Rapid in-solution equilibrium approach to KD determination of biotherapeutic drug candidates
During our latest webinar, Johan Engström, Senior Scientist at Gyros Protein Technolgies gave a short introduction to Gyrolab technology and how you to set up and evaluate classical in-solution equilibrium experiments for determination of sub-nanomolar KD’s and active concentrations of interactants. Johan also gave examples of determinations of picomolar affinities for several marketed TNFα antagonists.
Bioanalytical laboratories often need to quickly develop robust immunoassays to support studies with tight timelines. Immunoassay performance is governed by many factors, such as reagent concentrations and sample dilution, and the classic experimental approach to improving assay performance involves changing one factor at a time. This approach is time consuming, misses important interactions between factors, and seldom leads to an optimal assay. There is an alternative, very powerful approach that has been used in many industries and applications to optimize processes – Design of Experiments (DOE). DOE has been successfully applied to immunoassay development by a number of research groups, including those using Gyrolab technology.
Pharmacokinetic (PK) studies of therapeutic proteins in early preclinical development demand great flexibility in bioanalytical methods. This is a real challenge during lead candidate selection when, for example, limited amounts of specific reagents must be labeled for immunoassays. Gregor Jordan and his colleagues at the Pharma Research & Early Development (pRED) department at Roche in Munich, Germany saw the advantages of transferring their ELISAs to the Gyrolab™ platform – low reagent consumption, semi-automation and fast assay turnaround – but wanted to go one step further in conserving critical resources. They therefore developed a generic detection reagent that would allow them to use the same antibody reagents on both ELISA and Gyrolab platforms.
Determining the immunogenic potential of a therapeutic antibody is a regulatory requirement during clinical development since anti-drug antibodies (ADA) can cause undesirable effects, ranging from loss of drug exposure and efficacy to serious adverse events. Justine Brose and her colleagues at Novimmune SA, Geneva, Switzerland evaluated four platforms to determine which platform and sample pre-treatment procedures could deliver a fit-for-purpose assay for ADA analysis to determine the immunogenicity of the therapeutic antibody Novimab. Of the platforms tested (ELISA and Gyrolab, Meso Scale Discovery, and AlphaLISA platforms) only Gyrolab and MSD platforms met all of their pre-specified assay requirements.
It is not unusual for a pharmacokinetics assay used early on in a study proves to lack the robustness needed to see the drug candidate through to more advanced clinical studies. A team at Genentech experienced just that and they solved the problem by transferring their ELISA to the Gyrolab platform.
Therapeutic proteins have revolutionized the treatment of many diseases and have a promising future with more than forty monoclonal antibodies and Fc-fusion therapeutics already on the US market and over 400 more in clinical development stages. Therapeutic proteins are, however, hit hard by a high attrition rate, mainly to poor efficacy, which results in only one in ten making it to market. Increasing the success rate relies on many factors, including the study of absorption, distribution, metabolism and excretion (ADME), as Jay Tibbitts at UCB Celltech, UK, and his coauthors point out in their extensive review on the subject.
High-throughput immunoassays underpin efficiency and productivity
Antibody-Drug Conjugates (ADCs) offer great potential in directing therapeutic agents directly to the target, but the complexity of their structure and dynamics in vivo present many bioanalytical challenges. In a recent review article, Seema Kumar and her colleagues at Pfizer Global R&D in the USA present a comprehensive analysis of how Ligand Binding Assays (LBAs) can be used in ADC analysis in preclinical studies.
While many preclinical and clinical studies require analysis of plasma, serum or blood, some involve more challenging matrices, such as vitreous and aqueous humor, tears, synovial fluid, and bronchoalveolar lavage. These matrices often have high viscosity, high protein concentration and low analyte concentration. Added to that, the samples are almost invariably very small, expensive and difficult to source. Analytical methods must therefore match the matrix’s complex qualities and also the small volumes available. 
