Method Development and Qualification of pH-Based CEX UPLC Method for Monoclonal Antibodies.

Area of Science:

• Biopharmaceutical Analysis
• Protein Chemistry
• Analytical Chemistry

Background:

• Post-translational modifications (PTMs) in monoclonal antibodies (mAbs) impact biological efficacy and safety, necessitating robust characterization.
• Charge variants are critical quality attributes for mAb product quality, stability, and consistency.
• Current charge variant characterization is a time-consuming, multi-step process involving extensive sample preparation.

Purpose of the Study:

• To develop a simplified, on-line analytical method for charge variant characterization of mAbs compatible with mass spectrometry (MS).
• To enable the detection of PTMs using a low ionic strength mobile phase suitable for MS detection.
• To establish a method for Quality Control (QC) release testing of therapeutic mAbs.

Main Methods:

• Development of a charge variant analytical method utilizing volatile buffers with low ionic strength.
• Application of pH gradient cation exchange chromatography coupled with on-line MS detection.
• Method qualification adhering to pharmacopeia guidelines.

Main Results:

• High-resolution separation of acidic and basic mAb charge variants achieved.
• Demonstrated linearity (2.4-3.6 mg/mL, %RSD < 2.0%) and a Limit of Quantitation of 0.15 mg/mL.
• Method exhibited precision, accuracy, robustness, and specificity with no matrix interference.

Conclusions:

• A simple, accurate, robust, and precise pH gradient cation exchange chromatography method was developed and qualified for native mAb charge variant characterization.
• The developed method facilitates on-line MS detection, enabling rapid analysis of PTMs on intact proteins.
• This approach streamlines QC release testing and can support biosimilar product profiling.