The criticality of high-resolution N-linked carbohydrate assays and detailed characterization of antibody effector function in the context of biosimilar development

Abstract

Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays. This work highlights the challenges of developing high-quality biosimilar candidates and the need for modern biotechnology capabilities. These results show that high-quality analytics, combined with sensitive cell-based assays to study in vivo mechanisms of action, is an essential part of biosimilar development.

The immunoglobulin G1 N-glycan composition affects binding to each low affinity Fc γ receptor

“…Our Fc constructs do not provide an opportunity to measure cell activation, although our results are quantitatively consistent with a recent report of Fc N-glycan composition on CD16A-expressing NK92 cell activation. Connell-Crowley and coworkers altered the N-glycan composition of 2 mAbs with glycosidase digestions, chromatography and antibody production techniques, 54 and determined that a 1% decrease in fucosylation resulted in a 24% increase in antibody-dependent cell-mediated cytotoxicity (ADCC). Reducing galactose levels showed -determined from fitting intensity data at equilibrium à -determined from fitting a kinetic model to the sensorgrams n.d.-not determined Figure 7.…”

Analysis of Tubespins as a suitable scale‐down model of bioreactors for high cell density CHO cell culture

“…HMW also correlated, suggesting molecular properties that lead to aggregation can be observed in both scales. HM predictability was acceptable for five cell lines but not for all, indicating TS results may need to be confirmed in BR when HM levels are critical in process development . Finally, product quality attributes that change as a function of culture time including deamidation, fragmentation and C‐terminal lysine levels are not predicted well by the TS scale‐down model, and further evaluation is needed in instrumented BR when studying these.…”

“…HM predictability was acceptable for five cell lines but not for all, indicating TS results may need to be confirmed in BR when HM levels are critical in process development. 19 Finally, product quality attributes that change as a function of culture time including deamidation, fragmentation and C-terminal lysine levels are not predicted well by the TS scale-down model, and further evaluation is needed in instrumented BR when studying these. This discrepancy applies in particular to our studies where HCD BR culture is only replenishing fresh medium but retaining both cells and product inside the BR for the whole process duration.…”

Real-time product attribute control to manufacture antibodies with defined N-linked glycan levels

“…Correspondence concerning this article should be addressed to C. Zupke at zupkeca@amgen.com. to control High Mannose levels to match antibody-dependent cell cytotoxicity (ADCC) activity 11 . However, obtaining similar levels of High Mannose glycans from one process to another may not be readily achievable when using differing cell-lines and process conditions.…”