Using lysine-reactive fluorescent dye for surface characterization of a mAb

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biopharmaceutical Science

Background:

• The biopharmaceutical industry requires robust methods for protein characterization to ensure product quality and consistency.
• Understanding protein conformation and dynamics is crucial for developing safe and effective therapeutics.

Purpose of the Study:

• To develop and validate a high-throughput, chromatography-based method for characterizing protein conformation and dynamics at peptide level resolution.
• To demonstrate the utility of the method in assessing the impact of deglycosylation on a recombinant monoclonal antibody (mAb) and in studying mAb-excipient interactions.

Main Methods:

• A novel method involving fluorescent labeling of surface lysine residues followed by enzyme digestion and HPLC analysis with fluorescence detection.
• Peptide mapping using fluorescence peak area to indicate solvent accessibility and identify conformational changes.
• Optional peptide identification using mass spectrometry.

Main Results:

• The method successfully probed protein surface changes due to deglycosylation of a recombinant IgG1 mAb.
• Application to mAb-excipient interaction studies revealed that polysorbate-20 (PS-20) increased fluorescent labeling of specific lysine residues.
• This provides peptide-level insights into the interaction between the mAb and PS-20.

Conclusions:

• The developed chromatography-based method offers a high-throughput approach for detailed protein conformation and dynamics analysis.
• This technique is valuable for biopharmaceutical characterization, quality control, and understanding drug-excipient interactions.
• The findings offer initial insights into the molecular interactions between mAbs and excipients like PS-20.