Lipid-linked oligosaccharide (LLO) profiles. The glycans from each sample were acid hydrolyzed from your lipid carriers, 2-AB labeled and detected by HILIC. (Glc Man and GlcNAc ?). B. Separation of EG2 antibodies on reduced 8-16% SDS-PAGE gel. The purified antibody in lane 8 was isolated from your culture prior to the 24 h incubation. Upper bands in lanes 1 to 4 correspond to glycosylated antibodies, and the lower bands were decided to be non-glycosylated antibodies. C. HPLC profiles of N-glycans isolated from EG2 antibodies produced by CHO cells with numerous initial glucose concentrations during a 24 h incubation. D. The effect of exposure time of cells to media depleted of glucose around the galactosylation (GI; Obatoclax mesylate (GX15-070) |) and the sialylation (SI; ?) indices of EG2 antibodies produced by CHO cells. LLO with a completed glycan structure Glc3Man9GlcNAc2.
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