Development of a Ligand binding assay for detection of Neutralizing antibodies against Monoclonal Antibody X in Human Serum

Abstract

INTRODUCTION: Biotherapeutics or large molecules such as Monoclonal Abs when administered in the patient can generate unwanted immune response by the production of neutralizing Abs.The Assessment of the Neutralizing Ab becomes important to understand the immunogenic response of a Drug/ Biopharmaceutic for which the Ligand Binding Neutralizing Assay can be developed. With the help of Nab assay,the presence of Neutralizing Ab production against the Therapeutics can be detected. HYPOTHESIS: 1. Neutralizing Abs maybe present complexed with Therapeutic MAb in the serum. The acid dissociation of the samples will dissociate the Therapeutic Monoclonal Ab X from the Neutralizing Abs (Immune complex) in the serum. Further by Affinity purification, the Neutralizing Ab specifically will be obtained in the free form from the matrix. 2. The Developed Ligand Binding Neutralizing Ab Assay will show detect Neutralizing Abs in the patient serum by preventing the binding of Therapeutic Monoclonal Ab X with VEGF, thereby confirming the presence or Absence of Neutralizing Ab in patient with the help of this assay. OBJECTIVE: 1. To Develop a Ligand binding assay for detection of neutralizing antibodies against Monoclonal Antibody X in Human Serum. 2. To check the stability and precision of the assay by running different Pre-Validation Parameters. DESIGN: To obtain the Neutralizing Ab in the serum free medium monoclonal Ab X coated sepharose beads were prepared and the double time affinity purification method was employed to prepare the samples which further can be analyzed with the help of ELISA. And once the Assay was developed the 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 normal human serum. The stability of the assay was proven with the help of different Pre-Validation parameters such as Accuracy and Precision, sensitivity, Selectivity and Drug-tolerance. CONCLUSION: The Developed Ligand Binding Assay for the detection of Neutralizing Ab which is produced against the therapeutic Monoclonal Ab X was found to be having the sensitivity of 300 ng/ml and the successful Matrix free Isolation of Neutralizing Ab was obtained by the double time affinity purification and Acid dissociation method.