Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by‐product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase‐2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6‐phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and
With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site‐specific N‐ and O‐glycan profiles of the recombinant erythropoietin Fc (EPO‐Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX‐CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO‐Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N‐ and O‐glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N‐linked glycopeptides. Using intact glycopeptide characterization, the EPO‐Fc was observed to maintain their individual EPO and Fc N‐glycan characteristics in which the EPO region presented bi‐, tri‐, and tetra‐branched N‐glycan structures, while the Fc N‐glycan displayed mostly biantennary glycans. EPO‐Fc protein generated in EX medium produced more complex tetra‐antennary N‐glycans at each of the three EPO N‐sites while IA medium resulted in a greater fraction of bi‐ and tri‐antennary N‐glycans at these same sites. Interestingly, the sialylation content decreased from sites 1–4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O‐glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site‐specific glycosylation profile and provide a more detailed characterization of N‐ and O‐glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest.
more » « less- NSF-PAR ID:
- 10102667
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- Volume:
- 116
- Issue:
- 9
- ISSN:
- 0006-3592
- Page Range / eLocation ID:
- p. 2303-2315
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract N ‐glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2‐deoxy‐d ‐glucose (2DG) and 5‐thio‐d ‐glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%–45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO‐Fc titers peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO‐Fc titers. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes.N ‐glycan analysis of EPO‐Fc revealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG‐supplemented cultures, respectively. Inhibitor supplementation also led to a decrease in bi‐, tri‐, and tetra‐antennary structures and up to 50% lower EPO‐Fc sialylation. Interestingly, addition of 2DG led to the incorporation of 2‐deoxy‐hexose (2DH) on EPO‐FcN ‐glycans and addition of 5TG resulted in the first‐ever observedN ‐glycan incorporation of 5‐thio‐hexose (5TH). Six percent to 23% ofN ‐glycans included 5TH moieties, most likely 5‐thio‐mannose and/or 5‐thio‐galactose and/or possibly 5‐thio‐N ‐acetylglucosamine, and 14%–33% ofN ‐glycans included 2DH moieties, most likely 2‐deoxy‐mannose and/or 2‐deoxy‐galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism,N ‐glycosylation processing, and formation of alternative glycoforms. -
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Glycosylation is a critical quality attribute of monoclonal antibody (mAb) therapeutics. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is an invaluable technology for the characterization of protein glycosylation. HILIC/MS-based glycan analysis relies on the library search using Glucose Units (GU) and accurate mass (AM) as the primary search parameters for identification. However, GU-based identifications are gradient-dependent and are not suitable for applications where separation gradients need to be optimized to analyze complex samples or achieve higher throughput. Additionally, the workflow requires calibration curves (using dextran ladder) to be generated for each analysis campaign, which in turn, are used to derive the GU values of the separated glycan species. To overcome this limitation, we employed a two-step strategy for targeted glycan analysis of a mAb expressed in Chinese Hamster Ovary (CHO) cells. The first step is to create a custom library of the glycans of interest independent of GU values (thereby eliminating the need for a calibration curve) and instead uses AM and retention time (RT) as the primary search variables. The second step is to perform targeted glycan screening using the custom-built library. The developed workflow was applied for targeted glycan analysis of a mAb expressed in CHO for 1) cell line selection 2) characterizing the day-wise glycan evolution in a model mAb during a fed-batch culture, 3) assessing the impact of different media conditions on glycosylation, and 4) evaluating the impact of two different process conditions on glycosylation changes in a model mAb grown in a bioreactor. Taken together, the data presented in this study provides insights into the sources of glycan heterogeneity in a model mAb that are seen during its commercial manufacturing.more » « less
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