On-Line Ion Exchange Liquid Chromatography as a Process Analytical Technology for Monoclonal Antibody Characterization in Continuous Bioprocessing

Abstract

Combining process analytical technology (PAT) with continuous production provides a powerful tool to observe and control monoclonal antibody (mAb) fermentation and purification processes. This work demonstrates on-line liquid chromatography (on-line LC) as a PAT tool for monitoring a continuous biologics process and forced degradation studies. Specifically, this work focused on ion exchange chromatography (IEX), which is a critical separation technique to detect charge variants. Product-related impurities, including charge variants, that impact function are classified as critical quality attributes (CQAs). First, we confirmed no significant differences were observed in the charge heterogeneity profile of a mAb through both at-line and on-line sampling and that the on-line method has the ability to rapidly detect changes in protein quality over time. The robustness and versatility of the PAT methods were tested by sampling from two purification locations in a continuous mAb process. The PAT IEX methods used with on-line LC were a weak cation exchange (WCX) separation and a newly developed shorter strong cation exchange (SCX) assay. Both methods provided similar results with the distribution of percent acidic, main, and basic species remaining unchanged over a 2 week period. Second, a forced degradation study showed an increase in acidic species and a decrease in basic species when sampled on-line over 7 days. These applications further strengthen the use of on-line LC to monitor CQAs of a mAb continuously with various PAT IEX analytical methods. Implementation of on-line IEX will enable faster decision making during process development and could potentially be applied to control in biomanufacturing.

Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry

“…Recently CVA, applying both weak cation exchange (WCX) and strong cation exchange (SCX) chromatography, was used in a process analytical technology (PAT) framework as a powerful tool to control and optimise upstream and downstream processing. 14 While well suited for comparability studies, one limitation is the necessity to perform elaborate sample preparation procedures and follow-up experiments for initial identification of the peaks and in case of any out-of-specification events. These experimental procedures usually involve peak collection followed by buffer exchange to allow further analysis, which can be time consuming and costly.…”

Recent Advances in Chromatography for Pharmaceutical Analysis

“…According to the authors, implementation of on-line IEX also has the potential to be applied to biomanufacturing. 171 Last but not least, a similar PAT instrument was also recently applied as an on-line tool for the crystallization of three active pharmaceutical ingredients (i.e., caffeine, acetylsalicylic acid and a patented drug from GSK). Indeed, concentration is an important process parameter in pharmaceutical crystallization processes, and the real-time multicomponent concentration was monitored in pharmaceutical crystallization 172 with PAT technology.…”

Technology outlook for real‐time quality attribute and process parameter monitoring in biopharmaceutical development—A review

“…Liquid chromatography technologies have also been tailored to facilitate PAT applications. Waters PATROL Process Analysis System® is capable of extracting samples from the process stream in an automated fashion for analysis on a given chromatographic method (Patel et al, 2017). The Agilent two‐dimensional liquid chromatography (2D‐LC) system allows running of two chromatography methods in a sequential fashion, enabling the monitoring of two product quality attributes from a given sample (Buckenmaier, 2015).…”