Production of pharmaceutical proteins by transgenic animals

Abstract

Proteins started being used as pharmaceuticals in the 1920s with insulin extracted from pig pancreas. In the early 1980s, human insulin was prepared in recombinant bacteria and it is now used by all patients suffering from diabetes. Several other proteins and particularly human growth hormone are also prepared from bacteria. This success was limited by the fact that bacteria cannot synthesize complex proteins such as monoclonal antibodies or coagulation blood factors which must be matured by post-translational modifications to be active or stable in vivo. These modifications include mainly folding, cleavage, subunit association, gamma-carboxylation and glycosylation. They can be fully achieved only in mammalian cells which can be cultured in fermentors at an industrial scale or used in living animals. Several transgenic animal species can produce recombinant proteins but presently two systems started being implemented. The first is milk from farm transgenic mammals which has been studied for 20 years and which allowed a protein, human antithrombin III, to receive the agreement from EMEA (European Agency for the Evaluation of Medicinal Products) to be put on the market in 2006. The second system is chicken egg white which recently became more attractive after essential improvement of the methods used to generate transgenic birds. Two monoclonal antibodies and human interferon-beta 1a could be recovered from chicken egg white. A broad variety of recombinant proteins were produced experimentally by these systems and a few others. This includes monoclonal antibodies, vaccines, blood factors, hormones, growth factors, cytokines, enzymes, milk proteins, collagen, fibrinogen and others. Although these tools have not yet been optimized and are still being improved, a new era in the production of recombinant pharmaceutical proteins was initiated in 1987 and became a reality in 2006. In the present review, the efficiency of the different animal systems to produce pharmaceutical proteins are described and compared to others including plants and micro-organisms.

Fig. 1

Comparison of the glycosylation of recombinant proteins produced by different systems.

Fig. 2

Different methods to generate transgenic animals. (1) DNA transfer via direct microinjection into a pronucleus or cytoplasm of embryo; (2) DNA transfer via a transposon: the gene of interest is introduced in the transposon which is injected into a pronucleus; (3) DNA transfer via a lentiviral vector: the gene of interest is inserted into a lentiviral vector which is injected between zona pellucida and membrane of oocyte or embryo; (4) DNA transfer via sperm: sperm is incubated with the foreign gene and injected into oocyte cytoplasm for fertilization by ICSI (Intracytoplasmic Sperm Injection); (5) DNA transfer via pluripotent cells: DNA is introduced into pluripotent cell lines (ES: embryonic stem cells: lines established from early embryo, EG: embryonic germ cells: lines established from the primordial germ cells of foetal gonads). The pluripotent cells containing DNA are injected into early embryos to generate chimeric animals harbouring the foreign gene; (6) DNA transfer via cloning: the foreign gene is introduced into somatic cells, the nucleus of which are introduced into the cytoplasm of enucleated oocytes to generate transgenic clones. Methods 4, 5 and 6 allow random gene addition and targeted gene integration via homologous recombination for gene addition or gene replacement including gene knock out and knock in.