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Biotechnol. Appl. Biochem. (1999) 30, 109–112 (Printed in Great Britain)

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REVIEW Towards molecular farming in the future: using plant-cell-suspension cultures as bioreactors

Rainer Fischer*1 , Neil Emans*, Flora Schuster*, Stephan Hellwig* and Ju rgen Drossard*

*Institut fu r Biologie I (Botanik Molekulargenetik), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany, and Fraunhofer Department for Molecular Biotechnology, IUCT, Grafschaft, Auf dem Aberg 1, D-57392 Schmallenberg, Germany

Plant-suspension cells are an in vitro system that can be used for recombinant protein production under care- fully controlled certified conditions. Plant-suspension cells can be grown in shake flasks or fermenters to produce secondary metabolites, like vincristine and vinblastine, and to produce recombinant proteins after transformation. This review article focuses on dis- cussing the generation of transformed suspension-cell lines expressing recombinant proteins, like antibodies, and recombinant-protein downstream processing and purification.

Introduction

The utilization of plant cells for the production of natural or recombinant compounds of commercial interest has gained increasing attention over past decades [1,2]. Of the various systems used for cultivation in vitro of plant cells, such as hairy roots [3], immobilized cells [4] and free cell suspensions [5], the latter is generally regarded to be the most suitable for large-scale applications in the biotechnology industry [6,7]. Compared with the more conventional expression systems like bacteria, yeast and mammalian-cell cultures, the number of applications is still relatively low [5]. However, it is anticipated that the advances in the genetic engineering of plants and the demand for large quantities of new or improved diagnostics and therapeutics in the healthcare and pharmaceutical markets will lead to a rapid expansion in this research field. A number of plant species have been used for generation and propagation of cell-suspension cultures, ranging from model systems like Arabidopsis [8], Catharanthus [9] and Taxus [10], to important monocotyledon or di- cotyledon crop plants like rice [11], soya bean [12], alfalfa [13] and tobacco [14]. Plant-cell suspensions are normally derived from calli cultivated on solidified media. Transfer of friable callus clumps to liquid medium and agitation on rotary shakers or in fermenters results in cultures of single cells or small aggregates of 10–20 cells. If a homogenous culture can be generated and maintained, in principle the fermentation of

plant cells requires quite similar techniques and equipment

to the fermentation of lower eukaryotes, although they are

not comparable in terms of generation times, obtainable cell densities or nutritional requirements. It is possible to cultivate plant-cell suspensions using conventional fermenter equipment with minor adjustments and to apply standard

modes like batch, fed-batch, perfusion and continuous fermentation [6,15,16]. Large-scale fermentations up to a volume of 100000 litres have been performed successfully. The limitations of plant-cell fermentation are related to poor growth rates, relatively low production rates of secondary metabolites, somaclonal variation and gene silencing, inhibition of product formation at high cell densities leading to a low volumetric productivity, formation of aggregates and wall growth and, at least for some species, shear-sensitivity of the cells [6,17–19]. Some of these problems have been addressed by improved fermenter design and agitation conditions [20,21], optimization of nutrient supply [22,23] and feeding of precursors or elicitors [24–28]. Others depend on the plant species used and on

a careful selection of the callus cell lines with respect

to product formation, growth characteristics and genetic stability. Most applications of plant-cell-suspension cultures in biotechnology are aimed at the production of naturally occurring secondary metabolites. This has included pro- duction of shikonin [29], anthocyanins [23,30], ajmalicine [31,32] and, recently, important anti-tumour agents like taxol [10,33], vinblastine and vincristine [34]. In our lab- oratory, we focus on the production of recombinant proteins and antibodies in plants and plant-cell cultures. Today, the expression of recombinant antibodies and antibody fragments in plants is a well-established technique [35,36], and the advantages of plants over bacterial or mammalian production systems have been reviewed [37]

Abbreviations used: IMAC, metal-ion affinity chromatography; DTT, dithiothreitol. 1 To whom correspondence should be addressed, at: Institut fu r Biologie I (Botanik/Molekulargenetik), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany.

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and are discussed later in detail. In particular, the capability of plant cells to synthesize, process and target large complex mammalian proteins in a manner very similar to their natural hosts makes them an attractive alternative for recombinant- protein production. Further benefits of using plants are the reduced technical, ethical and safety issues and costs compared with mammalian-cell cultures or transgenic animals. The advantages of intact plants lie in the fast biomass build-up, the low cultivation costs and the easy storage and distribution of transgenic seed material. When clinical use of recombinant proteins is intended, their production under defined, controllable and sterile conditions with straight- forward purification protocols may be advantageous. There- fore, we express full-size antibodies, antibody fragments and fusion proteins in our model transgenic-plant-cell-suspen- sion systems, Nicotiana tabacum cv. SR-1 [38] and BY-2 [14], but also in pea, wheat and rice using shake-flask or fermentation cultures.

Tobacco suspension-cell lines as model systems for recombinant-protein production

Plant transformation and protein targeting

Transfer of the foreign gene into the plant cells can be performed using Agrobacterium-mediated transformation [39,40], particle bombardment [41], electroporation of protoplasts [42] or viral vectors [43]. For the transformation of tobacco plants or suspension cells, we routinely use Agrobacterium-mediated transformation. Whereas trans- genic N. tabacum cv. SR-1 are regenerated from transformed leaf discs, the BY-2 cell line can be directly transformed by co-cultivation of suspension cells and Agrobacteria [44], because of its unique growth characteristics. The latter procedure has the significant advantage that transient expression of the foreign gene can be detected 2–3 days after co-cultivation, the selection of transgenic callus via kanamycin resistance takes approx. 3 weeks and the initiation of suspension cell lines from this callus can be performed within less than 2 months. In comparison, leaf- disc transformation, selection, regeneration and assay of transgenic SR-1 plants, followed by establishment of callus- and suspension cultures requires approx. 1 year. However, we generally obtain higher production levels in cell sus- pensions derived from transgenic SR-1 plants. Recombinant proteins expressed in plant-cell-suspen- sion cultures are found in the culture supernatant or retained within the cells. This localization depends on two factors: (i) the presence of targeting leader peptides (of plant or heterologous origin) in the recombinant protein and (ii) on the permeability of the plant cell wall for macromolecules [45]. Targeting signals can be used to direct the protein for

