Chinese hamster ovary mutants for glycosylation engineering of biopharmaceuticals

Abstract

The Chinese hamster ovary (CHO) cell has long been the cell of choice for the biopharmaceutical industry. The reasons for this are partly historical and partly because CHO cells produce glycoproteins with a glycan complement similar to that synthesized by human cells. The CHO-DUKX-B11 line was established early in the development of the biotechnology industry and has the advantage of allowing selection for high expression of transgenes. In addition, CHO cells glycosylate glycoproteins with a subset of glycans typical of human cells and essentially lacking antigenic sugar residues like α(1,3)Gal, β(1,2)xylose or N-glycolylneuraminic acid, typical of rodent or plant cells. In addition, CHO cells have a pseudo-haploid genome allowing stable mutants with desirable properties to be readily isolated. It is becoming increasingly evident that the manipulation of glycosylation pathways to produce secreted glycoprotein therapeutics with glycans appropriate for optimal half-life, cellular targeting and functional activity, is very important. This editorial will discuss CHO glycosylation mutants that are currently available, and the potential for glycosylation engineering to produce optimal biopharmaceuticals in the future. CHO cell mutants with altered glycosylation were initially isolated by selection for resistance to the cytotoxicity of plant lectins [1]. Cells selected for resistance to one lectin, were often resistant to others with related glycan binding properties, and also hypersensitive to lectins recognizing sugars that became newly terminal due to the nature of the glycosylation defect. By comparing resistance to a panel of plant lectins, lectin-resistant mutants could be grouped into lectin resistance phenotypes. In addition, somatic cell hybridization was used to define genetic complementation groups. Once genes encoding glycosylation activities were cloned, mutations giving rise to each complementation group were identified and numerous allelic series defined. There are now a large number of CHO glycosylation mutants with known genetic and biochemical defects that may be used to engineer glycoproteins in order to optimize their properties. These include mutants that generate glycoproteins with modified N-glycans and/or O-glycans [2], glycosaminoglycans [3] or glycophosphotidylinositol-linked glycans [4]. However, for these mutants to be useful in the industrial level production of glycoprotein therapeutics, a specific glycosylation mutant must be employed from the very beginning of engineering a cell line for high productivity. While it is often stated that existing CHO mutant lines are not high producers, this may Pamela Stanley Department of Cell Biology, Albert Einstein College of Medicine, New York, NY 10461, USA pamela.stanley@einstein.yu.edu “...manipulation of glycosylation to enhance the properties of glycoproteins can be performed at any stage of the development of a production cell line using CRISPR/Cas9 technology.” SPECIAL FOCUS y An ‘omics approach to Chinese hamster ovary-based pharmaceutical bioprocessing