An analytical method that allows determination of free amino acids in bioprocessed broth during the bioprocess would help to optimize and adjust its composition on-line. This could lead a significant impact on improvement of the bioprocess parameters and the quality of the final product. For this purpose, we tested the method, where amino acids are derivatized with reagents ortho-phthalaldehyde (OPA) and (9-Fluorenylmethyl) chloroformate (FMOC), followed by separation of the derivatives by reverse phase chromatography on an ultra high pressure liquid chromatography (UHPLC) system. The method was optimized in the way to achieve the highest possible level of automation, therefore all steps (dilution, derivatization, separation and detection) are performed directly in the autosampler compartment of the UHPLC system. The suitability of the method was evaluated according to the pre-defined validation parameters. Our results are showing that we successfully optimized and achieved targeted level of automation of the method, and we fulfilled the validation parameters via standard solutions. Analysis of bioprocess samples confirmed the suitability of OPA and FMOC method for the determination of primary amino acids. However quantification of secondary amino acids is more challenging due to non-specific chromatographic peaks and chromatogram variability of amino acids derivatized with FMOC reagent. The cause of the most problematic nonspecific peaks was identified and confirmed to be due to the OPA reagent itself and the reactions of the OPA reagent with unknown components in the bioprocess media. Nevertheless, the main issue remains determination of secondary amino acids, whereas the chromatographic peaks are not optimally separated and their detection is variable. The method needs further optimization to achieve all the acceptance criteria for determining the full spectrum of amino acids (primary and secondary amino acids) with the desired level of automation.
|
