期刊
出版社
ELSEVIER
DOI: 10.1016/j.jchromb.2021.122731
关键词
Lectin chromatography; polyHIPE monolith; Glycosylated proteins; Perfusion bioprocess monitoring
资金
- Slovenian Research Agency (ARRS) [P1-0153, J2-9440]
- European Regional Development Fund
- Slovenian Ministry of Education, Science, and Sport (project BioPharm.Si)
An affinity column based on lectins was developed for monitoring glycosylated protein concentration in perfusion bioprocesses. The immobilized lectin showed improved binding kinetics towards Fc-fusion protein with the introduction of a spacer and complete desorption using 0.25 M galactose was achieved. The affinity column exhibited linearity in a specific concentration range and flow-unaffected binding for certain flow rates, along with long-term stability and no unspecific binding of culture media components.
In-process monitoring of glycosylated protein concentration becomes very important with the introduction of perfusion bioprocesses. Affinity chromatography based on lectins allows selective monitoring when carbohydrates are accessible on the protein surface. In this work, we immobilized lectin on polyHIPE type of monoliths and implemented it for bioprocess monitoring. A spacer was introduced to lectin, which increased binding kinetics toward Fc-fusion protein, demonstrated by bio-layer interferometry. Furthermore, complete desorption using 0.25 M galactose was shown. Affinity column exhibited linearity in the range between 0.5 and 8 mg/ml and flow-unaffected binding for the flow-rates between 0.5 and 8 ml/min. Long-term stability over at least four months period was demonstrated. No unspecific binding of culture media components, including host cell proteins and DNA, was detected. Results obtained by affinity column matched concentration values obtained by a reference method.
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