Development of a generic reversed-phase liquid chromatography method for protein quantification using analytical...

Area of Science:

• Biopharmaceutical manufacturing
• Analytical chemistry
• Protein biochemistry

Background:

• Biopharmaceutical drug substances require monitoring of critical quality attributes (CQAs) like protein titer and purity during production.
• Existing methods may lack the speed or robustness needed for real-time monitoring in upstream and downstream bioprocessing.
• Intracellular protein production in E. coli presents unique challenges for analytical quantification.

Purpose of the Study:

• To develop a novel, rapid, robust, and mass spectrometry-compatible reversed-phase liquid chromatography (RPLC) method.
• To enable accurate quantification and baseline resolution of target proteins during biopharmaceutical manufacturing.
• To establish a versatile method applicable to a broad range of intracellularly produced proteins.

Main Methods:

• Analytical Quality by Design (AQbD) workflow for method development.
• Selection of stationary phase to minimize protein adsorption and maximize efficiency.
• Optimization of gradient conditions and column temperature using chromatographic modeling.
• Method validation including linearity, limit of quantification, and repeatability.
• In-silico and experimental robustness testing using Design of Experiments (DoE).

Main Results:

• A novel RPLC method was successfully developed and validated for protein quantification.
• The method demonstrates high efficiency, robustness, and compatibility with mass spectrometry.
• Baseline resolution and quantification of target proteins were achieved.
• The method proved effective for monitoring protein titers in both upstream and downstream processing.
• Comparison against established methods confirmed its accuracy and reliability.

Conclusions:

• The developed RPLC method provides a versatile tool for rapid recombinant protein titer measurement.
• The AQbD approach offers a structured protocol for generating new analytical methods in biopharmaceutical development.
• This method facilitates improved process monitoring and control of critical quality attributes in biopharmaceutical manufacturing.