Critical Quality Attributes
Daryl Fernandes, D.Phil., founder and CEO of Ludger, led a workshop on comparability issues related to glycosylation throughout the life cycle of a biopharmaceutical product at the “Comparability for Biologics” meeting.
Ludger performs glycoprofiling for the biopharmaceutical industry and develops methods to measure and control glycosylation during biotherapeutics production. The company is also developing a framework for glycosylation analysis that combines HPLC and MS technologies for separation, quantification, and identification of carbohydrates.
Dr. Fernandes emphasized the importance of adopting Quality by Design (QbD) strategies that rely on the identification of critical glycosylation parameters early in the product life cycle. “A sensible interpretation of QbD will help determine significant differences in comparability of glycosylation,” he said.
Virtually every batch of a glycoprotein will have variation in the glycan composition, but some variation will have no effect on the safety or efficacy of the product. “QbD offers an opportunity to deal effectively with the complexity and variability of drug glycosylation,” he added. Each biotherapeutic product is different, so each requires an individualized model to define consistency.
Manufacturers of glycoprotein therapeutics should identify glycosylation critical quality attributes (GCQAs) early in the product life cycle, in Dr. Fernandes’ view. GCQAs can be used to determine which glycosylation features should be eliminated or modified to optimize a drug’s safety and efficacy profile. They can help define the ideal glycosylation profile of a biotherapeutic and guide critical decision-making, related for example to selection of an expression system or of a particular cell line or clone that will yield optimal glycosylation.
Identifying and prioritizing GCQAs for process design and small-scale process development, or in scale-down process modeling, can assist in predicting variability in glycosylation that may occur as a result of changes in cell-culture conditions during scale-up. Selection of glycoprofiling methods and analytical tools for monitoring glycosylation during process design and development and throughout production should take into consideration the most current regulatory guidelines.
M-Scan presented workshops on strategies and analytical techniques used for biopharmaceutical characterization and comparability studies at the recent “BioProduction” meeting in Barcelona and at the “BioAnalytical Congress” in Brussels.
A contract services provider that specializes in PTM characterization and protein and carbohydrate structural analysis, M-Scan utilizes MALDI-MS, electrospray (ES)-MS, and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) technology for glycosylation-site analysis and glycan profiling.
Regulatory requirements for glycosylation are “becoming more stringent,” said Fiona Greer, global director of quality assurance, a result of the growing recognition that the carbohydrate portion of a glycoprotein may affect the molecule’s ability to fold properly, its stability, its ability to communicate with surrounding cells, and various other factors that can impact the safety and efficacy of a biotherapeutic agent including its circulating half-life, function, and immunogenic potential.
As PTMs are highly product-specific, glycosylation analysis is necessarily product-dependent, and a glycoprofiling strategy must be developed for each individual biotherapeutic product. M-Scan designs a step-wise analytical strategy that includes MS-based and non-MS methods to provide an overview of the total carbohydrate structure of a glycoprotein.
Monosaccharide composition is identified and quantified by MS and/or HPAEC-PAD, with particular attention paid to sialic acid analysis. Gas chromatography (GC)-MS with methylation provides information about the linkages of individual sugars, and MALDI-MS and electrospray LC-MS techniques can be used for glycan population analysis, to obtain structural data, and to identify sites of glycosylation. For a more in-depth analysis, the company relies on MS-MS techniques to perform antennal profiling analysis.