385

Plant-based Expression of

.. .. Jorg Knablein Schering AG, Berlin, Germany

1 Introduction 387

2 Alternative Expression Systems 387

3 History of Plant Expression 389

4 Current Status of Plant-based Expression 390 4.1 SWOT Analysis Reveals a Ripe Market for Plant Expression Systems 390 4.2 Risk Assessment and Contingency Measures 392

5 The Way Forward: Moving Plants to Humanlike Glycosylation 396

6 Three Promising Examples: Tobacco (Rhizosecretion, Transfection) and (Glycosylation) 398 6.1 Harnessing Tobacco Roots to Secrete Proteins 398 6.2 High Protein Yields Utilizing Viral Transfection 399 6.3 Simple Moss Performs Complex Glycosylation 401

7 Other Systems Used for Plant Expression 404

8 Analytical Characterization 405

9 Conclusion and Outlook 406

Acknowledgments 407

Bibliography 407 Books and Reviews 407 Primary References 407

Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2nd Edition. Volume 10 Edited by Robert A. Meyers. Copyright  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30552-1 386 Plant-based Expression of Biopharmaceuticals Keywords

GMP Good Manufacturing Practice (GMP) was established by WHO in 1968 to guarantee the optimum degree of quality during production and processing of pharmaceuticals (cGMP means under the current regulations of the authorities).

Transgenic Organisms that have externally introduced foreign DNA/genes stably integrated into their genome to, for example, produce desired substances like human insulin.

Plant-based Expression Transgenic plants can be genetically modified with a gene of interest to produce a of interest.

Glycosylation It is the addition of polysaccharides to a certain molecule such as a protein. The majority of proteins are synthesized in the rough endoplasmic reticulum (ER) where they undergo glycosylation.

Bioreactor It is a vessel in which a (bio)chemical process that involves organisms or biochemically active substances (e.g. enzymes) derived from such organisms is carried out.

 Biopharmaceuticals are currently the mainstay products of the biotechnology market and represent the fastest growing and, in many ways, the most exciting sector within the pharmaceutical industry. The term ‘‘biopharmaceutical’’ was originated in the 1980s, when a general consensus evolved that it represented a class of therapeutics produced by means of modern biotechnologies. Already a quarter of a century ago, ‘‘humulin’’ (recombinant human insulin, produced in E. coli and developed by Genentech in collaboration with Eli Lilly) was approved and received marketing authorization in the United States of America in 1982. Since then the market for biopharmaceuticals has been steadily growing and currently nearly 150 biopharmaceuticals have gained approval for general human use (EU and USA). Over this period it became obvious that production capacities for biopharmaceuticals with ‘‘conventional’’ would be a bottleneck and that worldwide fermentation capacities are limited. One exciting solution to these ‘‘capacity crunches’’ is the use of transgenic plants to produce biopharmaceuticals. This article describes different plant expression systems, their advantages and limitations, and concludes by considering some of the innovations and trends likely to influence the future of plant-based biopharmaceuticals. Plant-based Expression of Biopharmaceuticals 387

1 are competing (see ref Knablein¨ (2004), Introduction review). To circumvent this capacity crunch, it Biopharmaceuticals, which are large mole- is necessary to look into other technolo- cules produced by living cells, are currently gies rather than the established ones, like, the mainstay products of the biotechnology for example, Escherichia coli or CHO (Chi- industry. Indeed, biologics such as Genen- nese hamster ovary) cell expression. One tech’s (Vacaville, CA, USA) human growth solution to avoid these limitations could factor somatropin or Amgen’s (Thousand be the use of transgenic plants to ex- Oaks, CA, USA) recombinant erythropoi- press recombinant proteins at low cost, etin (EPO) have shown that biopharma- in GMP (good manufacturing practice) ceuticals can benefit a huge number of quality greenhouses (with purification and patients and also generate big profits for fill finish in conventional facilities). Plants these companies at the same time. The therefore provide an economically sound single most lucrative product is EPO and source of recombinant proteins, such as in- combined sales of the recombinant EPO dustrial enzymes, and biopharmaceuticals. products ‘‘Procrit’’ (Ortho biotech) and Furthermore, using the existing infrastruc- ‘‘Epogen’’ (Amgen) have reportedly sur- ture for crop cultivation, processing, and storage will reduce the amount of capital passed the $6.5 billion mark. But it has investment required for commercial pro- also become obvious over the last couple duction. For example, it was estimated that of years that current fermentation capaci- the production costs of recombinant pro- ties will not be sufficient to manufacture all teins in plants could be between 10 and biopharmaceuticals (in the market already 50 times lower than those for producing or in development), because the market thesameproteininE. coli and Alan Dove and demand for biologics is continuously describes a factor of thousand for cost of and very rapidly growing; for antibodies protein (US dollar per gram of raw mate- alone (with at least 10 monoclonal an- rial) expressed in, for example, CHO cells tibodies approved and being marketed), compared to transgenic plants. So, at the the revenues are predicted to expand to dawn of this new millennium, a solution is US$3 billion in 2002 and US$8 billion in imminent to circumvent expression capac- 2008. The 10 monoclonal antibodies on ity crunches and to supply mankind with the market consume more than 75% of the the medicines we need. Providing the right industry’s manufacturing capability. And amounts of biopharmaceuticals can now there are up to 60 more that are expected be achieved by applying our knowledge of to reach the market in the next six or seven modern life sciences to systems that were years. Altogether, there are about 1200 on this planet long time before us – plants. protein-based products in the pipeline with a 20% growth rate and the market for 2 current and late stage (Phase III) is es- Alternative Expression Systems timated to be US$42 billion in 2005 and even US$100 billion in 2010. But, there Currently, CHO cells are the most widely are obvious limitations of large-scale man- used technology in biomanufacturing be- ufacturing resources and production ca- cause they are capable of expressing pacities – and pharmaceutical companies eukaryotic proteins (processing, folding, 388 Plant-based Expression of Biopharmaceuticals

and posttranslational modifications) that produce them for the final round of clinical cannot be provided by E. coli.Along trials, in order to guarantee bioequivalence track record exists for CHO cells, but (e.g. toxicity, bioavailability, pharmacoki- unfortunately they bring some problems netics, and pharmacodynamics) of the along when it comes to scaling up pro- molecule. So, companies have to choose duction. Transport of oxygen (and other between launching a product manufac- gases) and nutrients is critical for the fer- tured at a smaller development facility mentation process, as well as the fact that (and struggling to meet market demands) heat must diffuse evenly to all cultured or building larger, dedicated facilities for a cells. According to the Michaelis–Menten drug that might never be approved! equation, the growth rate depends on the Therefore, alternative technologies are oxygen/nutrient supply; therefore, good used for the expression of biopharma- mixing and aeration are a prerequisite ceuticals, some of them also at lower for the biomanufacturing process and are costs involved (see Fig. 2). One such al- usually achieved by different fermentation ternative is the creation of transgenic modes (see Fig. 1). But the laws of physics animals (‘‘’’), but this suffers set strict limits on the size of bioreac- from the disadvantage that it requires a tors. For example, an agitator achieves long time to establish such animals (ap- good heat flow and aeration, but with in- proximately 2 years). In addition to that, creased fermenter size, shear forces also some of the human biopharmaceuticals increase and disrupt the cells – and build- could be detrimental to the mammal’s ing parallel lines of bioreactors multiplies health, when expressed in the mammary the costs linearly. A 10 000-L glands. This is why ethical debates some- costs between US$ 250 000 to 500 000 times arise from the use of transgenic and takes five years to build (concep- mammals for production of biopharma- tual planning, engineering, construction, ceuticals. Although there are no ethical validation, etc.). An error in estimating de- concerns involved with plants, there are mand for, or inaccurately predicting the societal ones that will be addressed later. approval of, a new drug can be incredibly Another expression system (see Fig. 2) uti- costly. To compound the problem, regu- lizes transgenic chicken. The eggs, from lators in the United States and Europe which the proteins are harvested, are demand that drugs have to be produced natural protein-production systems. But for the market in the same system used to production of transgenic birds is still

Mechanic: Agitator Pneumatic: Gassing Hydrodynamic: Pumps (a) (b) (c) (d) .. Fig. 1 Different fermentation modes for (d) airlift reactor. Source: Knablein J. bioreactors. In order to achieve best aeration and (2002) Transport Processes in Bioreactors and mixing and to avoid high shear forces, different Modern Fermentation Technologies,Lectureat fermentation modes are applied. (a) mechanical, University of Applied Sciences, (b) pneumatical, (c) hydrodynamic pumps, Emden, Germany. Plant-based Expression of Biopharmaceuticals 389

