Ligand Binding Methodologies for the Assessment of Pharmacodynamics and Immunogenicity of an mRNA-LNP Therapeutic Encoding for Multiple Proteins

This case study describes the bioanalytical and immunogenicity methodologies employed to support the development of an mRNA-LNP therapeutic encoding multiple distinct proteins, designed to work synergistically to achieve a distinct therapeutic effect. The discussion emphasizes bioanalytical method development, assay lifecycles, and the challenges encountered in development for this complex modality. The development of surrogate reference materials was essential for enabling accurate evaluations of the mRNA-derived proteins. This process involved expressing the proteins through cell culture and purification then generating anti-idiotypic antibodies to serve as critical reagents for both assay types. Due to the multi-domain nature of the proteins, antibodies specific to individual functional domains were created, along with purified versions of these domains to support domain-specific assessments in the immunogenicity assays. These efforts required extensive planning and development, ensuring reliable, specific assays for evaluating therapeutic performance and immunogenicity. The development of mRNA-derived protein assays required ultra-sensitive methods capable of detecting low pg/mL levels of protein. Multiple anti-idiotypic antibodies were screened and carefully selected to achieve this sensitivity while minimizing cross-reactivity among the co-expressed mRNA-derived proteins. The assays were designed to quantify intact, full-length proteins with high specificity, ensuring reliable measurements of the therapeutic's pharmacodynamic effects. In immunogenicity evaluations, particular focus was placed on detecting and characterizing anti-drug antibodies (ADAs) against the mRNA-derived proteins, which contained multiple functional domains. The structural intricacy of these proteins required a multi-tiered approach for detecting and confirming ADA specificity, with an emphasis on assessing antibodies targeting individual functional domains. These methods provided a robust evaluation of immunogenicity risks, supporting a comprehensive understanding of therapeutic safety. This study underscores the value of flexible, data-driven strategies in overcoming the unique challenges of mRNA therapeutics, contributing to their clinical success, and the evolution of next-generation bioanalytical methodologies.