During a small-scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small-scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production.
Oxidative damage to proteins is one of the most prominent chemical degradation pathways that are of concern for drug product development in the biotechnology industry. Especially susceptible to oxidation are the Met and Trp residues in proteins. While L-Met and L-Trp have been shown to act as antioxidants typically protecting proteins against Met and Trp oxidation, respectively, l-Trp has been shown to be particularly sensitive to light, thereby producing various reactive oxygen species (ROS), including H2O2. There is hence a need to identify nonphotosensitive molecules that can protect Trp oxidation in proteins so that they can be easily handled under drug product manufacturing conditions. A combination of screening methods, namely, cyclic voltammetry (CV) and hydrogen peroxide generation upon photoirradiation, was used to screen several molecules to identify compounds that can act as antioxidants. Specifically, indole and tryptophan with hydroxy groups on the six-membered aromatic ring were found to have lower oxidation potentials than the parent compounds and produced the least amount of H2O2 upon light exposure. These derivatives were also found to sufficiently protect tryptophan oxidation in mAb1 against a variety of reactive oxygen species such as alkyl peroxides, hydroxyl radicals, and singlet oxygen and may be useful as part of the formulation toolkit to protect against protein degradation via oxidation.
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