Hyphenation of multi-dimensional chromatography and mass spectrometry for the at-line-analysis of the integrity of recombinant protein drugs

Title data

Canarelli, Stéphane ; Fisch, Igor ; Freitag, Ruth:
Hyphenation of multi-dimensional chromatography and mass spectrometry for the at-line-analysis of the integrity of recombinant protein drugs.
In: Journal of Chromatography B. Vol. 775 (2002) Issue 1 . - pp. 27-35.
ISSN 1873-376X
DOI: https://doi.org/10.1016/S1570-0232(02)00286-6

Official URL:

Abstract in another language

A robust tool is proposed for the rapid at-line verification of the identity and integrity of (recombinant) proteins, namely the hyphenation of multidimensional chromatography and mass spectrometry (MS). A recombinant human antibody produced in Chinese hamster ovary cells is taken as pertinent example. The recombinant human antibody is first captured from the production environment by affinity chromatography (rProtein A, isolation/concentration of the target molecule) and automatically transferred to an enzyme reactor (immobilized trypsin column) for digestion, thereby yielding different peptides corresponding to the protein sequence. The peptides are then separated on a reversed-phase column before being analyzed and identified by MS. This step does not require a fine resolution since the mass spectrometer can identify a variety of substances at the same time. The results are then analyzed in silico with suitable bio-informatic tools. When the gene sequence of the protein product is known, proteolytic cleavages can be predicted and the exact mass and hence the amino acid sequence of each peptide can thereby be deduced. Fitting experimental data and reference peptide sequences then provides important information about the integrity of the protein and more particularly about its sequence. In our case, the integrity of 45 of the light and 75 of the heavy chain sequences of the antibody could be verified within minutes.

Further data

Item Type:	Article in a journal
Refereed:	Yes
Keywords:	Recombinant protein drugs
Institutions of the University:	Faculties > Faculty of Engineering Science > Chair Process Biotechnology > Chair Process Biotechnology - Univ.-Prof. Dr. Ruth Freitag Faculties Faculties > Faculty of Engineering Science Faculties > Faculty of Engineering Science > Chair Process Biotechnology
Result of work at the UBT:	No
DDC Subjects:	500 Science 500 Science > 500 Natural sciences 600 Technology, medicine, applied sciences 600 Technology, medicine, applied sciences > 620 Engineering
Date Deposited:	25 Feb 2016 15:17
Last Modified:	30 Jun 2022 10:25
URI:	https://eref.uni-bayreuth.de/id/eprint/31068