Development of a Ligand binding assay for Detection of Neutralizing Antibodies against Monoclonal Antibody X in Human Serum

Abstract

INTRODUCTION: The small organic molecules as a drug for treatment of diseases had been widely used in pharmaceutical industries. The mode of action of small molecules is mainly through cell penetration, which resulted into a desired cellular response but it can also penetrates into the healthy cells which has many adverse side effects. Due to which biologics or biopharmaceuticals, have emerged as the next-generation of therapeutic molecules in the pharmaceutical industry like monoclonal antibody which has the desired cellular response when administered in the patient. Due to the large structure and more complexity, protein-based drugs have the potential to induce immune responses. Due to unwanted immune response, it can produce an antibody against a drug known as antidrug antibody (ADA).The assessment of antidrug antibody (ADA) is carried out by a multi-tiered testing approach in which screening, confirmatory and titration assays are done to identify ADA positive samples. Finally characterization assay is carried out, in which bioassays or ligand-binding assays are used to identify neutralizing antibodies. Among ADA, neutralizing antibody has been reported to reduce the efficacy of the drug by binding to the active site of it and also has many other complications like neutralization of the endogenous proteins. Therefore assessment of the Neutralizing Ab becomes important to understand the immunogenic response of a Drug/ Bio therapeutics. OBJECTIVE: 1. To Develop a Ligand binding assay for detection of neutralizing antibodies against Monoclonal Antibody X (drug) in Human Serum. 2. To check the performance of the assay by running different Pre-Validation Parameters like sensitivity, specificity, drug tolerance and accuracy & precision. DESIGN: To obtain the Neutralizing Ab from the serum, free from the complex with the Therapeutic Mab, acid dissociation and double step affinity purification method was employed and detection of the samples containing Neutralizing antibodies were checked with the help of an competitive ligand binding assay. After the development of the assay, different Pre-Validation Parameters were checked. RESULT: A ligand binding assay was successfully developed to detect the presence of neutralizing Abs produced against the therapeutic Monoclonal Ab X in XI normal human serum. The different Pre-Validation parameters such as Accuracy and Precision, sensitivity, selectivity and drug-tolerance of the assay were checked by performing various experiments. CONCLUSION: The Neutralizing antibodies were successfully isolated from the Antibody–MAb (drug) complex in the serum with help of acid dissociation and double step affinity purification with Monoclonal Ab X coated SepharoseTM beads. The Ligand Binding Assay (LBA) was sensitive enough to detect a low level of isolated neutralizing Ab i.e. 250ng/mL. The developed assay was passed successfully in different pre-validation parameters and was ready for validation and study samples analysis.