BioPharm

www.biopharminternational.com October 2018 BioPharm International eBook 17 Biopharma Laboratory Best Practices Glycan Analysis positions of glycosylation patterns and the specifications are equations describing contours of acceptability. BioPharm: What are the recom- mended technologies and/or pro- cedures for consistent analytical results? Jones (ProZyme): Out-of-the box glycan sample preparation k its can be used to prepare released N-glycans labeled w ith a f luo - rophore for separation by liquid chromatography or capillary elec- trophoresis, with detection by flu- orescence or mass spectrometry. These kits speed up the sample preparation with consistent results and may be automated. Fernandes (Ludger): You will invari- ably encounter inconsistent results when you're developing your gly- can analysis toolkit. These generally manifest as distortions in stoichiom- etry of analyte species, degradation of analytes, or fluctuations in signal intensity. It's important to remove all potential sources of such variability in a systematic way. The first step is to examine the symptoms then use tools such as Ishikawa diagrams together with a risk analysis matrix to (a) deter- mine likely problems and (b) help you prioritize your troubleshoot- ing process. Make sure you cast your net wide when performing root cause analysis—inconsisten- cies can arise from many sources. Next, ensure your QMS is appropri- ately designed for glycan work and is followed closely. Ludger's QMS, which we call 'Jura', is based on an ISO [International Organization for Standardization] 9001:2015 frame- work with additional modules for analytical work. These include ele- ments from [International Council for Harmonization] Q2(R1) for method validation (but with adapta- tions for multiple analytes encoun- tered in drug glycoprofiling), ISO 17025, OECD GLP, and GMP. The next considerations are the physico-chemical properties of your samples and the GCQAs you're mea- suring. You must choose methods that would be sufficiently robust and reliable for those samples and their GCQAs. For example, drugs such as EPO and FSH bear sialylated N-glycans that contribute to long serum half-lives. However, these are vulnerable to acid-catalysed de-siay- lation. So, you should eliminate or mitigate risky steps that involve low pH, heat, or drying (which can accel- erate de-siaylation) then check that the sialic acids are safe throughout the remaining steps. Rev iew t he qua l it y of you r reagents. Avoid assembling reagents from big bottles on the fly. You'll save money on the chemicals, but it will cost you much more because your results are likely to be unreli- able. Instead, use pre-made, properly QC'd kits and reagents. Ensure that your sample prep equipment and analytical instru- ments are well-maintained, up to spec, and properly calibrated. This includes things like changing the oil in vacuum pumps, ensuring integ- rity and cleanliness of seals for cen- trifugal evaporators, and calibration of liquid handlers. Ensure your protocols are straight- forward and, where possible, stan- dardized. If there can be variations in any steps, then give clear guid- ance on how to choose different pro- tocol routes. Make sure all your lab staff are for- mally trained and that they interpret the protocols uniformly. Lastly, ensure that you have appropriate operational checks. These include the use of qualitative and quantitative analytical stan- dards, system suitability standards, and a matrix of positive and negative controls. Widdowson (Thermo Fisher Scientific): Top-of-the-range instrumentation is at the heart of a robust and consistent analytical solution. Liquid chroma- tography is the recommended tech- nology for consistent glycan analysis, using hydrophilic interaction liquid chromatography (HILIC) separation of released and derivatized glycans. [Ultra-high pressure liquid chro- matography] instruments allow for faster and more efficient separations of analytes with excellent retention time precision and highly reproduc- ible peak areas. These systems also offer unrivaled sensitivity for low abundant analytes and their bio- compatibility maximizes long-term instrument performance. All of these are critical features for consistent analysis, particularly if the methods are to be used in QC laboratories. REFERENCES 1. P. Zhang, et al., Drug Discovery Today, 21(5); 740-765 (2016). 2. M. Thaysen-Andersen, et al., RSC Adv. 3; 22683-22705 (2013). 3. A. D. Eckard, et al., "Novel Automated and Manual N-Glycan Sample Preparation Workflow for High Throughput Screening for Characterisation of Biotherapeutics," American Pharmaceutical Review, December 22, 2017. 4. A. M. Goetze, et al., Glycobiol. 21(7); 949-959 (2011). 5. M. Butler and M. Spearman, Curr. Opin. Biotechnol. 30; 107-112 (2014). BP "For all types of biopharmaceutical glycoprotein, safety and efficacy are critical, and the specification limits should be set with this in mind." —Philip Widdowson, Thermo Fisher Scientific

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