Tailoring antibody glycosylation via integrating genome and protein engineering to generate preferred glycoforms on the Fc region

One critical quality attribute of therapeutic antibodies is the glycosylation pattern at the Fc region.We combined genome editing of CHO cells and protein engineering of the IgG Fc region to allow antibodies presenting high level of galactosylation or exclusively α-2,6 sialylation. To generate IgG with high α-2,6 sialylation, we combined amino acid mutations in the Fc region of IgG and introduction of α-2,6 sialyltransferase in CHO to produce IgGs with significant levels of both α-2,6 and α-2,3 sialylation. Furthermore, to produce exclusively α-2,6 sialylation IgG in CHO, CRISPR/Cas9 was implemented to disrupt two dominant α-2,3 sialyltransferase genes (ST3GAL4 and ST3GAL6), then α-2,6 sialyltransferewas introduced in a α-2,3 sialylation knockout cell line. Notably, no α-2,3 linked sialic acids of IgG produced from the α-2,3 sialyltransferase knockout-α-2,6 sialyltransferase overexpression pools were detected by HPLC sialic acid quantification after the α-2,3 linkage specific sialidase cleavage. Finally, glycosylation analysis of IgG with four amino acid mutations generated by an α-2,3 sialyltransferase knockout-α-2,6 sialyltransferase overexpression stable CHO clone rendered \u3e75% of sialylated glycans, among which 62.5 % was biantennary disialylated glycans. Interestingly, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from CHO cells in conjunction with protein engineering of the Fc region produced IgGs with a great majority of bigalactosylated and fucosylated (G2F) glycoforms. Expression of the IgG with engineered Fc region (F241A) in triple gene knockout (FuT8-/-, ST3GAL4-/- and ST3GAL6-/-) CHO cells lowered the galactosylation content to 65% bigalactosylated glycoform (G2). However, overexpression of IgGs with four amino acid substitutions from the α-2,3 sialyltransferases knocked out CHO cells reconstituted the fraction of G2 glycoform back up to approximately 80%. Collectively, this study, to our knowledge, is the first attempt for generating highly galactosylated or solely α-2,6 sialylated N-glycans on antibodies in vivo, allowing researchers in both academia and industry to evaluate the significance of tailoring glycosylation on IgGs in biomedicine and biotechnology applications. References: Chung CY, Wang Q, Yang S, Ponce SA, Kirsch BJ, Zhang H, Betenbaugh MJ. Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells. Biotechnol Bioeng. 2017 Jul 7 Chung CY, Wang Q, Yang S, Yin B, Zhang H, Betenbaugh M. Integrated Genome and Protein Editing Swaps α-2,6 Sialylation for α-2,3 Sialic Acid on Recombinant Antibodies from CHO. Biotechnol J. 2017 Feb;12(2

Intact glycopeptide analysis of recombinant protein from CHO cells

The quality of recombinant glycoproteins including antibodies and other biologics is dictated by their glycan profiles. What is missing is how to analyze these glycans rapidly for process improvement and control applications. Conventional glycan analysis involves the release of glycans, which rarely captures the glycan site-specific information. Intact glycopeptide analysis in which glycans are retained on the peptide provides insights into the glycan structure and the glycosylation site information simultaneously. This information can reveal additional details about site occupancy and cellular glycosylation of proteins. Avoiding glycan release and some modifications and labeling steps in our intact glycopeptide analysis can result in a shorter sample preparation time than conventional glycan analysis methods. Compared to peptide mapping using LC-MS to decipher protein amino acid sequence in proteomics, this analysis focuses on glycopeptide profiling following protease-digestion. With the aid of LC-MS/MS, we are able to obtain targeted glycoprotein sequence information, glycan profiles and glycan distribution at specific sites. Here we present the application of glycopeptide analysis for model AMBIC and other proteins from CHO-GS and CHO-K1 cells. The site-specific glycosylation patterns of our model proteins EPO-Fc and EPO are characterized. Further, we examine the impact of media formulation and additives on the glycan profiles for these proteins. Please click Additional Files below to see the full abstract

Model-based analysis of N-glycosylation in Chinese hamster ovary cells

The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower likelihood of causing immunogenic responses. Because glycan structures are the product of the concerted action of intracellular enzymes, it is difficult to predict a priori how the effects of genetic manipulations alter glycan structures of cells and therapeutic properties. For that reason, quantitative models able to predict glycosylation have emerged as promising tools to deal with the complexity of glycosylation processing. For example, an earlier version of the same model used in this study was used by others to successfully predict changes in enzyme activities that could produce a desired change in glycan structure. In this study we utilize an updated version of this model to provide a comprehensive analysis of N-glycosylation in ten Chinese hamster ovary (CHO) cell lines that include a wild type parent and nine mutants of CHO, through interpretation of previously published mass spectrometry data. The updated N-glycosylation mathematical model contains up to 50,605 glycan structures. Adjusting the enzyme activities in this model to match N-glycan mass spectra produces detailed predictions of the glycosylation process, enzyme activity profiles and complete glycosylation profiles of each of the cell lines. These profiles are consistent with biochemical and genetic data reported previously. The model-based results also predict glycosylation features of the cell lines not previously published, indicating more complex changes in glycosylation enzyme activities than just those resulting directly from gene mutations. The model predicts that the CHO cell lines possess regulatory mechanisms that allow them to adjust glycosylation enzyme activities to mitigate side effects of the primary loss or gain of glycosylation function known to exist in these mutant cell lines. Quantitative models of CHO cell glycosylation have the potential for predicting how glycoengineering manipulations might affect glycoform distributions to improve the therapeutic performance of glycoprotein products

Elucidating dipeptide utilization and metabolism by CHO cells for improved cell culture performance

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Dynamic regulation of mitochondrial metabolism as a strategy to maximize mAb production in industrial CHO cell cultures

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Characterizing the effect of glutamine supplementation on asparagine and glutamine metabolism using 13C metabolic flux analysis

Upstream development efforts often focus on improved productivity. Among those efforts, improvements in medium formulations have translated into greater titers. To continue this historical trend, a better understanding of the cell metabolism is warranted for guiding efficient utilization of medium components to improve titer while minimizing byproducts. 13C Metabolic Flux Analysis (13C MFA) offers opportunities to study metabolic phenotypes by applying isotope tracers to estimate the intracellular fluxes through metabolic pathways. In this work, 13C MFA was applied to study the effects of glutamine supplementation by 13C parallel labelling of cultures with [U-13C]asparagine, [U-13C]glutamine and an a mixture of [U-13C]glucose with [1,2-13C]glucose. The study was focused on two metabolic states characterized by glutamine consumption in the early exponential phase and glutamine production in the late exponential phase of a fed-batch culture. To quantify individual metabolic pathway activity, metabolic flux maps were generated for the glutamine supplemented feeds compared to a control case with glutamine in the initial medium. The glutamine supplementation condition resulted in redistribution of the fluxes in the TCA cycle. Furthermore, measurements of the enrichment of cell protein indicate different allocations of the fed nutrients into generated biomass for the glutamine supplemented condition. Comparison between the early and the late exponential phases provided novel insights on how glutamine modulates CHO central carbon metabolism and supports the important role of glutamine as a major source of energy for cell proliferation. These findings contribute towards an improved characterization of the metabolism of industrial cells with useful implications for optimizing medium and feed development

Amino acid analysis to support a genome-scale nutrient minimization forecast algorithm for controlling essential amion acid levels in CHO cell cultures

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Metabolic engineering of high-productivity CHO host lines for biomanufacturing

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Design of experiments guided control of waste inhibitory by-products in high density CHO cell cultures

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