Photodegradation of Rituximab and Critical Evaluation of Its Sensibility to Electromagnetic Radiation.

Rituximab is a monoclonal antibody used in the treatment of lymphoma non-Hodgkin. This mAb is photosensitive as it is administrated to the patient by infusion/perfusion; therefore, photostability is a decisive factor in the efficacy of this biologic. To better understand the photodegradation mechanisms of Rituximab, this biologic was exposed to different irradiance conditions. We show in this study that this mAb photodegrade proportionally to irradiance intensities. The main modifications of Rituximab by irradiance correlate to the increase in aggregates, decrease in its Tm, acidic charge variants, oxidation of the Trp (36) in the heavy chain, and decrease in complement-dependent cytotoxicity (CDC) potency. To understand the relationship between real-life photo-exposition conditions and ICH standardized light tests, a full characterization was set up. Worst photo-stress cases were evaluated, 1 and 2 h under direct sunlight through a window, mimicking the ID65 electromagnetic radiation profile. Our results show that only exposition to direct sun irradiance during 2 h, (≈ 150 kluxes•h), increases critically soluble and insoluble aggregates, diminishing Tm, increasing acidic charge variants, and the photooxidation of the Trp (36) in the heavy chain measured by peptide mapping-RP-UPLC-MS. A decrease in CDC below 80% resulted under this condition, which correlates with physicochemical analyses. While inside light-room exposition (similar to ICH test) and ICH conditions do not have any contribution to the degradation of Rituximab measured by these physicochemical and biological analytical methods. These results indicate that exposition of Rituximab to below ≈ 75 kluxes•h of sun light cannot photodegrade critically this biologic inside of its primary container.

Cysteine in cell culture media induces acidic IgG1 species by disrupting the disulfide bond network.

A high degree of charge heterogeneity is an unfavorable phenomenon commonly observed for therapeutic monoclonal antibodies (mAbs). Removal of these impurities during manufacturing often comes at the cost of impaired step yields. A wide spectrum of posttranslational and chemical modifications is known to modify mAb charge. However, a deeper understanding of underlying mechanisms triggering charged species would be beneficial for the control of mAb charge variants during bioprocessing. In this study, a comprehensive analytical investigation was carried out to define the root causes and mechanisms inducing acidic variants of an immunoglobulin G1-derived mAb. Characterization of differently charged species by liquid chromatography-mass spectrometry revealed the reduction of disulfide bonds in acidic variants, which is followed by cysteinylation and glutathionylation of cysteines. Importantly, biophysical stability and integrity of the mAb are not affected. By in vitro incubation of the mAb with the reducing agent cysteine, disulfide bond degradation was directly linked to an increase of numerous acidic species. Modifying the concentrations of cysteine during the fermentation of various mAbs illustrated that redox potential is a critical aspect to consider during bioprocess development with respect to charge variant control.

Elucidating the effects of pH shift on IgG1 monoclonal antibody acidic charge variant levels in Chinese hamster ovary cell cultures.

Charge variants, especially acidic charge variants, in recombinant monoclonal antibodies are critical quality attributes, which can affect antibodies' properties in vitro and in vivo. Meanwhile, charge variants are cumulative effects of various post-translational modifications and chemical degradations on antibody. In this work, to investigate the effect of lowering culture pH in the stationary phase on acidic charge variant contents in fed-batch cultures and its mechanism, cell culture experiments in 2-L bioreactors were firstly performed to explore the changes in the charge distribution under the pH downshift condition using weak cation exchange chromatography. It is found that acidic charge variant contents were significantly decreased by pH downshift. Then, to reveal the mechanism by which the content of acidic charge variants is reduced under pH downshift condition, the variation of post-translational modifications and chemical degradations under the pH downshift condition was explored. Meanwhile, the structure of the acidic charge variants was characterized. Several analysis experiments including size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate under non-reducing conditions, tryptic peptide map, and reduced antibody mass were applied in this study. The results show that the mechanism by which the content of acidic charge variants is reduced is that the contents of disulfide bond reduction, galactosylation, and asparagine deamination of the HC-N388 in the Fc domain were reduced by pH downshift.

Investigation of Metal-Catalyzed Antibody Carbonylation With an Improved Protein Carbonylation Assay.

Protein carbonylation is a posttranslational modification referring to the occurrence of aldehydes and ketones in proteins. The current understanding of how carbonylation, in particular, metal-catalyzed carbonylation, occurs in recombinant mAbs during production and storage is very limited. To facilitate investigations into mAb carbonylation, we developed a protein carbonylation assay with improved assay robustness and precision over the conventional assays. We applied this assay to investigate mAb carbonylation under production, storage, and stress conditions and showed that iron, hydrogen peroxide, and polysorbate 20 at pharmaceutically relevant levels critically influence the extent of mAb carbonylation. In addition, we found that while carbonylation correlates with mAb aggregation in several cases, carbonylation cannot be used as a general indicator for aggregation. Furthermore, we observed that mAb carbonylation level can decrease during storage, which indicates that carbonylation products may not be stable. Finally, we report for the first time a positive correlation between carbonylation and acidic charge heterogeneity of mAbs that underwent metal-catalyzed oxidation. This finding shows that the impact of protein carbonylation on product quality for mAbs is not limited to aggregation but also extends to charge heterogeneity.

Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture.

During the production of therapeutic monoclonal antibodies (mAbs), not only enhancement of mAb productivity but also control of quality attributes is critical. Charge variants, which are among the most important quality attributes, can substantially affect the in vitro and in vivo properties of mAbs. During process development for the production of mAbs in a Chinese hamster ovary cell line, we have observed that an improvement in mAb titer is accompanied by an increase in the content of acidic charge variants. Here, to help maintain comparability among mAbs, we aimed to identify the process parameters that controlled the content of acidic charge variants. First, we used a Plackett-Burman design to identify the effect of selected process parameters on the acidic charge variant content. Eight process parameters were selected by using a failure modes and effects analysis. Among these, temperature shift was identified from the Plackett-Burman design as the factor most influencing the acidic charge variant content. We then investigated in more detail the effects of shift temperature and temperature shift timing on this content. The content decreased with a shift to a lower temperature and with earlier timing of this temperature shift. Our observations suggest that Plackett-Burman designs are advantageous for preliminary screening of bioprocess parameters. We report here for the first time that temperature downshift is beneficial for effective control of the acidic peak variant content.