Development of a generic reversed-phase liquid chromatography method for protein quantification using analytical quality-by-design principles

This paper details the development of a novel reversed-phase liquid chromatography (RPLC) method designed for the facile and robust quantification of proteins during the upstream and downstream processing of intracellularly produced proteins in *Escherichia coli* (*E. coli*). The primary objective was to establish a fast, robust, and mass spectrometry-compatible method capable of baseline resolving and quantifying target proteins. The method development followed an Analytical Quality by Design (AQbD) workflow, systematically optimizing parameters such as stationary phase selection, gradient conditions, and column temperature. The study utilized a wide-pore superficially porous particle (SPP) column with a high coverage phenyl bonding, which addressed common challenges in RPLC protein analysis like on-column adsorption and poor selectivity for closely related proteins. The established Method Operable Design Region (MODR) was rigorously tested for robustness through in-silico and experimental Design of Experiment (DoE) approaches. The RPLC method was validated for key parameters including linearity, limit of quantification (LOQ), and repeatability, demonstrating its suitability for accurate protein quantification. This versatile method is applicable for rapid and straightforward recombinant protein titer measurement, facilitating the detection of a broad range of proteins in biopharmaceutical production.

This paper details the development of a novel reversed-phase liquid chromatography (RPLC) method designed for the facile and robust quantification of proteins during the upstream and downstream processing of intracellularly produced proteins in Escherichia coli ( E. coli ). The primary objective was to establish a fast, robust, and mass spectrometry-compatible method capable of baseline resolving and quantifying target proteins. The method development followed an Analytical Quality by Design (AQbD) workflow, systematically optimizing parameters such as stationary phase selection, gradient conditions, and column temperature. The study utilized a wide-pore superficially porous particle (SPP) column with a high coverage phenyl bonding, which addressed common challenges in RPLC protein analysis like on-column adsorption and poor selectivity for closely related proteins. The established Method Operable Design Region (MODR) was rigorously tested for robustness through in-silico and experimental Design of Experiment (DoE) approaches. The RPLC method was validated for key parameters including linearity, limit of quantification (LOQ), and repeatability, demonstrating its suitability for accurate protein quantification. This versatile method is applicable for rapid and straightforward recombinant protein titer measurement, facilitating the detection of a broad range of proteins in biopharmaceutical production. Research Background and Significance Protein quantification is a cornerstone in biopharmaceutical development, especially for recombinant proteins produced intracellularly in microbial hosts such as Escherichia coli . Traditional protein quantification methods like SDS-PAGE and ELISA often lack the throughput, robustness, or specificity required for rapid process monitoring