Major technology Mammalian (CHO) cells Transgenic mammal milk Transgenic chicken eggs Transgenic plants companies Amgen GTC Biotherapeutics Avigenics Croptech (Thousand Oaks, CA) (Framingham, MA) (Athens, GA) (Blacksburg, VA) Genentech PPL Therapeutics Origen Therapeutics Epicyte (S. San Francisco, CA) (Edinburgh, UK) (Burlingame, CA) (San Diego, CA) other current biologics BioProtein TranXenoGen Large Scale Biology manufacturers: (Paris, France) (shrewsbury, MA) (Owensboro, KY) Crucell Viragen Meristem Therapeutics (Leiden, Netherlands) (Plantation, FL) (Clermont-Ferrand, France) uses human cells GeneWorks Prodigene (Ann Arbor, MI) (College Station, TX) Vivalis (Nantes, France) Estimated cost − − . (cost/g raw material)* $150$1 $2 $1 $2 $0 05

*Company estimates Fig. 2 Companies and technologies in for CHO cells to 0.05 US$ per gram for biomanufacturing. A comparison of different transgenic plants. Source: Dove, A. expression systems shows the big differences in (2002) Uncorking the biomanufacturing terms of costs, ranging from 150 US$ per gram bottleneck, Nat. Biotechnol. 20, 777–779. several years behind transgenic mammal have been primarily small molecules, technology. Intensive animal housing con- however. One of the most popular ex- straints also make them more susceptible amples is aspirin (acetylsalicylic acid) to to disease (e.g. Asia 1997 or Europe 2003: relieve pain and reduce fever. A French killing of huge flocks with thousands of pharmacist first isolated natural salicin chicken suffering from fowl pest). In the (a chemical relative of the compound light of development time, experience, used to make aspirin) from white wil- costs, and ethical issues, plants are there- low bark in 1829. Advances in genetic fore the favored technology, since such engineering are now allowing for the systems usually have short gene-to-protein production of therapeutic proteins (as op- times (weeks), some are already well es- posed to small molecules) in plant tissues. tablished, and as mentioned before, the Expression of recombinant proteins in involved costs are comparatively low. This plants has been well documented since low cost of goods sold (COGS) for plant- the 1970s and has slowly gained credi- derived proteins is mainly due to low bility in the biotechnology industry and capital costs: greenhouse costs are only regulatory agencies. The first proof of US$ 10 per m2 versus US$ 1000 per m2 concept has been the incorporation of for mammalian cells. insect and pest resistance into grains. For example, ‘‘Bt corn’’ contains genes 3 from Bacillus thuringensis and is currently History of Plant Expression being grown commercially. Genetic engi- neering techniques are now available for Plants have been a source of medicinal the manipulation of almost all commer- products throughout human evolution. cially valuable plants. Easy transforma- Theseactivepharmaceuticalcompounds tion and cultivation make plants suitable 390 Plant-based Expression of Biopharmaceuticals

for production of virtually any recombi- purposes. These antibodies can serve in nant protein. health care and medicinal applications, ei- Plants have a number of advantages ther directly by using the plant as a food over microbial expression systems, but ingredient or as a pharmaceutical or diag- oneofthemisofoutmostimportance: nostic reagent after purification from the they can produce eukaryotic proteins in plant material. In addition, antibodies may their native form, as they are capable improve plant performance, for example, of carrying out posttranslational modifi- by controlling plant disease or by modify- cations required for the biological activity ing regulatory and metabolic pathways. of many such proteins (see Fischer Schill- berg (2004), books). These modifications can be acetylation, phosphorylation, and 4 glycosylation, as well as others. Per se, Current Status of Plant-based Expression there is no restriction to the kind of pro- 4.1 teins that can be expressed in plants: SWOT Analysis Reveals a Ripe Market for vaccines (e.g. pertussis or tetanus toxins), Plant Expression Systems serum proteins (e.g. albumin), growth fac- tors (e.g. vascular endothelial growth factor When I analyzed the different expres- (VEGF), erythropoietin), or enzymes (e.g. sion systems regarding their strengths, urokinase, glucose oxidase, or glucocere- weaknesses, opportunities, and threats brosidase). However, enzymes sometimes (SWOT), the advantages of plants and their have very complex cofactors, which are es- potential to circumvent the worldwide ca- sential for their catalytic mode of action, pacity limitations for protein production butcannotbesuppliedbymostexpression became quite obvious (see Fig. 3). Compar- systems. This is why, for the expression of ison of transgenic animals, mammalian some enzymes, expression systems with cell culture, plant expression systems, special features and characteristics need yeast, and bacteria shows certain advan- to be developed. Another very important tages for each of the systems. In the classofproteinsistheantibodies(e.g. order in which the systems were just men- scFv, Fab, IgG, or IgA). More than 100 tioned, we can compare them in terms antibodies are currently used in clinical of their development time (speed). Trans- trials as therapeutics, drug delivery vehi- genic animals have the longest cycle time cles, in diagnostics and imaging, and in (18 months to develop a goat), followed drug discovery research for both screen- by mammalian cell culture, plants, yeast, ing and validation of targets. Again, plants and bacteria (one day to transform E. are considered as the system of choice coli). If one looks at operating and capi- for the production of antibodies (‘‘plan- tal costs, safety, and scalability, the data tibodies’’) in bulk amounts at low costs. show that plants are beneficial: therefore, Since the initial demonstration that trans- in the comparison (see Fig. 3), they are genic tobacco (Nicotiana tabacum)isable shown on the right-hand side already. to produce functional IgG1 from mouse, But even for glycosylation, multimeric as- full-length antibodies, hybrid antibodies, sembly and folding (where plants are not antibody fragments (Fab), and single-chain shown on the right-hand side, meaning variable fragments (scFv) have been ex- other systems are advantageous), some pressed in higher plants for a number of plant expression systems are moving in Plant-based Expression of Biopharmaceuticals 391

Strengths Minus Plus • Access new manufacturing facilities Speed • High production rates/high protein yield • Relatively fast 'gene to protein' time Operating • Safety benefits;no human pathogens/no TSE cost • Stable cell lines/high genetic stability • Simple medium (water, minerals & light) Captial costs • Easy purification (ion exchange vs protA) Glyco- Weaknesses sylation • No approved products yet (but Phase III) Multimeric • No final guidelines yet (but drafts available) assembly Opportunities Folding • Reduce projected COGS • Escape capacity limitations • Achieve human-like glycosylation Safety

Threats Scalability • Food chain contamination • Segregation risk TRENDS in Biotechnology Bacteria Yeast Plants Vol.20 No.12, 2002 Mammalian Transgenic cell culture animals Fig. 3 SWOT analysis of plant expression assembly, and folding, where plants are not systems. Plant expression systems have a lot of shown on the right-hand side (meaning other advantages (plus) over other systems and are systems are advantageous), some plant therefore mostly shown on the right-hand side of expression systems are moving in that direction the picture (Raskin, I., Fridlender, B., et al. (as will be shown exemplarily in the section on (2002) Plants and human health in the moss). Also the weaknesses and threats can be dealt with, using the appropriate plant twenty-first century, Trends Biotechnol. 20, .. 522–531). Herein different systems (transgenic expression system. Source: Knablein J. (2003) animals, mammalian cell culture, plants, yeast, Biotech: A New Era In The New and bacteria) are compared in terms of speed Millennium – From Plant Fermentation To Plant (how quickly they can be developed), operating Expression Of Biopharmaceuticals,PDA and capital costs, and so on, and plants are International Congress, Prague, Czech Republic. obviously advantageous. Even for glycosylation, that direction. An example of this is the any pathogens, which are known to harm moss system from the company greenova- animal cells (as opposed to animal cell cul- tion Biotech GmbH (Freiburg, Germany), tures and products), nor do the products which will be discussed in detail in the contain any microbial toxins, TSE (Trans- example section. This system performs missible Spongiform Encephalopathies), proper folding and assembly of even such prions, or oncogenic sequences. In fact, complex proteins like the homodimeric humans are exposed to a large, con- VEGF. Even the sugar pattern could suc- stant dose of living plant viruses in the cessfully be reengineered from plant to diet without any known effects/illnesses. humanlike glycosylation. Plant production of protein therapeutics In addition to the potential of perform- also has advantages with regard to their ing human glycosylation, plants also enjoy scale and speed of production. Plants the distinct advantage of not harboring can be grown in ton quantities (using 392 Plant-based Expression of Biopharmaceuticals

