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Production of therapeutic monoclonal antibodies using genetically modified plants may provide low cost, high scalability and product safety; however, antibody purification from plants presents a challenge due to the large quantities of biomass that need to be processed. Protein A column chromatography is widely used in the pharmaceutical industry for antibody purification, but its application is limited by cost, scalability and column fouling problems when purifying plant-derived antibodies. Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds, are relatively inexpensive to produce and provide a new approach for the capture of monoclonal antibodies from plants. When Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase while impurities remain in the aqueous phase. This is followed by repeated partitioning of oilbody phase against an aqueous phase via centrifugation to remove impurities before purified antibody is eluted from the oilbodies. We have developed this purification process to recover trastuzumab, an anti-HER2 monoclonal antibody used for therapy against specific breast-cancers that over express HER2 (human epidermal growth factor receptor 2), from transiently infected Nicotiana benthamiana. Protein A-OB overcomes the fouling problem associated with traditional Protein A chromatography, allowing for the development of an inexpensive, scalable and novel high-resolution method for the capture of antibodies based on simple mixing and phase separation.

Apolipoprotein AI Milano (ApoAI(Milano) ) was expressed as a fusion protein in transgenic safflower seeds. High levels of expression corresponding to 7 g of ApoAI(Milano) per kilogram of seed have been identified in a line selected for commercialization. The ApoAI(Milano) fusion protein was extracted from seed using an oilbody-based process and matured in vitro prior to final purification. This yielded a Des-1,2-ApoAI(Milano) product which was confirmed by biochemical characterization including immunoreactivity against ApoAI antibodies, isoelectric point, N-terminal sequencing and electrospray mass spectrometry. Purified Des-1,2-ApoAI(Milano) readily associated with dimyristoylphosphatidylcholine in clearance assays comparable to Human ApoAI. Its biological activity was assessed by cholesterol efflux assays using Des-1,2-ApoAI(Milano) :1-palmitoyl-2-oleoyl phosphatidylcholine complexes in vitro and in vivo. This study has established that high levels of biologically functional ApoAI(Milano) can be produced using a plant-based expression system.

The evolution of the seed system provides enormous adaptability to the gymnosperms and angiosperms, because of the properties of dormancy, nutrient storage and seedling vigour. Many of the unique properties of seeds can be exploited in molecular farming applications, particularly where it is desirable to produce large quantities of a recombinant protein. Seeds of transgenic plants have been widely used to generate a raw material for the extraction and isolation of proteins and polypeptides, which can be processed into valuable biopharmaceuticals. The factors that control high-level accumulation of recombinant proteins in seed are reviewed in the following paragraphs. These include promoters and enhancers, which regulate transcript abundance. However, it is shown that subcellular trafficking and targeting of the desired polypeptides or proteins play a crucial role in their accumulation at economically useful levels. Seeds have proven to be versatile hosts for recombinant proteins of all types, including peptides or short and long polypeptides as well as complex, noncontiguous proteins like antibodies and other immunoglobulins. The extraction and recovery of recombinant proteins from seeds is greatly assisted by their dormancy properties, because this allows for long-term stability of stored products including recombinant proteins and a decoupling of processing from the growth and harvest cycles. Furthermore, the low water content and relatively low bioload of seeds help greatly in designing cost-effective manufacturing processes for the desired active pharmaceutical ingredient. The development of cGMP processes based on seed-derived materials has only been attempted by a few groups to date, but we provide a review of the key issues and criteria based on interactions with Food and Drug Administration and European Medicines Agency. This article uses 'case studies' to highlight the utility of seeds as vehicles for pharmaceutical production including: insulin, human growth hormone, lysozyme and lactoferrin. These examples serve to illustrate the preclinical and, in one case, clinical information required to move these plant-derived molecules through the research phase and into the regulatory pathway en route to eventual approval.

Expression of recombinant proteins as translational fusions is commonly employed to enhance stability, increase solubility and facilitate purification of the desired protein. In general, such fusion proteins must be cleaved to release the mature protein in its native form. The usefulness of the procedure depends on the efficiency and precision of cleavage and its cost per unit activity. We report here the development of a general procedure for precise and highly efficient cleavage of recombinant fusion proteins using the protease chymosin. DNA encoding a modified pro-peptide from bovine chymosin was fused upstream of hirudin, carp growth hormone, thioredoxin and cystatin coding sequences and expressed in a bacterial Escherichia coli host. Each of the resulting fusion proteins was efficiently cleaved at the junction between the pro-peptide and the desired protein by the addition of chymosin, as determined by activity, N-terminal sequencing and mass spectrometry of the recovered protein. The system was tested further by cleavage of two fusion proteins, cystatin and thioredoxin, sequestered on oilbody particles obtained from transgenic Arabidopsis seeds. Even when the fusion protein was sequestered and immobilized on oil-bodies, precise and efficient cleavage was obtained. The precision, efficiency and low cost of this procedure suggest that it could be used in larger scale manufacturing of recombinant proteins which benefit from expression as fusions in their host organism.

The advantages and limitations of plant-based systems for the production of recombinant biopharmaceutical proteins are discussed in the context of other host production systems presently available. A novel approach for the purification of recombinant proteins expressed in oilseeds is described that is based on the targeting of these proteins to seed oil bodies. The utility of this approach is demonstrated by the purification of the anticoagulant protein hirudin, produced in seeds of Brassica napus. Drug Dev. Res. 42:172–181, 1997. © 1997 Wiley-Liss, Inc.

