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Application of a PEG/Salt Aqueous Two-Phase Partition System for The Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract Dimitris Platis, Nikolaos Labrou To cite this version: Dimitris Platis, Nikolaos Labrou. Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract. Biotechnology Journal, Wiley-VCH Verlag, 2009, 4 (6), pp.1320. 10.1002/biot.200800359. hal-00495061 HAL Id: hal-00495061 https://hal.archives-ouvertes.fr/hal-00495061 Submitted on 25 Jun 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biotechnology Journal Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract For Peer Review Journal: Biotechnology Journal Manuscript ID: BIOT-2008-0359.R2 Wiley - Manuscript type: Research Article Date Submitted by the 30-Mar-2009 Author: Complete List of Authors: Platis, Dimitris; Agricultural University of Athens, Agricultural Biotechnology Labrou, Nikolaos; Agricultural University of Athens, Agricultural Biotechnology Aqueous two phase system, molecular farming, downstream Keywords: processing, therapeutic monoclonal antibody, transgenic plants Wiley-VCH Page 2 of 26 Biotechnology Journal 1 1 2 3 4 Research Article ((4760 words)) 5 6 7 Application of a PEG/salt aqueous two-phase partition 8 9 10 system for the recovery of monoclonal antibodies from 11 12 13 unclarified transgenic tobacco extract 14 15 16 17 18 D. Platis and N.E. Labrou* 19 20 For Peer Review 21 22 23 24 25 Enzyme Technology Laboratory, Department of Agricultural Biotechnology, 26 27 28 Agricultural University of Athens, 75 Iera Odos, GR 118 55 Athens, Greece 29 30 31 32 33 34 35 36 37 38 39 40 41 42 * To whom all correspondence should be addressed 43 44 45 Tel and fax: +30 210 5294308, e-mail: [email protected] 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Wiley-VCH Biotechnology Journal Page 3 of 26 2 1 2 3 Keywords 4 5 6 Aqueous two phase system; downstream processing; molecular farming; therapeutic 7 8 monoclonal antibody; transgenic plants 9 10 11 12 13 Abbreviations 14 15 CCD, central composite design; ELISA, enzyme-linked immunosorbent assay; mAb 16 17 18 2G12/4E10, human anti-HIV 2G12/4E10 monoclonal antibody; PEG, 19 20 polyethylenoglycol;For Pi, phosphate;Peer SDS-PAGE,Review sodium sulphate dodecyl 21 22 polyacrylamide gel electrophoresis; TBS, Tris buffered saline; TCA, trichloroacetic 23 24 25 acid 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Wiley-VCH Page 4 of 26 Biotechnology Journal 3 1 2 3 1. Abstract 4 5 6 Aqueous two phase partition systems (ATPS) have been widely used for the separation 7 8 of large variety of biomolecules. In the present report, the application of a 9 10 11 polyethyleneglycol/phosphate (PEG/Pi) ATPS for the separation of anti-HIV 12 13 monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified 14 15 transgenic tobacco crude extract was investigated. Optimal conditions that favour 16 17 18 opposite phase partitioning of plant debris/mAb as well as high recovery and 19 20 purification were foundFor to be Peer 13.1% w/w [PEGReview 1500], 12.5% w/w [Pi] at pH 5 with a 21 22 phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these 23 24 25 conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase 26 27 with 85 and 84% yield and purification 2.4 and 2.1fold, respectively. The proposed 28 29 ATPS was successfully integrated in an affinity-based purification protocol, using 30 31 32 Protein A, yielding antibodies of high purity and yield. From this study, ATPS was 33 34 shown to be suitable for initial protein recovery and partial purification of mAbs from 35 36 37 unclarified transgenic tobacco crude extract. 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Wiley-VCH Biotechnology Journal Page 5 of 26 4 1 2 3 2. Introduction 4 5 6 The application of aqueous twophase systems (ATPS) in protein recovery has 7 8 received considerable attention. ATPS offer low-cost, easily scalable, environmentally 9 10 11 benign as well as multifunctional operations [1-4] . PEG/salt systems, in particular, 12 13 are widely applied for bioseparation of proteins because they are cost-effective low 14 15 viscocity systems, suitable for continuous large scale operations, present shorter 16 17 18 sepration times and able to handle high biomass load [5-8] . The bulk of both phases 19 20 consist mainly of Forwater (80–90%). Peer PEG has Review a stabilising effect on the protein tertiary 21 22 23 structure, offering