Project: Production, process development and quality control of recombinant plant-made proteins using hydrophobins.

The goal of the project is to assess and optimize new downstream processing technology for industrial barley harvest containing high-value recombinant proteins for life science- and biomedical market._x000D__x000D_ORF Genetics (Iceland) has developed a new technology platform for the production of high value recombinant proteins in plants and is a leader within the field. This technology directs accumulation of recombinant proteins into the grains of barley and opens means for economical ways of producing cleaner high-value recombinant proteins of superior quality (biorisk-free products). New dedicated high-tech greenhouses are used for the hydroponic cultivation of barley lines producing valuable recombinant proteins and provide controlled conditions to ensure consistency of the raw material used for processing in this project._x000D_ORF Genetics successfully launched globally its product line of ISOkine™ growth factors and cytokines in 2008. Applications are in molecular and cell biology, such as stem cell research, pharmaceuticals, regenerative medicine, tissue engineering, cellular therapy and clinical transplantation._x000D__x000D_ORF Genetics would like to strengthen its downstream processing capacities to be able to expand further and COtain its competitive edge for years to come._x000D__x000D_Preliminary data from VTT demonstrates that fusion of a target protein to hydrophobin (HFBI) can increase accumulation of the a marker protein in a plant based expression system as well as simplifying and increasing yield from the purification procedure by enabling a 2-phase extraction of proteins from crude extracts. _x000D_To study whether this also applies for recombinant proteins with high market value and the barley based Orfeus™ expression system, five different proteins were chosen as candidate molecules for fusion with HFBI. These target proteins differ with regards to size, complexity, function and glycosylation and should therefore, in addition to answering the general question of feasibility of using HFBI fusions in barley, also be able to clarify whether HFBI can be used for a broad spectrum of proteins. Furthermore, this variety of proteins will be helpful when studying the possible immunogenicity of HFBI. The market value and demand of the candidates was of course kept in mind as they were chosen and is described in the market section. _x000D__x000D_The project serves as a feasibility study to address the following obstacles facing manufacturing of recombinant proteins:_x000D_- enhance stability of fusion proteins with the proprietary tag_x000D_- establish purification and processing protocols using the proprietary tag_x000D_- increase yields and minimize yield losses_x000D_- improve efficiency of downstream processing_x000D_- make overall production of recombinant proteins in plants even more economical_x000D_- assess the purity, quality and safety of the fusion proteins containing the tag_x000D_- scale up successful project derived processes to industrial scale._x000D__x000D_Five recombinant proteins will be tested to obtain full range of information on the effect of the fusion tag on protein-protein interactions, receptor binding and purification protocols:_x000D_The proteins differ in size, post translational modification, biological function, application and market potential. The markets cover industrial processes, biomanufacturing processes and biopharmaceutical applications. These markets place different demands for quality, validation requirements, purity, quantity and ultimately price. The principles of processing are, however, very similar irrespective of the final market destination. Therefore, successful downstream process development in this project will be instrumental for the entrance of the respective recombinant proteins to all these different markets._x000D__x000D__x000D_ _x000D_ORF Genetics has expertise in all aspects of manufacturing of recombinant proteins. ORF will produce the transgenic plants accumulating the recombinant proteins for the project. ORF will be the end user of the successful project and will introduce improvements into its manufacturing operations as it finds suitable, by leasing or licensing of hydrophobin technology from VTT. _x000D_VTT is the inventor and developer of hydrophobin separation technology and has expertise in process development._x000D_Nordic BioPharma specializes in biochemical and biophysical analysis of proteins. The safety of the biomedical candidate products will be assessed using cell-based assays that assess immunogenicity and bioactivity._x000D_The complementary efforts, expertise and technology of the high tech project Ps have the possibility to promote and strengthen an enabling technology for the manufacturing of some of the most valuable compounds in the world in a uniquely economical way._x000D__x000D_

