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Disulfide bond reduction, which commonly occurs during monoclonal antibody (mAb) manufacturing processes, can result in a drug substance with high levels of low molecular weight (LMW) species that may fail release specifications because the drug's safety and the efficiency may be affected by the presence of this material. We previously studied disulfide reoxidation of mAbs and demonstrated that disulfide bonds could be reformed from the reduced antibody via redox reactions under an optimal redox condition on Protein A resin. The study here implements a redox system in a manufacturing setting to rescue the reduced mAb product and to further eliminate LMW issues in downstream processing. As such, we incorporate the optimized redox system as one of the wash buffers in Protein A chromatography to enable an on-column disulfide reoxidation to form intact antibody in vitro. Studies at laboratory scale (1 cm (ID) x 20 cm (Height), MabSelect SuRe LX) and pilot scale (30 cm (ID) x 20 cm (Height), MabSelect SuRe LX) were performed to demonstrate the effectiveness and robustness of disulfide formation with multiple mAbs using redox wash on Protein A columns. By applying this rescue strategy using ≤50 g/L-resin loading, the intact mAb purity was improved from <5% in the Protein A column load to >90% in the Protein A column elution with a product yield of >90%. Studies were also done to confirm that adding the redox wash has no negative impact on process yield or impurity removal or product quality. The rescued mAbs were confirmed to form complete interchain disulfide bonds, exhibiting comparable biophysical properties to the reference material. Furthermore, since the redox wash is followed by a bridging buffer wash before the final elution, no additional burden is involved in removing the redox components during the downstream steps. Due to its ease of implementation, significant product purity improvement, and minimal impact on other product quality attributes, we demonstrate that the on-column reoxidation using a redox system is a powerful, simple, and safe tool to recover reduced mAb during manufacturing. Moreover, the apparent benefits of using a high-pH redox wash may further drive the evolution of Protein A platform processes.

A novel fluorescence-based immunochromatographic assay (ICA) for rapid detecting 4(5)-methylimidazole (4-MI) is presented in this study. In our work, the conjugates of fluorescent microspheres (FMs) and 4-MI monoclonal antibody were used as probe for ICA. Under optimal conditions, a standard curve of ICA-based detection of 4-MI was developed, linear detection ranged from 0.50 to 32.0 mg/L. The cross-reactivities were observed less than 3.93% by detecting 6 selected structural analogues of 4-MI. The recoveries of 4-MI in caramels detection were ranged from 82.85% to 102.31%, with the coefficient of variation (n = 3) below 9.06%. Quantitative comparison of the established fluorescence-based ICA with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) analysis of real caramel colour samples indicated a good correlation among the methods. Therefore, our developed fluorescence-based ICA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of 4-MI in food safety control., (Copyright © 2015 Elsevier Ltd. All rights reserved.)