An ability to produce commercially useful levels of pharmaceutically active proteins in plants in early generations is important to support pre-clinical and clinical trials of such proteins. Traditional commercial manufacture of pharmaceutically active proteins in plants requires transformation of a desired transgenic plant with the nucleic acid molecule of interest and subsequent selection and breeding over multiple generations. Such an approach often fails to deliver commercially useful quantities of the pharmaceutically active protein in early generations such as the R0, R1 and R2 generations. Moreover, many traditional approaches require partial destruction of valuable seed stock to provide protein and to determine either the genotype or phenotype of the seed during the breeding process.
Traditional methods for the manufacture of pharmaceutically active proteins in plants often required transformation, growing the R0 generation with subsequent selfing and selection to generate at least two to three more generations, up to the R3, that could be bulked up for commercial production of the desired pharmaceutically active protein. After the appropriate transgenic events have been selected, large scale production of the active proteins becomes the focus of the process. The use of such corn hybrids for protein production is common. The development of corn hybrids requires the development of inbred lines, the crossing of these lines, and the evaluation of the crosses. A hybrid corn variety includes those varieties that are the cross of two or more such inbred lines, each of which may have one or more desirable characteristics lacked by the other or which complement the other. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which have commercial potential. There are several alternatives to produce hybrids from two, three and four inbred lines. They are known as single, triple and double hybrids respectively. The progeny of the first generation produced by such crosses are designated as F1. In the development of hybrids only the F1 hybrid plants are sought, as an F1 hybrid is more vigorous than its inbred parents.
Nonetheless, transgenic plants are potentially one of the most economical systems for large-scale production of recombinant proteins for industrial and pharmaceutical uses (Austin et al., Ann. NY Acad. Sci., 721:235-244 (1994); Krebbers et al., In: P. R. Shewry and S. Gutteridges (eds.), Plant Protein Engineering, pp. 315-325, Cambridge University Press, London (1992); Pen et al., In: A. Hiatt (ed.), Transgenic Plants fundamentals and Applications, pp. 239-251, Marcel Dekker, New York (1993); Whitelam et al., Biotechnol. Genet. Eng. Rev., 11:1-29 (1993)). Advantages of plant systems often include, the low cost of growing plants on large acreage; the ease in scale-up (increase of planted acreage); the availability of natural protein storage organs; and the established practices for the efficient harvesting, transporting, storing, and processing (Whitelam et al., supra; Goddijn and Pen, Trends Biotechnol., 13:379-387 (1995); Ponstein et al., Ann. NY Acad. Sci, 792:92-98 (1996)).
There is a need to provide commercially significant quantities of pharmaceutically active proteins in early plant generations such as R0, R1 and R2. The methods of the present invention provide approaches to deliver commercially significant quantities of pharmaceutically active proteins in such generations. Moreover, the methods of the present invention provide methods that do not require destruction of valuable stock to produce commercially significant quantities of pharmaceutically active proteins.