Validation of model virus removal and inactivation capacity of an erythropoietin purification process.

Human erythropoietin (hEpo) production requires mammalian cells able to make complex post-translational modifications to guaranty its biological activity. As mammalian cell can be reservoir of pathogenic viruses and several animal origin components are usually used in the cultivation of mammalian cells, hEpo contamination with viruses is something of great concern. As consequence, this study investigated the viral removal and inactivation capacity of a recombinant-hEpo (rec-hEpo) purification process. Canine parvovirus, Human poliovirus type-2, Bovine viral diarrhea virus and Human immunodeficiency virus type-1 were used for measuring process viral removal and inactivation capacities. In conclusion, this study corroborated that the assessed rec-hEpo purification process has enough capacity (5.0-19.4 Logs) for removing and inactivating these model viruses and sodium hydroxide demonstrated to be a robust sanitization solution for chromatography columns (5.0 (PV-2)-6.7 (CPV) Logs).

Expression and characterization of an anti-(hepatitis B surface antigen) glycosylated mouse antibody in transgenic tobacco (Nicotiana tabacum) plants and its use in the immunopurification of its target antigen.

Transgenic plants expressing recombinant immunoglobulins have arisen as an alternative technology for the large-scale production of antibodies useful in therapeutics and in industrial processes. In the present paper we report the expression in transgenic tobacco ( Nicotiana tabacum ) of an anti-HBsAg [anti-(hepatitis B virus surface antigen)] mouse IgG1 mAb (monoclonal antibody), currently used for the industrial purification of the recombinant vaccine antigen. Using the sweet potato sporamin signal peptide, a KDEL (Lys-Asp-Glu-Leu) ER (endoplasmic reticulum) anchorage domain, and a heavy- and light-chain gene tandem construction, we generated F1 plants in which the expression of the antibody accounted for 0.5% of the total soluble proteins. The 'plantibody' (functional IgG antibody produced in plants) was easily purified by Protein A-Sepharose chromatography with a yield of approximately 35 microg/g of fresh leaf material, and its glycosylation indicated that, irrespective of the KDEL signal, the molecule is modified in both the ER and Golgi. Finally, a successful comparison of the plantibody with the ascites-derived mAb in the immunoaffinity purification of the vaccine recombinant HBsAg was performed. Taken as a whole, our results show that the large-scale production of this antibody of industrial relevance in transgenic tobacco is feasible.