Integrated Process Analytical Platform for Automated Monitoring of Monoclonal Antibody N-Linked Glycosylation.

The biopharmaceutical industry is transitioning toward the adoption of continuous biomanufacturing practices that are often more flexible and efficient than traditional batch processes. Federal regulatory agencies are further urging the use of advanced process analytical technology (PAT) to analyze the design space to increase the process knowledge and enable high-quality biologic production. Post-translational modifications of proteins, such as N-linked glycosylation, are often critical quality attributes that affect biologics' safety and efficacy, requiring close monitoring during manufacturing. Here, we developed an online sequential-injection-based PAT system, called N-GLYcanyzer, which can rapidly monitor mAb glycosylation during upstream biomanufacturing. The key innovation includes the design of an integrated mAb sampling and fully automated sample derivation system for antibody titer and glycoform analysis within 3 h. The N-GLYcanyzer process includes mAb capture, deglycosylation, released glycan labeling with fluorescent dyes, and labeled glycan enrichment for direct injection/analysis on an integrated high-performance liquid chromatography system. Different fluorescent tags and reductants were tested to maximize glycan labeling efficiency under aqueous conditions, while porous graphitized carbon (PGC) was used for optimizing glycan recovery and enrichment. We found that 2-aminobenzamide labeling of glycans with 2-picoline borane as a reducing agent, using the N-GLYcanyzer workflow, shows higher glycan labeling efficiency under aqueous conditions, leading upward to a 5-fold increase in fluorescent product intensity. Finally, we showcase how the N-GLYcanyzer platform can be implemented at-/online in an upstream bioreactor for automated and near-real-time glycosylation monitoring of a Trastuzumab biosimilar produced by Chinese hamster ovary cells.

Evaluation of single-use optical and electrochemical pH sensors in upstream bioprocessing.

Culture pH is a critical process parameter during CHO cell bioreactor operations that is key for proper cell growth, protein production, and maintaining the critical quality attributes of a monoclonal antibody drug substance. The traditional means of measuring pH in bioreactors is with an electrochemical probe that can withstand and maintain accuracy through repeated sterilization cycles. An alternative technique for measuring pH is an optical sensor composed of a fluorescent dye that is sensitive to the hydrogen ion concentration. In this work we explore single-use electrochemical and single-use optical pH sensors in stirred-tank and rocking bioreactors, respectively, to understand how their overall performance compares to traditional electrochemical probes in benchtop glass stirred tank bioreactors. We found that the single-use optical pH sensors were generally less accurate than the electrochemical probes, especially in detecting large pH drifts from the setpoint. The single-use electrochemical probes were increasingly accurate as pH was increased from <7.0 to 7.5 but tended to decrease in accuracy as the batch age increased. In conclusion, single-use pH sensors offer a convenient means to measure pH during an upstream bioprocess, but the limitations of these sensors should be built into process control such that deviations in process pH, and consequently potential fluctuations in product quality, can be avoided.

Recent Advances in Biologic Therapeutic N-Glycan Preparation Techniques and Analytical Methods for Facilitating Biomanufacturing Automation.

N-glycosylation is a post-translational modification that occurs during the production of monoclonal antibody (mAb) therapeutics. During production of mAb based therapeutics the use of various hosts and cell culture additives attribute to glycan heterogeneity. The safety and efficacy of monoclonal antibodies with mechanism of actions that utilize Fc effector functions can be negatively impacted by glycan heterogeneity and thus is often considered a critical quality attribute (CQA). In this mini review, we discuss recent advances in mAb sample preparation specifically focused on denaturation, enzymatic processing, and released glycans derivatization methods. Additionally, we review the recent advances in characterization of released and intact N-glycans using chromatography, capillary electrophoresis, and mass spectrometry techniques with a focus on rapid, automated approaches that support analysis of glycosylation profiles of biopharmaceuticals. We delve into advances within sample preparation techniques that allow for rapid and robust sample preparation as well as how these techniques are being used for innovative at-line high-throughput screening and process analytical technology (PAT). The future of biomanufacturing is focused on decreasing process costs while increasing process understanding and quality for novel biologic candidates and biosimilars. Therefore, advances in PAT for biotherapeutics will positively influence current manufacturing practices and enable further bioprocess automation.