existing plant/crop technology, like com- applying advanced containment technolo- mercial greenhouses), be extracted with gies (GMP greenhouses, bioreactors) and industrial-scale equipment, and produce avoiding open-field production. kilogram-size yields from a single plot of Owing to the obvious strengths of plant cultivation. These economies of scale are expression systems, there has been explo- expected to reduce the cost of production sive growth in the number of start-up of pure pharmaceutical-grade therapeutics companies. Since the 1990s, a number by more than 2 orders of magnitude ver- of promising plant expression systems sus current bacterial fermentation or cell have been developed, and in response to culture reactor systems (plus raw material this ‘‘blooming field’’ big pharmaceutical COGS are estimated to be as low as 10% companieshavebecomemoreinterested. of conventional cell culture expenses). Now, the plant expression field is ‘‘ripe’’ Although a growing list of heterolo- for strategic alliances, and, in fact, the gous proteins were successfully produced last year has seen several major biotech in a number of plant expression systems companies begin partnerships with such with their manifold advantages, there are plant companies. The selection of several also obvious downsides. One weakness is such partnerships shown in Table 1 clearly that no product has been approved for demonstrates that, in general, there has been sufficient experimentation with var- the market yet (but will be soon, since ious crops to provide the overall proof of some are in Phase III clinical trials al- concept that transgenic plants can pro- ready, see Table 1). The other weakness duce biopharmaceuticals. However, and is that no final regulatory guidelines ex- this can be seen in the table as well, the ist. But as mentioned before, regulatory commercial production of biopharmaceu- authorities (Food and Drug Administra- ticals in transgenic plants is still in the tion (FDA), European Medicine Evaluation early stages of development and yet the Agency (EMEA), and Biotechnology Regu- most advanced products are in Phase III latory Service (BRS) and the Biotechnology clinical development. Industry Organization (BIO) have drafted guidelines on plant-derived biopharma- 4.2 ceuticals (see Table 2) and have asked Risk Assessment and Contingency the community for comments. The FDA Measures has also issued several PTC (Points To Consider) guidelines about plant-based bi- For a number of reasons, including the ologics, and review of the July 2002 PTC knowledge base developed on genetically confirms that the FDA supports this field modifying its genome, industrial pro- and highlights the benefits of plant ex- cesses for extracting fractionated products pression systems – including the absence and the potential for large-scale produc- of any pathogens to man from plant ex- tion, the preferred plant expression system tracts. The main concerns of using plant has been corn. However, the use of expression systems are societal ones about corn touches on a potential risk: some environmental impacts, segregation risk, environmental activist groups and trade and contamination of the food chain. associations are concerned about the effect But these threats can be dealt with, us- on the environment and possible contam- ing nonedible plants (nonfood, nonfeed), ination of the food supply. These issues Plant-based Expression of Biopharmaceuticals 393 Issued: Sep 6, 2002 Issued: Mar 13, 2002 Released: Oct 22, 2002 Released: Mar 01, 2001 Released: May 17, 2002 Released: Mar 5, 2001 ce of Science and Technology sgene expression in higher plants’’ dical Devices Derived from Bioengineered Plants for derived biologics’’ for Offi ographic Restrictions for Plant-made Pharmaceuticals and nce Anti-ideotype antibody Tobacco Phase I cument for Confinement and Development of Plant-made ce Confidential Potato Preclinical uticals Gastric lipase Corn Phase II biotechs Antibodies Potato Preclinical + cor Anti-HSV antibody Corn Phase I substances produced by stable tran Use in Humans and Animals’’ Industrials’’ Products (CPMP) Points to ConsiderManufacture on of the Biological Use Medicinal of Products Transgenic for Plants Human in Use’’ the Pharmaceuticals in the United States’’ Policy/Council on Environmental Quality ‘‘Points To Consider Quality Aspects of Medicinal Products containing active ‘‘Concept Paper on the Development of a Committee for Proprietary Medicinal Plant-derived biopharmaceuticals in clinical trials. Drafted guidelines on plant-derived biopharmaceuticals. (European Medicine Evaluation (European Medicine Evaluation (Food and Drug Administration) ‘‘Drugs, Biologics, and Me (Biotechnology Regulatory Services ‘‘Case study on plant- (Biotechnology Industry Organization) ‘‘BIO Position on Ge (Biotechnology Industry Organization) ‘‘Reference Do Agency) Agency) EMEA EMEA FDA BIO BIO Meristem TherapeuticsMPB Cologne GmbH CNRS Aventis CropScien Human lactoferrin Corn Preclinical Tab. 1 CompanyMonsantoLarge Scale BiologyMeristem TherapeuticsLarge Scale BiologyMonsantoProdiGeneEpicyte Pharmaceutical Solvay Pharmace Partner Own product Guy’s Hospital ProdiGene, London Plant Bioscie Dow, Cento Tab. NeoRx 2 Own productAgency Anticaries antibodyBRS scFv (non-Hodgkin) Protein/indication Corn Tobacco TGEV vaccine Antitumor antibody Host Guideline Phase Phase IIIs III Corn Stage Corn Phase I Phase I Status Crop TechCrop TechAltaGen Bioscience Inc. U.S. Army 3 Immunex Amgen Enbrel (arthritis) Therapeutic antibodies Tobacco Tobacco Preclinical Preclinical 394 Plant-based Expression of Biopharmaceuticals

are reflected in the regulatory guidelines to the widespread death of Monarch but- and have been the driving force to inves- terflies. Although this was eventually not tigate other plants as well. While many found to be the case, the public outcry mature and larger companies have been over the incident was a wake-up call to working in this area for many years, there the possible dangers of transgenic food are a number of newcomers that are de- technology. To avoid the same bad per- veloping expertise as well. These smaller ception for biopharmaceuticals expressed companies are reacting to the concerns in plants, there is the need for thor- by looking at the use of nonedible plants ough risk assessment and contingency that can be readily raised in greenhouses. planning. One method is the employ- All potential risks have to be assessed ment of all feasible safety strategies to and contingency measures need to be es- prevent spreading of engineered DNA (ge- tablished. Understanding the underlying netic drift), like a basic containment in issues is mandatory to make sophisticated a greenhouse environment. Although no decisions about the science and subse- practical shelter can totally eradicate insect quently on the development of appropriate and rodent intrusion, this type of isolation plant expression systems for production of is very effective for self-pollinators and biopharmaceuticals. those plants with small pollen dispersal Ongoing public fears from the food patterns. The use of species-specific, frag- industry and the public, particularly in ile, or poorly transmissible viral vectors Europe (‘‘Franken Food’’) could have is another strategy. Tobacco mosaic virus spillover effects on plant-derived pharma- (TMV), for example, usually only infects a ceuticals. Mistakes and misunderstand- tobacco host. ings have already cost the genetically It requires an injury of the plant to enhanced grain industry hundreds of mil- gain entry and cause infection. Destruction lions of dollars. The only way to prevent of a field of TMV-transformed tobacco plant expression systems from suffer- requires only plowing under or application ing the same dilemma is to provide the of a herbicide. These factors prevent both public with appropriate information on horizontal and vertical transmission. In emerging discoveries and newly developed addition, there is no known incidence of production systems for biopharmaceuti- plant viruses infecting animal or bacterial cals. Real and theoretical risks involve cells. Another approach is to avoid stable the spread of engineered genes into wild transgenic germlines and therefore most plants,animals,andbacteria(horizontal uses of transforming viruses do not transmission). For example, if herbicide involve the incorporation of genes into resistance was transmitted to weeds, or the plant cell nucleus. By definition, it is antibiotic resistance was to be transmitted almost impossible for these genes to be to bacteria, superpathogens could result. If transmitted vertically through pollen or these genetic alterations were transmitted seed. The engineered protein product is to their progeny (vertical transmission), an produced only by the infected generation explosion of the pathogens could cause ex- of plants. Another effective way to reduce tensive harm. An example of this occurred the risk of genetic drift is the use of several years ago, when it was feared that plants that do not reproduce without pest-resistant genes had been transmit- human aid. The modern corn plant ted from Bt corn to milkweed – leading cannot reproduce without cultivation and Plant-based Expression of Biopharmaceuticals 395 the purposeful planting of its seeds. spreading antibiotic resistance from one If a plant may sprout from grain, it (transgenic donor) plant to other wild- still needs to survive the wintering-over type plants or bacteria in the environment. process and gain access to the proper Although prokaryotic promoters for an- planting depth. This extinction process is tibiotic resistance are sometimes used so rapid, however, that the errant loss of in the fabrication and selection of trans- an ear of corn is very unlikely to grow genic constructs, once a transgene has a new plant. Another very well-known been stably incorporated into the plant example of self-limited reproduction is genome, it is under the control of plant the modern banana. It propagates almost (eukaryotic) promoter elements. Hence, exclusively through vegetative cloning (i.e. antibiotic-resistance genes are unable to via cuttings). pass from genetically altered plants into Pollination is the natural way for most bacteria and remain functional. As stated plants to spread their genetic information, earlier, another common fear is the cre- make up new plants, and to deliver their ation of a ‘‘super bug.’’ The chance of offspring in other locations. The use of creating a supervirulent virus or bacterium plants with limited range of pollen dis- from genetic engineering is unlikely, be- persal and limited contact with compatible cause the construction of expression cas- wild hosts therefore is also very effective settes from viral or bacterial genomes to prevent genetic drift. Corn, for exam- involves the removal of the majority of ple, has pollen, which survives for only genes responsible for the normal function 10 to 30 min and, hence, has an effective of these organisms. Even if a resul- fertilizing radius of less than 500 m. In tant organism is somewhat functional, North America, it has no wild-type rela- it cannot compete for long in nature tives with which it could cross-pollinate. with normal, wild-type bacteria of the In addition to being spatially isolated from same species. nearby cornfields, transgenic corn can be As one can see from the aforementioned ‘‘temporally isolated’’ by being planted safety strategies, considerable effort is at least 21 days earlier or 21 days later put into the reduction of any potential than the surrounding corn, to ensure risk from the transgenic plant for the that the fields are not producing flowers environment. In general, the scientific at the same time. Under recent USDA risk can be kept at a minimum, if (U.S. Department of Agriculture) regula- common sense is applied – in accordance tions, the field must also be planted with with Thomas Huxley (1825–1895) that equipment dedicated to the genetically ‘‘Science is simply common sense at its modified crop. For soybeans, the situa- best.’’ For example, protein toxins (for tion is different, since they are virtually vaccine production) should never be grown 100% self-fertilizers and can be planted in in food plants. very close proximity to other plants without Additionally, the following can be em- fear of horizontal spread. Another option ployed as a kind of risk management to is the design of transgenic plants that have prevent the inappropriate or unsafe use of only sterile pollen or – more or less only genetically engineered plants: applicable for greenhouses – completely prevent cross-pollination by covering the • An easily recognized phenotypic char- individual plants. One public fear regards acteristic can be coexpressed in an 396 Plant-based Expression of Biopharmaceuticals

