Best Practices for Development and Validation of Enzymatic Activity Assays to Support Drug Development for Inborn Errors of Metabolism and Biomarker Assessment.

Aberrant or dysfunctional cellular enzymes are responsible for a wide range of diseases including cancer, neurodegenerative conditions, and metabolic disorders. Deficiencies in enzyme level or biofunction may lead to intracellular accumulation of substrate to toxic levels and interfere with overall cellular function, ultimately leading to cell damage, disease, and death. Marketed therapeutic interventions for inherited monogenic enzyme deficiency disorders include enzyme replacement therapy and small molecule chaperones. Novel approaches of in vivo gene therapy and ex vivo cell therapy are under clinical evaluation and provide promising opportunities to expand the number of available disease-modifying treatments. To support the development of these different therapeutics, assays to quantify the functional activity of protein enzymes have gained importance in the diagnosis of disease, assessment of pharmacokinetics and pharmacodynamic response, and evaluation of drug efficacy. In this review, we discuss the technical aspects of enzyme activity assays in the bioanalytical context, including assay design and format as well as the unique challenges and considerations associated with assay development, validation, and life cycle management.

Neutralizing Antibody Sample Testing and Report Harmonization.

Immunogenicity testing and characterization is an important part of understanding the immune response to administration of a protein therapeutic. Neutralizing antibody (NAb) assays are used to characterize a positive anti-drug antibody (ADA) response. Harmonization of reporting of NAb assay performance and results enables efficient communication and expedient review by industry and health authorities. Herein, a cross-industry group of NAb assay experts have harmonized NAb assay reporting recommendations and provided a bioanalytical report (BAR) submission editable template developed to facilitate agency filings. This document addresses key bioanalytical reporting gaps and provides a report structure for documenting clinical NAb assay performance and results. This publication focuses on the content and presentation of the NAb sample analysis report including essential elements such as the method, critical reagents and equipment, data analysis, study samples, and results. The interpretation of immunogenicity data, including the evaluation of the impact of NAb on safety, exposure, and efficacy, is out of scope of this publication.

Multifaceted Approach for Quantification and Enzymatic Activity of Iduronate-2-Sulfatase to Support Developing Gene Therapy for Hunter Syndrome.

Mucopolysaccharidosis type II, commonly called Hunter syndrome, is a rare X-linked recessive disease caused by the deficiency of the lysosomal enzyme iduronate-2-sulphatase (I2S). A deficiency of I2S causes an abnormal glycosaminoglycans accumulation in the body's cells. Although enzyme replacement therapy is the standard therapy, adeno-associated viruses (AAV)-based gene therapy could provide a single-dose solution to achieve a prolonged and constant enzyme level to improve patient's quality of life. Currently, there is no integrated regulatory guidance to describe the bioanalytical assay strategy to support gene therapy products. Herein, we describe the streamlined strategy to validate/qualify the transgene protein and its enzymatic activity assays. The method validation for the I2S quantification in serum and method qualification in tissues was performed to support the mouse GLP toxicological study. Standard curves for I2S quantification ranged from 2.00 to 50.0 µg/mL in serum and 6.25 to 400 ng/mL in the surrogate matrix. Acceptable precision, accuracy, and parallelism in the tissues were demonstrated. To assess the function of the transgene protein, fit-for-purpose method qualification for the I2S enzyme activity in serum was performed. The observed data indicated that the enzymatic activity in serum increased dose-dependently in the lower I2S concentration range. The highest I2S transgene protein was observed in the liver among tissue measured, and its expression level was maintained up to 91 days after the administration of rAAV8 with a codon-optimized human I2S. In conclusion, the multifaceted bioanalytical method for I2S and its enzymatic activity were established to assess gene therapy products in Hunter syndrome.

Anti-drug Antibody Sample Testing and Reporting Harmonization.

A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication.

Fragmentation of biotinylated cyclic peptides.

Electrospray ionization coupled with tandem mass spectrometry (MS/MS) was used to determine the preferred binding site(s) of biotin NHS ester with a series of cyclic peptides with antibiotic properties. The peptides investigated are polymyxins, cyclic peptides produced by Bacillus polymyxa. In spite of the 1:1 stoichiometry used in the labeling reaction, multiple biotin molecules were incorporated into intact polymyxin peptides. Given the amine specificity of the activated biotin and the large number of amino acids with primary amines in the polymyxins, it was not clear by inspection which binding sites were more reactive than others. MS/MS was used to characterize the structure of the biotinylated peptides. MS/MS spectra of cyclic peptides often lead to ambiguous structure determinations due to the potential for multiple ring openings which result in the generation of multiple ion series. The MS/MS spectra of polymyxin peptides are especially difficult to characterize due to the lack of variety in their amino acids; however, the added complexity of the biotin aided the elucidation of the fragmentation pathways. MS/MS spectra of the species with biotin additions were used to rationalize the preferential binding sites of these molecules.