Rapid Antibody Structural Characterization and Quantification via Microdroplet Trypsin Digestion

Area of Science:

• Biochemistry and Proteomics
• Analytical Chemistry
• Biopharmaceutical Analysis

Background:

• Protein structural characterization is crucial for biopharmaceuticals.
• Monoclonal antibodies (mAbs) require rapid and accurate analytical methods.
• Existing methods for protein digestion and quantitation can be time-consuming.

Purpose of the Study:

• To develop a fast antibody characterization and quantitation method.
• To utilize online microdroplet trypsin digestion for ultrafast protein analysis.
• To enable precise peptide mapping, modification pinpointing, and absolute quantitation of mAbs.

Main Methods:

• Online microdroplet trypsin digestion for ultrafast protein digestion (<1 ms, >90% efficiency).
• Mass spectrometry (MS) and MS/MS for peptide mapping and modification analysis.
• Spiking with standard peptides and heavy isotope-labeled antibodies for absolute quantitation.

Main Results:

• Identified major deamidation at N387 in NIST mAb using MS/MS.
• Successfully quantified methionine oxidation using absolute quantitation with improved accuracy.
• Achieved sensitive and accurate absolute antibody quantification (R²=0.99, LOD=1.2 ng, 0.6-10% error).

Conclusions:

• Microdroplet digestion offers a rapid and efficient approach for antibody characterization.
• The developed method provides accurate absolute quantitation of post-translational modifications and antibody levels.
• This technique is valuable for biopharmaceutical analysis, offering speed, sensitivity, and accuracy.