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.
Approaches to Humanized Proteins
Numerous efforts have emerged to generate “humanized” biotherapeutics with glycosylation patterns similar to those found in native proteins. Monoclonal antibodies are a favorite target for this strategy, which reduces immunogenicity and may improve pharmacokinetics as well, but any glycosylated protein is fair game. Early in 2008, AlphaMed Pharmaceuticals introduced a humanized version of alpha 1-antitrypsin for treating emphysema and autoimmune diseases. AlphaMed produces the molecule in yeast and claims it is “virtually identical” to the human enzyme.
Glycosylation-related services are similarly significant toward the goal of humanized proteins. Glycart’s GlycoMAB® technology overexpresses glycosyltransferase genes in antibody-producing cells, so that the proteins produced are 50 times more attractive to immune effector cells (natural killer cells and macrophages) that kill antibody targets such as cancer cells and infectious microbes. GlycArt also has five therapeutic antibodies in development for cancer and autoimmune diseases.
According to the company, GlycoMAB expands the “therapeutic window” for antibody drugs, can improve the success rate of mAb therapeutics, provides life-cycle management through second-generation products, and improves cost of goods.
Yves Durocher, Ph.D., of the Institute for Biotechnology Research (BRI) at the University of Montreal, investigates the effect of cellular expression system on (among many other things) protein glycosylation. Dr. Durocher, who uses both CHO and HEK cells to create transiently and stably expressing clones, recently reported on the O-glycosylation of interferon.
Native interferon has one glycosylation sites, but the hormone is normally manufactured in nonglycosylating E. coli. Non glycosylated interferon clears rapidly from the body, so it is often PEGylated to improve its pharmacokinetics.
Dr. Durocher believes that O-glycosylation of interferon in mammalian cells, while not as long-lasting as the PEGylated version, would be an improvement over bacterially-generated interferon.
HEK, the human cell line most widely used in biomanufacturing, naturally produces human-like glycosylation patterns. In July 2008, Selexis entered a partnership with NRC-BRI (National Research Council of Canada’s Biological Research Institute) to offer a variant of HEK, NRC-BRI HEK293, as part of the company’s rapid cell line development services. HEK cells are one of the very few FDA-approved human cell lines.
Eli Lilly’s Xigrils®, a recombinant activated protein C sepsis drug, is manufactured using HEK293 cells. Lilly uses this platform because the drug requires full gamma carboxylation of glutamic acid residues, which HEK carries out but CHO cells do only with difficulty. Both cell types perform N-glycosylation.
The groundwork for large-scale HEK manufacturing was laid by Amine Kamen, Ph.D., at BRI. The program is expected to lead to rapid development of high-performing human cells as an option to CHO.