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Investigation of the interaction mechanism of the recombinant human antibody MDJ8 and its fragments with chromatographic apatite phases

Title data

Schubert, Sven ; Freitag, Ruth:
Investigation of the interaction mechanism of the recombinant human antibody MDJ8 and its fragments with chromatographic apatite phases.
In: Journal of Chromatography A. Vol. 1216 (May 2009) Issue 18 . - 3831 - 3840.
ISSN 1873-3778
DOI: https://doi.org/10.1016/j.chroma.2009.02.074

Official URL: Volltext

Abstract in another language

The chromatographic behaviour of a recombinant human antibody (IgG1-subtype, κ-light chain, MW: 149.5 kD, pI: 9.3) was investigated as a function of the buffer pH and buffer type (HEPES, phosphate, borate) on fluoroapatite and hydroxyapatite stationary phases. {HEPES} buffer was used at pH 7.0, phosphate buffer at pH 8.2 and borate buffer between pH 8.5 and 11. Elution was by a double gradient method of first a salt gradient from 0 to 1 M NaCl in the corresponding buffer, followed by a step gradient to 0.4 M sodium phosphate. Regardless of the pH and buffer type, the antibody eluted in the NaCl gradient; capacity factors decreased with increasing pH. At pH 11 the antibody eluted in the flow-through. Retention was thus dominated by electrostatic interaction throughout the investigated pH-range. Investigation of antibody fragments obtained by papain digestion (fc- and fab-fragments) and deglycosylated fc-fragments showed that the sugar structures had no influence on the chromatographic behaviour. Instead the chromatographic behaviour was dominated by that of the fab-fragment. ζ-Potential measurements verified that the apatite surface bore a negative surface charge in the investigated pH range, while the antibody net surface charge switched from positive to negative as the pH increased. The corresponding isoionic point was a function of both the buffer concentration and the buffer species. However, above a pH of 8.3 the ζ-potential of the antibody generally was negative. Simulations of the molecular electrostatic potential of the antibody and the two fragments revealed the presence of a positively charged patch within the fab-fragment, which only disappeared above a pH of 10. Most likely this patch was responsible for the observed behaviour.

Further data

Item Type: Article in a journal
Refereed: Yes
Keywords: Zeta potential
Institutions of the University: Faculties > Faculty of Engineering Science
Faculties > Faculty of Engineering Science > Chair Process Biotechnology
Faculties > Faculty of Engineering Science > Chair Process Biotechnology > Chair Process Biotechnology - Univ.-Prof. Dr. Ruth Freitag
Faculties
Result of work at the UBT: Yes
DDC Subjects: 600 Technology, medicine, applied sciences
600 Technology, medicine, applied sciences > 620 Engineering
Date Deposited: 24 Feb 2016 13:32
Last Modified: 24 Feb 2016 13:32
URI: https://eref.uni-bayreuth.de/id/eprint/31034