BioPharm

14 BioPharm International eBook October 2018 www.biopharminternational.com Biopharma Laboratory Best Practices Glycan Analysis glycoform heterogeneity. The sec- ond is glycosylation site complex- ity, so drugs like EPO have several glycosylation sites, each with its own pattern of glycans. And the third is the inherent structural complexity of the glycans themselves. W i d d o w s o n ( T h e r m o F i s h e r Scientific): Biopharmaceuticals are produced from living cells and, so, are highly susceptible to changes in the production process. The com- bination of the cell line and the specific growth conditions used is primarily responsible for the pro- ductivity of the cells and the titer of the final product. For glycoproteins, this process is much more complex. Since glycosylation is a non-template driven process, the relative expres- sion and activity of the specific host cell glycosylation enzymes is respon- sible driving the final glycan com- position and structure. With this in mind, even a subtle change in growth conditions can ultimately have a significant effect on the safety and efficacy of the final product (1). BEST PRACTICES IN GLYCAN ANALYSIS BioPharm: What are best practices for transferring glycan analysis from site to site? Fernandes (Ludger): Transferring your glycan analysis toolset and the knowledge and skills to use it effectively is not trivial. It requires a practical, well-thought-out plan and real cohesion between the people involved. Our approach, when trans- ferring glycan analysis workflows to partner labs such as clients or collab- orators, is to start with co-develop- ment of the tech transfer plan. This involves checking, and if necessary upgrading, lab infrastructure, analyt- ical platforms, other equipment, and resources for the target lab as well as implementing a comprehensive training program. Standardization is important. The workflows we transfer will have been developed and validated up to GMP level so that they work for us as well as partners without needing signifi- cant changes. Full training and support would cover the following: strategy for the optimization, measurement and control of the glycosylation of our partner's drug, the principles of the analytical methods, technological workflows, analysis and interpreta- tion of results, practicals, adaption of workflows for the target site (if needed) then re-testing, and finally, formal validation according to the QMS [quality management system] of the new site. Jones (ProZyme): Method transfer can be challenging. Using commer- cial sample preparation kits may simplify that aspect, together with the appropriate documentation, training, and change control. For instrumentation, glycan standards and well-characterized glycoprotein controls may be used for system suit- ability testing and instrument quali- fication. W i d d o w s o n ( T h e r m o F i s h e r Scientific): A fully optimized glycan analysis solution is essential for suc- cessful transfer between sites. Due to the number of steps involved, there are many sources of potential variation that could lead to incon- sistencies in results. Fully standard- ized sample preparation workflows and analytical platforms (chroma- tography columns and instrumenta- tion) remove many sources of this variation and should be matched between sites as closely as possible. In addition, a well-characterized panel of reference and suitability standards, which are available to all sites involved in the transfer, and an agreed set of robust acceptance crite- ria will lead to the greatest chance of successful transfer. BioPharm: What are best practices for sample preparation? W i d d o w s o n ( T h e r m o F i s h e r Scientific): Sample preparation for gly- can analysis is crucial for ensuring consistent and reproducible results. This is particularly important for methods that are to be performed in the [quality control] laboratory. There are a number of steps involved in the enzymatic release, fluorescent labeling, and purification of glycans, and each step must be highly opti- mized. Protein denaturation, which is particularly important for mono- clonal antibody deglycosylation, must be reproducible to ensure run- to-run consistency. Incubation steps should be as short as possible and at as low a temperature as possible, without having a detrimental effect on efficiency, to prevent the loss of labile components such as sialic acid (2). Sample clean-up and purification prior to analysis should not be biased towards individual glycan structures or glycan types (3). Fernandes (Ludger): The quality of your sample preparation greatly impacts the reliability of your glycan analyses. The main types of analytes for biopharmaceutical glycoprofiling are intact drug glycoforms or frag- ments thereof, glycopeptides, N- and O-glycans, and monosaccharides. "Transferring your glycan analysis toolset and the knowledge and skills to use it effectively is not trivial." —Daryl Fernandes, Ludger Ltd

• Cover