Review


Nature Biotechnology 22, 1415 - 1422 (2004)
Published online: 4 November 2004 | doi:10.1038/nbt1027

Plant cell cultures for the production of recombinant proteins

Stephan Hellwig1, Jürgen Drossard1, Richard M Twyman2 & Rainer Fischer1,3


The use of whole plants for the synthesis of recombinant proteins has received a great deal of attention recently because of advantages in economy, scalability and safety compared with traditional microbial and mammalian production systems. However, production systems that use whole plants lack several of the intrinsic benefits of cultured cells, including the precise control over growth conditions, batch-to-batch product consistency, a high level of containment and the ability to produce recombinant proteins in compliance with good manufacturing practice. Plant cell cultures combine the merits of whole-plant systems with those of microbial and animal cell cultures, and already have an established track record for the production of valuable therapeutic secondary metabolites. Although no recombinant proteins have yet been produced commercially using plant cell cultures, there have been many proof-of-principle studies and several companies are investigating the commercial feasibility of such production systems.


Techniques for the propagation of plant cells were developed in the 1950s when it was realized that plant cell cultures had the potential to synthesize a variety of useful, low molecular weight molecules1. Although the use of plant cells to produce such molecules has been studied extensively, only two secondary metabolites—shikonin and paclitaxel (Taxol)—have thus far been produced on a commercial scale. Part of the reason for this is the difficulty in achieving even moderate yields of most target compounds, a bottleneck that has stimulated a significant interest in the metabolic engineering of a wide variety of medicinal and nonmedicinal plants2.

In contrast, the application of plant cell culture to recombinant protein production has focused only on a small number of well-characterized plant cell lines, the most popular of which are derived from the tobacco cultivars Bright Yellow 2 (BY-2) and Nicotiana tabacum 1 (NT-1). The first recombinant protein produced in plant cells was reported nearly 15 years ago3. Since this initial demonstration, over 20 different recombinant proteins have been produced in plant cell cultures, including antibodies, enzymes, hormones, growth factors and cytokines (Table 1).


In this review, we consider how plant cell cultures are now being developed as production systems for recombinant proteins, and highlight recent achievements that identify the unique benefits of this safe, flexible and efficient production platform.

Why plant cell culture systems?

Although there has been considerable interest in the development of whole plants for the production of recombinant proteins, the advantages of agricultural-scale production (low capital equipment costs and scalability) can be outweighed by the long development times, variations in product yield and quality, and the difficulty in applying good manufacturing practice (GMP) to the early stages of production4, 5, 6. In whole plants, the possibility of contamination with agrochemicals and fertilizers must be considered, as well as the impact of pests and diseases, and the variable cultivation conditions due to local differences in soil quality and microclimate.

Plant cell culture as an expression system for recombinant proteins avoids these problems while retaining the advantages. Like microbes, plant cells are inexpensive to grow and maintain, but because they are higher eukaryotes they can carry out many of the post-translational modifications that occur in human cells. Plant cells are also intrinsically safe, because they neither harbor human pathogens nor produce endotoxins.

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Plant cells, like microbes, can be maintained in simple, synthetic media, but like animal cells they can synthesize complex multimeric proteins and glycoproteins, such as immunoglobulins7, 8 and interleukins9. Recombinant human glycoproteins synthesized in plants show much greater similarity to their native counterparts in terms of N-glycan structure compared to the same proteins produced in yeast, bacteria and filamentous fungi10. Unlike field-grown plants, the performance of cultured plant cells is independent of the climate, soil quality, season, day length and weather. There is no risk of contamination with mycotoxins, herbicides or pesticides11 and there are fewer by-products (e.g., fibers, oils, waxes, phenolics and adventitious agents). Perhaps the most important advantage of plant cells over whole plants is the much simpler procedure for product isolation and purification11, 12, 13 especially when the product is secreted into the culture medium. This means that GMP can be implemented throughout the production pipeline.

Principles of plant cell culture

Several approaches can be used for the in vitro cultivation of plant cells, including the derivation of hairy roots14, shooty teratomas15, immobilized cells16 and suspension cell cultures17. Even so, researchers have focused their attention on suspension cells because these are the most amenable to GMP procedures and they can be cultivated relatively easily in large-scale bioreactors18, 19. Suspension cell cultures have been prepared from several different plant species, including Arabidopsis thaliana20, Taxus cuspidata21, Catharanthus roseus22 and important domestic crops such as tobacco, alfalfa, rice, tomato and soybean23, 24, 25, 26, 27. The popularity of these different systems for recombinant protein manufacture is discussed below.

