ARTICLE IN PRESS JID: CHROMA [m5G;November 21, 2020;2:28] Journal of Chromatography A xxx (xxxx) xxx Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Three-dimensional chromatography for purification and characterization of antibody fragments and related impurities from Escherichia coli crude extracts Clemens Schimek a,1 , Matthias Kubek a , David Scheich a , Mathias Fink a , Cécile Brocard b , Gerald Striedner a , Monika Cserjan-Puschmann a , Rainer Hahn a,∗ a 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 b Biopharma Austria Process Science, Boehringer Ingelheim RCV GmbH & Co KG, Dr.-Boehringer-Gasse 5-11, A-1120 Wien a r t i c l e i n f o Article history: Received 17 September 2020 Revised 5 November 2020 Accepted 9 November 2020 Available online xxx Keywords: Fab Affinity chromatography Protein L Protein G automation mass spectrometry a b s t r a c t Antibody fragments (Fab) are often produced by recombinant methods in Escherichia coli as no glycosy- lation 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 spectrome- try 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 col- umn. 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 an- other 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 character- izing Fab and Fab-related impurities, which significantly facilitated strain selection and optimization of cultivation conditions. © 2020 Elsevier B.V. All rights reserved. 1. Introduction Antigen binding fragments (Fab) offer certain advantages over full-length monoclonal antibodies. Compared to antibodies, the tis- sue penetration and targeting epitopes of Fab, parameters which are difficult to access for antibodies, are enhanced because of the reduced molecular size of Fab [1]. Along with the smaller size, immunogenicity is also reduced [1,2]. For most Fabs, effec- tor functions are not required or are not even beneficial, there- fore glycosylation is not needed [3]. Consequently, microbial ex- pression systems which feature high growth rates and cell den- sities are attractive for Fab production [4,5]. Among these sys- tems, E. coli is often the first choice because it is generally well ∗ Corresponding author at: Muthgasse 18, A-1190 Vienna, Austria. E-mail address: rainer.hahn@boku.ac.at (R. Hahn). 1 Clemens Schimek and Matthias Kubek contributed equally to this work. understood and easy to cultivate [6]. However, recombinant Fab production in E. coli can still be challenging compared to other proteins [5,7-11]. To generate soluble functional Fab, light chain (LC) and heavy chain (HC) antibody precursors must be expressed correctly and transported into the periplasm where the assembly into the whole molecule takes place [12-14]. Besides the correctly processed Fab, free LC and variants or modified versions thereof are common by-products during the fermentation process [12,13]. These product-related impurities must be removed during the pu- rification process. Ideally, their formation should be prevented dur- ing cell cultivation. To obtain detailed information on the produc- tion process, the time-resolved occurrence of Fab over the course of fermentation is required [15]. However, determination of the aforementioned by-products in the context of fermentation pro- cess monitoring has not yet been reported. Thus, methods are needed to separate and identify the various protein species aris- ing from crude E. coli homogenates and fermentation broth. This https://doi.org/10.1016/j.chroma.2020.461702 0021-9673/© 2020 Elsevier B.V. All rights reserved. Please cite this article as: C. Schimek, M. Kubek, D. Scheich et al., Three-dimensional chromatography for purification and characterization of antibody fragments and related impurities from Escherichia coli crude extracts, Journal of Chromatography A, https://doi.org/10.1016/ j.chroma.2020.461702