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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 sec⁻¹ m⁻²; λ = 400 - 700 nm) and a 25/22 °C light/dark temperature regime.
3. Harvest all leaves except the four cotyledon leaves, which are located at t....

Results

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.......

Discussion

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.......

Materials

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