engineered product (e.g. tomatoes that 5 contain a therapeutic protein can be The Way Forward: Moving Plants to selected to grow in a colorless variety Humanlike Glycosylation of fruit). • Protein expression can be induced only As discussed earlier, plant production of after harvesting or fruit ripening. For therapeutic proteins has many advantages example, CropTech’s (Blacksburg, VA, over bacterial systems. One very impor- USA) inducible expression system in tant feature of plant cells is their capability tobacco, MeGA-PharM, leads to very of carrying out posttranslational modifica- efficient induction upon leaf injury (har- tions. Since they are eukaryotes (i.e. have a vest) and needs no chemical inducers. nucleus), plants produce proteins through an ER (endoplasmatic reticulum) pathway, This system possesses a fast induction adding sugar residues also to the pro- response and protein synthesis rate, and tein – a process called glycosylation.These thus leads to high expression levels with carbohydrates help determine the three- no aged product in the field (no environ- dimensional structures of proteins, which mental damage accumulation). are inherently linked to their function and • Potentially antigenic or immunomodu- their efficacy as therapeutics. This glycosy- latory products can be induced to grow lation also affects protein bioavailability in, or not to grow in, a certain plant tis- and breakdown of the biopharmaceuti- sue (e.g. root, leaf/stem, seed, or pollen). cal; for example, proteins lacking terminal In this way, for example, farmers can be sialic acid residues on their sugar groups protected from harmful airborne pollen are often targeted by the immune system or seed dusts. and are rapidly degraded. The glycosyla- • Although no absolute system can pre- tion process begins by targeting the protein vent vandalism or theft of the transgenic to the ER. During translation of mRNA plants, a very effective, cheap solution (messenger RNA) into protein, the ribo- has been used quietly for many years some is attached to the ER, and the nascent now in the United States. Plots of these protein fed into the lumen of the ER as modified plants are being grown with translation proceeds. Here, one set of gly- absolutely no indication that they are cosylation enzymes attaches carbohydrates different from a routine crop. In the to specific amino acids of the protein. Midwest, for example, finding a trans- Other glycosylation enzymes either delete genic corn plot among the millions of or add more sugars to the core structures. acres of concurrently growing grain is This glycosylation process continues into virtually impossible. The only question the Golgi apparatus, which sorts the new here is, if this approach really helps proteins, and distributes them to their final facilitating a fair and an open discus- destinations in the cell (see Fig. 4). Bacte- sion with the public. Asking the same ria lack this ability and therefore cannot question for the EU is not relevant: be used to synthesize proteins that require owing to labeling requirements, this ap- glycosylation for activity. Although plants proach would not be feasible, as, in have a somewhat different system of pro- general, it is much more difficult to tein glycosylation from mammalian cells, perform open-field studies with trans- the differences usually prove not to be a genic plants. problem. Some proteins, however, require Plant-based Expression of Biopharmaceuticals 397

Fig. 4 The glycosylation pathway via ER and Golgi apparatus. In the cytosol, carbohydrates are attached to a lipid precursor, which is then transported into the lumen of the ER to finish core glycosylation. This glycan is now attached to the nascent, folding polypeptide chain (which is synthesized by ribosomes attached to the cytosolic side of the ER from where it translocates into the lumen) and subsequently trimmed and processed before it is folded and moved to the Golgi apparatus. Capping of the oligosaccharide branches with sialic acid and fucose is the final step on the way to a mature glycoprotein. Source: Dove, A. (2001) The bittersweet promise of glycobiology, Nat. Biotechnol. 19, 913–917.

Native Transgenic Bacteria Yeast Transgenic glycoproteins animals plants Peptide Galactose Mannose N-glycolylneuraminic acid

Xylose Fucose N-acetylglucosamine N-acetylneuraminic acid

Fig. 5 Engineering plants to humanlike glycosylation. The first step to achieve humanlike glycosylation in plants is to eliminate the plant glycosylation pattern, that is, the attachment of β 1–2 linked xylosyl- and α 1–3 linked fucosyl sugars to the protein. Because these two residues have allergenic potential, the corresponding enzymes Xylosyl- and Fucosyl Transferase are knocked out. In case galactose is relevant for the final product, Galactosyl Transferase is inserted into the host genome. Galactose is available in the organism so that this single gene insertion is .. sufficient to ensure galactosylation. Source: Knablein J. (2003) Biotech: A New Era In The New Millennium – Biopharmaceutic drugs manufactured in novel expression systems, DECHEMA-Jahrestagung der Biotechnologen, Munich, Germany, 21. 398 Plant-based Expression of Biopharmaceuticals

humanlike glycosylation (see Fig. 5) – they needs only simple medium, and can be must have specific sugar structures at- keptinagreenhouse(seeFig.6).Opti- tached to the correct sites on the molecule mized antibody expression can be rapidly to be maximally effective. Therefore, some verified using transient expression assays efforts are being made in modifying host (short development time) in the plants plants in such a way that they provide before creation of transgenic suspension the protein with human glycosylation pat- cells or stable plant lines (longer devel- terns. One example of modifying a plant opment time). Different vector systems, expression system in this way is the trans- harboring targeting signals for subcellular genic moss, which will be discussed in the compartments, are constructed in parallel next section. and used for transient expression. Apply- ing this screening approach, high express- ing cell lines can rapidly be identified. For 6 example, transgenic tobacco plants, trans- Three Promising Examples: Tobacco formed with an expression cassette con- (Rhizosecretion, Transfection) and Moss taining the GFP (Green Fluorescent Pro- (Glycosylation) tein) gene fused to an aps (amplification- promoting sequence), had greater levels of To further elaborate on improving glycosy- corresponding mRNAs and expressed pro- lation and downstream processing, three teins compared to transformants lacking interesting plant expression systems will aps. Usually, downstream processing (iso- be discussed. All systems share the advan- lation/extraction and purification of the tage of utilizing nonedible plants (nonfood target protein) is limiting for such a sys- and nonfeed) and can be kept in either a tem, for example, if the protein has to greenhouse or a fermenter to avoid any be isolated from biochemically complex segregation risk. Another obvious advan- plant tissues (e.g. leaves), this can be a tage is secretion of the protein into the laborious and expensive process and a medium so that no grinding or extrac- major obstacle to large-scale protein man- tion is required. This is very important in ufacturing. To overcome this problem, light of downstream processing: protein secretion-based systems utilizing trans- purification is often as expensive as the genic plant cells or plant organs aseptically biomanufacturing and should never be un- cultivated in vitro would be one solution. derestimated in the total COGS equation. However, in vitro systems can be expen- sive, slow growing, unstable, and relatively 6.1 low yielding. This is why another inter- Harnessing Tobacco Roots to Secrete esting route was followed. Secretion of Proteins molecules is a basic function of plant cells and organs in plants, and is espe- Phytomedics (Dayton, NJ, USA) uses to- cially developed in plant roots. In order bacco plants as an expression system for to take up nutrients from the soil, inter- biopharmaceuticals. Besides the advantage act with other soil organisms, and defend of being well characterized and used in themselves against numerous pathogens, agriculture for some time, tobacco has plant roots have evolved sophisticated a stable genetic system, provides high- mechanisms based on the secretion of density tissue (high protein production), different biochemicals (including proteins Plant-based Expression of Biopharmaceuticals 399

