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www.biopharminternational.com July 2021 BioPharm International eBook 5 Challenges with respect to func- tional analyses often revolve around proper, timely, development of the nec- essary cells and antibodies, Fitzgibbons adds. Similarly, extractables and leach- ables assessments require a large panel of extraction and analytical techniques to be undertaken to assure detection, identif ication, and quantitation of a potential toxic, unknown material. Mea nwh i le, st r uc t u re –f u nc t ion assessments have been known to be c ha l leng i ng b ec au s e t he a mou nt of material available to address all orthogonal methods is often limited, says Pedro Morales, director, Catalent Biologics. "For example, it becomes challenging to isolate and analyze variants that are present in only small a mou nts w it h i n t he f rac t ionated sample," he says. Mora les a lso e x pla ins t hat t he e x t rac t ion a nd /or pu r i f icat ion of v a r ia nts needs to be done wh i le avoiding the introduction of addi- tional—or exacerbation of—post-trans- lationa l modif ications (P T Ms) or impacting the material so as to create aggregates. He says that experimental design should include appropriate con- trols needed for analysis in parallel to the analyte(s) of interest. "It is always challenging to develop a robust func- tional assay that will ensure a solid structure–function correlation that can be unequivocally determined," he states. Jana Hersch, scientif ic consultant, Genedata, adds that correct predic- tion of how a complex biological will behave in vivo remains the biggest challenge during the development of complex biotherapeutic molecules. She points out that antibody-drug conju- gates (ADCs), bispecif ics, and even engineered cell-surface receptors, such as chimeric antigen receptors (CARs), rely on the scientists' ability to assess how these molecules will behave once inside the patient. "It is important to predict not just whether they bind with the desired aff inity and specif icity to their tar- get in vitro, but also that they bind in the right tissues, or that they have the appropriate level of clearance from the body to achieve the desired therapeu- tic effect and cause minimal harm," Hersch says. She also explains that many different versions of a complex biological molecule are often tested in iterative pharmacokinetic and phar- macody namic ana lyses before the preferred format as well as the f inal formulation are selected. ESTABLISHING ANALYTICAL STUDIES Campbell Bunce, chief scientific offi- cer at Abzena, emphasizes the point that all drug developers should look to establish the right set of analytical methods and workf lows to assess the developability of their individual drug candidate and ensure that those they take forward have a reduced risk of inherent liabilities at an early stage. He states a holistic approach that applies design of experiment principles is rec- ommended to capture a wide range of parameters to focus across several char- acteristics, such as function, manufac- turability, immunogenicity, and safety. "For example, through developability assessment, methods can be applied that will provide insights into the rela- tive stability of the biological molecules and allow them to be ranked against comparators to help select the best lead candidate for development," he says. Bunce notes that often, novel bio- therapeutic candidates are unique with little information that can be drawn on to help develop the right assays and comparative assessment parameters, as can be done with antibodies, where a signif icant amount of historical data exists. Certain aspects of novel bio- logic molecules can be pieced together, however, directed by experience-led trial and error to generate developa- bility profiles that lead to better out- comes, he explains. "There may be occasions where lia- bilities cannot be designed out using protein engineering techniques and thus, need to be managed through dif- ferent approaches. Consequently, other mitigating options can be evaluated and applied," Bunce states. Biologics, for example, may be affected by one or more degradation pathways that can be evaluated and addressed through the formulation development process to maximize the stability of the biologic drug candidate. Because of the complexity of bio- logical molecules, a panel of analytical methods is used to analyze the thera- peutic proteins for lot release to ensure consistent product quality, safety, and eff icacy, adds Gang Huang, senior v ice-president, ana ly tica l sciences and clinical qualit y control, Wu X i Biologics. Though enormous work is done in the clone selection and process development stages, it is still expected that a final drug product is composed of a mixture of hundreds to thousands of variants that differ in PTMs and higher order structure, he emphasizes. As a result, state-of-the-art analytical methods have been used to thoroughly characterize various PTMs and degra- dation pathways of these proteins. Bakewell notes that mass spectrom- etry (MS)-based analyses are a criti- cal component of determining primary str uct ure for conjugated proteins, monoclonal antibodies (mAbs), and cell and gene therapy (CGT) products. He further explains that intact mass and peptide mapping-based MS meth- ods allow for confirmation of protein sequence as well as identif ication of any disulfide bonds, oxidation/deam- idation events, glycosylation patterns, and PTMs, all of which are key struc- tural critical quality attributes (CQAs). Meanwhile, information about sec- ondary and tertiary structure can be determined through techniques such as circular dichroism, isothermal calo- rimetry, size exclusion chromatography, and multi-angle light scattering. "Complementa r y to t hese pro- tein-level analyses, sequencing of the Biopharmaceutical Analysis Analytical Evolution