Acronym HydroPro (Reference Number: 5320)
Duration 01/09/2010 - 31/12/2013
Project Topic The project will assess and implement new downstream processing technology (VTT) for novel molecular farming technology for production of high-value recombinant proteins for life science- and biomedical market (ORF Genetics). _x000D_ The quality and safety of the resulting products are tested by (NBPh)
Project Results
(after finalisation)
The project progress is evaluated in the light of objectives presented in the original research plan: “The project serves as a feasibility study to address the following obstacles facing manufacturing of recombinant proteins:“ The summary is made on the level of whole project and includes also the activieties from other project Ps than VTT._x000D__x000D_1) enhance stability of fusion proteins with the proprietary tag_x000D__x000D_It has been show in N. benthamiana that hydrophobin fusion technology can improve target protein expression as well as that novel purification protocols have been developed to optimize target protein recovery (VTT). In the barley system there is a documented expression of the HFB-fusion target proteins. However, although the ATPS was demonstrated to work for the barley produced HFB-fusion proteins, the expression levels and the yield where so low that it was not possible to purify the target products from the barley system for further analysis of their properties. Unfortunately, data from N. benthamiana now suggest that one reason for low expression is, surprisingly, negative effect of codon optimization of HFB1 and also that the orientation of hydrophobin fusion P can have a major effect for the expression yield. For future characterisation of activity and immunosafety, the expression system developed in N. benthamiana was used._x000D__x000D__x000D_2) establish purification and processing protocols using the proprietary tag_x000D__x000D_Aqueous two phase separation-based purification protocols have been established for HFB1-protein A, HFB2-protein A, HFB4-transferrin and VEGF165-HFB1 (VTT). The activities of protein A and transferrin are retained after the ATPS purification processes and with the Hydrophobin tags (VTT, NBP). Also activity of VEGF165-HFB1 was confirmed in cell-based assay.As a conlusion from the developed protocols is that there is no general setting that is appropriate for all target proteins it is rather target protein dependent._x000D__x000D_3) increase yields and minimize yield losses_x000D__x000D_Fusion and coexpression strategies have been shown to improve accumulation of target proteins in N. benthamiana (VTT). _x000D__x000D_4) improve efficiency of downstream processing_x000D__x000D_Series of ten different hydrophobins have been tested (VTT) and so far HFB1, HFB2 and HFB4 behave best at 2-phase separation. A big set of different surfactants have been tested in order to improve the efficacy of downstream processing. The best all-round surfactant has been Triton x-114. Also it seems that the ATPS needs to be optimized for each target protein separately. _x000D__x000D_5) make overall production of recombinant proteins in plants even more economical_x000D__x000D_See 1, 3, 4. Advances have been made, but protein specific issues influence the final outcome._x000D__x000D_6) assess the purity, quality and safety of the fusion proteins containing the tag._x000D__x000D_The target proteins and HFB1 tag have been analyzed in silico for possible T-cell epitopes (NBP). In vitro cultivation of five different cell lines in the presence of HFB1 or GFP-HFB1 fusion protein did not show any toxicity effects. In fact, a stimulation of cell proliferation was observed in the precence of these proteins (NBP)._x000D__x000D_Transferrin and protein A have been tested for activity and are functional at the same level as commerciable avalable proteins (NBP, VTT). The purity of the proteins is high and after the final purification step one major band is present on denaturing gels (VTT)._x000D__x000D_VEGF 165 (ORF) was evaluated for immunogenicty both in silico and in vitro (NBP). The procedure developed by NBP had a population coverage (Europe) above 90% and did not reveal any immunogenic responses in the donors tested. The biological activity of VEGF 165 was evaluated (ORF). Activity was found but it was lower than the unfused VEGF165 counterpart._x000D__x000D__x000D_7) scale up successful project derived processes to industrial scale_x000D__x000D_The N. benthamiana system has been scaled up to produce more biomass with recombinant proteins by use of vacuum delivery o
Network Eurostars
Call Eurostars Cut-Off 3

Project partner

Number Name Role Country
3 Nordic BioPharma AB Partner Sweden
3 Technical Research Centre of Finland Partner Finland
3 ORF Genetics Coordinator Iceland