A framework for real‐time glycosylation monitoring (RT‐GM) in mammalian cell culture

Abstract

Glycosylation is a critical characteristic of biotherapeutics because of its central role in in vivo efficacy. Multiple factors including medium composition and process conditions impact protein glycosylation and characterizing cellular response to these changes is essential to understand the underlying relationships. Current practice typically involves glycosylation characterization at the end of a fed-batch culture, which in addition to being an aggregate of the process, reflects a bias towards the end of the culture where a majority of the product is made. In an attempt to rigorously characterize the entire time-course of a fed-batch culture, a real-time glycosylation monitoring (RT-GM) framework was developed. It involves using the micro sequential injection (μSI) system as a sample preparation platform coupled with an ultra-performance liquid chromatography (UPLC) system for real-time monitoring of the antibody glycan profile. Automated sampling and sample preparations were performed using the μSI system and this framework was used to study manganese (Mn)-induced glycosylation changes over the course of a fed-batch culture. As expected, Mn-supplemented cultures exhibited higher galactosylation levels compared to control while the fucosylation and mannosylation were consistent for both supplemented and control cultures. Overall, the approach presented in the study allows real time monitoring of glycosylation changes and this information can be rapidly translated into process control and/or process optimization decisions to accelerate process development.

A View on the Importance of “Multi-Attribute Method” for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy

“…Control of the glucose feeding based on real-time monitoring of the culture at the bioreactor stage using a Raman probe allows a more precise control of the glucose concentration in the culture than conventional periodic glucose measurements and therefore a more consistent level of glycation in mAb products manufactured (35). Similarly, others have reported real-time glycosylation monitoring of bioreactor using a micro-sequential injection system coupled with U/HPLC Nglycan analysis (36), which supported the use of the method for process monitoring and control. In another report, Zupke et al showcased direct control of high-mannose N-glycosylation levels by controlling mannose feed to the bioreactor (37).…”

The glycosylation aspects of biosimilarity

“…Tharmalingam et al [71] developed a real-time N-glycosylation monitoring (RT-GM) framework to characterize the entire time-course of a fed-batch culture. The sample preparation platform utilized microsequential injection (mSI), which was coupled with an ultraperformance liquid chromatography (UPLC) system based real-time glycan profiling for therapeutic antibody manufacturing.…”

Control of IgG glycosylation by in situ and real‐time estimation of specific growth rate of CHO cells cultured in bioreactor

“…Chinese hamster ovary (CHO) cells are the most commonly used cell lines for mAb production due to their capability of performing human‐like posttranslational modifications (PTMs; Cole, Demont, & Marison, ). Glycosylation is one of the most important PTMs for mAbs, as it impacts effector functions, pharmacokinetics, antigenicity, safety, stability, and solubility of the mAbs produced (Tharmalingam, Wu, Callahan, & T. Goudar, ). Therefore, glycosylation is a critical quality attribute (CQA) for mAb production processes that should be taken into consideration in all manufacturing steps, as outlined in the quality by design (QbD) approach initiated by the Food and Drug Administration (FDA) in 2004(ICH, ).…”