Jürgen Drossard

Polyclonal antibodies for specific detection of tobacco host cell proteins can be efficiently generated following RuBisCO depletion and the removal of endotoxins

The production of biopharmaceutical proteins in plants requires efficient downstream processing steps that remove impurities such as host cell proteins (HCPs) and adventitious endotoxins produced by bacteria during transient expression. We therefore strived to develop effective routines for endotoxin removal from plant extracts and the subsequent use of the extracts to generate antibodies detecting a broad set of HCPs. At first, we depleted the superabundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) for which PEG precipitation achieved the best results, preventing a dominant immune reaction against this protein. We found that a mixture of sera from rabbits immunized with pre-depleted or post-depleted extracts detected more HCPs than the individual sera used alone. We also developed a powerful endotoxin removal procedure using Polymyxin B for extracts from wild type plants or a combination of fiber-flow filtration and EndoTrap Blue for tobacco plants infiltrated with Agrobacterium tumefaciens. The antibodies we generated will be useful for quality and performance assessment in future process development and the methods we present can easily be transferred to other expression systems rendering them useful in the field of plant molecular farming.

Facing the Future with Pharmaceuticals from Plants

Plants are the ultimate source of many of today’s pharmaceutical compounds, but most of our protein drugs are derived from animal The biopharmaceutical industry has developed with mammalian cells treated as the gold standard for production, hence the regulations governing biopharmaceutical production have been tailored for these systems. This has had a negative impact on the use as plants for biopharmaceutical production, despite their many potential advantages which include the prospect of inexpensive, large-scale biopharmaceutical production without sacrificing product quality or safety. The first plant-derived pharmaceutical products have now been approved but these represent a tiny proportion of the products in development, products which could have a profound impact on the cost and availability of medicines to those most in need. In this review, we summarize the state-of-the-art in plant-based production systems and discus the development issues which remain to be addressed before plants become an acceptable mainstream production technology. sources, and are produced either in cultured animal cells or microbes.

Modulation of Plant Function and Plant Pathogens by Antibody Expression

The expression of antibodies or antibody fragments in plants that bind to functional domains of plant or pathogen derived proteins, is a novel approach to elucidate and alter primary or secondary metabolism in plants and also to engineer pathogen resistance by inactivating targets inside the plant cell through immunomodulation. The feasibility of this approach either to interfere in plant metabolism or in plant pathogen infections has been shown for several antibodies that bind to key plant metabolites or to viral target proteins. Questions such as how cellular targeting alters the expression and accumulation of these proteins/molecules in plants remain to be answered before antibody technology can be used commercially. Alternatively, plants provide an excellent source for biomass production making it feasible to produce high amounts of valuable recombinant antibodies for diagnostic and therapeutic applications. This approach has yet to be evaluated in terms of expression levels, genetic stability in the field and downstream processing.

Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial.

Innovative Produktion pharmazeutischer Proteine in Pflanzen

The release of the pharmaceutical product Elelyso showed that plantderived biopharmaceuticals can compete economically with products from conventional expression systems. Improvements are required in terms of automation, consistency and purification as well as quality control and regulatory compliance to ensure a successful commercialization of biopharmaceuticals from plants and plant cells. Here we discuss possible improvements and challenges, which will affect the development of the molecular farming approach.

Expression of Recombinant Antibodies in Plants and Cell Suspension Cultures

Monoclonal antibodies (mAbs)7 are extremely useful tools in medicine, biology and biotechnology. Combining hybridoma and recombinant DNA technology gave access not only to full-size molecules but also to various recombinant antibody fragments (rAbs) and fusion proteins19, broadening the range of possible applications. Recent improvements in heterologous gene expression systems5,9,10,11,15 and development of phage display technologies19 make it possible to design and express rAbs against almost any antigen and to fine tune these molecules for improved performance.