Brown drug substance color investigation in cell culture manufacturing using chemically defined media: A case study

Xu, Jinying; Jin, Mi Sun; Song, Hangtian; Huang, Chao; Xu, Xuankuo; Tian, Jun; Qian, Nan‐Xin; Steger, Kirby; Lewen, Nancy; Tao, Li; Li, Zheng Jian

doi:10.1016/j.procbio.2013.10.015

Cited by 35 publications

(55 citation statements)

References 28 publications

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“…Back in 2011, it was shown that the oxidation kinetics of amino acids are drastically changed in the presence of the photosensitizer riboflavin through production of singlet oxygen species . Furthermore, reaction products of tryptophan have been identified to affect both cell growth and product quality . Chumsae et al .…”

Section: Introductionmentioning

confidence: 99%

Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC‐MS/MS

Krattenmacher

Heermann

Calvet

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUND Chemically defined media (CDM) are used routinely in industrial settings for the production of biopharmaceuticals from mammalian cells, so attention has shifted to understanding their chemical behavior and impact on process robustness. In this context, one particular problem is the risk of cross reactivity and instability in complex mixtures of chemicals at high concentrations. RESULTS In order to characterize potentially unstable medium compounds in feed media, we developed and validated a novel liquid chromatography tandem mass spectrometry (LC‐MS/MS) method to simultaneously quantify the majority of medium compounds. We used this application advantage to provide the following insights into molecular processes occurring in CDM prepared at different temperatures and during storage: Concentration profiles of critical compounds in CDM are independent of preparation temperature and storage material but show significant alterations over time. In particular l‐cysteine, l‐proline, vitamin B6, thiamine and cyanocobalamin were found to be rather unstable. Using mixed mode chromatography allows expansion of the method to cover additional analytes of interest, as demonstrated by inclusion of seven additional CDM reaction products. CONCLUSION The successful development of a new LC‐MS/MS method allowed us to show the impact of storage duration on the stability of CDM compounds. Furthermore, the results suggest that essential parameters for medium preparation (e.g. temperature) and storage upscaling (e.g. vessel material) are not impacting upon the chemical composition of CDM. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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Section: Introductionmentioning

confidence: 99%

Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC‐MS/MS

Krattenmacher

Heermann

Calvet

et al. 2018

J of Chemical Tech & Biotech

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“…Analytical techniques suggest that approximately 1% of the tryptophan was degraded in the cell culture medium yet this was still enough to reduce cell growth by 10%. Our data suggests that screening for the formation in NFK would ensure process robustness including but not limited to cell culture performance and drug substance color …”

Section: Discussionmentioning

confidence: 88%

Tryptophan oxidation catabolite,N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance

McElearney

Ali

Gilbert

et al. 2015

Biotechnol Progress

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Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC-MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N-formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance.

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“…The color may not affect safety or efficacy, however, color variation in a therapeutic product may imply process inconsistency and poor understanding of the process. Normal mAb product manufacturing commonly has some color variation which is acceptable (Derfus et al, ; Prentice et al, ; Song et al, ; Vijayasankaran et al, ; Xu et al, ). However, color variation due to process related impurities can be a major concern as a symptom of lack of process control.…”

Section: Introductionmentioning

confidence: 99%

“…Pink coloration of biologics has been reported during manufacturing process (Derfus et al, ; Prentice et al, ; Vijayasankaran et al, ; Xu et al, ) with the hypothesis that the color is correlated with the cobalt (vitamin B 12 ) concentration in the final pools. Two critical factors were identified, disulfide reduction of antibody molecules (Derfus et al, ) and the chemical form of HO—Cbl (Prentice et al, ).…”

Section: Introductionmentioning

confidence: 99%

Vitamin B₁₂ association with mAbs: Mechanism and potential mitigation strategies

Cheng

Martin

Huang

et al. 2017

Biotech & Bioengineering

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Process control for manufacturing biologics is critical for ensuring product quality, safety, and lot to lot consistency of therapeutic proteins. In this study, we investigated the root cause of the pink coloration observed for various in-process pools and drug substances in the antibody manufacturing process. Vitamin B is covalently bound to mAbs via a cobalt-sulfur coordinate bond via the cysteine residues. The vitamin B was identified to attach to an IgG4 molecule at cysteine residues on light chain (Cys-214), and heavy chain (Cys-134, Cys-321, Cys-367, and Cys-425). Prior to attachment to mAbs, the vitamin B needs to be in its active form of hydroxocobalamin. During culture media preparation, storage and cell culture processing, cyanocobalamin, the chemical form of vitamin B added to media, is converted to hydroxocobalamin by white fluorescence light (about 50% degradation in 11-14 days at room temperature and with room light intensity about 500-1,000 lux) and by short-wavelength visible light (400-550 nm). However, cyanocobalamin is stable under red light (wavelength >600 nm) exposure and does not convert to hydroxocobalamin. Our findings suggests that the intensity of pink color depends on concentrations of both free sulfhydryl groups on reduced mAb and hydroxocobalamin, the active form of vitamin B . Both reactants are necessary and neither one of them is sufficient to generate pink color, therefore process control strategy can consider limiting either one or both factors. A process control strategy to install red light (wavelength >600 nm) in culture media preparation, storage and culture processing areas is proposed to provide safe light for biologics and to prevent light-induced color variations in final products.

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Brown drug substance color investigation in cell culture manufacturing using chemically defined media: A case study

Cited by 35 publications

References 28 publications

Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC‐MS/MS

Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC‐MS/MS

Tryptophan oxidation catabolite,N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance

Vitamin B₁₂ association with mAbs: Mechanism and potential mitigation strategies

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Brown drug substance color investigation in cell culture manufacturing using chemically defined media: A case study

Cited by 35 publications

References 28 publications

Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC‐MS/MS

Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC‐MS/MS

Tryptophan oxidation catabolite,N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance

Vitamin B12 association with mAbs: Mechanism and potential mitigation strategies

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Vitamin B₁₂ association with mAbs: Mechanism and potential mitigation strategies