Plant suspension cells are prepared by the agitation of friable callus tissue in shaker flasks or fermenters to form single cells and small aggregates. Callus is undifferentiated tissue obtained by cultivating explants on solid medium containing the appropriate mixture of plant hormones to maintain the undifferentiated state. The cells are grown in liquid culture medium containing the same hormones to promote rapid growth and prevent differentiation28, 29, 30.

If transgenic plants expressing the recombinant protein of interest are used as the source of callus tissue, further genetic manipulation is unnecessary (that is, the callus and/or suspension does not have to be selected for transformed cells). Alternatively, wild-type cell suspensions can be transformed with recombinant plasmids either by cocultivation with Agrobacterium tumefaciens or particle bombardment (Fig. 1)

Figure 1: Overview of biopharmaceutical production in plant cells.

Figure 1 : Overview of biopharmaceutical production in plant cells.

For secreted proteins, the product is recovered directly from the culture supernatant, whereas for intracellular proteins, homogenization and filtration to remove coarse debris is necessary. DSP, downstream processing; QA, quality assurance; QC, quality control.

Full size image (83 KB)

The principles applied to the culture of microbial cells apply also to plant cells, although cell densities and growth rates are lower31. The growth rates of microbial cultures range from 0.1 h−1 to 1 h−1, whereas the growth rate of C. roseus suspension cells ranges from 0.019 h−1 to 0.028 h−1, with wild-type cells usually showing faster growth than transformed cells32, 33. The popular tobacco cell line BY-2 has a particularly high growth rate for plant cells (up to 0.044 h−1) and shows growth synchronicity and very low levels of nicotine compared with whole tobacco plants27.

Oxygen uptake rates (and thus the oxygen transfer rates the bioreactor has to deliver) are also relatively low in plant cells. For example, Taticek et al.31 reported an oxygen uptake rate (OUR) of 1–3.5 mmol l−1 h−1 in plant cell cultures, compared with ~5–90 mmol l−1 h−1 in bacterial cultures. Despite these differences, conventional fermenter equipment can be modified easily to work with plant cells, and many of the fermentation strategies applied to microbial cultures can also be applied to plants34, 35.

The cryocultivation of plant cell cultures is still in its infancy and is therefore not widely used except in commercial processes such as the production of paclitaxel. However, since this procedure is vital for reproducible processes, it will have to be developed before recombinant proteins can be produced on a commercial basis using cultured plant cells. In our laboratory, a protocol has been established for the cryocultivation of transformed BY-2 cells.

Recombinant protein production

Tobacco suspension cells, particularly those from the closely related cultivars BY-2 and NT-1, are frequently chosen as host cell lines because transformation and propagation are simple and well-established, and they have favorable growth characteristics. Although intact plants can be a useful source of suspension cells, freshly initiated plant suspension cultures may take a long time to acquire the favorable growth characteristics of BY-2 and NT-1. Other plant suspension cultures used for the production of recombinant proteins include rice36, 37, 38, 39, soybean40 and tomato26. Such cell lines have been studied because of the possibility that they could be more favorable than tobacco in terms of by-product levels and, since they are derived from food crops, regulatory compliance. Other anticipated benefits of exploring diverse cell lines include faster growth, higher expression levels, more efficient secretion and other advantages concerning process compatibility. Some researchers focus on plants with a higher protein content, for example, soybean and lupin, assuming that these might more readily facilitate higher expression levels.

The design of the construct used to express the recombinant protein is a key factor in determining yield. Promoter choice affects the yield by determining the rate of transcription. The most commonly used promoter is the cauliflower mosaic virus (CaMV) 35S promoter or its enhanced version (Table 1) but a number of alternative constitutive promoters can be used, including the hybrid (ocs)3mas promoter (constructed from octopine synthase (ocs) and mannopine synthase (mas) promoter sequences) and the ubiquitin promoters from maize and A. thaliana33, 37, 40, 41. In contrast to these constitutive promoters, the rice α-amylase RAmy3D promoter is induced by sugar deprivation36.

