(T1330-04-22) Cell Culture Media Monitoring in Cell and Gene Therapy Supporting Upstream Bioprocessing

Purpose: In viral vector-based gene therapy, modified viruses are used as drug-delivery vehicles to introduce specific DNA sequences into cells. The HEK293 cell line, from immortalized human embryonic kidney cells, is commonly used in the production of viral vectors such as adenoviral (AV), adeno-associated viral (AAV), and retroviral vectors. Cellular media conditions, specifically the ability to measure key media components and monitor their levels during cellular proliferation and growth is critical to generating desired product at high yield. A good handle on concentration of consumed nutrients and metabolite generated during growth provide an window into cellular processes and information on critical metabolic markers of success or potential failure. The goal was to develop an easy to deploy methodology for cell culture media analysis based on a high-resolution mass spectrometry platform, with minimal sample preparation, for the analysis of commercial HEK 293 viral vector media analysis

Methods: A UPLC reversed phase chromatographic method was developed on a C18 column for the separation of components present in DMEM and IMDM media. A single stage HRMS system in +VE and -VE ion mode was used to acquire mass spectrometry data on the media components and the cellular metabolites, which was used to generate a 200+ compound library used in identification. Amino acid standards were used to generate a calibration curve to assess the quantification range, accuracy and precision of the assay. HEK293 media and DMEM media were diluted 1:100, 1:200, 1:500, and 1:1000 fold and amino acids in the media were quantified using the amino acid calibration curve.

Results: A single UPLC method was developed to separate HEK293 media and DMEM media components. Results show that the media contains typical classes of cell culture media compounds such as amino acids and vitamins. Chromatography was optimized to separate critical isobaric pairs such as leucine/isoleucine and citrate/isocitrate (Figure 1). Nucleic acids and nucleosides not typically observed in other mammalian media were also detectable using this method. Different media show different profile for media components—isoleucine, pyridoxine and hypoxanthine distribution profiles are shown in Figure 2. Amino acid quantification showed excellent precision and accuracy for underivatized analysis of amino acids (Table 1).

Conclusion: A comprehensive library-based LC-MS method and workflow have been developed for key component of cell media analysis on a LC-MS HRMS system and was applied to HEK 293 media analysis used in viral vector production. The method allowed accurate measurement of amino acids, vitamins, nucleobases in a complex IMDM media. The assay’s accuracy and precision for amino acids were within +/- 15% and +/- 5%, respectively, making this method suitable for amino acid quantitation under underivatized conditions. The method enables bioprocessing groups to quickly gain insight during bioprocess development in viral vector-based gene therapy.

Figure 1. Overlaid XIC of top 35 compounds observed in HEK 293 media under ESI+ conditions. For display purposes, amino acids are extracted from 1:2000 diluted sample, Vitamins and other compounds are extracted from 1:100 diluted sample. Vitamin, myo-inositol is observed in ES- (data not shown).

Figure 2. Representative bar trend plots of compound response. (A) amino acid example, isoleucine, showing standard calibration curve at the beginning and end of injection, and sample injections. (B) vitamin example, pyridine, showing the compound is present in HEK293 media and DMEM media, and not present in IMDM media as expected. (C) nucleobase example, hypoxanthine, showing the compound is present in HEK293, and not in DMEM and IMDM media.

Table 1. Amino acid quantitation. Summary of linearly, precision and accuracy of QC samples from 3 replicate injections.