Posts Tagged ‘biosimilars’

PBVAB 5 – Part 3

21 August, 2013

PBVAB 5 Verona, June 2013 – Part 3

Technically, Sue Huddy’s piece should have been Part 3; however, it reports things that happened after what I am reporting on, so I’ll keep that label!

This post will report on Sessions 3 & 4, namely, Technology Advances and Perspectives.

I opened Session 3 with a talk on ‘Virus-derived ssDNA vectors for the expression of foreign proteins in plants’, focusing mainly on geminiviruses (naturally).  I wrote this a couple of years ago as a chapter for a book which seemed to not be forthcoming; however, I was assured during my talk by Yuri Gleba – the co-Editor with Kenneth Palmer of a “Current Topics in Microbiology and Immunology” issue on “Plant Viral Vectors” – that this offering is now in fact available, so here’s a link for anyone who wants to buy it.

Current Topics in Microbiology and Immunology 2011,

Virus-Derived ssDNA Vectors for the Expression of Foreign Proteins in Plants

Edward P. RybickiDarrin P. Martin

Plant viruses with ssRNA genomes provide a unique opportunity for generating expression vehicles for biopharming in plants, as constructs containing only the replication origin, with the replication-associated protein (Rep) gene provided in cis or in trans, can be replicationally amplified in vivo by several orders of magnitude, with significant accompanying increases in transcription and expression of gene(s) of interest. Appropriate replicating vectors or replicons may be derived from several different generic geminiviruses (family Geminiviridae) or nanoviruses (family Nanoviridae), for potential expression of a wide range of single or even multiple products in a wide range of plant families. The use of vacuum or other infiltration of whole plants by Agrobacterium tumefaciens suspensions has allowed the development of a set of expression vectors that rival the deconstructed RNA virus vectors in their yield and application, with some potential advantages over the latter that still need to be explored. Several modern applications of ssDNA plant vectors and their future potential will be discussed.

I noted that several firms are already using geminivirus-derived expression technology – like Kentucky Bioprocessing, who offer use of it as a service, and Medicago Inc, who use it in manufacturing vaccine products – and that it has considerable potential for improvement.  There is also the possibility of using other ssDNA virus-derived vectors, including from bacteria.

E.V. Sheshukova (N.I. Vavilov Institute of General Genetics RAS, Moscow) followed up with an account of how the use of antisense RNA to plant death factor (PDF) could modulate PDF level so as to avoid the necrotisation caused by rapid protein over-expression.  Their group used a TMV-based vector to co-express an antisense with the gene of interest, and got 4-5-fold increase in protein expression, equivalent to using the silencing suppressor p19 from a tombusvirus.

Diego Orzaez (IPMCP-CSIC, Valencia, Spain) spoke next, on the same technology I have previously described (with beautiful pictures from Diego) here: that is, the enabling of tools for multigene engineering of plants – and specifically in this case, the elegant use of superinfection exclusion phenomenon seen with RNA plant virus-derived vectors that are capable of movement, for the expression of polyclonal antibody mixtures in plant leaves.  They had successfully shown expression of 300+ individual clones from a camel VHH clonal library derived against a mixture of 3 snake venoms, in a mosaic on a single leaf.  This was seriously impressive for me: imagine, polyclonal “sera” from a leaf!

Diego noted that the FDA allows the 2-animal rule for products like antivenin, and things used for biodefence: that is, an efficacy trial in an animal, followed by Phase 1 trial in humans (=safety).  This could help expedite approval of such products.

We discussed the paper previously blogged on from this group in Journal Club today, incidentally, to much appreciation of the truly excellent work, and the colour Figures.  Thanks, Richard!

Reza Saberianfar (Agriculture and Agri-Food Canada, Ontario) described their investigations of protein body biogenesis in N benthamiana.  They had looked mainly at hydrophobin and elastin fusion proteins, in order to overcome the joint bottlenecks of inadequate accumulation, and difficulties in purification of recombinant proteins from plants.  He noted that hydrophobin and elastin PBs were different sizes: they had used protoplasts of infiltrated leaves and confococal microscopy and Imaris software to find every PB in individual cells, to determine that  shows hydrophobin-based PBs were 1-2 um, and ELP-based were 2-3 um in diameter, for the same amount of protein.  PBs made from  hydrophobin and ELP-linked proteins shared the same ER origin, but Zera-based PBs had a different origin and Zera fusions did not need a KDEL for ER retention.  An interesting observation was that PBs could form in the ER in the absence of fusion tags if expression levels were high.  One could also increase the expression of other proteins by coexpressing them with a fusion protein, as they get incorporated into PBs anyway – eg: EPO.

Lauri Reuter (VTT-Technical Research Centre, Finland) continued in the theme of fusion proteins with a talk on the production of hydrophobin fusions in tobacco BY-2 suspension cultured cells.  It was interesting to hear that WAVE bioreactors did not work well because they did not shake fast enough, but that conventional steel bioreactors did – with capacities of 20 – 600 litre, and even up to 20 m3.  The cells are apparently surprisingly tolerant to shear stresses, and yields of GFP::hydrophobin fusion from 600 litre reactors were as good or better as from a 50 ml shake flask – at 300 mg/litre.  Purification was simple, in that reactors could be pumped out onto a filter, and the cell “cake” pressed dry – for subsequent lyophilisation and storage at room temperature, for example.  French pressing of fresh cells was also an option.  Hydrophobin fusions allowed aqueous 2-phase separations, for simple and rapid enrichment.  Inclusion of a Tobacco etch virus self-cleaving motif allowed removal of the hydrophobin.

hphobinThe “Perspectives” Session was notable for two talks, and a proposal: the latter was by Julian Ma for a “Society for Molecular Farming”, which was well supported and will probably kick off sometime this year.

Jim Larrick (Panorama Research, Mountain View, California) gave a typically eclectic, wide-ranging and highly enthusiastic talk on ‘Anti-fragility: Big picture issues in pharmaceutical development’.  He used the “Black Swan” analogy repeatedly to explain how the enterprise funding and pharma research sectors embodied fragile or anti-fragile thinking – with the observation that it was easier to resist black swans (eg: the unexpected) with a raft of small projects, than to have a few big ones.  He also pointed out that the NIH liked big projects – and that a useful alternative name for them was “Not Invented Here”!  Right up there with “Not Real Funding” as the alternative name for our National Research Foundation….

IMG_0133

Matthew Paul (St. George’s University of London) presented a set of 15 case studies of commercial paths to introducing molecular farming, which was very interesting to us academic types.  More interesting was the fact that while innovative and protectable technology and products were important to start-ups, the majority of successful ones had their basis in platform development – and the average time from platform to product identification was about five years.  Venture capital firms were considered too greedy for early-stage start-ups, but their involvement later led to stability as their partnering was long term.

Another interesting feature was that many of the successful ventures sold “side products”: for example, Ventria sold cytokines and cosmetic formulations, while KBP sold cell culture reagents.  Several also licenced out technology platforms, but the revenue was not held to be so good.

There were three main indicators of success:

  • Management quality
  • A good lead product
  • Having a panel of products

IMG_0135A good strategy to stay alive was “maximum income / minimum burn” – and he held up the example of Medicago in this regard.  He noted that in the absence of major investment from Big Pharma, Phase 2 trial success was the driver for commercialisation.

PBVAB 5 Verona June 2013: Session 7

3 August, 2013

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 “BryotechnologyTM 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.


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