Multiplex analysis of muscle developing four monoclonal antibodies in human plasma with LC-HRMS

Abstract

The loss of skeletal muscle is a following defect of various diseases including muscular dystrophy, type 2 diabetes mellitus, and chronic cardiovascular disease. The purpose of current medication is to decrease muscle degeneration rate by treating nutritional supplements and appetite stimulants. However, the most plausible therapeutics for muscle wasting disorders is preventing the receptor signaling of the transforming growth factor beta (TGFβ) superfamily; myostatin, GDF-11, and activin A, because these are negative regulators of skeletal muscle growth. Inhibition of the myostatin receptor signaling pathway has shown to cause increased muscling, thereby, the abuse of inhibitors of this pathway as potential performance-enhancing agents in sports is prohibited by the World Anti-Doping Agency (WADA). In this study, a multiplex detection method for five prohibited monoclonal antibodies in doping; trevogrumab, landogrozumab, domagrozumab, garetosmab, and bimagrumab, which are designed for the inhibition of TGFβ superfamily, were developed. To reduce the abundant contaminants of plasma, ultra-low volume of plasma (5？L) was consumed with immuno-affinity purification by applying the protein G magnet beads. For the internal standards, dulaglutide, which is available to purify with protein G beads, was used to monitor technical errors during sample preparation. After capturing five antibodies and an internal standard, the tryptic digestion and LC-HRMS analysis allowed the detection of signature peptides from each antibody. As a result, the multiplex method was successfully validated with competent specificity, identification capacity, linearity, precision, and accuracy. All of five monoclonal antibodies had less than 1？g/mL of the limit of detection (LOD). Even though no anti-TGFβ superfamily antibodies have acquired clinical approval, the development of detection method for prohibited compounds is an imperative responsibility for doping control as an effective preventive measure. This presented approach can be applied or readily modified in numerous novel protein therapeutics for further target analysis.