Multilevel─Intact, Subunits, and Peptides─Characterization of Antibody-Based Therapeutics by a Single-Column LC-MS...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Biotechnology

Background:

• Regulatory mandates require comprehensive, multi-level characterization of biotherapeutics.
• Conventional mass spectrometry (MS) assays include intact protein, middle-up, and bottom-up analyses, often requiring multiple columns.
• Advancements in middle-down MS and reversed-phase liquid chromatography (LC) offer opportunities for streamlined workflows.

Purpose of the Study:

• To develop and evaluate a robust middle-down LC-MS method for biotherapeutic protein characterization.
• To assess a single-column C4 LC-MS setup for multi-level analysis of therapeutic proteins.
• To demonstrate the efficiency and comprehensiveness of the streamlined approach for product quality attribute (PQA) assessment.

Main Methods:

• Development of a middle-down LC-MS method utilizing a ZenoTOF 7600 instrument.
• Evaluation of a C4 column for LC-MS analyses.
• Application of the method for intact protein, middle-up/down, and bottom-up analyses of NISTmAb, bispecific antibody (bsAb), and etanercept.

Main Results:

• Successful multi-level characterization of therapeutic proteins using a C4 LC-MS setup.
• High sequence coverage and comprehensive post-translational modification profiling achieved via bottom-up C4 LC-MS.
• First report of middle-down analysis for etanercept subunits and detailed mapping of glycosylation sites.

Conclusions:

• A streamlined single-column LC-MS setup enables robust and efficient multi-level characterization of biotherapeutics.
• The developed method facilitates comprehensive PQA assessment, including complex glycosylation profiling.
• This approach simplifies the integration of LC-MS as a process analytical technology for biopharmaceutical applications.