Phytomedics (tobacco):

Root secretion, easy recovery Greenhouse contained tanks High density tissue Salts and water only Tobacco is well characterized Stable genetic system

Fig. 6 Secretion of the biopharmaceuticals via (left flask in panel lower left edge). The picture tobacco roots. The tobacco plants are genetically also shows a schematic drawing of the modified in such a way that the protein is hydroponic tank, as well as tobacco plants at secreted via the roots into the medium different growth stages, for example, callus, fully grown, and greenhouse plantation. Source: (‘‘rhizosecretion’’). In this example, the tobacco .. plant takes up nutrients and water from the Knablein J. (2003) Biotech: A New Era in the New medium and releases GFP (Green Fluorescent Millennium – Biopharmaceutic Drugs Protein). Examination of root cultivation medium Manufactured in Novel Expression Systems, by its exposure to near ultraviolet-illumination DECHEMA-Jahrestagung der Biotechnologen, reveals the bright green-blue fluorescence Munich, Germany, 21. (See color plate. p. xxv) characteristics of GFP in the hydroponic medium like toxins) into their neighborhood (rhi- of continuous protein production that in- zosphere). In fact, Borisjuk and coworkers tegrates the biosynthetic potential of a could demonstrate that root secretion can plant over its lifetime and might lead to be successfully exploited for the continu- higher protein yields than single-harvest ous production of recombinant proteins and extraction methods. Rhizosecretion is in a process termed ‘‘rhizosecretion.’’ Here, demonstrated in Fig. 6, showing a trans- an endoplasmic reticulum signal peptide genic tobacco plant expressing GFP and is fused to the recombinant protein, which releasing it into the medium. is then continuously secreted from the roots into a simple hydroponic medium 6.2 (based on the natural secretion from roots High Protein Yields Utilizing Viral of the intact plants). The roots of the to- Transfection bacco plant are sitting in a hydroponic tank (see Fig. 6), taking up water and ICON Genetics (Halle, Germany) has de- nutrients and continuously releasing the veloped a protein-production system that biopharmaceutical. By this elegant set up, relies on rapid multiplication of viral vec- downstream processing becomes easy and tors in an infected tobacco plant (see cost-effective, and also offers the advantage Fig. 7). Viral transfection systems offer 400 Plant-based Expression of Biopharmaceuticals

ICON Genetics (tobacco): • Viral transfection • Fast development Expression in plant tissue • High protein yields (a) (b) • Coexpression of genes

Coom assie gel

RbcL

GFP (c) (d)

CP RbcS

Fig. 7 Viral transfection of tobacco plants. This new generation platform for fast (1 to 2 weeks), high-yield (up to 5 g kg−1 fresh leaf weight) production of biopharmaceuticals is based on proviral gene amplification in a nonfood host. Antibodies, antigens, interferons, hormones, and enzymes could successfully be expressed with this system. The picture shows development of initial symptoms on a tobacco following the Agrobacterium-mediated infection with viral vector components that contain a GFP gene (a); this development eventually leads to a systemic spread of the virus, literally converting the plant into a sack full of protein of interest within two weeks (b). The system allows to coexpress two proteins in the same cell, a feature that allows expression of complex proteins such as full-length monoclonal antibodies. Panels (c) and (d) show the same microscope section with the same cells, expressing Green Fluorescent Protein (c) and Red Fluorescent Protein (d) at the same time. The yield and total protein concentration achievable are illustrated by a Coomassie gel with proteins in the system: GFP (protein of interest), CP (coat protein from wild-type virus), RbcS and RbcL (small and large subunit of ribulose-1,5-bisphosphate carboxylase). Source: .. Knablein J. (2003) Biotech: A New Era in the New Millennium – Biopharmaceutic Drugs Manufactured in Novel Expression Systems, DECHEMA-Jahrestagung der Biotechnologen, Munich, Germany, 21. (See color plate. p. xxv)

a number of advantages, such as very monoclonal antibodies, because they con- rapid (1 to 2 week) expression time, sist of the light and heavy chains, which possibility of generating initial milligram are expressed independently and are sub- quantities within weeks, high expression sequently assembled), low expression level levels, and so on. However, the existing in systemically infected leaves, and so on. viral vectors, such as TMV-based vectors ICON has solved many of these problems used by, for example, Large Scale Biology by designing a process that starts with Corp. (Vacaville, CA, USA) for production an assembly of one or more viral vec- of single-chain antibodies for treatment tors inside a plant after treating the leaves of non-Hodgkin lymphoma (currently in with agrobacteria, which deliver the nec- Phase III clinical trials, see Table 1), had essary viral vector components. ICON’s numerous shortcomings, such as inabil- proviral vectors provide advantages of fast ity to express genes larger than 1 kb, and high-yield amplification processes in a inability to coexpress two or more pro- plant cell, simple and inexpensive assem- teins (a prerequisite for production of bly of expression cassettes in planta,and Plant-based Expression of Biopharmaceuticals 401 full control of the process. The robustness 6.3 of highly standardized protocols allows the Simple Moss Performs Complex use of inherently the same safe protocols Glycosylation for both laboratory-scale as well as indus- trial production processes. In this system, Greenovation Biotech GmbH (Freiburg, the plant is modified transiently rather Germany) has established an innovative than genetically and reaches the speed and production system for human proteins. yield of microbial systems while enjoying The system produces pharmacologically active proteins in a bioreactor, utilizing posttranslational capabilities of plant cells. amoss()cellcul- De- and reconstructing of the virus adds ture system with unique properties (see some safety features and also increases ef- Fig. 8). It was stated before that posttrans- ficiency. There is no ‘‘physiology conflict,’’ lational modifications for some proteins because the ‘‘growth phase’’ is separated are crucial to gain complete pharmaco- from the ‘‘production phase,’’ so that no logical activity. Since moss is the only competition occurs for nutrients and other known plant system that shows a high components required for growth and also frequency of homologous recombination, for expression of the biopharmaceutical at this is a highly attractive tool for produc- thesametime. tion strain design. By establishing stable This transfection-based platform allows integration of foreign genes (gene knock- the production of proteins in a plant host at out and new transgene insertion) into the a cost of US$1 to 10 per gram of crude pro- plant genome, it can be programmed to tein. The platform is essentially free from produce proteins with modified glycosyla- limitations (gene insert size limit, inability tion patterns that are identical to animal to express more than one gene) of current cells. The moss is photoautotrophic and viral vector-based platforms. The expres- therefore only requires simple media for sion levels reach 5 g per kilogram of fresh growth, which consist essentially of wa- leaf tissue (or some 50% of total cellular ter and minerals. This reduces costs and protein!) in 5 to 14 days after inoculation. also accounts for significantly lower in- Since the virus process (in addition to su- fectious and contamination risks, but in perhigh production of its own proteins, addition to this, the system has some including the protein of interest) leads to more advantages: the shutoff of the other cellular protein • The transient system allows production synthesis, the amount of protein of inter- of quantities for a feasibility study est in the initial extract is extremely high within weeks – production of a stable (Fig. 7). It thus results in reduced costs of expression strain takes 4 to 6 months. downstream processing. Milligram quan- • On the basis of transient expression tities can be produced within two weeks, data, the yield of stable production lines gram quantities in 4 to 6 months, and is expected to reach 30 mg L−1 per the production system is inherently scal- day. This corresponds to the yield of able. A number of high-value proteins have a typical fed-batch culture over 20 days − been successfully expressed, including an- of 600 mg L 1. tibodies, antigens, interferons, hormones, • Bacterial fermentation usually requires and enzymes (see Klimyuk, Marillonnet, addition of antibiotics (serving as se- Knablein,¨ McCaman, Gleba (2005), books). lection marker and to avoid loss of the 402 Plant-based Expression of Biopharmaceuticals

Greenovation (moss system): • Simple medium (photoautotrophic plant needs only water and minerals) • Robust expression system (good expression levels from 15 to 25°C)

• Secretion into medium via human leader sequence (broad pH range: 4-8) • Easy purification from low salt medium via ion exchange

