Metabolic and Process Engineering to Control Glycan Structures for Biopharmaceuticals Produced in Cultured Mammalian Cells

Biopharmaceuticals (also known as biologics) play an increasing role in the treatment of a wide range of diseases, particularly cancer, autoimmune diseases, and infectious diseases. In 2017 and 2018, 11 of the 15 best-selling drugs worldwide were biopharmaceuticals, generally produced in cultured mammalian cells. The vast majority of biopharmaceuticals are glycoproteins, in which the attached glycan moieties play important and often critical roles in controlling activity, clearance, and immunogenicity. In addition to glycoproteins, carbohydrates, particularly glycosaminoglycans (GAGs) such as heparin, the most widely used anticoagulant drug in the world, are critically important biopharmaceutical products. Many blockbuster biopharmaceuticals such as adalimumab (Humira), trastuzumab (Herceptin), and bevacizumab (Avastin) have recently come off patent, providing an opportunity for production of biosimilar versions by companies other than the innovator. In addition, there is increasing interest in producing glycosaminoglycans from cultured mammalian cells, rather than the current purification from animal tissues with the attendant risks of contamination by adventitious agents and adulteration due to isolation under non-cGMP conditions. To successfully produce biosimilars and bioengineered GAGs, control of glycan composition and structures is critical. This control is challenging as glycan synthesis is a nontemplated process, which is controlled by a complex collection of factors including the glycoprotein being synthesized or the core protein to which the GAG is attached, production host, enzyme activities, and bioprocess conditions. In this chapter, we review some basics of glycoprotein and GAG biosynthesis with a particular focus on our current understanding of how glycan structures are controlled in vivo. We then review studies in which glycan structures for recombinant proteins and glycosaminoglycans have been optimized by cell line metabolic engineering and bioprocess manipulations. In particular, CRISPR has permitted exquisite editing of host cells, allowing tailored production of glycan structures, facilitating the production of biosimilars and bioengineered GAGs as well as setting the stage for “biobetters” in which improved functionality is obtained by glycoengineering. However, complicated new products including bispecific antibodies, intricate Fc-fusion proteins and molecules not yet envisioned may necessitate new hosts and further advances in glycoengineering.