Automated in-solution protein digestion using a commonly available high-performance liquid chromatography autosampler

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Therapeutic protein characterization is crucial for drug development and quality control.
• Monoclonal antibodies (mAbs) present unique challenges in peptide mapping due to their complexity and disulfide bonds.
• Existing methods for peptide mapping can be labor-intensive and time-consuming.

Purpose of the Study:

• To describe a fully automated peptide mapping liquid chromatography/mass spectrometry (LC/MS) system.
• To demonstrate the system's utility for characterizing therapeutic proteins, particularly monoclonal antibodies.
• To provide detailed protocols for automated sample preparation and LC/MS analysis.

Main Methods:

• Development of a completely automated system integrating sample preparation (denaturation, reduction, alkylation, digestion) and LC/MS analysis.
• Utilized a common high-performance liquid chromatography (HPLC) autosampler for all automated steps.
• Established protocols for Lys-C digestion of intact disulfide bonds and tryptic digestion post-reduction/alkylation for mAbs.

Main Results:

• The automated system successfully performed peptide mapping of monoclonal antibodies, achieving high peptide coverage.
• Automated Lys-C digestion followed by disulfide reduction enabled disulfide linkage analysis.
• The system demonstrated efficiency in preparing and analyzing multiple samples for protein characterization.

Conclusions:

• The automated peptide mapping LC/MS system offers significant advantages for therapeutic protein characterization.
• This system enhances the identification and quantification of post-translational modifications.
• It is valuable for process development, formulation studies, and routine quality control of therapeutic proteins.