• Easy genetic modifications to cell lines • Stable cell lines / high genetic stability

• Codon usage like human (no changes required) • Inexpensive bioreactors from the shelf

• Nonfood plant (no segregation risk) • Good progress on genetic modification of glycosylation pathways (plant to human)

Fig. 8 Greenovation use a fully contained moss bioreactor. This company has established an innovative production system for human proteins. The system produces pharmacologically active proteins in a bioreactor, utilizing a moss (Physcomitrella patens) .. cell culture system with unique properties. Source: Knablein J. (2003) Biotech: A New Era in the New Millennium – Biopharmaceutic drugs Manufactured in Novel Expression Systems, DECHEMA-Jahrestagung der Biotechnologen, Munich, Germany, 21.

expression vector). For moss cultivation, to eliminate the plant glycosylation, for no antibiotics are needed – this avoids example, the attachment of β-1-2-linked the risk of traces of antibiotics having xylosyl and α-1-3-linked fucosyl sugars to a significant allergenic potential in the the protein, because these two residues finished product. have allergenic potential. Greenovation • Genetic stability is provided by the was able to knockout the relevant glyco- fact that the moss is grown in small sylation enzymes xylosyl transferase and plant fragments and not as proto- fucosyl transferase, which was confirmed plasts or tissue cultures avoiding so- by RT-PCR (reverse transcriptase PCR). maclonal variation. And indeed, xylosyl and fucosyl residues • As a contained system, the moss biore- were completely removed from the glyco- actor can be standardized and validated sylation pattern of the expressed protein according to GMP standards mandatory as confirmed by MALDI-TOF (matrix as- in the pharmaceutical industry. sisted laser desorption ionization time of • Excretion into the simple medium is flight) mass spectroscopy analysis (see another major feature of the moss biore- Fig. 9). actor, which greatly facilitates down- A very challenging protein to express is stream processing. VEGF because this homodimer consists As discussed in detail, the first step to of two identical monomers linked via get humanlike glycosylation in plants is a disulfide bond. To produce VEGF in Plant-based Expression of Biopharmaceuticals 403

Successful knockout of Xylosyl transferase in moss XT-KO plants: RT-PCR and MALDI-TOF analysis

XT114F/ XT15R RT-PCR Xylosyl transferase R10/R11

Control: APS reductase (R10 and R11)

MALDI

Fig. 9 Knockout of Xylosyl Transferase in moss. To avoid undesired glycosylation, greenovation knocked out the Xylosyl and Fucosyl Transferase, as confirmed by RT-PCR. MALDI-TOF results show that indeed, xylosyl- and fucosyl-residues were completely removed from the glycosylation pattern of the expressed protein (data for knockout of .. Fucosyl Transferase not shown). Source: Knablein J. (2003) Biotech: A New Era in the New Millennium – Biopharmaceutic Drugs Manufactured in Novel Expression Systems, DECHEMA-Jahrestagung der Biotechnologen, Munich, Germany, 21.

kDa 5 ng 10 ng TPx TPy Stimulation of human vascular epithelial cells 130 37 VEGF121dimer 26

120 SDS PAGE

1000 WT 800 110 600 400 Counts 200 0 0 50 100 150

Fluorescence intensity Incorporation rate (% of control) 100 1000 RPMI - rh VEGF - P 27 P 31 tWTVEGF p31 −1 −1 800 Medium Control (1ng mL ) (2 ng mL ) − 600 (1ng mL 1) 400 Counts 200 FACS Biological activity of recombinant 0 0 50 100 150 analysis VEGF Fluorescence intensity Fig. 10 Greenovation could successfully linked via the disulfide-bond. The analytical express the biopharmaceutical VEGF. This assays clearly show that expression in moss growth factor is a very complex protein yielded completely active VEGF. Source: .. consisting of two identical monomers linked via Knablein J. (2003) Biotech: A New Era in the New a disulfide-bond. To produce VEGF in an active Millennium – from Plant Fermentation to Plant form, the monomers need to be expressed to the Expression of Biopharmaceuticals,PDA right level, correctly folded, assembled, and International Congress, Prague, Czech Republic. 404 Plant-based Expression of Biopharmaceuticals

30 L pilot reactor for moss Two weeks after incubation Fig. 11 Scaling of photobioreactors up to scaling of the photobioreactors up to several several 1000 L. The moss bioreactor is based on 1000 L. Adaptation of existing technology for the cultivation of Physcomitrella patens in a large-scale cultivation of algae is done in fermenter. The moss protonema is grown under cooperation with the Technical University of photoautotrophic conditions in a medium that Karlsruhe. Source: greenovation Biotech GmbH consists essentially of water and minerals. Light (Freiburg, Germany) and Professor C. Posten, and carbon dioxide serve as the only energy and Technical University (Karlsruhe, Germany). carbon sources. Cultivation in suspension allows

an active form, the following need to the optimum for the protein of interest. be provided: Adapting existing technology for large- scale cultivation of algae, fermentation of • Monomers need to be expressed to the moss in suspension culture allows scaling right level. of the photobioreactors up to several • Monomers need to be correctly folded. 1000 L (see Fig. 11). Finally, the medium is • Homodimer needs to be correctly as- inexpensive, since only water and minerals sembled and linked via a disulfide bond. are sufficient. • Complex protein needs to be secreted in its active form. And in fact, all this could be achieved 7 with the transgenic moss system as shown Other Systems Used for Plant Expression in Fig. 10. These results are very promising because they demonstrate that this system Several different plants have been used is capable of expressing even very complex for the expression of proteins in plants. proteins. In addition to that, the moss All these systems have certain advantages system adds no plant-specific sugars to the regarding edibility, growth rate, scalability, protein – a major step toward humanlike gene-to-protein time, yield, downstream glycosylation. Furthermore, moss is a processing, ease of use, and so on, which robust expression system leading to high I will not discuss in further detail here. A ◦ yields at 15 to 25 CandthepHcan selection of different expression systems be adjusted from 4 to 8 depending on is listed: Plant-based Expression of Biopharmaceuticals 405

Alfalfa Ethiopian Potatoes high percentage of water, which could re- mustard sult in unavoidable proteolysis during the Arabidopsis Lemna Rice process. Proteins stored in seeds can be Banana Maize Soybean desiccated and remain intact for long peri- Cauliflower Moss Tomatoes ods of time. The purification and extraction Corn Oilseeds Wheat of the protein is likely to be done by adapta- tions of current processes for the extraction Some of these systems have been used and/or fractionation. For these reasons, it for research on the basis of their ease is anticipated that large-scale commercial of transformation, well-known characteri- production of recombinant proteins will zation, and ease to work with. However, involve grain and oilseed crops such as they are not necessarily appropriate for maize, rice, wheat, and soybeans. On the commercial production. Which crop is ul- basis of permits for open-air test plots timately used for full-scale commercial issued by the USDA for pharmaceutical production will depend on a number of proteins and industrial biochemicals, corn factors including is the crop of choice for production with 73% of the permits issued. The other major • time to develop an appropriate system crops are soybeans (12%), tobacco (10%), (gene-to-protein); and rice (5%). • section of the plant expressing the In general, the use of smaller plants that product/possible secretion; can be grown in greenhouses is an effective • cost and potential waste products way of producing the biopharmaceuticals from extraction; and alleviating concerns from environ- • ‘‘aged’’ product/ease of storage; mental activist groups that the transgenic • long-term stability of the storage tissue; plant might be harmful to the environ- • quantities of protein needed (scale ment (food chain, segregation risk, genetic of production). drift, etc.).

