Development and implementation of analytical tools to analyze the glycosylation profile of monoclonal antibodies and correlate with their function

• Grant number: 25/06732-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: August 01, 2025
• End date: December 31, 2027
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Giuseppe Palmisano
• Grantee: Thais Mingatos de Toledo
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 24/02146-7 - Discovery and validation of de novo generated endogenous biomarkers for the diagnosis of Chagas disease, AP.R

Abstract

Biologics have become a rising treatment for many diseases, such as cancer, hormone repositioning, vaccines, and many others. The increased use of biologics raised attention to drug spending in this treatment niche. Thus, as patent exclusivities for a biologic expire, biosimilars are produced and gain room as a treatment alternative. However, subtle differences in the structure do not make a biosimilar an exact copy of the biological medicine but must possess a high degree of similarity, function, safety, and immunogenicity profiles as the originator. Therefore, a crucial point in biosimilar development is to ascertain its molecular structure and guarantee its comparability with the biologic of reference. Hence, the protein structure must be analyzed thoroughly, including post-translational modifications. Monoclonal antibodies (mAbs) are often used to restore, enhance, or mimic the immune response as a remedy. mAbs possess a unique structure that provides its highly specialized function in the organism, and one key aspect of mAbs function is protein glycosylation, which regulates its stability, half-life, conformation, and activity. Since glycans are chemically diverse, their structural micro and microheterogeneity is often overlooked. Low abundance glycan structures, site-specific glycosylation and glycan isomers impact the comprehensiveness of antibody characterization. Several available methodologies are needed to have a partial glycoproteomic characterization adding slow turnaround and low throughput. Moreover, these technologies are cumbersome making the harmonization of analytical methods a challenge. Glycosylation of therapeutic antibodies is subjected to changes that are dependent on the expression system variability, bioreactor conditions and batch-to-batch consistency. To fill these gaps, this project will develop and implement a sensitive, accurate, reproducible and high-throughput analytical strategy, from sample preparation to data analysis, to characterize the glycosylation dynamics of therapeutic antibodies (rituximab, trastuzumab, Infliximab, NISTmAb). The rationale combination of chemoenzymatic methods combined with deep and fast LC-MS/MS methods and accurate data analysis will be harmonized to provide a detailed characterization of therapeutic antibodies' glycosylation. This project will provide a platform to understand the antibody glycosylation structure and functions relationships. The optimal methodology will then be applied to analyze the glycosylation profile of commercial monoclonal antibodies and the glycoprofile will be correlated with their efficacy. (AU)