Another important aspect of construct design is the presence or absence of a leader peptide, which directs the recombinant protein to the secretory pathway. Leader peptides from plant and nonplant proteins appear to function equivalently, and many human secreted proteins have been expressed using their endogenous leaders (Table 1). Proteins directed to the secretory pathway in cultured cells will eventually reach the apoplast, from where they will diffuse through the cell wall and into the culture medium unless they become trapped in the cell wall matrix. This depends on the size of the protein as well as other physicochemical properties42, 43, 44. Proteins of less than 30 kDa tend to be secreted into the medium whereas larger proteins are quantitatively retained, but large proteins including full-sized antibodies can be secreted efficiently whereas some small proteins remain trapped, suggesting that charge and/or hydrophobicity may also be important determinants.

Specific challenges

As well as the slow growth rates of plant cells compared with microbial cells, other challenges associated with suspension cells include the formation of aggregates, the tendency for cells to adhere to the walls of the fermenter vessel, gene drift (somaclonal variation), gene silencing and in some cases shear sensitivity45, 46, 47. Many of these issues have been addressed through improved fermenter design and agitation or aeration conditions48, 49, optimization of the nutrient supply50, 51 or by the careful selection of callus cell lines with respect to product formation, growth characteristics and genetic stability38.

Another challenge is the relatively low protein yields from plant cells compared with plant storage organs. This partly reflects the undifferentiated state of the suspension cells and the lack of promoters that are very active under such conditions. Yields are also affected by protein stability, as there is significant degradation of proteins secreted into the culture medium because of proteolysis, aggregation and further unknown factors26, 52, 53. For example, up to 80% of a purified IgG1 added to sterile, conditioned Murashige & Skoog and Gamborg's B5 media was degraded within 2 h44.

Improving productivity

The productivity of plant cell cultures can vary considerably, with recombinant protein levels ranging from 0.0064% to 4% of total soluble protein (TSP) or from 0.5 μg l−1 to 200 mg l−1 based on the culture volume. However, different experiments are very difficult to compare because extraction procedures and the methods used to determine protein concentration are also variable and have not been standardized. In most cases, the expression and recovery levels obtained with plant cell cultures are approximately one or two orders of magnitude below the threshold where processes become economically feasible. One important factor in this respect is whether the product is a secreted protein that can be purified readily from the fermentation supernatant.

Separation of plant cells from the fermentation supernatant is simple compared with the same process in microbial fermentations and can often be accomplished by straightforward filtration steps. Moreover, plant cell culture media usually contain very few proteins compared with the supernatants of microbial cultures, which further facilitates recovery. Unfortunately, it has been shown that proteins such as antibodies can be very unstable in plant cell culture media15, 44, which is probably why proteins targeted to the endoplasmic reticulum using a retention signal accumulate to levels (based on cultivation volume) 10- to 100-fold higher than identical proteins targeted for secretion37, 54, 55, 56. However, the productivities achieved by some groups are very encouraging. The use of the inducible RAmy3D promoter in combination with rice cell lines36, 38, 39 has provided impressive yields, even though the growth rates and characteristics of rice cell lines are inferior to those of BY-2 and NT-1 cell lines. Recombinant protein concentrations >10 mg l−1 based on culture supernatant volume constitute promising starting points for process development36, 39, 57, 58.

The recovery of recombinant proteins from plant cell cultures can be enhanced by modifying the culture conditions or adding various agents to the growth medium (Table 2). Substances that have been tested include simple inorganic compounds59, amino acids to provide precursors for protein synthesis8, stabilizing agents such as dimethylsulfoxide60, polyethylene glycol9, 53, polyvinylpyrrolidone9, 15, 52, 53, 59, 61, gelatin9, 53, 59 and bovine serum albumin62 (these are thought to work by protecting the protein from degradation and preventing precipitation or adsorption to the vessel walls), gibberellic acid and haemin (for increasing the rate of protein synthesis)44 and Brefeldin A, which inhibits secretion15.


