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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 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.