Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells

• Published in: Biotechnology and bioengineering 2017-12, Vol.114 (12), p.2848-2856
• Main Authors: Chung, Cheng‐yu, Wang, Qiong, Yang, Shuang, Ponce, Sean A., Kirsch, Brian J., Zhang, Hui, Betenbaugh, Michael J.
• Format: Article
• Language: English
• Subjects: Amino acid composition; Amino acids; Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - genetics; antibody engineering; Biotechnology; Chinese hamster ovary cells; CHO Cells; Chromosome Mapping - methods; Combinatorial analysis; Combinatorial Chemistry Techniques - methods; Cricetulus; CRISPR/Cas 9 genome editing; Disruption; Fc receptors; Galactose; Galactose - metabolism; Genetic Enhancement - methods; Genomes; Glycan; glycoengineering; Glycosylation; Immunoglobulin G; Immunoglobulin G - genetics; Immunoglobulin G - metabolism; Immunoglobulins; Monoclonal antibodies; Polysaccharides; Protein engineering; Protein Engineering - methods; Proteins; Quality management; Toxicity
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.26375

One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities. In this study, the disruption of two α‐2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms standards for specific biomedical and biotechnology applications. This study focuses on producing IgG with glycoprofiles containing predominately biantennary structures terminating with two galactose residues (G2F and G2). Wild‐type IgGs expressed from CHO‐K1 cells typically produce glycoprofiles with zero or one galactose residues (G0F/G1F). To increase IgG galactosylation, we first generated IgG variants with one or more amino acid substitutions in the Fc region. These IgG variants were expressed in α‐2,3 sialyltransferases knock‐out CHO cells, which then exhibited a significant increase in the G2F/G2 glycoform content.