Three-dimensional chromatography for purification and characterization of antibody fragments and related impurities from Escherichia coli crude extracts.

Affiliation

Schimek C(1), Kubek M(1), Scheich D(1), Fink M(1), Brocard C(2), Striedner G(1), Cserjan-Puschmann M(1), Hahn R(3).
Author information:
(1)Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
(2)Biopharma Austria Process Science, Boehringer Ingelheim RCV GmbH & Co KG, Dr.-Boehringer-Gasse 5-11, A-1120 Wien.
(3)Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. Electronic address: [Email]

Abstract

Antibody fragments (Fab) are often produced by recombinant methods in Escherichia coli as no glycosylation is needed. Besides the correctly expressed Fab molecule, a multitude of host cell impurities and product related impurities are present in the crude sample. The identification and characterization of the product-related impurities, such as modified Fab-molecules or free light chain, are of utmost importance. The objective of this work was to design a purification strategy to isolate and characterize Fab and related impurities. A three-dimensional chromatography method was established, consisting of two affinity steps (Protein G and Protein L) and subsequent cation exchange chromatography, followed by mass spectrometry analysis of the purified samples. The procedure was automated by collecting the eluted target species in loops and directly loading the samples onto the high-resolution cation exchange chromatography column. As an example, four different Fab molecules are characterized. All four samples contained mainly the correct Fab, while only one showed extensive N-terminal pyroglutamate formation of the Fab. In another case, we found a light chain variant with uncleaved amino acids from the lead molecule, which was not used for the formation of whole Fab as only correct Fab was found in that sample. Impurities with lower molecular weights, which were bound on the Protein L column, were observed in all samples, and identified as fragments of the light chain. In conclusion, we have devised a platform for characterizing Fab and Fab-related impurities, which significantly facilitated strain selection and optimization of cultivation conditions.