Solving Complex Biologics Truncation Problems by Top-Down Mass Spectrometry.

Area of Science:

• Biopharmaceutical Manufacturing
• Analytical Chemistry
• Protein Therapeutics

Background:

• The development of non-monoclonal antibody (non-mAb) protein therapeutics requires advanced characterization techniques.
• Protein truncation is a significant manufacturing issue for non-mAb drug substances, often challenging to identify with standard methods.
• Traditional methods like intact mass spectrometry can yield ambiguous or incorrect results for complex truncation scenarios.

Purpose of the Study:

• To address the limitations of existing methods in identifying protein truncation in non-mAb drug substances.
• To develop and validate a novel analytical workflow for precise identification of protein clipping locations.
• To offer a robust solution for pharmaceutical companies facing challenges in therapeutic protein production.

Main Methods:

• Development of an online top-down liquid chromatography-mass spectrometry (LC-MS) based workflow.
• Application of the developed workflow to analyze complex cases of truncated drug substances.
• Comparison of the workflow's efficacy against traditional and other orthogonal analytical methods.

Main Results:

• Successfully identified specific clipping locations in two complex cases of truncated protein therapeutics.
• The online top-down LC-MS workflow demonstrated superior capability in resolving protein clipping problems.
• The method showed high compatibility with routine intact mass spectrometry, enhancing its practical utility.

Conclusions:

• The developed online top-down LC-MS workflow is a valuable tool for accurately identifying protein truncation sites.
• This method offers a unique and effective solution for characterizing truncated species in non-mAb drug substances.
• The workflow supports the pharmaceutical industry in overcoming critical challenges in therapeutic protein production and quality control.