Plants can be genetically modified in a way that enables them to produce pharmaceutically active proteins for various indications. The small moss Physcomitrella patens is able to produce more complex molecules than bacteria. Moreover, moss is less expensive to grow and less susceptible to pathogenic contaminants than Chinese hamster ovary (CHO) cells. greenovation Biotech GmbH, which has its headquarters in Heilbronn and a branch in Freiburg, uses its proprietary bryotechnology to produce and commercialise antibodies, enzymes and coagulation factors “made from moss”.
Founded in Freiburg in 1999 by Prof. Dr. Gunther Neuhaus and Prof. Dr. Ralf Reski from the University of Freiburg, greenovation Biotech GmbH has since expanded to become a company with a workforce of 20. Dr. Thomas Frischmuth, a member of the company’s advisory board since 2002, became its CEO in 2011. Frischmuth, who has focussed on plant biotechnology throughout his career, is also the CEO of five other companies, all in the drug production and analytics sector. Using greenovation’s proprietary bryotechnology, Frischmuth and his team have plans to use mosses for producing and optimising drugs. The team hopes to place the first drugs on the market in the near future. In contrast to molecular pharming, which uses whole plants, bryotechnology only employs haploid protonema cells (a thread-line chain of cells from the earliest stage of a moss’ life cycle).
Mosses, which have been colonising our planet for 450 million years and have barely changed in genetic terms, have many advantages over bacterial or CHO cultures for the cultivation and production of therapeutic proteins. While bacteria are only able to produce relatively simple proteins such as insulin, eukaryotic cells are able to produce relatively complex molecules such as antibodies and enzymes that even have specific surface glycosylation patterns. Glycosylation is a type of post-translational modification that involves the addition of carbohydrate structures to proteins. These carbohydrate structures determine the character and enzymatic activity of the target protein. In animal cultures, the attachment of carbohydrate structures leads to a heterogeneous population of glycoproteins, which creates a problem in the industrial production and regulatory approval of therapeutic proteins. “Genes only determine the primary sequence of a protein,” Frischmuth explains, going on to add, “different carbohydrates can be added after translation and may lead to undesired side effects.” Plants have a fairly homogeneous population of glycoproteins. The removal of plant-specific enzymes and the introduction of human-specific enzymes make the plant’s original glycosylation pattern more similar to that of humans, resulting in highly homogeneous and extremely pure pharmaceuticaly active products. In addition, stable moss lines can be established within a relatively short time as the haploid protonema is easy to transform and it integrates the transgene in a stable way.
Compared to animal cell cultures, which need to be replaced after around 20 days, moss cell lines can be cultured for many months. The sought-after product can easily be harvested, as signalling sequences ensure that the target protein is actively released into the culture medium. Since the plant cell cultures are kept in closed systems, outbreeding can be excluded, thus precluding yet another debate on safety issues. greenovation switched the production of active pharmaceuticals from reaction tubes to disposable wave reactors in 2009 in order to completely exclude any vulnerability in the culture. “The regulatory authorities have a huge problem with tube systems of this kind,” said the biologist, “because the cleaning of mechanical pumps and the GMP production plants is a time-consuming process (ed. note: GMP are “good manufacturing practice” guidelines that help ensure a high-quality product).
A wave reactor is a big table that can be tilted in two directions; a tray containing a transparent plastic bag is placed on the table and irradiated from above. The bags come in 100, 300 or 1000 l sizes. The bags contain the Physcomitrella cultures in a medium consisting of water, nutrient salts and carbon dioxide. “The introduction of the wave reactors represented a huge milestone for us. The movement of the table produces accurately calculated waves that guarantee that the cultures are mixed well and exposed to optimal light quantities,” said Frischmuth recalling the company’s decision to shift from tubes to wave reactor systems.
The handling of the wave reactor system, which was approved for application back in 2000, is simpler than for the tube reactors. In addition, it is a completely closed system and does not need to be cleaned, which is a decisive issue in terms of efficiency and contamination. “The bag is emptied with a large disposable pump and thrown away. The supernatant is further processed and a new bag is used for a new culture,” said Frischmuth explaining how the new wave reactor system works. Frischmuth can be certain that his protein production plant will never have to be closed down due to a viral infection.
With a financial injection from Zukunftsfond Heilbronn and L-EigenkapitalAgentur (L-EA), greenovation is working on transferring green biotechnology from the laboratory scale to industrial GMP production. In future, the company will have two pillars to lean on. greenovation is focussed on the development of a drug for patients suffering from Fabry disease, a rare genetic lysosomal storage disease. In people with Fabry disease, the activity of the enzyme alpha-galactosidase is dramatically reduced, resulting in the pathological accumulation of a particular metabolic product, pain and organ failure. Frischmuth has great faith in an enzyme replacement therapy that functionally compensates for alpha-galactosidase deficiency, for which greenovation produces the enzyme alpha-galactosidase. “Two alpha-galactosidase enzyme replacement therapies are available, but they are both produced with animal cell cultures,” said Frischmuth. “We are hoping that our moss will enable us to produce even better therapeutic proteins.” Biomeva GmbH and greenovation have recently signed an agreement for the GMP production of a biopharmaceutical protein to treat Fabry disease; the GMP alpha-galactosidase will be available for clinical trials in early 2014.
greenovation also plans to encourage pharmaceutical and biotechnology companies to test its bryotechnological approach on the large scale. Frischmuth is approaching customers who already use mammalian cell products and are seeking to optimise their products using moss cell cultures. greenovation’s BryoSpeed allows the rapid, transient production of small quantities for initial quality and feasibility assessments. Following successful evaluations, greenovation will be able to commence long-term large-scale production using its BryoMaster moss expression system. “We hope to use this to offer our moss platform as a service to our customers,” said Frischmuth.
PD Dr. Thomas Frischmuth
greenovation Biotech GmbH