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secretion [46] or to intracellular organelles (endoplasmic reticulum, chloroplast, vacuole, membranes) [47]. The cytosol is generally not suitable as a storage compartment for recombinant protein. When the secretory pathway is utilized, molecules 20–30 kDa will pass readily through the plant cell wall and be secreted into the culture medium, whereas larger proteins will be retarded to a certain extent, depending on their size. Targeting signals can be used to retain recombinant proteins within distinct compartments of the cells to preserve integrity, to protect them from proteolytic degradation and to increase accumulation levels [47,48]. However, this targeting makes the disruption of the cells necessary prior to protein purification. Cell disruption has several drawbacks; apart from requiring additional equipment and labour it causes the release of phenolic substances or proteases that can reduce protein yield. Thus the preferred approach is to target proteins for secretion and capture them from the culture supernatant or release them from the cell by mild enzymic cell-wall digestion. We have expressed a wide panel of recombinant antibodies intra- and extracellularly in tobacco-suspension cells, including full-size IgGs, Fab fragments, single chains, bispecific antibody fragments and fusion proteins. Using our standard plant-expression vector, we could obtain ex- pression levels of between 2 and 20 µg of recombinant antibody g of fresh cell weight; this level could be signifi- cantly increased by targeting of the protein to the en- doplasmic reticulum as well as by optimizing cultivation conditions by controlled amino acid supplementation or elicitation [52].

Fermentation

Using the rapidly dividing tobacco BY-2 cell line for fermentation, we have scaled-up the cultivation of transgenic suspension cells to a working volume of 40 litres. With a 10% (v v) inoculum, fermentation times of 150 h resulted in a yield of 7.5 kg of fresh cell weight, corresponding to 0.4 kg dry weight. Apart from some growth on the fermenter walls and impeller axis, we did not experience any engineering problems. The equipment used was a standard stirred-tank reactor with a three-bladed impeller run at 50 rev. min and an aeration rate of 0.1 vol. of air vol, of culture per min.

Protein extraction and purification

Compared with other expression systems, the major differences in purifying recombinant proteins from plant- suspension cells arise in the very first steps of the procedure. If the protein of interest is contained in the culture supernatant, removal of cell material can be achieved easily by vacuum filtration followed by clarification of the filtrate before initial purification steps. However, if the target protein is located intracellularly, a suitable method for gentle

Molecular farming in plant-suspension cells

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and efficient cell disruption is essential for large-scale protein production. Mechanical cell-disruption devices like bead mills, although very efficient, give rise to problems due to heat generation, disruption of subcellular organelles ac- companied by liberation of noxious chemicals (alkaloids, phenolics), and generation of fine cell debris, which can be difficult to remove. In general, when disruption of the cells is necessary, large buffer volumes and various additives, like polyvinylpolypyrrolidone (PVPP), dithiothreitol (DTT), ascorbic acid, EDTA and others, are often used to counteract oxidation and proteolytic activity, but these can limit the choice of initial purification methods. For example, using DTT or EDTA is incompatible with immobilized metal-ion affinity chromatography (IMAC). Our preferred methods for protein extraction are either cell sonication or cell-wall digestion using technical-grade pectinase [52]. Enzymic digestion has the advantage that it quantitatively releases secreted full-size antibodies that cannot cross the cell wall. After extraction, bulk cell material can be removed by centrifugation or filtration through cheesecloth and the filtrate clarified by cross-flow or hollow-fibre filtration. Alternatively, we use expanded-bed chromatography [49] to process the unclarified filtrate. For concentration of normally highly dilute recom- binant antibody solutions and efficient initial purification, we use affinity chromatography as a first capturing step. We use Protein A Protein G, antigen affinity or IMAC depending on the expressed recombinant proteins and, within the limita- tions mentioned above, have never encountered problems specific to the processing of plant suspension cell extracts. To minimize the time that the target proteins are exposed to the cell-extract components, the use of chromatography media allowing high flow rates is recommended. Using affinity chromatography for initial purification followed, if necessary, by an ion-exchange step and gel filtration for final purification, we generally obtain yields of 80% of intact functional recombinant antibody from the starting material.

Conclusions

The use of plant-cell suspension cultures for the production of recombinant proteins is clearly still in its infancy. The feasibility of this approach has been confirmed by the large- scale production of several high-value recombinant proteins and the production of natural secondary metabolites in suspension cells. A great advantage of plant-cell-suspension cultures is that recombinant proteins can be produced under certified conditions [certified Good Manufacturing Practice (‘cGMP’), certified Good Laboratory Practice (‘cGLP’)], but yields are low compared with stably transformed plants and yeast. Recent improvements in the design of novel pro- moters [50] and other control elements together with

deeper insights into the mechanisms of plant gene silencing [51] and gene targeting will lead to significant improvements in product yields. This will lay the basis for predicting the behaviour of transgenes in plant cell culture. The advent of new transformation technologies will allow us to extend production to a broader range of plant species that have better characteristics for protein expression than tobacco. Currently, a systematic evaluation of growth and elicitation conditions should result in higher productivity and more applications of plant-cell-suspension cultures in recom- binant-protein production.

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Received 29 April 1999; accepted 21 May 1999