Amino Acid Analysis for Cell and Gene Therapy Processes

Cell therapy and viral vector processes run in small volumes and often in serum-free media, and the spent medium is the most direct record of what the cells used. In a recent perfusion study of CAR-T expansion in stirred-tank bioreactors, viability fell below 90% when lactate stayed above about 15 mmol/L for more than 48 hours. The authors add that amino acid analyses, which they did not perform, "may also inform on potential targeted supplementation strategies" ³. A free amino acid and ammonia profile fills that gap.

What we measure in cell and gene therapy media

T and NK cells. In a 21-day CAR-T expansion, analysis of the supernatants showed significant consumption of all amino acids in the medium during the first 12 days, and minimal consumption after day 14 ². Ammonia can build up quickly. When T cells were held concentrated for 6 hours, ammonium rose to 3.5 mM at room temperature and 4 mM at 37 °C, and the ammonium level correlated with greatly decreased later expansion and day-13 viability ¹. In that study glutamine was not limiting: at most 0.5 mM was consumed ¹. In an NK-92 cell line grown in 2 L stirred bioreactors, ammonia ended at 1.95–2.48 mM, while glucose and glutamine did not reach limitation during growth ⁵.

Pluripotent stem cells. Human iPSCs in mTeSR1 medium consumed arginine, cystine, glutamine and serine heavily over 3 days, and tryptophan was the most consumed of all the amino acids tested ⁶. In stirred-tank expansion of human pluripotent stem cells, no amino acid ran out, glutamine included, while glutamate, alanine and to some extent glycine accumulated, more so under repeated-batch feeding ⁷. Perfusion in the same study raised the average density by 47% over repeated batch ⁷.

Viral vectors. In lentiviral vector production by stable producer clones induced at high density, HPLC analysis of spent media showed that amino acids and glucose were not depleted and that lactate and ammonia were in a normal range. That ruled those components out as the cause of lower productivity ⁸. Blending media and feeds from different suppliers then raised lentiviral productivity by up to fivefold ⁸. In AAV production by transfection, exchanging the medium at transfection correlated clearly with higher production. The authors proposed nutrient limitation or by-product accumulation as the cause but did not measure metabolites ⁹.

Analytes	Why they matter in these processes	Panel
Ammonia, glutamine, glutamate	Ammonium after a concentrated T-cell hold tracked poorer later expansion 1; NK-92 runs ended near 2–2.5 mM ammonia 5	AA-1
Arginine, histidine, tryptophan and the other essential amino acids	Consumed throughout the first 12 days of CAR-T expansion 2; tryptophan was the most consumed amino acid in iPSC culture 6	AA-1
Serine	Heavily consumed by iPSCs 6	AA-1
Alanine, glutamate, glycine	Accumulated in stirred-tank pluripotent stem cell culture, more under repeated-batch feeding 7	AA-1
Cysteine, cystine	Cysteine fell during a concentrated T-cell hold 1; cystine was heavily consumed by iPSCs 6	AA-3, on request

We derivatize free amino acids with AQC, separate them by reversed-phase HPLC and detect at 260 nm; see how AQC derivatization works. The same chemistry was used to measure 19 amino acids and ammonium in the T-cell study above ¹. Results come in µM and mg/L, with an internal standard in every sample and a QC standard in every run. Lactate and glucose are not on our panel, so read our results alongside your analyzer data. Our work supports research, development and process monitoring; the lab does not operate under GLP or GMP.

Questions this answers

• "Is our perfusion rate or feed limited by an amino acid, or only by lactate?"
• "Does ammonium build up during our wash, hold or expansion steps, and does that track with poorer expansion?"
• "On which day does amino acid uptake level off, so we can time the last feed or the harvest?"
• "Can our iPSC process move from daily full medium exchange to less frequent feeding without running short of glutamine, serine or tryptophan?"
• "When we transfect at high cell density, is lost vector productivity caused by a depleted amino acid, or do we need a medium exchange?"
• "Is the ammonia we measure coming from the cells, from warmed glutamine-containing medium, or from how the samples were stored?"

Caveats for this matrix

Application notes

Application notes for this matrix are in preparation. The methods library covers the chemistry in the meantime.

Key references

1. Impacts of transient exposure of human T cells to low oxygen, temperature, pH and nutrient levels relevant to bioprocessing for cell therapy applications — Kunitskaya A, Piret JM, Cytotherapy, 2025.
2. Mapping real-time metabolic kinetics of expanded CAR T cells using hyperpolarized 13C-glucose and metabolomics — Jensen PR, Mathiassen TBW, Hansen MR, et al., Scientific Reports, 2025.
3. Optimising and adapting perfusion feeds in serum-free medium to intensify CAR-T cell expansion in stirred-tank bioreactors — Springuel P, Hood T, Slingsby F, et al., Frontiers in Bioengineering and Biotechnology, 2025.
4. Automated, aseptic sampling with small-volume capacity from microbioreactors for cell therapy process analysis — Chan ZX, Chelvam SP, Sin WX, et al., Frontiers in Bioengineering and Biotechnology, 2025.
5. Homogeneous shear distribution improves NK-92 cell cytotoxicity in a clinically relevant 2 L membrane-stirred bioreactor — von Werz V, van Heuvel Y, Hadrbolec M, et al., Frontiers in Bioengineering and Biotechnology, 2026.
6. Tryptophan metabolism regulates proliferative capacity of human pluripotent stem cells — Someya S, Tohyama S, Kameda K, et al., iScience, 2021.
7. Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors — Kropp C, Kempf H, Halloin C, et al., Stem Cells Translational Medicine, 2016.
8. Culture media selection and feeding strategy for high titer production of a lentiviral vector by stable producer clones cultivated at high cell density — Shen CF, Tremblay S, Sabourin-Poirier C, et al., Bioprocess and Biosystems Engineering, 2022.
9. AAV process intensification by perfusion bioreaction and integrated clarification — Mendes JP, Fernandes B, Pineda E, et al., Frontiers in Bioengineering and Biotechnology, 2022.
10. Decomposition of L-glutamine and accumulation of ammonium in cell culture media inhibit infectivity of influenza viruses — Kegel NB, Kaufmann A, Matrosovich M, Bauer S, Dorna J, Virology, 2025.

OpenChemWorks Laboratory · Reviewed by the laboratory director, PhD chemist · Updated September 24, 2026