Depending on the genetic complexity and ease of manipulation, the develop- 8 ment time to produce an appropriate Analytical Characterization transgenic plant for milligram production of the desired protein can vary from 10 Validated bioanalytical assays are essential to 12 months in corn as compared to only and have to be developed to characterize weeks in moss. Estimates for full GMP the biopharmaceuticals during the produc- production in corn are 30 to 36 months tion process (e.g. in-process control) and and approximately 12 months for moss. to release the final product for use as a Expression of the protein in various tissues drug in humans. These assays are applied oftheplantcanresultinagreatvariationin to determine characteristics such as pu- yield. Expression in the seed can often lead rity/impurities, identity, quantity, stability, to higher yields than in the leafy portion of specificity, and potency of the recombi- theplant.Thisisanotherexplanationfor nant protein during drug development. the high interest in using corn, which has Since the very diverse functions of dif- a relatively high seed-to-leaf ratio. Extrac- ferent proteins heavily depend on their tion from leaf can be costly as it contains a structure, one very valuable parameter in 406 Plant-based Expression of Biopharmaceuticals

protein characterization is the elucidation could fall dramatically within the next of their three-dimensional structure. Al- decade because of the use of, for exam- though over the last couple of years a lot of ple, plant expression systems. Fears about effort was put into a method for improv- the risks of the plant expression technol- ing the elucidation of protein structures ogy are real and well founded, but with a (during my PhD thesis, I was also work- detailed understanding of the technology, ing in this fascinating field together with it is possible to proactively address these my boss Professor Robert Huber, Nobel safety issues and create a plant expres- Prize Laureate in 1988, ‘‘for the determi- sion industry almost free of mishaps. For nation of the three-dimensional structure this purpose, the entire set up, consisting of a photosynthetic reaction centre’’), it is of the specific plant expression system and still very time consuming to solve the 3-D the protein being produced, needs to be an- structure of larger proteins. This is why de- alyzed and its potential risks assessed on a spite the high degree of information that case-by-case basis. As plant-derived thera- canbeobtainedfromtheproteinstruc- peutics begin to demonstrate widespread, ture, this approach cannot be applied on tangible benefits to the population and as a routine basis. Therefore, tremendous ef- the plant expression industry develops a forts are put into the development of other longer safety track record, public accep- assays to guarantee that a potent biophar- tance of the technology is likely to improve maceutical drug is indeed ready for use continuously. Plants are by far the most in humans. abundant and cost-effective renewable re- source uniquely adapted to complex bio- chemical synthesis. The increasing cost of 9 energy and chemical raw materials, com- Conclusion and Outlook bined with the environmental concerns associated with conventional pharmaceu- The production of protein therapeutics tical manufacturing, will make plants even from transgenic plants is becoming a more compatible in the future. With the reality. The numerous benefits offered words of Max Planck (1858–1947) ‘‘How by plants (low cost of cultivation, high far advanced Man’s scientific knowledge biomass production, relatively fast gene- may be, when confronted with Nature’s to-protein time, low capital and operating immeasurable richness and capacity for costs, excellent scalability, eukaryotic post- constant renewal, he will be like a mar- translational modifications, low risk of veling child and must always be prepared human pathogens, lack of endotoxins, for new surprises,’’ we will definitely dis- as well as high protein yields) virtu- cover more fascinating features of plant ally guarantee that plant-derived proteins expression systems. But there is no need will become more and more common to wait: combining the advantages of some for therapeutic uses. Taking advantage of technologies that we already have in hand plant expression systems, the availability could lead to the ultimate plant expression of cheap protein-based vaccines in un- system. This is what we should focus on, derdeveloped countries of the world is because, then, at the dawn of this new mil- possible in the near future. The cost of lennium, this would for the first time yield very expensive hormone therapies (ery- large-enough amounts of biopharmaceuti- thropoietin, human growth hormone, etc.) cals to treat everybody on our planet! Plant-based Expression of Biopharmaceuticals 407

Acknowledgments cholera toxin B subunit vaccine, Nat. Biotechnol. 16, 292–297. I would like to thank the companies green- Arakawa, T., Yu, J., Chong, D.K., Hough, J., ovation Biotech GmbH (Freiburg, Ger- Engen, P.C., Langridge, W.H. (1998) A plant- based cholera toxin B subunit-insulin fusion many), ICON Genetics (Halle, Germany), protein protects against the development of and Phytomedics (Dayton, NJ, USA) for autoimmune diabetes, Nat. Biotechnol. 16, providing some data and figures to prepare 934–938. this manuscript. Arthur D. Little, Inc. (ADL), AgIndustries Research,Cambridge,MA,Copyright 2002. See also Bioprocess Engineering; Artsaenko, O., et al. (1998) Potato tubers as a biofactory for recombinant antibodies, Mol. Expression Systems for DNA Pro- Breeding 4, 313–319. cesses; Plant Gene Expression, Beachy, R.N., Fitchen, J.H., Hein, M.B. (1996) Use of Plant Viruses for Delivery of Vaccine Regulation of. Epitopes, in: Collins, G.B., Sheperd, R.J. (Eds.) Engineering Plants for Commercial Products and Applications, New York Academy of Sciences, Bibliography New York, pp. 43–49. Boothe, J.G., Parmenter, D.L., Saponja, J.A. (1997) Molecular farming in plants: oilseeds as Books and Reviews vehicles for the production of pharmaceutical proteins, Drug Develop. Res. 42, 172–181. Fischer, R., Schillberg S. (Eds.) (2004) Molecular Borisjuk, N.V., Raskin, I., et al. (1999) Produc- Farming: Plant-made Pharmaceuticals and tion of recombinant proteins in plant root Technical Proteins, Wiley, ISBN: 3-527-30786-9 exudates, Nat. Biotechnol. 17, 466–469. Fischer, R., et al. (2004) Plant-based production Borisjuk, N.V., Raskin, I., et al. (2000) Tobacco of biopharmaceuticals, Curr. Opin. Plant Biol. ribosomal DNA spacer element stimulates 7(2), 152–158. Horn, M.E., Woodard, S.L., Howard, J.A. (2004) amplification and expression of heterologous Plant molecular farming: systems and genes, Nat. Biotechnol. 18, 1303–1306. products, Plant Cell Rep. 22(10), 711–720. Cabanes-Macheteau, M., et al. (1999) N- Klimyuk,V.,Marillonnet,S.,Knablein,¨ J., Mc- glycosylation of a mouse IgG expressed Caman, M., Gleba, Y. (2005) Production of in transgenic tobacco plants, Glycobiology 9, Recombinant Proteins in Plants, in: Modern 365–372. Biopharmaceuticals – Design, Development and Chance, R.E., Frank, B.H. (1993) Research, Optimization,Wiley-VCH,inpress. development, production and safety of Knablein,¨ J. (2004) Biopharmaceuticals Ex- biosynthetic human insulin, Diabetes care pressed in Plants – A New Era in the New 16(3), 133–142. Millennium, in: Muller,¨ R., Kayser, O. (Eds.) Chaudhary, S., Parmenter, D.L., Moloney, M.M. Applications in Pharmaceutical Biotechnology, (1998) Transgenic Brassica carinata as a Wiley-VCH, ISBN 3-527-30554-8. vehicle for the production of recombinant Ma, J.K., Drake, P.M., Christou, P. (2003) The proteins in seeds, Plant Cell Rep. 17, 195–200. production of recombinant pharmaceutical Conrad, U., Fiedler, U. (1994) Expression of proteins in plants, Nat. Rev. Genet. 4(10), engineered antibodies in plant cells, Plant Mol. 794–805. Biol. 26, 1023–1030. Stoger, E., et al. (2004) Antibody production in Conrad, U., Fiedler, U., Artsaenko, O., Phillips, transgenic plants, Methods Mol. Biol. 248, J. (1998) High-level and stable accumulation 301–318. of single-chain Fv antibodies in plant storage organs, J. Plant Physiol. 152, 708–711. Primary References Cramer, C.L., Boothe, J.G., Oishi, K.K. (1999) Transgenic plants for therapeutic proteins: Arakawa, T., Chong, D.K.X., Langridge, W.H.R. linking upstream and downstream strategies, (1998) Efficacy of a food plant-based oral Curr. Top. Microbiol. Immunol. 240, 95–118. 408 Plant-based Expression of Biopharmaceuticals

