Isolation and characterization of rabies monoclonal antibody charge variants.

Current postexposure prophylaxis of rabies includes vaccines, human rabies immunoglobulin (RIG), equine RIG, and recombinant monoclonal antibodies (mAb). In the manufacturing of rabies recombinant mAb, charge variants are the most common source of heterogeneity. Charge variants of rabies mAb were isolated by salt gradient cation exchange chromatography (CEX) to separate acidic and basic and main charge variants. Separated variants were further extensively characterized using orthogonal analytical techniques, which include secondary and tertiary structure determination by far and near ultraviolet circular dichroism spectroscopy. Charge and size heterogeneity were evaluated using CEX, isoelectric focusing (IEF), capillary-IEF, size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and western blotting. Antigen binding affinity was assessed by enzyme linked immuno-sorbent assay and rapid florescence foci inhibition test. Results from structural and physicochemical characterizations concluded that charge variants are formed due to posttranslational modification demonstrating that the charge heterogeneity, these charge variants did neither show any considerable physicochemical change nor affect its biological function. This study shows that charge variants are effective components of mAb and there is no need of deliberate removal, until biological functions of rabies mAb will get affected.

Assessment of change in the basic variants composition of trastuzumab during dilution in saline for administration.

Postproduction handling of drug products during preparation or clinical use may affect the structure and efficacy of the drug and perhaps remain unnoticed. Since chemical modifications can impact the product's structure, stability, and biological activity, this study investigates the impact of elevated temperature and subtle shift in pH on the drug product post-dilution in saline. The mAb sample diluted in saline for administration was stressed at elevated temperature and slightly acidic pH condition. Extended stability studies were performed and monitored for size and charge heterogeneity. Size heterogeneity shows no significant changes, whereas charge heterogeneity shows an increase in basic variants and a reduction in main species. Further, basic variants were isolated and characterized to identify the type and site of chemical modification. Intact mass analysis and peptide mapping identify that the basic variants were attributed mainly to the isomerization of HC Asp102 into iso-Asp or its succinimide intermediate. Four basic variants were found to exhibit similar structural properties as the main and control samples. However, basic variants showed reduced binding affinity to HER2 receptor, while there was no significant difference in FcRn binding. The results indicate that modification in the HC Asp102, which is present in the CDR, affects antigen binding and thus can influence the potency of the drug product. Hence, with the conventional stability studies required to license the drug product, including in-use or extended stability studies to mimic the postproduction handling would be desirable.

Improving titer while maintaining quality of final formulated drug substance via optimization of CHO cell culture conditions in low-iron chemically defined media.

During biopharmaceutical process development, it is important to improve titer to reduce drug manufacturing costs and to deliver comparable quality attributes of therapeutic proteins, which helps to ensure patient safety and efficacy. We previously reported that relative high-iron concentrations in media increased titer, but caused unacceptable coloration of a fusion protein during early-phase process development. Ultimately, the fusion protein with acceptable color was manufactured using low-iron media, but the titer decreased significantly in the low-iron process. Here, long-term passaging in low-iron media is shown to significantly improve titer while maintaining acceptable coloration during late-phase process development. However, the long-term passaging also caused a change in the protein charge variant profile by significantly increasing basic variants. Thus, we systematically studied the effect of media components, seed culture conditions, and downstream processing on productivity and quality attributes. We found that removing ß-glycerol phosphate (BGP) from basal media reduced basic variants without affecting titer. Our goals for late-phase process development, improving titer and matching quality attributes to the early-phase process, were thus achieved by prolonging seed culture age and removing BGP. This process was also successfully scaled up in 500-L bioreactors. In addition, we demonstrated that higher concentrations of reactive oxygen species were present in the high-iron Chinese hamster ovary cell cultures compared to that in the low-iron cultures, suggesting a possible mechanism for the drug substance coloration caused by high-iron media. Finally, hypotheses for the mechanisms of titer improvement by both high-iron and long-term culture are discussed.

Effects of copper on CHO cells: cellular requirements and product quality considerations.

Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes.