Vitamin B₁₂ association with mAbs: Mechanism and potential mitigation strategies

Abstract

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.

Vitamins in cell culture media: Stability and stabilization strategies

“…Although there are many suggested additives that can stabilize cyanocobalamin, (see Macek, ; Kirschbaum, for comprehensive lists), the most recognized are ferric salts (e.g., FeCl 3 and saccharated iron oxide) (Macek, ; Newmark, ; Skeggs, ), phosphate buffer (Monajjemzadeh et al, ), and potassium ferrocyanide (Zuck & Conine, ). A considerably novel method for minimizing the cyano‐to‐hydroxocobalamin conversion is the application of red light filters in the working laboratory, to filter out the lower wavelengths of light (<600 nm) that contribute most to this reaction (Du et al, ). This was shown to be especially relevant to the biomanufacturing industry as it was demonstrated that the B 12 cobalamins can be incorporated into therapeutic mAbs as a result of thiolates in the protein reacting with hydroxocobalamin, forming a sulfide‐cobalt linkage, which is analogous to in vivo ligand‐exchange processes.…”

The Different Colors of mAbs in Solution

“…Association of vitamin B12, in its hydroxocobalamin form has been reported in a number of instances to contribute to a red/pink coloration of mAbs of different isotypes and Fc-Fusion purified material [8,[10][11][12]. Red colored mAbs were found to have absorbance at about 365 nm, which corresponds to one of the absorbance maximum of vitamin B12 (Table 2, Figure 6).…”

Control of Antibody Impurities Induced by Riboflavin in Culture Media During Production

“…12,16 More recently, covalent modification of photoexposed recombinant antibodies in the presence of cyanocobalamin (vitamin B12) in culture media has been observed. 17 While the exposure resulted in a pink coloration of process intermediates and final drug substance, no impact to binding or potency was reported. Product quality attributes that impact potency will have a direct influence on clinical performance of the drug product, elevating the need for effective process control and monitoring strategies during development.…”