Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Summary

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Abstract

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

Introduction

The production of biopharmaceutical proteins in plants is advantageous because plants are inexpensive to grow, the platform can be scaled up just by growing more plants, and human pathogens are unable to replicate ^1,2. Transient expression strategies based for example on the infiltration of leaves with Agrobacterium tumefaciens provides additional benefits because the time between the point of DNA delivery and the delivery of a purified product is reduced from years to less than 2 months ³. Transient expression is also used for functional analysis, e.g. to test genes for their ability to complement loss-of-function mutants or to....

Protocol

1. Planning a DoE Strategy

1. Identify relevant factors and responses for inclusion in the design.
   1. Define one or several responses for measurement. Here, 2G12 and DsRed expression levels were used (μg/ml), including the minimum detectable difference regarded as relevant (10 and 20 μg /ml, respectively) and an approximate value for the estimated standard deviation of the system (4 and 8 μg/ml, respectively) based on previous experiments.
   2. Use the available literature, data from previous experiments or specialized screening designs (e.g. a factorial design, see the introduction) to select significant factors whose impact on the re....

Results

A descriptive model for DsRed accumulation during transient expression using different promoters and 5'UTRs

DsRed fluorescence in leaf extracts was used to indicate the expression level of the recombinant protein and thus was used as the response in the DoE strategy. The minimum detectable difference we considered relevant was 20 μg/ml and the estimated standard deviation of the system was 8 μg/ml based on initial experiments. Factors included in the transient expression model.......

Discussion

Every experiment requires careful planning because resources are often scarce and expensive. This is particularly true for DoE strategies because errors during the planning phase (e.g. selecting a base model that does not cover all significant factor interactions) can substantially diminish the predictive power of the resulting models and thus devalue the entire experiment. However, these errors can easily be avoided by following basic procedures.

Considerations during DoE planning

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Design-Expert(R) 8	Stat-Ease, Inc.	n.a.	DoE software
Tryptone	Carl Roth GmbH	8952.2	Media component
Yeast extract	Carl Roth GmbH	2363.2	Media component
Sodium chloride	Carl Roth GmbH	P029.2	Media component
Ampicillin	Carl Roth GmbH	K029.2	Antibiotic
Agar-Agar	Carl Roth GmbH	5210.2	Media component
Escherichia coli K12 DH5a	Life Technologies	18263-012	Microorganism
pPAM	GenBank	AY027531	Cloning/expression vector;
NucleoSpin Plasmid	MACHEREY-NAGEL GmbH	740588.250	Plasmid DNA isolation kit
NucleoSpin Gel and PCR Clean-up	MACHEREY-NAGEL GmbH	740609.250	Plasmid DNA purification kit
NanoDrop 2000	Thermo Scientific	n.a.	Spectrophotometer
NcoI	New England Biolabs Inc.	R3193L	Restrictionendonuclease
EcoRI	New England Biolabs Inc.	R3101L	Restrictionendonuclease
AscI	New England Biolabs Inc.	R0558L	Restrictionendonuclease
NEB 4	New England Biolabs Inc.	B7004S	Restrictionendonuclease buffer
TRIS	Carl Roth GmbH	4855.3	Media component
Disodium tetraborate	Carl Roth GmbH	4403.3	Media component
EDTA	Carl Roth GmbH	8040.2	Media component
Agarose	Carl Roth GmbH	6352.4	Media component
Bromophenol blue	Carl Roth GmbH	A512.1	Color indicator
Xylene cyanol	Carl Roth GmbH	A513.1	Color indicator
Glycerol	Carl Roth GmbH	7530.2	Media component
Mini-Sub Cell GT Cell	BioRad	170-4406	Gel electrophoresis chamber
Agrobacterium tumefaciens strain GV3101:pMP90RK	DSMZ	12365	Microorganism
Electroporator 2510	Eppendorf	4307000.658	Electroporator
Beef extract	Carl Roth GmbH	X975.2	Media component
Peptone	Carl Roth GmbH	2365.2	Media component
Sucrose	Carl Roth GmbH	4621.2	Media component
Magnesium sulfate	Carl Roth GmbH	0261.3	Media component
Carbenicillin	Carl Roth GmbH	6344.2	Antibiotic
Kanamycin	Carl Roth GmbH	T832.3	Antibiotic
Rifampicin	Carl Roth GmbH	4163.2	Antibiotic
FWD primer	Eurofins MWG Operon	n.a.	CCT CAG GAA GAG CAA TAC
REV primer	Eurofins MWG Operon	n.a.	CCA AAG CGA GTA CAC AAC
2720 Thermal cycler	Applied Biosystems	4359659	Thermocycler
RNAfold webserver	University of Vienna	n.a.	Software
Ferty 2 Mega	Kammlott	5.220072	Fertilizer
Grodan Rockwool Cubes 10 x10 cm	Grodan	n.a.	Rockwool block
Greenhouse	n.a.	n.a.	For plant cultivation
Phytotron	Ilka Zell	n.a.	For plant cultivation
Omnifix-F Solo	B. Braun	6064204	Syringe
Murashige and Skoog salts	Duchefa	M 0222.0010	Media component
Glucose	Carl Roth GmbH	6780.2	Media component
Acetosyringone	Sigma-Aldrich	D134406-5G	Phytohormon analogon
BioPhotometer plus	Eppendorf	6132 000.008	Photometer
Osram cool white 36 W	Osram	4930440	Light source
Disodium phosphate	Carl Roth GmbH	4984.3	Media component
Centrifuge 5415D	Eppendorf	5424 000.410	Centrifuge
Forma -86C ULT freezer	ThermoFisher	88400	Freezer
Synergy HT	BioTek	SIAFRT	Fluorescence plate reader
Biacore T200	GE Healthcare	n.a.	SPR device
Protein A	Life Technologies	10-1006	Antibody binding protein
HEPES	Carl Roth GmbH	9105.3	Media component
Tween-20	Carl Roth GmbH	9127.3	Media component
2G12 antibody	Polymun	AB002	Reference antibody