Stefan Rasche

Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

Tobacco BY‐2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY‐2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred‐tank reactors, and orbitally‐shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale‐up study, using a 200‐L orbitally‐shaken bioreactor holding disposable bags, and BY‐2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200‐fold difference in cultivation volume. Final cell fresh weights of 300–387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75–85% and a purity of >95%. Our results demonstrate the suitability of orbitally‐shaken bioreactors for the scaled‐up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY‐2 culture medium. Biotechnol. Bioeng. 2015;112: 308–321.

The production of recombinant cationic α-helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum.

Synthetic linear antimicrobial peptides with cationic α-helical structures, such as BP100, are valuable as novel therapeutics and preservatives. However, they tend to be toxic when expressed at high levels as recombinant peptides in plants, and they can be difficult to detect and isolate from complex plant tissues because they are strongly cationic and display low extinction coefficient and extremely limited immunogenicity. We therefore expressed BP100 with a C-terminal tag which preserved its antimicrobial activity and demonstrated significant accumulation in plant cells. We used a fluorescent tag to trace BP100 following transiently expression in Nicotiana benthamiana leaves and showed that it accumulated in large vesicles derived from the endoplasmic reticulum (ER) along with typical ER luminal proteins. Interestingly, the formation of these vesicles was induced by BP100. Similar vesicles formed in stably transformed Arabidopsis thaliana seedlings, but the recombinant peptide was toxic to the host during latter developmental stages. This was avoided by selecting active BP100 derivatives based on their low haemolytic activity even though the selected peptides remained toxic to plant cells when applied exogenously at high doses. Using this strategy, we generated transgenic rice lines producing active BP100 derivatives with a yield of up to 0.5% total soluble protein.

Tackling Heterogeneity: A Leaf Disc-Based Assay for the High-Throughput Screening of Transient Gene Expression in Tobacco

Transient Agrobacterium-mediated gene expression assays for Nicotiana tabacum (N. tabacum) are frequently used because they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein production. However, for three model proteins, we found that recombinant protein accumulation (rpa) was significantly influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation (max/min ratio) was found to be as high as 11. Therefore, construct-based impacts on the rpa level that are less than 11-fold will be masked by background noise. To address this problem, we developed a leaf disc-based screening assay and infiltration device that allows the rpa level in a whole tobacco plant to be reliably and reproducibly determined. The prototype of the leaf disc infiltration device allows 14 Agrobacterium-mediated infiltration events to be conducted in parallel. As shown for three model proteins, the average max/min rpa ratio was reduced to 1.4 using this method, which allows for a sensitive comparison of different genetic elements affecting recombinant protein expression.

One-Step Protein Purification: Use of a Novel Epitope Tag for Highly Efficient Detection and Purification of Recombinant Proteins

We used the interaction between an epitope of the Tobacco mosaic virus 54K replicase (tag54) and its corre- sponding monoclonal antibody 54 (mAb54) for the design of a new epitope-tagging system. We fused the DNA sequence of tag54 and two elongated derivates thereof to the C-terminus of the chloramphenicol acetyltransferase (CAT) gene and produced the tagged proteins in tobacco. Immunoblot and ELISA analysis demonstrated that the tagged proteins were de- tected with high sensitivity in plant extracts. Moreover, the tag54 system enabled the full recovery of the recombinant CAT with excellent purity through immunoaffinity chromatography.

An Immunohistochemical Assay on Human Tissue using a Human Primary Antibody

Non human antibodies administered to human patients often generate anti-antibody responses, leading in extreme cases to anaphylactic shock. Completely human antibodies are therefore favored over their murine, chimeric and humanized counterparts. However, the accurate evaluation of human antibodies on human tissue samples cannot be achieved using indirect immunohistochemical methods because of endogenous immunoglobulins that are co-detected by the secondary antibodies. Direct detection is often used instead, but this lacks the signal amplification conferred by the secondary antibody and is therefore less sensitive. We developed a simple fluorescence-based indirect immunohistochemical method that allows human primary antibodies bound specifically to their target antigens in human tissue samples to be detected clearly and without interfering background staining. This approach involves a biotinylated human primary antibody (H10Biotin) and Cy3-conjugated streptavidin (StrepCy3). We tested the protocol using a human carcinoembryonic antigen (CEA) specific IgG1 (H10). We identified an exposure time threshold that allowed the elimination of low StrepCy3 background staining, yet achieved sufficient signal amplification to make our approach four times more sensitive than comparable direct immunohistochemical procedures. The principle of this indirect immunohistochemical assay should be transferable to other species allowing the specific and sensitive detection of any primary antibody on homologous tissues.

More for less: Improving the biomass yield of a pear cell suspension culture by design of experiments

Plant cell suspension cultures are widely used for the production of recombinant proteins and secondary metabolites. One of the most important steps during process development is the optimization of yields by testing different cultivation parameters, including the components of the growth medium. However, we have shown that the biomass yield of a cell suspension culture derived from the pear cultivar Pyrus communis cv. Champagner Bratbirne can be significantly improved solely by varying the temperature, inoculum density, illumination, and incubation time. In contrast to medium optimization, these simple physical factors are easily controlled and varied, thereby reducing the effort required. Using an experimental design approach, we improved the biomass yield from 146 g fresh weight (FW)/L to 407 g FW/L in only 5 weeks, simultaneously reducing the costs of goods sold per kg biomass from €125 to €45. Our simple approach therefore offers a rapid, efficient and economical process for the optimization of plant cell suspension cultures.

Statistical experimental designs for the production of secondary metabolites in plant cell suspension cultures.

Statistical experimental designs, also known as the “design of experiments” (DoE) approach, are widely used to improve not only technical processes but also to answer questions in the agricultural, medical and social sciences. Although many articles have been published about the application of DoE in these fields, few studies have addressed the use of DoE in the plant sciences, particularly in the context of plant cell suspension cultures (PCSCs). Compounds derived from PCSCs can be developed as pharmaceuticals, chemical feedstocks and cosmetic ingredients, and statistical experimental designs can be used to improve the productivity of the cells and the yield and/or quality of the target compounds in a cost efficient manner. In this article, we summarize recent findings concerning the application of statistical approaches to improve the performance of PCSCs and discuss the potential future applications of this approach.

Plant Cell-Based Recombinant Antibody Manufacturing with a 200 L Orbitally Shaken Disposable Bioreactor

Tobacco BY-2 cells are an attractive platform for the manufacture of a variety of biopharmaceutical proteins, including antibodies. Here, we describe the scaled-up cultivation of human IgG-secreting BY-2 cells in a 200 L orbitally shaken disposable bioreactor, resulting in cell growth and recombinant protein yields that are proportionately comparable with those obtained from cultivations in 500 mL shake flasks. Furthermore, we present an efficient downstream process for antibody recovery from the viscous spent culture medium using expanded bed adsorption (EBA) chromatography.