Validation of Protein A chromatography: orthogonal method with size exclusion chromatography validation for mAb titer analysis

Abstract Objectives After reviewing the reference product specifications, found that they include the SEC HPLC result but do not specify Protein A chromatography as a test. Although there is no record of reference product specifications, alternative methods are necessary, particularly for routine analysis of biotechnological products. Methods The SEC mobile phase consisted of 0.1 M sodium phosphate and 0.2 M saline buffer pH 6.80 run isocratically at a flow rate of 0.5 mL/min. The Protein A chromatography column was equilibrated (60 mM sodium phosphate buffer, pH 7.4 (binding buffer), 60 mM sodium phosphate buffer, pH 2.5 (elution buffer) gradient system) at 1 mL/min. Results The SEC HPLC method validation study was conducted, ranging from (0.01–2.0 mg/mL) 1.0 % (LLOQ level) to 200 %. The Protein A chromatography method between (0.0078 and 4.0 mg/mL) 0.78 % (LLOQ level) and 400 % linearity levels. In the linearity study, both ANOVA and Graphpad results were shown. Both bioanalytical methods were performed by establishing validation parameters for the mAb reference product in accordance with the ICH M10 guideline. Conclusions Additionally, the findings shed light on routine titer analysis of biotechnological products, as there is no documentation in the literature regarding Protein A chromatography validation.