ViroBlogy

Suzanne Huddy, a postdoc in our lab, kindly took some notes in a session I moderated at the 5th PBVAB in Verona this year.

Little did she know this is just my way of easing her in to doing this more often…B-)  Thanks, Sue!

Session 7: Manufacturing and Production Systems Developments

Moderator: EP Rybicki

Andreas Schaaf from Greenovation Biotech GmbH presented on “Bryotechnology^TM en route to the clinic”, highlighting a production platform based on the moss Physcomitrella patens.  The overriding advantage of this system is that the moss is haploid and therefore genome modification is fairly straight forward with timelines for modifications similar to that of yeast systems.  Physcomitrella patens is also fairly unique since it has a very high occurring rate of homologous recombination (HR).  These traits along with the fact that the genome is sequenced and annotated allow fairly simple customization of the genomic background.  Using this, they have glyco-engineered strains and have removed plantized glycosylation completely.

Other than the products mentioned on their website (www.greenovation.com), they are currently working on α-galactosidase for treating Fabry disease.  Fabry disease is a rare genetic lysosomal storage disorder which results in the accumulation of lipids in the kidney, autonomic nervous system and cardiovascular system cells.  They are also working on the production of recombinant human β glucocerebrosidase for the treatment of Gaucher disease.  Interestingly, these are the same products produced by Protalix Therapeutics.

Stefan Schillberg from the Fraunhofer IME presented on “Co-MoFarm- Contained molecular farming: Controlled contained systems for high yield consistency”.  The CoMoFarm project has been funded for 3.5 years under the European Commission 7th Framework programme.  This project focused on the development of high-yielding plant-based production systems for recombinant proteins.

The presentation initially contrasted the production capability of the various plant platforms employed by this group using both HA (influenza hemagglutinin) and the human M12 antibody as protein products.  The production platforms included Arabidopsis and rice suspension cells, tobacco plants, roots and suspension cells, and moss suspension cultures.  The results presented highlighted the fact that one production platform is not necessarily optimal for all recombinantly expressed proteins, although the traditional tobacco leaves and BY-2 suspension cultures did produce the highest expression levels.  By further optimization of cultivation parameters (including media components), expression levels could be increased by up to 30 fold.  The presentation also showed that expression could also be improved by co-expression of the target protein with a fluorescent marker, DsRed.  In short, this allows the development of higher expressing lines through the non-invasive selection single elite expressing cells by flow-cytometry.  Stephan Schillberg also presented on the groups development of non-invasive monitoring systems for plant cell health and productivity.

The presentation was ended with a comparison on the cost of production of M12 antibody in either tobacco plants or BY-2 cells grown in 200 L bioreactors.  While the cost of producing this product in tobacco plants was less per gram of the product, the time for production in BY-2 cells was much shorter.  Details of the costing can be found at http://comofarm.org/useruploads/files/CoMoFarm_2013-6.pdf, where CoMoFarm have kindly made the presentation given in Verona available.

Pascal Drake from St. George’s University of London presented on “Hydroponic cultivation of tobacco for the production of recombinant pharmaceutical proteins by rhizosecretion”.  This presentation looked at the production and optimization of antibodies and Cyanovirin-N (CV-N) (a cyanobacterial protein which displays virucidal activity) in hydroponically cultivated tobacco plants.  Data was shown that suggested the inclusion of PGRs (plant growth regulators) and a nitrate source in the hydroponic medium could increase the concentration of the protein of interest in the medium.  Hydroponic cultivation has some advantages over traditional cultivation of tobacco plants.  Plants are cultivated in chemically defined media, therefore there is better control over the process and in this way this system approaches cell fermentation processes.  Additionally, fully processed secreted proteins can be harvested over the lifetime of the plant and purification can be simplified since the medium does not contain as many proteins as a whole leaf extract.  A “nifty” way of doing a western blot was also shown- basically, transgenic plants are germinated on nitrocellulose paper; this paper can then be used directly for a western blot since the protein of interest would have been secreted directly from the roots of the plant onto the membrane.  After development of the blot, the presence of the protein is seen in “root-shaped” pattern.

Bertrand Magy from the Institute of Life Sciences at the University catholique de Louvain, Belgium presented on the “Development of suspension cells as a competitive production system for antibodies”.  This research looked at designing an optimized antibody scaffold that can be combined with different variable regions in order to produce high levels of functional antibodies.  Initially, the expression of different IgG isotypes (human, rat and mouse) with the same variable region was investigated in tobacco and Arabidopsis thaliana suspension cells.  Bertrand showed that while antibodies accumulated in the extracellular medium, degradation occurred according to the isotype.  In this case, A. thaliana was also shown to be the better producer.  As is the case with many other cell suspension-based expression, the yield of antibody could be optimized by manipulating the growth medium.  Levels of antibody production of >30 mg/L could be achieved.