Modeling of the Biological Activity of Monoclonal Antibodies Based on the Glycosylation Profile.

The influence of the glycosylation profile of IgG on biological activity is known, but it is not clear which glycoforms have the highest impact on the main mechanism of action. The aim of this study was to design a mathematical model for predicting the antibody-dependent cellular cytotoxicity (ADCC) activity and the Fc gamma IIIa receptors' (FcɣRIIIa) relative binding of rituximab drug products based on their glycosylation profile. An additional goal was to identify the glycoforms that have the greatest impact on these mechanisms of action. For these purposes, the glycosylation profile was examined by hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC), ADCC was assessed using a Promega kit, and FcɣRIIIa's binding affinity was assessed by surface plasmon resonance (SPR) analysis of a group of >50 rituximab drug products. Based on the results, mathematical models for the ADCC and FcɣRIIIa binding affinity prediction were designed using JMP 13.2.0. The quality of the model and the influence of sample size and heterogeneity on the reliability were verified. The results allow for the evaluation of rituximab drug products' activity based on their glycosylation profile and show that with a sufficiently large and differentiated dataset, it is possible to generate models for different monoclonal antibodies.

Toward Shortened the Time-to-Market for Biopharmaceutical Proteins: Improved Fab Protein Expression Stability Using the Cre/lox System in a Multi-Use Clonal Cell Line.

Stable gene integration and rapid selection of high-expressing clones are important when developing biopharmaceutical systems to produce a protein of interest. According to regulatory guidelines, the final production clones should be stable through multiple cell generations. To achieve long-term stable expression of Fab genes via recombinase-mediated cassette exchange (RMCE), we modified mutual configurations of the lox sequences. By inversion of the spacer orientation, we avoided the loss of the integrated gene after several dozen cycles of cell division. This feature also prevents reversible transgene integration. Although the RMCE allows us to generate transgenic lines rapidly relative to current methods, it remains difficult to obtain stable industrial cell lines for long-term culturing and for the initial development stage. In this study, we present an approach to shortening the timeline for therapeutic protein development. Our approach provides easy access to the same clonal cell line in the initial development phase, and also for the production of biopharmaceutical proteins.