Mon. Dec 9th, 2019

Simultaneous metal chelate affinity purification and endotoxin clearance of recombinant antibody fragments

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international center for biotechnology information11

international center for biotechnology information11


Endotoxins are frequent contaminants of recombinant proteins produced inEscherichia coli. Due to their adverse effects, endotoxins have to be removed from recombinant proteins prior their use in cell-based assays or parenteral application. Reduction of endotoxin to less than 10 EU mg−1is, however, one of the most problematic steps during protein purification fromE. coli and often associated with substantial loss of biological materials. The present paper describes the use of a single step procedure enabling metal chelate affinity purification and endotoxin clearance from antibody fragments produced inE. coliusing a non-ionicdetergent. Endotoxin content was as low as 5 to 9 EU mg−1with a recovery of antibody fragments of over 90%. Non-ionicdetergent treatment did not compromise integrity and functionality of these multimeric molecules. Furthermore, recombinantantibody fragments did not stimulate endotoxin-sensitive cell lines confirming the low endotoxin content. In conclusion, this one-step protocol is a rapid, cost effective and automation-compatible procedure suitable for recombinant antibody fragments.© 2006 Elsevier B.V. All rights reserved


The ability to express functional antibody fragmentsinEscherichia coli(Better et al., 1988; Skerra andPluckthun, 1988) enables the production of significantquantities of material for in vitro experiments, cell-basedassays and in vivo applications. Unfortunately,E. coliderived products are prone to contamination with endotoxins. Endotoxins are toxic components of the outer membrane of gram-negative bacteria. Exposure to these soluble substances may affect the membrane structure of mammalian cells (Jacobs, 1984; Portoles etal., 1987), inhibit cell growth (Dudley et al., 2003) and decrease tissue culture cell viability (Cotten et al., 1994).Endotoxin induces the activation of monocytes, macro-phages (Gao and Tsan, 2003b) and endothelial cells(Munshi et al., 2002). LPS-activated cells releasemediators such as pro-inflammatory cytokines, TNF-αand interleukin-1 (Raetz and Whitfield, 2002). Finally,in humans and animals, exposure to endotoxin causes fever and may result in septic shock (Fiuza and Suffredini, 2001; Martich et al., 1993).The incidence of contaminating endotoxin can complicate the interpretation of experiments or lead tothe misinterpretation of results (Dudley et al., 2003; Gao and Tsan, 2003a), highlighting the benefits to eliminate endotoxin from E. coli derived products. Achieving a low level of endotoxin content together with an excellent recovery rate of the target protein is, however,a difficult task and often dependent on the physico-chemical properties of the target molecule (Petsch and Anspach, 2000). Several methods have been applied to remove endotoxin from biological products (Petsch andAnspach, 2000). Phase separation using non-ionicdetergent takes advantage of the hydrophobic propertiesof endotoxin trapping them in the detergent phase,whereas the hydrophobic proteins remain in the aqueousphase (Aida and Pabst, 1990). It is, however, time consuming and can lead to protein denaturation.Ultrafiltration is an efficient method to eliminate endotoxin from protein solution. It can, however, lead to a substantial loss of the target molecule (Petsch andAnspach, 2000). Anion exchange resins and selective affinity sorbents, which are based on the differentialinteraction of endotoxin and proteins with functional groups immobilized on the resin, often lead to a loss of biological material as multiple rounds of binding are needed to eliminate substantial amounts of endotoxins (Anspach and Hilbeck, 1995). A method for endotoxin removal from his-tagged monomer recombinant proteins using affinity chromatography in combination withthe non-ionic detergent Triton X-114 has been describedpreviously (Reichelt et al., 2006). However, non-ionicdetergent can cause change in protein conformation andimpair binding properties (Hsu and Youle, 1997; Hsuand Youle, 1998; Tan and Ting, 2000).We tested whether this one-step protocol using non-ionic detergent could be applied to antibody fragmentscomposed of two non-covalently linked polypeptidechains (Rauchenberger et al., 2003) and to bivalentantibody fragments containing a small homodimeriza-tion domain (dHLX) (Pack and Pluckthun, 1992). Ascase study, we selected a fully human monoclonalantibody MOR102 (#5) targeting the intercellularadhesion molecule 1 (ICAM-1) derived from theHuman Combinatorial Antibody Library (HuCAL®)(Boehncke et al., 2005; Knappik et al., 2000). The antibody MOR102 (#5) was expressed in two different antibody formats: (i) non-covalently linked Fab frag-ments and (ii) bivalent Fab-dHLX fragments ashistidine-tagged protein.We demonstrate that Triton X-114 is effective at eliminating endotoxin using Ni-NTA affinity chromato-graphy from antibody fragments. Protein recovery was at least 90% with endotoxin content as low as 5 to 9 EUmg−1for the two antibody formats tested. Binding activity and functionality were not compromised as assessed in binding and cell-based assays, respectively.Furthermore, antibody fragments purified using Triton-X114 did not activate endothelial cells, which corrobo-rates with low endotoxin content. Therefore, including Triton X-114 washing step in the purification protocol isa straightforward methodology for the removal of endotoxin from recombinant antibody produced in bacteria

Reference:   T. Zimmerman et al.   Simultaneous metal chelate affinity purification and endotoxin clearance of recombinant antibody fragment Journal of Immunological Methods 314 (2006) 67–73

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