Product yields can be improved not only by medium optimization but also through process development (Table 3). All major fermentation strategies (batch, fed-batch, repeated batch or draw/fill, continuous culture and perfusion culture) can be applied to plant cell culture35, 63, 64. Although the growth of plant cells in batch culture in many cases does not follow the simple growth kinetics of microbes12, 27, plant cells are suitable for continuous fermentation as described for secondary metabolite production35, 64, 65, 66. Problems associated with the continuous culture of plant cells include genetic instability and non-growth-coupled production35, although the latter can be circumvented using two-stage bioreactors, as described, for example, by Sahai and Shuler67. Even so, it remains to be seen whether simple batch processes or long-term draw-fill, semicontinuous or continuous perfusion processes will emerge as the most suitable fermentation strategies for the production of proteins using plant cell cultures.


Downstream processing and regulatory considerations

Downstream processing is an integral part of every biomanufacturing process and is concerned with the isolation and purification of the product from the raw biomass. Regardless of the production system, downstream processing represents up to 80% of overall production costs, although this depends on the required level of purity, which is highest for clinical-grade materials. In many cases, it is necessary to develop specific processing steps for each product, although certain classes of product can be isolated using a standardized approach (e.g., the use of affinity chromatography to isolate recombinant antibodies or recombinant proteins expressed with integral epitope tags68). Several aspects of downstream processing have to be customized specifically for whole-plant systems, including adding steps for the removal of fibers, oils and other by-products, and process optimization for the treatment of different plant species and tissues.

Such issues do not apply so much to plant cell cultures, where the main choice is between the extraction of proteins from the wet biomass and purification of secreted proteins directly from the culture medium (Fig. 1). Secreted proteins are advantageous because they circumvent several unit operations during the purification process (that is, cell disruption and removal of debris) and provide a starting material, the culture supernatant, which has a much lower content of contaminating proteins and other metabolites than whole cell extracts. The downside of this approach is that the recombinant protein is highly diluted, so large volumes of liquid must be processed, and that the proteins can undergo significant degradation for the reasons discussed above.

If plant cell cultures are used to produce clinical-grade proteins, then downstream processing steps need to meet the standards that have been set for other biopharmaceutical production systems, including a strict regime of quality assurance and quality control to achieve approval of regulatory agencies69. Regulatory guidance for biopharmaceutical production in plants currently exists only as draft legislation, published in 2002 by both the Food and Drug Administration70 and the European Agency for the Evaluation of Medicinal Products71. Although not explicitly excluding plant cell cultures as production systems, both documents focus on intact plants as production hosts, making the situation regarding cultured cells less than clear. In addition to the applicable parts of these documents, other nonplant-specific GMP guidelines for biotechnological production (e.g., Annex 18 of the EU Guide to Good Manufacturing Practice) will probably be relevant for pharmaceutical production in plant cell cultures. One of the most important requirements is a thorough description of the genetic background and genetic stability of the host cell as well as the precise documentation of all events associated with the introduction of the transgene into the plant cell. Many of the plant cell lines currently used for recombinant protein expression have a long history in the public domain (including the BY-2 and NT-1 cell lines) and have not been characterized or banked sufficiently to fulfill GMP requirements. Cell banking is a prerequisite for the reliable supply of well-defined starting material, and a routine procedure for the cryopreservation of plant cells will have to be developed and validated.

The initial stages of processing display the greatest variability and have to be optimized in a system-specific manner. The secreted protein of interest, even though highly dilute, should form the major proteinaceous component of the harvest broth, and an important task is to find the appropriate harvesting window, taking into consideration the dynamics of protein expression and stability with respect to the production cycle. For example, in batch or fed-batch processes, peak production of the recombinant protein often occurs in the exponential phase, and begins to decline at the point where the cell mass and total protein content of the culture reach their maximum levels15. Once the appropriate window has been determined, clarification, concentration and product capture are the next processing steps. Large-scale clarification is generally carried out by dead-end or cross-flow filtration (or a combination thereof), sometimes preceded by bulk cell mass removal using a decanter, disk-stack separator or a semicontinuous or continuous centrifuge. Of these methods, cross-flow filtration provides the best clarified feed for packed-bed chromatography, but it is also the most demanding technique for method development and optimization. Experiments in our laboratory (unpublished data) have shown that considerable amounts of extracellular polysaccharides are produced by some transformed BY-2 cell lines and lead to the rapid formation of a gel layer on membrane surfaces, drastically reducing permeate flow and trapping the protein of interest within the layer. Hollow-fiber systems may therefore be a more suitable choice for these initial microfiltration steps.