thus a gentle biocompatible nontoxic environment for labile 24 25 biomolecules at which biological activity and protein solubility is maintained [9] . A 26 27 variety of operational conditions, such as choice and concentration of polymer, salt, 28 29 30 pH can be manipulated in order to achieve differential partitioning of the protein of 31 32 interest. Differential partitioning mainly depends on surface properties (such as the 33 34 size, charge and hydrophobicity) of the protein as well as on the physicochemical traits 35 36 37 of the two phases [10-12] . Despite the fact that recovery of monoclonal antibodies 38 39 from complex biological tissues has been reported in several studies, the wide-scale 40 41 42 application of ATPS has been hindered by the poorly-understood partitioning 43 44 mechanism [13,11] . 45 46 47 48 49 Future progress in utilizing transgenic plants for biopharmaceuticals production will 50 51 depend on the efficiency of the purification methods, since purification represents 52 53 54 most of the cost of biopharmaceutical production in transgenic plants [14,15] . 55 56 Therefore, new approaches are necessary to minimize or eliminate purification costs. 57 58 Several chromatographic strategies have been investigating so far. These include 59 60 cation exchange chromatography [16] , anion exchange chromatography [17] , Wiley-VCH Page 6 of 26 Biotechnology Journal 5 1 2 3 metal chelate affinity chromatography [18,19] , polyelectrolyte precipitation [20] , 4 5 6 Protein A affinity chromatography [21] , and biomimetic affinity chromatography 7 8 [22,23] . ATPS is a pre-purification/concentration method that offers potential for 9 10 11 product capture integrated with solids removal. ATPS has been evaluated for 12 13 purifying egg white lysozyme [24] and mAb 2F5 and 2G12 using clarified tobacco 14 15 16 extracts [25,26] . In this latter study, the effect of PEG molecular weight, pH and 17 18 tie line length, as well as the overall potential use of PEG/phosphate aqueous two 19 20 phase systems forFor the recovery Peer and purification Review of mAbs 2F5 and 2G12 were 21 22 23 examined in Platis et al. , 2006 [25] . Similarities in the partitioning behaviour of 24 25 monoclonal antibodies, especially of the same subclass (IgG1) have been observed in 26 27 28 the literature probably due to overall similarities in general structural and 29 30 physicochemical characteristics [27,28] . Therefore, in the present study, ATPS 31 32 optimization was achieved by analyzing the factors influencing the partitioning of 33 34 35 mAb 2G12 and tobacco proteins, based on initial parameter range discovery as 36 37 published previously [25] . This allowed the use of a standard two-parameter Central 38 39 40 Composite Design (CCD) for a narrow range of PEG and phosphate concentrations on 41 42 multiple PEG MWs, in order to predict conditions of optimal differential plant debris 43 44 separation, purification and yield [29,30] . All pertaining experiments were 45 46 47 conducted at pH 5, which was found to be optimal for extraction and partitioning 48 49 [25] . 50 51 52 53 54 Protein isolation and purification from plants is a difficult task, owing to the 55 56 complexity of the plant system and the presence of phenolic compounds, pigments, 57 58 59 and mucilages [14,31] . The presence of such compounds is potential interfering 60 factor in downstream processing and has to be addressed early in the isolation process. Wiley-VCH Biotechnology Journal Page 7 of 26 6 1 2 3 For example, the presence of phenols and anionic polysaccharides can cause problems 4 5 6 during chromatographic purification since they bind strongly to the chromatographic 7 8 matrices and may give them ionexchange characteristics [32-34] or potentially 9 10 11 reduce their half-life [25] . The presence of these compounds may strongly bind, 12 13 inactivate and modify recombinant proteins expressed in transgenic plants [35,36] . 14 15 16 This can have a detrimental effect on the structural integrity, purity and function of the 17 18 isolated protein. The economic impact of these metabolites on the downstream 19 20 process, especiallyFor on an industrial Peer scale, has Review not been adequately addressed. 21 22 23 24 25 Plant biotechnology has demonstrated that transgenic plants are suitable hosts for 26 27 expressing recombinant biomolecules.
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