Cramer, C.L., et al.. (1996) Bioproduction of Pharmaceutical Proteins, John Wiley & Sons, Human Enzymes in Transgenic Tobacco, in: London, UK, 281–297.. Collins, G.B., Sheperd, R.J. (Eds.) Engineering Garber, K. (2001) Biotech industry faces new Plants for Commercial Products and bottleneck, Nat. Biotechnol. 19, 184–185. Applications, New York Academy Of Sciences, Giddings, G., Allison, G., Brooks, D., Carter, C. NewYork,62–71. (2000) Transgenic plants as factories for Dalsgaard, K., et al. (1997) Plant-derived vaccine biopharmaceuticals, Nat. Biotechnol. 18, protects target animals against a viral disease, 1151–1155. Nat. Biotechnol. 15, 248–252. Goddijn, O.J.M., Pen, J. (1995) Plants as Davies, L., Plieth, J. (2001) The challenge of bioreactors, Trends Biotechnol. 13, 379–387. meeting the escalating demand for proteins, Hamamoto, H., et al. (1993) A new tobacco Scr Mag 10, 25–29. mosaic virus vector and its use for Della-Cioppa, G., Grill, L.K. (1996) Production the systematic production of angiotensin-I- of Novel Compounds in Higher Plants by converting enzyme inhibitor in transgenic Transfection with RNA Viral Vectors, in: tobacco and tomato, Biotechnology 11, Collins, G.B., Sheperd, R.J. (Eds.) Engineering 930–932. Plants for Commercial Products and Hiatt, A., Cafferkey, R., Bowdish, K. (1989) Applications, New York Academy of Sciences, Production of antibodies on transgenic plants, New York, pp. 57–61. Nature 342, 76–78. Dieryck, W., et al. (1997) Human haemoglobin Hood, E.E., Jilka, J.M. (1999) Plant-based from transgenic tobacco, Nature 386, 29–30. production of xenogenic proteins, Curr. Opin. Doran, P.M. (2000) Foreign protein production Biotechnol. 10, 382–386. in plant tissue cultures, Curr. Opin. Biotechnol. Johnson, E. (1996) Edible plant vaccines, Nat. 11, 199–204. Biotechnol. 14, 1532–1533. Dove, A. (2001) The bittersweet promise of Knablein,¨ J. (2003) Biotech: A new era in glycobiology, Nat. Biotechnol. 19, 913–917. the new millennium – fermentation and Dove, A. (2002) Unkorking the biomanufactur- expression of biopharmaceuticals in plants, ing bottleneck, Nat. Biotechnol. 20, 777–779. SCREENING – Trends Drug Discov 4, 14–16. Drake, P.M., Chargelegue, D., Vine, N.D., Van Knablein,¨ J., McCaman, M. (2003) Modern Dolleweerd, C.J., Obregon, P., Ma, J.K. (2002) biopharmaceuticals-recombinant protein ex- Transgenic plants expressing antibodies: a pression in transgenic plants, SCREEN- model for phytoremediation, FASEB J. 16(14), ING – Trends Drug Discov 6, 33–35. 1855–1860. Knablein,¨ J., Huber, R., et al. (1997) Drug & Market Development Publications, [Ta6Br12]2+, a tool for phase determination Antibody Engineering: Technologies, Applications of large biological assemblies by X-ray crystal- and Business opportunities, Westborough, MA, lography, J. Mol. Biol. 270, 1–7. Copyright 2003. Kumagai, M.H., et al. (1993) Rapid, high- Evangelista, R.L., Kusnadi, A.R., Howard, J.A., level expression of biologically active alpha- Nikolov, Z.L. (1988) Process and economic trichosanthin in transfected plants by an RNA evaluation of the extraction and purification of viral vector, Proc. Natl. Acad. Sci. USA 90, recombinant glucouronidase from transgenic 427–430. corn, Biotechnol. Prog. 14, 607–614. Kusnadi, A., Nikolov, Z.L., Howard, J.A. (1997) Fischer, R., Emans, N. (2000) Molecular farming Production of recombinant proteins in of pharmaceutical proteins, Transgenic Res. 9, transgenic plants: practical considerations, 279–299. Biotechnol. Bioeng. 56, 473–484. Fischer, R., Hoffmann, K., Schillberg, S., Ma, J.K.C. (2000) Genes, greens, and vaccines, Emans, N. (2000) Antibody production by Nat. Biotechnol. 18, 1141–1142. molecular farming in plants, J. Biol. Regul. Ma, J.K.C., Hein, M.B. (1995) Plant antibodies Homeost. Agents 14, 83–92. for immunotherapy, Plant Physiol. 109, Ganz, P.R., et al.. (1996) Expression of Human 341–346. Blood Proteins in Transgenic Plants: The Ma, J.K.C., Hein, M.B. (1996) Antibody Cytokine GM-CSF as a Model Protein, Production and Engineering in Plants, in: in: Owen, M.R.L., Pen, J. (Eds.) Transgenic Collins, G.B., Sheperd, R.J. (Eds.) Engineering Plants: A Production System for Industrial and Plants for Commercial Products and Plant-based Expression of Biopharmaceuticals 409

Applications, New York Academy of Sciences, Richter, L.J., Thanavala, Y., Arntzen, C.J., Ma- New York, pp. 72–81. son, H.S. (2000) Production of hepatitis Ma, J.K.C., Hiatt, A. (1996) Expressing Anti- B surface antigen in transgenic plants bodies in Plants for Immunotherapy, in: for oral immunization, Nat. Biotechnol. 18, Owen, M.R.L., Pen, P. (Eds.) Transgenic Plants: 1167–1171. A Production System for Industrial and Pharma- Ruggiero, F., et al. (2000) Triple helix assembly ceutical Proteins, John Wiley & Sons, London, and processing of human collagen produced UK, pp. 229–243. in transgenic tobacco plants, FEBS Lett. 469, Ma, J.K.C., Vine, N.D. (1999) Plant expression 132–136. systems for the production of vaccines, Curr. Sijmons, P.C., et al. (1990) Production of Top. Microbiol. Immunol. 236, 275–292. correctly processed human serum albumin in Ma, J.K.C., et al. (1998) Characterization of transgenic plants, Biotechnology 8, 217–221. a recombinant plant monoclonal secretory Smith, M.D. (1996) Antibody production in antibody and preventive immunotherapy in plants, Biotechnol. Adv. 14, 267–281. humans, Nat. Med. 4(5), 601–606. Smith, M.D., Glick, B.R. (2000) The production Ma, S.W., et al. (1997) Transgenic plants of antibodies in plants, Biotechnol. Adv. 18, expressing autoantigens fed to induce oral 85–89. immune tolerance, Nat. Med. 3, 793–517. Stoger, E., et al. (2000) Cereal crops as McCormick, A.A., et al. (1999) Rapid production viable production and storage systems for of specific vaccines for lymphoma by pharmaceutical scFv antibodies, Plant Mol. expression of the tumor-derived single-chain Biol. 42, 583–590. Fv epitopes in tobacco plants, Proc. Natl. Acad. Tacket, C.O., Mason, H.S. (1999) A review of Sci. USA 96, 703–708. oral vaccination with transgenic vegetables, McGarvey, P.B., et al. (1995) Expression of Microbes Infect. 1, 777–783. the rabies virus glycoprotein in transgenic Tacket, C.O., et al. (1998) Immunogenicity in tomatoes, Biotechnology 13, 1484–1487. humans of a recombinant bacterial antigen Moloney, M.M. (1995) ‘‘Molecular farming’’ delivered in a transgenic potato, Nat. Med. 4, in plants: achievements and prospects, 607–609. Biotechnol. Eng. 9, 3–9. Technology Catalysts International Corporation, Morrow, K.J. (2002) Economics of antibody Biopharmaceutical Farming, Falls Church, VA, production, Genet. Eng. News 22, 34–39. Copyright 2002. Mushegian, A.R., Shepard, R.J. (1995) Genetic Thanavala, Y., et al. (1995) Immunogenicity of elements of plant viruses as tools for genetic transgenic plant-derived hepatitis B surface engineering, Microbiol. Rev. 59, 548–578. antigen, Proc. Natl. Acad. Sci. USA 92, Parmenter, D.L., et al. (1995) Production of 3358–3361. biologically active hirudin in plant seeds The Context Network, Biopharmaceutical sing oleosin partitioning, Plant Mol. Biol. 29, Production in Plants, Biopharma Prospectus, 1167–1180. West Des Moines, IA, Copyright 2002. Pen, J. (1996) Comparison of Host Systems Tomsett, B., Tregova, A., Garoosi, A., Cad- for the Production of Recombinant Proteins, dick, M. (2004) Ethanol-inducible gene expres- in: Owen, M.R.L., Pen, J. (Eds.) Transgenic sion: first step toward a new green revolution? Plants: A Production System for Industrial and Trends Plant Sci. 9(4), 159–161. Pharmaceutical Proteins, John Wiley & Sons, Valdes, R., et al. (2003) Hepatitis B surface London, UK, pp. 149–167. antigen immunopurification using a plant- Ponstein, A.S., Verwoerd, T.C., Pen, J. (1996) derived specific antibody produced in large Production of Enzymes for Industrial scale, Biochem. Biophy. Res. Commun. 310, Use, in: Collins, G.B., Sheperd, R.J. (Eds.) 742–747. Engineering Plants for Commercial Products and Vandekerckhove, J., et al. (1989) Enkephalines Applications, Vol. 792, New York Academy of produced in transgenic plants using modified Sciences, New York, pp. 91–98. 2S storage proteins, Biotechnology 7, 929–932. Raskin, I., Fridlender, B., et al. (2002) Plants and Whitelam, G.C. (1995) The production of human health in the twenty-first century, recombinant proteins in plants, J. Sci. Food Trends Biotechnol. 20, 522–531. Agric. 68, 1–9. 410 Plant-based Expression of Biopharmaceuticals

Whitelam, G.C., Cockburn, W. (1996) Antibody Zhong, G.Y., et al. (1999) Commercial produc- expression in transgenic plants, Trends Plant tion of aprotinin in transgenic maize seeds, Sci. 1, 268–272. Mol. Breeding 5, 345–356.