Of the essential post-translational modifications (PTMs) that proteins undergo before their synthesis is complete, glycosylation is arguably the best known. The medical and economic significance of monoclonal antibodies and other glycoproteins has spurred a resurgence of interest in glycans, particularly in their analysis and ways to direct their biosynthesis in desirable ways.
The U.S. Pharmacopeia (USP), for example, develops both documentary and physical standards for analyzing and characterizing glycoproteins. The standards are critical for assuring the purity and quality of these products.
USP defines standards as “horizontal” if they are not linked to a particular product but to a procedure in a General Chapter, for example, the bacterial endotoxins test in USP <85>. “Vertical” standards are linked to a specific product, i.e., a monograph for a drug substance or product and associated reference materials.
By the time you read this, USP will have published a new standard, “USP Chapter <1084>, Glycoprotein and Glycan Analysis—General Considerations,” in Pharmacopeial Forum (PF36(2)). The standard, which will be subject to a 90-day public comment period, will contain general guidance for glycan and glycoprotein analysis.
Future, procedure-driven tests and chapters will be linked to physical reference standards currently in development. In that regard, Chapter <1084> is an introductory or “umbrella” document. USP has pursued a similar approach with offerings on nucleic acid based techniques (e.g., USP General Information Chapters <1125>-<1130>, now official).
Tina S. Morris, Ph.D., vp for biologics and biotechnology at the UPS, describes the chapter as an “overview on how to approach the analysis of a glycosylation product in light of regulatory expectations.” For example, the chapter will use flow charts or “decision trees” that guide users through analytical options, depending on whether the focus is an intact glycoprotein or cleaved glycans.
According to Dr. Morris, USP enjoys “very good” relations with FDA, which routinely sends liaisons to USP standards committees. Chapter <1084> was written by an ad hoc advisory panel composed of experts from industry, academia, the U.K.’s National Institute for Biological Standards and Control, and USP. “Regulators usually provide very helpful input,” she says.
PTM research is increasingly regulation-driven, says Elizabeth Higgins, Ph.D., CEO of GlycoSolutions, which provides analytic support for developers of glycoproteins. As evidence mounts that glycosylation affects glycoprotein (particularly mAb) safety and efficacy, biopharmaceutical manufacturers use glycosylation as a surrogate, to monitor lot-to-lot consistency. “Companies are increasingly interested in measuring galactosylation or core fucosylation of the Fc glycosylation,” observes Dr. Higgins.
GlycoSolutions’ business is growing, in no small part due to interest in biosimilars. Knowing a priori which glycans confer optimal efficacy and safety has been a goal of glycoprotein research, but hard-and-fast rules that apply to all classes of protein have been difficult to formulate due to a scarcity of applicable data. For biosimilars, however, developers are aiming for glycosylation patterns that are similar to the existing drug.
GlycoSolutions worked on 20 projects in 2008, 25 in 2009, and expects 30 customers in 2010. “Biosimilars represent our largest growth area right now. We’ve also been expanding our services,” Dr. Higgins says. GlycoSolutions’ workload last year involved mAbs (29%), non-mAb glycoproteins (17%), biosimilars (33%), one gene- therapy project, and one biofuel project. Ninety-four percent of their projects involved development-stage proteins.
Pauline Rudd, Ph.D., a professor at the NIBRT Dublin-Oxford Glycobiology Laboratory, has spent much of the past decade developing chromatographic methods for analyzing the glycosylation of therapeutic proteins. Her technique involves cleaving the sugars enzymatically and attaching fluorescent labels to the glycans. This is followed by analysis by HPLC and fluorescence detection.
Dr. Rudd employs dedicated computer software to compare peaks with entries in a glycan database, which she has constructed by cleaving dozens of glycosylated proteins and analyzing the glycans released. The software/database analyze the exoglycosidase array digestions for final structure assignment, which includes monosaccharide sequence and linkage information.
Recently, Dr. Rudd’s group acquired liquid-handling capability for automating the analysis, which she says takes around eight hours from taking a sample from cell culture to data analysis. The idea is to complete the analysis within the time frame of one biomanufacturing shift.
This standard analysis will not reveal where the glycans were originally located on the protein. That requires site analysis, an extremely time- and labor-intensive process. “Site analysis isn’t easy. You really need a good reason to justify carrying one out,” says Dr. Rudd.
Two interesting applications of Dr. Rudd’s technique are quality by design and process analytic technology. Due to the complexity and time involved in the analysis (Dr. Rudd claims a five-hour turnaround for immunoglobulins), it is not suitable for real-time quality monitoring. However, it is rapid enough to enable tweaking conditions or for making changes to feeds during the time frame of a several week-long cell culture.
In 2008, Dr. Rudd’s group at NIBRT entered into a research collaboration with Lilly and in 2009, a collaboration with Roche was set up for cell-culture monitoring providing Roche with access to NIBRT’s glycan database and software. Also in 2009, the institute announced a research collaboration with the FDA on the characterization of glycosylation on theraputic enzymes under different bioprocessing conditions.