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Abstract

Introduction

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Acknowledgements

Materials

References

Biology

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Published: August 8th, 2016

DOI:

10.3791/54343

1Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V., 2Institute for Molecular Biotechnology, RWTH Aachen University, 3Department of Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT)

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 °C.

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 °C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Modern healthcare systems increasingly depend on biopharmaceutical proteins 1. Producing these proteins in plants is advantageous due to the low pathogen burden and greater scalability compared to conventional expression systems 2-4. However, the downstream processing (DSP) of plant-derived pharmaceuticals can be challenging because the disruptive extraction procedures result in a high particle burden, with turbidities exceeding 5,000 nephelometric turbidity units (NTUs), and host cell protein (HCP) concentrations often exceeding 95% [m/m] 5,6.

Elaborate clarification procedures are required to remove disper....

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1. Cultivate the Tobacco Plants

  1. Flush each mineral wool block with 1 to 2 L of deionized water and subsequently with 1 L of 0.1% [w/v] fertilizer solution. Place one tobacco seed in each mineral wool block and gently flush with 0.25 L of fertilizer solution without washing away the seed 16.
  2. Cultivate the tobacco plants for 7 weeks in a greenhouse with 70% relative humidity, a 16 hr photoperiod (180 µmol sec1 m2; λ = .......

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Heat precipitation of tobacco host cell proteins by blanching
The blanching procedure described in section 2. was successfully used to precipitate HCPs from tobacco leaves with 70 °C, reducing the TSP by 96 ± 1% (n = 3) while recovering up to 51% of the Vax8 target protein, thus increasing its purity from 0.1% to 1.2% before chromatographic separation 16. It was also possible to recover 83 ± 1% (n =3) of the fluorescent protein DsRed, increasing it.......

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The three methods for heat precipitation described above can effectively remove tobacco HCPs prior to any chromatographic purification step 16,17. They complement other strategies that aim to increase initial product purity, e.g., guttation 29, rhizosecretion 30 or centrifugal extraction 31,32, all of which are limited to secreted proteins. However, the heat-based methods can only be used in a meaningful way if the target protein to be purified can withstand the minimu.......

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We would like to acknowledge Dr. Thomas Rademacher, Alexander Boes and Veronique Beiß for providing the transgenic tobacco seeds, and Ibrahim Al Amedi for cultivating the tobacco plants. The authors wish to thank Dr. Richard M. Twyman for editorial assistance as well as Güven Edgü for providing the MSP1-19 reference. This work was funded in part by the European Research Council Advanced Grant ''Future-Pharma'', proposal number 269110, the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation) and Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.

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Name Company Catalog Number Comments
2100P Portable Turbidimeter Hach 4650000 Turbidimeter
Amine Coupling Kit GE Healthcare BR100050  SPR chip coupling kit
Autoclaving basket Nalgene 6917-0230 Basket for leaf blanching
Biacore T200 GE Healthcare 28-9750-01 SPR device
Bio Cell Analyser BCA 003 R&D with 3D ORM Sequip n.a. Particle size analyzer
Blender Waring 800EG Blender
BP-410 Furh 2632410001 Bag filter
Centrifuge 5415D Eppendorf 5424 000.410 Centrifuge
Centrifuge tube 15 mL Labomedic 2017106 Reaction tube
Centrifuge tube 50 mL self-standing Labomedic 1110504 Reaction tube
CM5 chip GE Healthcare BR100012  Chip for SPR measurements
Cuvette 10x10x45 Sarsted 67.754 Cuvette for Zetasizer Nano ZS
Design-Expert(R) 8 Stat-Ease, Inc. n.a. DoE software
Disodium phosphate Carl Roth GmbH  4984.3  Media component
Ferty 2 Mega Kammlott 5.220072 Fertilizer
Forma -86C ULT freezer ThermoFisher 88400 Freezer
Greenhouse n.a. n.a. For plant cultivation
Grodan Rockwool Cubes 10x10cm Grodan 102446 Rockwool block
Twentey-loop heat exchanger (4.8 m length) n.a. (custom design) n.a. Heat exchanger
HEPES Carl Roth GmbH 9105.3 Media component
K200P 60D Pall 5302303 Depth filter layer
KS50P 60D Pall B12486 Depth filter layer
Lauda E300 Lauda Dr Wobser GmbH Z90010 Water bath thermostat
L/S 24 Masterflex SN-06508-24 Tubing
mAb 5.2 American Type Culture Collection HB-9148 Vax8 specific antibody
Masterflex L/S Masterflex HV-77921-75 Peristaltic pump
Miracloth Labomedic 475855-1R Filter cloth
MultiLine Multi 3410 IDS WTW WTW_2020 pH meter / conductivity meter
Osram cool white 36 W Osram 4930440 Light source
Phytotron Ilka Zell n.a. For plant cultivation
Sodium disulfit Carl Roth GmbH 8554.1 Media component
Sodium chloride Carl Roth GmbH P029.2 Media component
Stainless-steel vessel; 0.7-kg 2.0-L; height 180 mm; diameter 120 mm n.a. (custom design) n.a. Container for heat precipitation
Synergy HT BioTek SIAFRT Fluorescence and spectrometric plate reader
VelaPad 60 Pall VP60G03KNH4 Filter housing
Zetasizer Nano ZS Malvern ZEN3600 DLS particle size distribution measurement
Zetasizer Software v7.11 Malvern n.a. Software to operate the Zetasizer Nano ZS device

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