By Dr Marek Zarzycki, Process Development Scientist
Global production of therapeutic recombinant antibodies is worth over 100 billion US dollars. These antibodies can be used to treat a wide variety of diseases and disorders including cancer, inflammatory disease and passive immunisation against infectious agents. Today, the majority of therapeutic recombinant antibodies are produced in mammalian cell lines, mainly to reduce the risk of immunogenicity caused by non-human glycosylation, and a clear regulatory path has been established. The patents for many of these antibodies are beginning to expire allowing for the production of lower cost biosimilar antibody analogues.
To meet growing demand for recombinant antibodies and the ever-decreasing margins on biosimilars, alternative expression systems account for an increasing market share. Plant expression provides the opportunity to develop biosimilars quickly and transfer them onto the regulatory path or to respond rapidly and effectively to biothreats and disease outbreaks.
At Leaf Expression Systems we have recently been working on the plant-based expression of several therapeutic antibodies – including an HIV antibody and a bevacizumab biosimilar. We are generating very positive data which will be published later this year.
The process to produce therapeutic recombinant antibodies in plants is now established. Starting from the heavy and light chain antibody protein sequence, DNA is synthesised with a plant optimised codon. The native signal peptide, which is required to direct the protein to the endoplasmic reticulum for glycosylation and secretion, can be replaced with a plant signal peptide to improve efficiency. This synthetic construct is cloned into a plasmid suitable for Agrobacterium-based plant transformation, with an expression cassette capable of high level protein expression, such as our proprietary Hypertrans® system that we use at Leaf Expression Systems. The heavy and light chains can be introduced in the same plasmid. Once the plasmid is introduced into Agrobacterium, plants are transiently transformed and grown until harvest (usually one week after transformation). Following the harvest, the antibody is purified using a number of optimised filtration and chromatographic steps, not dissimilar to that used for mammalian cell purification. This whole process can be completed in under 12 weeks, allowing the rapid development and optimisation of products.
One of the major advantages of plant transient expression is its efficiency in terms of time and cost in the scale up of production. Taking that into account, the Defense Advanced Research Projects Agency sponsored the development and validation of pilot scale production of H1N1 flu antigen to demonstrate the feasibility of rapid reaction for epidemic outbreaks. In this study Holtz et al. (2015) demonstrated that the process development and transfer to cGMP takes up to 6 months and the production of one batch (3.5t leaf material resulting in 250g of product) takes a week. It enables the completion of the whole campaign within 20 months with the cost of $0.1-0.12 per dose of vaccine.
One of the major hurdles for non-mammalian systems in the expression of antibodies is the type of glycosylation produced. While certain antibodies can be effective without glycosylation, many require this modification for activity and serum half-life. Plants are capable of complex glycosylation, however the types of patterns produced are different to that produced in human cells. Some of these modifications can lead to immunogenicity, such as β1,2-xylose and α1,3-frucose (Jin et al., 2008),while other important modifications, such as sialylation, are absent in plants. Fortunately, plants are highly amenable to modification, and genes can be knocked-down, out or in. This has been demonstrated for removing β1,2-xylose and α1,3-frucose by knocking-down or out the transferases responsible for this modification (Strasser et al., 2008). In addition, the introduction of the mammalian genes required for sialylation has been demonstrated in plants (Castilho et al. 2010). Demonstration of humanised glycosylation will allow plants to compete with mammalian systems.
It is increasingly accepted that plant transient expression offers major improvements over conventional production techniques, notably in terms of speed, cost-effectiveness and scalability. The technology therefore lends itself to responding rapidly to disease outbreaks, or meeting demanding production timelines.
If you’d like to find out more about our plant expression work, or to discuss potential collaboration, don’t hesitate to get in touch with us – [email protected] +44 (0) 1603 859 379