Keto leucine and keto isoleucine are bioavailable precursors of their respective amino acids in cell culture media

Schmidt, Corinna; Seibel, Ronja; Wehsling, Maria; Mignon, Maxime Le; Wille, Gregor; Fischer, Markus; Zimmer, Aline

doi:10.1016/j.jbiotec.2020.06.013

Cited by 6 publications

(3 citation statements)

References 41 publications

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“…They showed that valine and isoleucine (together as a feed) addition had the best positive impact on ammonium level, and the speci c production of ammonium was decreased by 35%. In another study (Schmidt et al 2020), it was shown that the CHO cell culture with the highest valine concentration produced less ammonium that is consistent with our ndings.…”

Section: Discussionsupporting

confidence: 92%

Amino acid feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells

Savizi

Maghsoudi

Motamedian

et al. 2021

Preprint

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Ammonia is a toxic byproduct of CHO cell metabolism, which inhibits cell growth, reduces cell viability, alters glycosylation, and decreases recombinant protein productivity. In an attempt to minimize the ammonium accumulation in cell culture media, different amino acids were added individually to the culture medium before the production phase to alleviate the negative effects of ammonium on cell culture performance. Among all the amino acids examined in this study, valine showed the most positive impact on CHO cell culture performance. When the cultured CHO cells were fed with 5 mM valine, EPO titer was increased by 25% compared to the control medium, and ammonium and lactate production were decreased by 23 and 26%, respectively, relative to the control culture. Moreover, the sialic acid content of the EPO protein in valine-fed culture was higher than in the control culture, most likely because of the lower ammonium concentration. Flux balance analysis (FBA) results demonstrated that the citric acid cycle was enriched by valine feeding. The measurement of TCA cycle activity supported this finding. The analysis revealed that there might be a link between promoting tricarboxylic acid (TCA) cycle metabolism in valine-fed culture and reduction in lactate and ammonia accumulation. Furthermore, in valine-fed culture, FBA outcomes showed that alanine was excreted into the medium as the primary mechanism for reducing ammonium concentration. It was predicted that the elevated TCA cycle metabolism was concurrent with an increment in recombinant protein production. Taken together, our data demonstrate that valine addition could be an effective strategy for mitigating the negative impacts of ammonium and enhancing glycoprotein production in both quality and quantity.

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Section: Discussionsupporting

confidence: 92%

Amino acid feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells

Savizi

Maghsoudi

Motamedian

et al. 2021

Preprint

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“…HCFs were cultured in Eagle's minimum essential media supplemented with 15% FBS, 200 U/ml penicillin, 200 μg/ ml streptomycin and 2 mM glutamine, in a humidified atmosphere at 37 °C and 5% CO 2 . Cysteine/cystine have been reported as common contaminants in culture media [25]. The culture media used herein was not modified to be cystine-free.…”

Section: Cystinotic Fibroblast Cell Culturementioning

confidence: 99%

Evaluation of NACA and diNACA in human cystinosis fibroblast cell cultures as potential treatments for cystinosis

Hector

Cairns

Wall³

2022

Orphanet J Rare Dis

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Background Cystinosis is a rare autosomal recessive lysosomal storage disease, associated with high morbidity and mortality. Mutations in the CTNS gene disable a membrane protein responsible for the transport of cystine out of the lysosome. Loss of transporter function leads to intralysosomal cystine accumulation and long-term damage to various tissues and organs, including the kidneys, eyes, liver, muscles, pancreas, and brain. The only cystine-depletion therapy for treatment of cystinosis is cysteamine which requires frequent administration of high doses and often causes gastrointestinal pain as well as pungent sulfurous odor in patients. The current in vitro study evaluated antioxidants, N-acetylcysteine amide (NACA; NPI-001) and (2R,2R′)-3,3′-disulfanediyl bis(2-acetamidopropanamide) (diNACA; NPI-002), as potential treatments for cystinosis. Methods Cytotoxicity of cysteamine, NACA and diNACA was evaluated in cultured human cystinotic fibroblasts (HCFs). HCFs were cultured in 96 well plates incubated for 0–72 h in the presence of 25, 50 or 75 μM each of either cysteamine, NACA or diNACA along with an untreated control. Media was removed and cell viability assessed. Next, cystine-depleting activities of cysteamine, NACA and diNACA were screened in HCFs cell culture utilizing an inexpensive, proven colorimetric assay. HCFs were seeded and allowed to reach approximately 80% confluence before the addition of the test articles: 50 μM of either cysteamine, NACA or diNACA in media along with an untreated control. HCFs were incubated, harvested, and cystine was reduced to cysteine, the concentration of which was then determined per quantity of protein compared to a cysteine standard. Statistically significant cystine depletion was determined by paired t-test versus untreated control (p < 0.05). Results Neither cysteamine, NACA nor diNACA at 25, 50 or 75 μM caused cytotoxicity in HCFs. Treatment with all tested concentrations (25, 50 or 75 µM) of either NACA or diNACA at 48 or 72 h resulted in statistically significant increases in cell viability, relative to untreated control, whereas the higher concentrations (50 or 75 µM) of cysteamine achieved statistical significance at both timepoints but not the lowest concentration (25 µM). All test articles depleted cystine from HCFs compared to control. NACA depletion of cystine was statistically superior to cysteamine at 6, 24 and 48 h and numerically greater at 72 h. DiNACA depletion of cystine was statistically superior to cysteamine at 6 and 48 h, slightly numerically greater at 24 h and slightly less at 72 h. Conclusions NACA and diNACA were non cytotoxic to HCFs and significantly increased cell viability. Cystine reduction was determined as percent of control after incubation with 50 µM of NACA, diNACA or cysteamine in HCFs cell culture for 6, 24, 48 and 72 h. Of the three test articles, NACA exhibited most rapid and greatest potency in cystine reduction. Rank order potency for cystine reduction over time was observed, NACA > diNACA ≥ cysteamine. Therefore, further study of NACA and diNACA as potential treatments for cystinosis is warranted.

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“…However, several amino acids such as cystine, tyrosine and branched‐chain amino acids (BCAA) present an intrinsic low solubility in CCM (Carta & Tola, 1996 ; Salazar et al, 2016 ). Several studies report utilization of noncanonical amino acids for biotechnological applications to support growth and productivity of CHO cells while presenting increased solubility properties in CCM (Chevallier et al, 2021 ; Hecklau et al, 2016 ; Schmidt et al, 2020 ; Wu et al, 2008 ; Zimmer et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media

Schmidt

Wehsling

Mignon

et al. 2021

Biotech & Bioengineering

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Increasing demands for protein‐based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed‐batch processes. N‐lactoyl‐leucine and N‐lactoyl‐isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N‐lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.

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Keto leucine and keto isoleucine are bioavailable precursors of their respective amino acids in cell culture media

Cited by 6 publications

References 41 publications

Amino acid feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells

Amino acid feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells

Evaluation of NACA and diNACA in human cystinosis fibroblast cell cultures as potential treatments for cystinosis

Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media

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