Isotope labeling experiments (ILEs) and (13)C flux analysis provide actionable information for metabolic engineers to identify knockout, overexpression, and/or media optimization targets. ILEs have been used in both academic and industrial labs to increase product formation, discover novel metabolic functions in previously uncharacterized organisms, and enhance the metabolic efficiency of host cell factories. This review highlights specific examples of how ILEs have been used in conjunction with enzyme or metabolic engineering to elucidate host cell metabolism and improve product titer, rate, or yield in a directed manner. We discuss recent progress and future opportunities involving the use of ILEs and (13)C flux analysis to characterize non-model host organisms and to identify and subsequently eliminate wasteful byproduct pathways or metabolic bottlenecks.

Refers to the functionality of the protein in a pharmaceutical product. The extent, type and consistency of glycosylation are used synonymously with quality here, as it is one of the most commonly studied properties affecting protein quality. Glycosylation: Post-translational modification undergone by proteins in which sugar chains (glycans) are added to the polypeptide chains of the proteins. Without proper glycosylation, the protein will not function correctly. Disulfide bonds: Covalent bonds between two cysteine residues that maintain the 3D structure of a protein. Intrachain disulfide bonds link cysteine residues of the same peptide chain whereas interchain disulfide bonds link separate peptide chains together (e.g., the light and heavy chains of an antibody). Galactosylation: Step preceding sialylation. The addition of a galactose molecule to the terminal site of the oligosaccharides. The terminal galactose provides the binding site for the terminal sialic acid added during sialylation. Sialylation: Final step in glycosylation where sialic acid groups are added onto the glycans in the terminal position. This step is crucial for the functionality and half-life of biotherapeutic proteins and their acceptance by patients.