HILIC-LC/MS METHOD FOR NON-DERIVATIZED AMINO ACID ANALYSIS IN SPENT MEDIA

摘要

Spent media analysis is vital to vaccine upstream process development and optimization. Determination of amino acid (AA) concentrations in culture media is essential to understand the changes in cell culture conditions over time and also to design optimal feed strategy to improve production economics. Amino acids are highly polar and most have low UV absorbance. Therefore, derivatization by Ortho Phthalaldehyde (OPA) and Fluorenylmethoxy Chloroformate (FMOC) is necessary to improve retention on reverse phase columns and to enhance sensitivities on UV/florescence detectors. This process is labor intensive and time consuming. In the current approach, we applied hydrophilic interaction chromatography (HILIC) and triple quadrupole mass spectrometry (MS) to quantitate non-derivatized AAs extracted from culture media. Samples are collected from the bioreactor at different time points. Prior to extraction, a mixture of C~(13)/N~(15) labeled AAs is spiked as internal standards (IS) to normalize variabilities in extraction recoveries. AAs and IS are extracted by adding 4 parts of 50% acetonitrile in 20mM Ammonium formate in water (pH=3) to 1 part of culture media sample. The samples are vortexed and centrifuged at 12000 RPM for 5 min to remove the cells and protein precipitates. Supernatants are directly injected on to HILIC-Z column (Agilent Technologies, 2.1X100mm; 2.7uM) connected to SCIEX API 4000 triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. The method uses 20 minute normal phase gradient (buffer system: 20mM Ammonium formate in water and 20mM Ammonium formate in 90% acetonitrile) at a flow rate of 0.5ml/min to resolve 17 amino acids. Data analysis is performed by Analyst software. Ratios of AA and corresponding IS peak areas vs. concentrations are plotted to generate calibration curves for each AA. Our method demonstrated nano-molar detection sensitivities and a linear range of 75nM to 2.5uM for most of the AAs. This method also exhibited excellent resolution between isobaric AAs Leucine and Isoleucine. In the poster presentation, we will present method qualification data in detail. We will also discuss application of this method to two independent vaccine upstream process development studies, one is associated with microbial fermentation process and another involved mammalian cell culture process.