No abstract

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

Intracellular dialysis of NIH/3T3 cells with a commercially available anti‐ClC‐3 polyclonal antibody (Ab) for ≈30 min completely inhibited expressed guinea‐pig ClC‐3 currents (IgpClC‐3), while intracellular dialysis with antigen‐preabsorbed anti‐ClC‐3 Ab failed to affect IgpClC‐3. Anti‐ClC‐3 Ab was used as a selective probe to examine the relationship between endogenous ClC‐3 expression and native volume‐sensitive outwardly rectifying anion channels (VSOACs) in guinea‐pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti‐ClC‐3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes. In contrast, native VSOAC function was unaltered by antigen‐preabsorbed anti‐ClC‐3 Ab. It is suggested that endogenous ClC‐3 represents a major molecular entity responsible for native VSOACs in cardiac and smooth muscle cells and Xenopus oocytes. Anti‐ClC‐3 Ab should be a useful experimental tool to directly test the relationship between endogenous ClC‐3 expression and native VSOAC function, and help resolve existing controversies related to the regulation and physiological role of native VSOACs in a wide variety of different cells.

Endothelial cells express receptors for ATP and UTP, and both UTP and ATP elicit endothelial release of vasoactive compounds such as prostacyclin and nitric oxide; however, the distinction between purine and pyrimidine nucleotide signaling is not known. We hypothesized that UTP plays a more important role in endothelial mitogenesis and chemotaxis than does ATP and that UTP is angiogenic. In cultured endothelial cells from guinea pig cardiac vasculature (CEC), both UTP and vascular endothelial growth factor (VEGF) were significant mitogenic and chemotactic factors; in contrast, ATP demonstrated no significant chemotaxis in CEC. In chick chorioallantoic membranes (CAM), UTP and VEGF treatments produced statistically significant increases in CAM vascularity compared with controls. These findings are the first evidence of chemotactic or angiogenic effects of pyrimidines; they suggest a role for pyrimidine nucleotides that is distinct from those assumed by purine nucleotides and provide for the possibility that UTP serves as an extracellular signal for processes such as endothelial repair and angiogenesis.

Abstract. The American Association of Pharmaceutical Scientists (AAPS) biosimilar focus group on nonclinical and clinical assays has developed this manuscript to guide the industry on best practices and testing strategies when developing neutralizing antibody (NAb) assays for biosimilar programs. The immunogenicity assessment to biosimilar and originator drug products is one of the key aspects of clinical programs for biosimilars to demonstrate biosimilarity. Establishing that there are no clinically meaningful differences in immune response between a proposed product and the originator product is a key element in the demonstration of biosimilarity. It is critical to collect, evaluate, and compare the safety and immunogenicity data from the clinical pharmacology, safety, and/or efficacy studies especially when the originator drug product is known to have potential for immune-mediated toxicity. This manuscript aims to provide a comprehensive review and recommendations on assay formats, critical reagents, approaches to method development, and validation of the neutralizing antibody assays in extrapolation within the scope of biosimilar drug development programs. Even if there are multiple options on the development and validation of NAb assays for biosimilar programs, the type of drug and its MoA will help determine the assay format and technical platform for NAb assessment (e.g., cell-based or non-cell-based assay). We recommend to always perform a one-assay approach as it is better to confirm the biosimilarity using one-assay for NAb. If a one-assay approach is not feasible, then a twoassay format may be used. This manuscript will provide all the details necessary to develop NAb assays for biosimilars.