Post-translational modifications (PTM)—chemical transformations that occur after a protein’s translation from RNA—include numerous changes, some well known and others quite obscure. Modifications based on the addition of molecules or functional groups are the most apparent and probably the most significant in terms of biological activity. These include acylation/deacylation, amidation, methylation, phosphorylation, sulfation, oxidation, and even PEGylation (the synthetic addition of polyethylene glycol residues).
The best-known PTM is glycosylation, the addition of sugar residues to amino acids bearing amino or hydroxyl groups. Sugar moieties can be short, a few hundred Daltons, or longer and more branched, up to several thousand Daltons.
Although sugars are ubiquitous in biological systems, analyzing and generally working with these essential building blocks is not everyone’s cup of tea. Organic chemists learn to dread sugars for the dizzying diversity of individual residues, and particularly for their limitless combinatorial possibilities. Consequently, specialists in carbohydrate chemistry and analysis are in high demand.
Glycan analysis occurs after cleaving sugars with an endoglycosidase, which leaves a pool of carbohydrates of various sizes and compositions for offline analysis. Alternately, one can use trypsin to cleave peptide bonds and generate smaller peptides or individual amino acids, and analyze them with the sugars in place by LC/MS.
N-linked glycans also tend to follow rules of construction. It is possible to piece together their structures based on those rules and their fragmentation patterns in a mass spectrometer. Verne Reinhold at the University of New Hampshire pioneered these techniques and coined the term “glycome” to define the sugar makeup of an organism or family of proteins. In terms of complexity and combinatorial possibilities, the glycome is to the proteome in complexity what the proteome is to the genome—that is, up to an order of magnitude more complex.
Three-year-old Bluestream Laboratories specializes in analyzing biopharmaceuticals, including proteins and antibodies glycosylated at multiple sites. “Our customers want to know types and locations of glycans present in their molecules,” says Mario DiPaola, Ph.D., CSO. The vast majority are N-glycosylations. “Predicting where these will occur is relatively straightforward. The question is whether the site is actually occupied by a glycan.” Serine and threonine glycosylations, which occur on oxygen, are more difficult to predict.
Even within a well-defined biological product, one sees significant heterogeneity in the glycome of a protein drug, not only from glycosylation site to site, but among identical locations. “You can build a picture of the types of glycans you expect to see, or actually observe,” says Dr. DiPaola, “but it is difficult to see a single answer. You often obtain a range of structures, some quite unusual.” Glycan analysis takes anywhere from a few days for a well-characterized antibody, to several weeks for a new molecule or fusion protein. “Proteins have their own personalities and quirks.”
Dr. DiPaola advises sponsors of new protein drugs to learn all they can about glycosylation as early in development as possible, since regulators will be interested in glycan type, degree, location, and degree of sialylation. Glycosylation often controls a molecule’s activity, immunogenicity, and, in the case of sialic acid, its pharmacokinetics. Because a range of process conditions affect glycosylation, regulators look to patterns as an indicator of batch-to-batch consistency.