February 1, 2009 (Vol. 29, No. 3)

Angelo DePalma Ph.D. Writer GEN

Glycosylation, in Particular, Will Play a Critical Role in the Approval of Biosimilars

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.

One of the driving forces behind transgenic animal development has been the desire for mammalian- or human-like protein glycosylation.

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.

PTMs and Biosimilars

PTMs, particularly glycosylation, will play a critical role in how biosimilars (also at times called follow-on biologics and generic biotherapeutics) are eventually approved.

The Food and Drug Act, under which small molecule drugs are approved, does not apply to biologicals. Neither does the Hatch-Waxman act of 1984, which created the generic drug industry (although earlier generic bio-drugs like insulin and human growth hormone were grandfathered under the drug law).

Since the idea of biosimilars arose more than a decade ago, the major stakeholders —with the exception of potential developers of generic biotherapeutics—have done their best to cloud the scientific issues surrounding these products. Since innovator companies are not obliged to share information with potential competitors, the latter are left to their own devices in designing processes that will faithfully recreate blockbuster biotherapeutics. But as everyone knows, in biotech the product is the process, and nowhere is this more evident than in PTMs.

Genzyme’s recent experience illustrates how seriously the FDA takes the issue of glycosylation and bioequivalence. Earlier this year, Genzyme was informed by the agency that the company’s Myozyme® enzyme replacement therapy for Pompe disease produced at 2,000 L scale was not equivalent to the drug manufactured in 160 L bioreactors. FDA asked for a separate Biologics License Application for the larger-scale product even though it is already approved in 40  countries, and had been shown to be “clinically effective and safe” in a 900 patient clinical study.

FDA based its decision on differences in glycosylation between products produced at the two scales. It is noteworthy that in public statements comparing products manufactured at the two scales, Genzyme merely said they were both safe and effective, avoiding any implication that the two were equivalent or bioequivalent, a term that bristles U.S. regulators. An FDA committee recommended in October 2008 that Myozyme 2000 be granted accelerated approval.

Katheryn Symank, an analyst with Frost and Sullivan, says that the Myozyme experience illustrates how far FDA will go to drive home the point that PTMs matter.

“FDA is still trying to invent an approval pathway for these drugs. Even the name they currently use, biosimilars, suggests inherent differences.” Symank is hopeful that the incoming administration, which promises national science initiatives, will help get the ball rolling with biosimilar approvals.

Evolution of Biosimilars

Glycosylation is a “huge scientific issue” in the evolution of biosimilars, says Bob Roth, M.D. Ph.D., medical director at the Weinberg Group, an international scientific and regulatory consulting firm. “It’s virtually impossible to make a protein with the same glycosylation patterns in two different processes. Even cell culture conditions can change glycosylation patterns.”

FDA is undoubtedly conscious here of other instances where seemingly trivial differences in PTMs cause serious side effects. One striking example is pure red cell aplasia, a rare, serious immune reaction to erythropoietin believed to arise from reactions to “unfriendly” glycosylation and/or sialylation patterns.

Harry M. Meade, Ph.D., svp for R&D at GTC Biotherapeutics, agrees that PTMs are at the heart of the biosimilars issue. GTC pioneered the production of therapeutic proteins in the milk of transgenic animals. “The PTMs in goat milk are, of course, mammalian, basically of the same structure as that found on human proteins, which puts them way ahead of plant sugars.”

As proof, he posits that there have never been adverse immune responses to recombinant proteins from milk (although issues have arisen with contaminating native animal proteins). Despite the fact that “goat milk proteins are just as human-like as those produced from CHO cells,” transgenics has yet to deliver on its promise, mostly due to regulators’ lack of familiarity with the technique and safety concerns.

Most experts agree that the impact of process on PTMs, and the potential of modifications to affect efficacy and safety, demands precisely the level of scrutiny that biosimilars are experiencing today from U.S. regulators. There are simply too many unknowns to act otherwise.

Dr. Roth feels that diligent oversight will eventually lead to a regulatory pathway for biosimilars that considers products on a case-by-case basis. “It will certainly not be the same as for generic drugs,” he says. Demonstrating what the differences are, quantifying them, and correlating them to clinical (safety or efficacy) outcomes will help the approval process.

“Developers will need to show which PTM differences are trivial before arguing which clinical development steps can safely be skipped.” It is almost certain, moreover, that any shortcuts will need to be justified empirically, in animal models and human subjects. “Presumably, for certain products, some early human testing might be avoided, and perhaps some of the late-stage testing as well, but nobody will be able to predict because nobody knows for sure.”

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