A frontal analysis combined with a simultaneous chromatographic analysis of macromolecules using a single chromatographic system

• Published in: Journal of Chromatography A 2020-01, Vol.1610, p.460571, Article 460571
• Main Authors: Ambrožič, Rok, Modic, Petra, Hribar, Gorazd, Podgornik, Aleš
• Format: Article
• Language: English
• Subjects: Monoclonal antibodies; On-line chromatographic analysis; PAT; pDNA; Preparative chromatography; Process monitoring
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2019.460571

•Monitoring the effluent of the preparative column by rapid chromatography on a single chromatographic system.•Fast chromatographic analysis was allowed at low pressure using convection based chromatographic columns.•Limit of detection can be tuned by sample loop and variable number of injections•Typical method accuracy is in the range of few percent or below. A chromatographic system was adapted to allow monitoring of eluent of preparative column via absorbance and with the chromatographic analysis of the target macromolecule on the same chromatographic system. The proposed approach was tested on important macromolecules, such as monoclonal antibodies, monoclonal antibody aggregates and plasmid DNA (pDNA). A frontal analysis was made on the preparative column, while a chromatographic on-line analysis was performed by sequentially injecting the preparative column outlet on a convection-based analytical column, operating on the same chromatographic system. Cation and/or anion exchangers were used as the chromatographic media (along with a protein A), depending on the sample to be purified. The method was found to be robust and reproducible. To adjust the limit of detection, an algorithm varying the number of injections was used, enabling accurate monitoring of an early breakthrough for concentrations below 1% of the feed concentration. The accuracy varies according to the applied flow rate, but it is typically in the range of few percent, or even below. Due to its simplicity and flexibility, the proposed method can be easily adapted to a pharmaceutical environment.