Optimization of two high-performance size-exclusion chromatography methods with diode-array detection for protein quantification and purity assessment

Abstract: Background: There is a need to develop methods of determining concentration and purity for two new affinity ligands, protein T and protein H, in order to ensure the quality of an industrial production of said proteins. Aims: To develop a High Performance Size Exclusion Chromatography, HPSEC, method to determine concentration and purity of protein T and to improve an existing HPSEC method for concentration and purity determination of protein H that show selectivity, display linearity between peak area and injection amount and give repeatable results. Methods: For protein T, Xbridge Premier Protein and Superdex Increase columns were used of the lengths 15 and 30 cm together with 50mM and 0.3M Sodium phosphate buffers and PBS and mobile phases. For protein H, only a 15 cm Superdex Increase column was used with 0.3M Sodium phosphate buffer. Different reducing conditions during sample preparation were tested. The linearity and measuring ranges were determined by injecting different volumes of reference standard and evaluating . Repeatability was tested with three triplicates of reference standards across three occasions with the same equipment and running conditions. Results: For protein T the 15 cm Xbridge column with 50mM Sodium Phosphate buffer was selected. Little difference between reduction conditions with exception of reduction at high temperature which resulted in degradation or aggregation for both proteins. Acceptable selectivity and separation between main peak and impurities was demonstrated for both methods. Both methods were linear and gave repeatable results in the measuring ranges 2.43 – 16.98µg, 4.85 – 16.98µg and 2.56 – 15.37µg for purity determination of protein T, concentration determination of protein T and both purity and concentration determination protein H, respectively. Conclusion: Two methods, one for protein T and one for protein H, were developed which met qualification criteria use in quality control.