Hydroxocobalamin association during cell culture results in pink therapeutic proteins

Prentice, K. M.; Gillespie, Ronald; Lewis, Nathan E.; Fujimori, Kiyoshi; McCoy, Rebecca; Bach, Julia; Connell-Crowley, Lisa; Eakin, Catherine M.

doi:10.4161/mabs.25921

Cited by 23 publications

(27 citation statements)

References 27 publications

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“…Cyanocobalamin is the vitamin B 12 form typically included in cell culture media as a cofactor required for DNA synthesis. Under cell culture conditions, however, cyanocobalamin is readily converted to hydroxocobalamin due to light exposure, 12 with up to 80% converted after 10 d in culture 13 . While both forms of vitamin B 12 are red-colored, 7 LC/MS results showed that the form remaining in red colored mAb pools is hydroxocobalamin, consistent with a previous report for an IgG2 product 13 .…”

Section: Discussionsupporting

confidence: 89%

“…Under cell culture conditions, however, cyanocobalamin is readily converted to hydroxocobalamin due to light exposure, 12 with up to 80% converted after 10 d in culture 13 . While both forms of vitamin B 12 are red-colored, 7 LC/MS results showed that the form remaining in red colored mAb pools is hydroxocobalamin, consistent with a previous report for an IgG2 product 13 . Since the majority of the experimental pools were from glass bioreactors exposed to light for the 14 d culture duration, it can be assumed that the hydroxocobalamin form was present in both C-HCCF and RP-HCCF; therefore, hydroxocobalamin availability alone was not sufficient to cause red color for these mAbs.…”

Section: Discussionmentioning

confidence: 99%

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Red colored IgG4 caused by vitamin B12 from cell culture media combined with disulfide reduction at harvest

Derfus

Dizon-Maspat

Broddrick

et al. 2014

mAbs

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While many antibody therapeutics are formulated at low concentration (~10–20 mg/mL) for intravenous administration, high concentration (> 100 mg/mL) formulations may be required for subcutaneous delivery in certain clinical indications. For such high concentration formulations, product color is more apparent due to the higher molecular density across a given path-length. Color is therefore a product quality attribute that must be well-understood and controlled, to demonstrate process consistency and enable clinical trial blinding. Upon concentration of an IgG4 product at the 2000 L manufacturing scale, variability in product color, ranging from yellow to red, was observed. A small-scale experimental model was developed to assess the effect of processing conditions (medium composition and harvest conditions) on final bulk drug substance (BDS) color. The model was used to demonstrate that, for two distinct IgG4 products, red coloration occurred only in the presence of disulfide reduction-mediated antibody dissociation. The red color-causing component was identified as vitamin B12, in the hydroxocobalamin form, and the extent of red color was correlated with the cobalt (vitamin B12) concentration in the final pools. The intensity of redness in the final BDS was modulated by changing the concentration of vitamin B12 in the cell culture media.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Red colored IgG4 caused by vitamin B12 from cell culture media combined with disulfide reduction at harvest

Derfus

Dizon-Maspat

Broddrick

et al. 2014

mAbs

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“…Recently, the concentration of B vitamins has been shown to impact the color of the drug substance [9,10]. Hence, it is critical to ensure vitamins are not depleted, while also not added in excess.…”

mentioning

confidence: 99%

Nutrient and metabolite analysis: understanding cell culture

Vijayasankaran¹

2014

Pharmaceutical Bioprocessing

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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%

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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Hydroxocobalamin association during cell culture results in pink therapeutic proteins

Cited by 23 publications

References 27 publications

Red colored IgG4 caused by vitamin B12 from cell culture media combined with disulfide reduction at harvest

Red colored IgG4 caused by vitamin B12 from cell culture media combined with disulfide reduction at harvest

Nutrient and metabolite analysis: understanding cell culture

Vitamin B₁₂ association with mAbs: Mechanism and potential mitigation strategies

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Hydroxocobalamin association during cell culture results in pink therapeutic proteins

Cited by 23 publications

References 27 publications

Red colored IgG4 caused by vitamin B12 from cell culture media combined with disulfide reduction at harvest

Red colored IgG4 caused by vitamin B12 from cell culture media combined with disulfide reduction at harvest

Nutrient and metabolite analysis: understanding cell culture

Vitamin B12 association with mAbs: Mechanism and potential mitigation strategies

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Product

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Vitamin B₁₂ association with mAbs: Mechanism and potential mitigation strategies