Noninvasive method for determination of immobilized protein A

Mravljak, Rok (author), Stantič, Metka (author), Bizjak, Ožbej (author), Podgornik, Aleš (author)

The pH transition method, developed for the determination of the ion-exchange group density on chromatographic stationary phase, was used for the quantification of immobilized protein A. Monolithic epoxy polyHIPE and particulate CNBr-Sepharose supports were used for immobilization. A lactate buffer was selected, having a buffer capacity peak approximately 0.5 pH units below the maximum buffer capacity of protein A. The pH transition measurements were performed at pH 4.3, where protein A exhibits maximum buffer capacity, with a lactate buffer concentration of 1 mM for protein A immobilized on polyHIPE monoliths and of 5 mM for protein A immobilized on CNBr-Sepharose. The pH transition height and full width at half maximum for the particulate support and the height for the polyHIPE matrix, showed a linear correlation with the amount of immobilized protein A determined from the absorbance difference before and after immobilization for both supports. The developed method allows a simple, non-invasive on-line determination of immobilized protein A using biological buffers, even for chromatographic columns with an amount of immobilized protein A as low as 0.25 mg. In addition, its sensitivity and duration can be easily adjusted by varying the buffer concentration and pH.

• Language: English
• Keywords: direct noninvasive immobilized protein, quantification, protein A, pH transition method, polyHIPE, CNBr-Sepharose
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Publication status: Published
• Publication version: Version of Record
• Year: 2022
• Number of pages: 7 str.
• Numbering: Vol. 1671, art. 462976
• PID: 20.500.12556/RUL-137650
• UDC: 66.08:577.112
• ISSN on article: 0021-9673
• DOI: 10.1016/j.chroma.2022.462976
• COBISS.SI-ID: 103441667
• Publication date in RUL: 24.06.2022

Record is a part of a journal

• Title: Journal of chromatography. A
• Shortened title: J. chromatogr., A
• Publisher: Elsevier
• ISSN: 0021-9673
• COBISS.SI-ID: 6398471

Licences

• License: CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
• Link: http://creativecommons.org/licenses/by-nc-nd/4.0/
• Description: The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.

Secondary language

• Language: Slovenian
• Keywords: direktna neinvazivna imobilizacija proteina, kvantifikacija, protein A, metoda pH prehoda, poliHIPE, CNBr-sefaroza

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: J2-9440
• Name: Karakterizacija fraktalnih struktur in povečevalni kriteriji njihove sinteze

• Funder: ARRS - Slovenian Research Agency
• Project number: P1-0153
• Name: Raziskave in razvoj analiznih metod in postopkov