Recommendations on qPCR/ddPCR assay validation by GCC.

Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.

Recommendations on ELISpot assay validation by the GCC.

Gene therapy, cell therapy and vaccine research have led to an increased need to perform cellular immunity testing in a regulated environment to ensure the safety and efficacy of these treatments. The most common method for the measurement of cellular immunity has been Enzyme-Linked Immunospot assays. However, there is a lack of regulatory guidance available discussing the recommendations for developing and validating these types of assays. Hence, the Global CRO Council has issued this white paper to provide a consensus on the different validation parameters required to support Enzyme-Linked Immunospot assays and a harmonized and consistent approach to Enzyme-Linked Immunospot validation among contract research organizations.

Recommendations for the content and management of Certificates of Analysis for reference standards from the GCC for bioanalysis.

The 13th Global CRO Council (GCC) closed forum for bioanalysis was held in New Orleans, LA, USA on 5 April 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. While ICH M10 will cover requirements for reference standards, one of the biggest challenges facing the CRO community is the lack of consistency and completeness of Certificates of Analysis for reference standards used in regulated bioanalysis. Similar challenges exist with critical reagents (e.g., capture and detection antibodies) used for assays supporting biologics. The recommendations provided in this publication are the minimum requirements for the content that GCC members believe should be included in Certificates of Analysis for reference standards obtained from commercial vendors, sponsors and compendial suppliers, for use in regulated bioanalytical studies. In addition, recommendations for internal standards, metabolites and critical reagents are discussed.

GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline.

The 13th GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5th, 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the ICH M10 Bioanalytical Method Validation Draft Guideline and to build unified comments to be provided to the ICH.

Ligand binding assay critical reagents and their stability: recommendations and best practices from the Global Bioanalysis Consortium Harmonization Team.

The L4 Global Harmonization Team on reagents and their stability focused on the management of critical reagents for pharmacokinetic, immunogenicity, and biomarker ligand binding assays. Regulatory guidance recognizes that reagents are important for ligand binding assays but do not address numerous aspects of critical reagent life cycle management. Reagents can be obtained from external vendors or developed internally, but regardless of their source, there are numerous considerations for their reliable long-term use. The authors have identified current best practices and provided recommendations for critical reagent lot changes, stability management, and documentation.

Protective meningococcal capsular polysaccharide epitopes and the role of O acetylation.

Previous studies with group C meningococcal polysaccharide-tetanus toxoid (GCMP-TT) conjugates had suggested that the GCMP O-acetyl group masked the protective epitope for group C meningococci through steric hindrance or altered conformations. For this report, we confirmed this phenomenon and performed comparative studies with group Y meningococcal polysaccharide (GYMP)-TT to determine whether it might extend to other serogroups. The de-O-acetylated (dOA) polysaccharides (PSs) resulted in higher serum bactericidal activities (SBA) towards the O-acetylated (OA) meningococcal strains from the respective serogroups. High-resolution H-nuclear magnetic resonance spectroscopy at 500 MHz and competitive inhibition serum bactericidal assays were used to characterize the nature of the protective epitope. In head-to-head comparisons with OA PSs as SBA inhibitors, the dOA PSs provided 10 to 1,000 times better inhibition for GCMP in human and mouse antisera and 6 to 13 times better inhibition for GYMP in mouse antisera, using OA strains in all assays. In addition, the SBA for OA strains was highly correlated with dOA PS-specific immunoglobulin G (r=0.72 to 0.98) for both GCMP and GYMP. The results suggest that there may be a generalized role for the O-acetyl group to provide an epitope of misdirected immunogenicity for meningococcal PS capsules, enabling escape from immune surveillance. In addition to greater chemical consistency, the dOA forms of GCMP and GYMP conjugate vaccines endow greater immunologic competence to the PSs, rendering them capable of eliciting higher levels of functional antibodies toward the protective epitopes.

Specificity of the immune response to a modified group B meningococcal polysaccharide conjugate vaccine.

Antibodies to a modified group B meningococcal polysaccharide vaccine were examined for antigenic and functional specificities. Bactericidal determinants were investigated by using immunoaffinity columns and competitive inhibition of bactericidal activity in an in vitro killing assay. We conclude that nearly all of the vaccine-induced bactericidal activity is specific for the native polysaccharide.