Alternatively, advanced chromatographic technologies such as expanded bed adsorption (EBA) may help to capture the target protein from particulate feed material. This technique has been used to capture recombinant proteins from several industrial-scale microbial and animal cell cultures, and a recent publication describes its use with plant cells although there is need for further improvements in column hardware and adsorbent material design72. If these hurdles can be overcome, EBA may be an efficient method for simultaneous clarification, concentration and initial purification of proteins from plant cell fermentation broth or cell extracts73. Another alternative is the use of aqueous two-phase systems for cultivation and, eventually, in-situ extraction from plant cell cultures74.

The initial purification of intracellular recombinant proteins expressed in plant cells requires downstream procedures similar to those used in whole transgenic plants. In both cases, disruption of cell walls and membranes is the first post-harvesting step, but the range of techniques available for plant suspension cells is wider that that used for whole plant tissue because there are no constraints reflecting different tissue types (leaves, seeds, fruits). Therefore, wet milling, sonication, pressure homogenization and enzymatic treatment have all been used to process plant cells, and the method used depends primarily on equipment availability (although it should be noted that chemical treatment adds another component to the feed material that has to be removed in subsequent steps). Wet milling and sonication are also the most difficult to scale up.

After cell disruption, clarification of the extract is carried out as described above, although cell debris and fines generated during homogenization may be harder to remove than intact cells. The advantages of intracellular expression for initial downstream processing lie in the smaller volume of the starting material and the generally higher concentration of the target protein, whereas the major disadvantage is the more complex composition of the feedstream and the liberation of proteolytic and oxidizing substances.

Several liquid chromatography steps are included in a full purification protocol, and again the initial chromatographic steps require the most development of the specific production system. In industrial processing, robust and inexpensive chromatography media are used in the initial steps, accepting that there will be some loss of selectivity and resolution75. However, important exceptions include the use of Protein A or Protein G affinity chromatography for antibody purification, and the use of affinity tags and their respective capture agents (e.g., His6 and Ni-NTA resin), which are highly selective initial capturing methods.

Conclusions

Although the advantages of plant cell culture are widely recognized and appreciated, improvements are required in several areas before the platform becomes acceptable and commercially feasible. Most importantly, promoters need to be identified that provide yields comparable to other expression platforms, that is, 10%–20% of total mRNA. Further work must address the reasons for protein instability and identify strategies to enhance protein recovery.

There is also much interest in addressing the differences in post-translational modification between mammalian and plant cells. Whereas the glycosylation steps occurring in the endoplasmic reticulum are conserved, they diverge in the late Golgi apparatus so that core α(1,6)-linked fucose and terminal sialic acid residues are added in mammals, whereas bisecting β(1,2)-xylose and core α(1,3)-fucose residues are added in plants. Although these differences have not thus far been shown to affect the biological activity of recombinant proteins, some reports have identified pharmacokinetic differences between plant-derived recombinant proteins and their native counterparts, for example, the significantly reduced half-life of plant-derived α1-antitrypsin, which Huang, J. et al. speculated was due to the lack of sialic acid39.

Nonmammalian glycans are furthermore regarded as potential immunogens or allergens particularly when the recombinant protein is intended for injection into human patients. Much emphasis has been placed on this issue by the regulatory authorities, which is understandable given conflicting reports on immunogenicity76, 77. Efforts to avoid nonmammalian glycosylation patterns include the prevention of late glycosylation by directing proteins to the endoplasmic reticulum through addition of KDEL-tags (a strategy incompatible with protein secretion), the introduction of point mutations to eliminate glycosylation completely, or the humanization of the plant glycosylation machinery. As an example of the last strategy, Palacpac et al.78 have expressed human β(1,4)galactosyltransferase in BY-2 cells, showing that plant glycan structures could be modified successfully.

The overall goal should be to produce in plant cells human proteins that are structurally and functionally equivalent to their native counterparts. If these challenges can be met, plant cells have the potential to compete with other expression platforms for the commercial production of recombinant proteins, as has already been achieved with small-molecule drugs.



Competing interests statement

The authors declare no competing financial interests.

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  1. Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Worringerweg 1, D-52074 Aachen, Germany.
  2. Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom.
  3. Institut für Biologie VII (Molekulare Biotechnologie), Aachen Technical University, D-52056 Aachen, Germany.

Correspondence to: Rainer Fischer1,3 e-mail: fischer@molbiotech.rwth-aachen.de