The production of therapeutic proteins in plant seed augments alternative production platforms such as microbial fermentation, cell-based systems, transgenic animals, and other recombinant plant production systems to meet increasing demands for the existing biologics, drugs under evaluation, and undiscovered therapeutics in the future. We have developed upstream purification technologies for oilseeds which are designed to cost-effectively purify therapeutic proteins amenable to conventional downstream manufacture. A very useful tool in these endeavors is the plant model system Arabidopsis thaliana. The current chapter describes the rationale and methods used to over-express potential therapeutic products in A. thaliana seed for evaluation and definitive insight into whether our production platform, Safflower, can be utilized for large-scale manufacture.

Gamma linolenic acid (GLA; C18:3Δ6,9,12 cis), also known as γ-Linolenic acid, is an important essential fatty acid precursor for the synthesis of very long chain polyunsaturated fatty acids and important pathways involved in human health. GLA is synthesized from linoleic acid (LA; C18:2Δ9,12 cis) by endoplasmic reticulum associated Δ6-desaturase activity. Currently sources of GLA are limited to a small number of plant species with poor agronomic properties, and therefore an economical and abundant commercial source of GLA in an existing crop is highly desirable. To this end, the seed oil of a high LA cultivated species of safflower (Carthamus tinctorius) was modified by transformation with Δ6-desaturase from Saprolegnia diclina resulting in levels exceeding 70% (v/v) of GLA. Levels around 50% (v/v) of GLA in seed oil was achieved when Δ12-/Δ6-desaturases from Mortierella alpina was over-expressed in safflower cultivars with either a high LA or high oleic (OA; C18:1Δ9 cis) background. The differences in the overall levels of GLA suggest the accumulation of the novel fatty acid was not limited by a lack of incorporation into the triacylgylcerol backbone (>66% GLA achieved), or correlated with gene dosage (GLA levels independent of gene copy number), but rather reflected the differences in Δ6-desaturase activity from the two sources. To date, these represent the highest accumulation levels of a newly introduced fatty acid in a transgenic crop. Events from these studies have been propagated and recently received FDA approval for commercialization as Sonova™400.

The evolution of the seed system provides enormous adaptability to the gymnosperms and angiosperms, because of the properties of dormancy, nutrient storage and seedling vigour. Many of the unique properties of seeds can be exploited in molecular farming applications, particularly where it is desirable to produce large quantities of a recombinant protein. Seeds of transgenic plants have been widely used to generate a raw material for the extraction and isolation of proteins and polypeptides, which can be processed into valuable biopharmaceuticals. The factors that control high-level accumulation of recombinant proteins in seed are reviewed in the following paragraphs. These include promoters and enhancers, which regulate transcript abundance. However, it is shown that subcellular trafficking and targeting of the desired polypeptides or proteins play a crucial role in their accumulation at economically useful levels. Seeds have proven to be versatile hosts for recombinant proteins of all types, including peptides or short and long polypeptides as well as complex, noncontiguous proteins like antibodies and other immunoglobulins. The extraction and recovery of recombinant proteins from seeds is greatly assisted by their dormancy properties, because this allows for long-term stability of stored products including recombinant proteins and a decoupling of processing from the growth and harvest cycles. Furthermore, the low water content and relatively low bioload of seeds help greatly in designing cost-effective manufacturing processes for the desired active pharmaceutical ingredient. The development of cGMP processes based on seed-derived materials has only been attempted by a few groups to date, but we provide a review of the key issues and criteria based on interactions with Food and Drug Administration and European Medicines Agency. This article uses 'case studies' to highlight the utility of seeds as vehicles for pharmaceutical production including: insulin, human growth hormone, lysozyme and lactoferrin. These examples serve to illustrate the preclinical and, in one case, clinical information required to move these plant-derived molecules through the research phase and into the regulatory pathway en route to eventual approval.

The increased incidence of diabetes, coupled with the introduction of alternative delivery methods that rely on higher doses, is expected to result in a substantial escalation in the demand for affordable insulin in the future. Limitations in the capacity and economics of production will make it difficult for current manufacturing technologies to meet this demand. We have developed a novel expression and recovery technology for the economical manufacture of biopharmaceuticals from oilseeds. Using this technology, recombinant human precursor insulin was expressed in transgenic plants. Plant-derived insulin accumulates to significant levels in transgenic seed (0.13% total seed protein) and can be enzymatically treated in vitro to generate a product with a mass identical to that of the predicted product, DesB(30)-insulin. The biological activity of this product in vivo and in vitro was demonstrated using an insulin tolerance test in mice and phosphorylation assay performed in a mammalian cell culture system, respectively.

Publisher Summary This chapter describes some of the critical factors in the design of transgenic plant expression systems and discusses the features of commercial production systems currently being developed. It also examines the range of applications of the technology and the commercial opportunities that exist and reviews the preliminary data from the large-scale recovery of recombinant proteins from plants, along with strategies for simplifying downstream purification. Economic analysis of preliminary data from recombinant protein production in high-yielding crop species and process simulations of downstream recovery operations have provided support for the predicted cost advantage of plant systems. Therefore, it seems likely that plants will be increasingly considered as the primary production vehicle, particularly for recombinant proteins required in large volumes or where high production costs now limit their application.