Development of an efficient LC-MS peptide mapping method using accelerated sample preparation for monoclonal...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Protein Analysis

Background:

• Peptide mapping using liquid chromatography-mass spectrometry (LC-MS) is crucial for characterizing monoclonal antibodies (mAbs) and their post-translational modifications (PTMs).
• Conventional protease digestion methods, often requiring several hours to overnight incubation, can introduce artifacts like deamidation and isomerization, leading to inaccurate quantification of modifications.
• Shortened digestion times can result in incomplete cleavage, compromising sequence coverage and reproducibility.

Purpose of the Study:

• To evaluate the efficacy of pressure cycling technology (PCT) in accelerating tryptic digestion and PNGase F deglycosylation for mAb analysis.
• To compare PCT-assisted methods with conventional incubation techniques regarding digestion efficiency, artifact generation, and chromatographic profile quality.

Main Methods:

• Applied PCT, involving alternating cycles of atmospheric and high pressure (30 kpsi) at 37°C, for a 0.5-hour tryptic digestion and PNGase F deglycosylation.
• Compared the 0.5-hour PCT methods with conventional 4-hour and 18-hour (overnight) digestions at 37°C under atmospheric pressure.

Main Results:

• The 0.5-hour PCT-assisted tryptic digestion minimized modification artifacts and reduced chromatographic interferences, leading to cleaner profiles and improved quantitation.
• PCT-assisted deglycosylation efficiently removed N-glycans from Asn301 in mAb heavy chains without negatively impacting the tryptic digest's chromatographic profile.
• PCT significantly reduced digestion time while maintaining or improving analytical quality compared to conventional methods.

Conclusions:

• PCT is a highly effective technology for rapid and accurate peptide mapping of mAbs.
• This accelerated approach mitigates common artifacts associated with prolonged incubations, enhancing the reliability of PTM analysis.
• PCT offers a valuable tool for improving the efficiency and accuracy of mAb characterization workflows.