Growth of BY‐2 Suspension Cells and Plantibody Production in Single‐Use Bioreactors

Raven, Nicole; Schillberg, Stefan; Kirchhoff, Janina; Brändli, Johanna; Imseng, Nicole; Eibl, Regine

doi:10.1002/9780470909997.ch21

Cited by 18 publications

(14 citation statements)

References 44 publications

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“…Transgenic tobacco ( Nicotiana tabacum cv. Petite Havana SR1) plants expressing the heavy chain (HC) and the light chain (LC) of the M12 antibody were generated by Agrobacterium ‐mediated transformation (Raven et al, ). The M12 HC and LC coding sequences were present as separate expression cassettes within one T‐DNA, separated by scaffold attachment regions.…”

Section: Methodsmentioning

confidence: 99%

“…The M12 HC and LC coding sequences were present as separate expression cassettes within one T‐DNA, separated by scaffold attachment regions. Gene expression was driven by the double‐enhanced Cauliflower mosaic virus (CaMV) 35S promoter, and signal peptide sequences were included for secretion to the apoplast (Raven et al, ). Hairy roots were established from the T1 generation of transgenic plants by infecting the leaves with wild‐type Agrobacterium rhizogenes LBA9402.…”

Section: Methodsmentioning

confidence: 99%

“…Monoclonal antibodies are the largest class of biologics because of their functional diversity, but their stoichiometric mechanism of action means they are often needed in substantial amounts. We have therefore developed tobacco hairy root cultures for the production of monoclonal antibody M12, which was derived from a single‐chain variable fragment (scFv) selected from a naïve human phage display library using a breast carcinoma cell line (Raven et al, ). M12 binds to vitronectin, which may influence tumor hemostasis and malignancy (Bloemendal et al, ; Kirchhoff et al, ).…”

Section: Introductionmentioning

confidence: 99%

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Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody

Häkkinen

Raven

Henquet³

et al. 2013

Biotech & Bioengineering

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Recombinant pharmaceutical proteins expressed in hairy root cultures can be secreted into the medium to improve product homogeneity and to facilitate purification, although this may result in significant degradation if the protein is inherently unstable or particularly susceptible to proteases. To address these challenges, we used a design of experiments approach to develop an optimized induction protocol for the cultivation of tobacco hairy roots secreting the full-size monoclonal antibody M12. The antibody yield was enhanced 30-fold by the addition of 14 g/L KNO3 , 19 mg/L 1-naphthaleneacetic acid and 1.5 g/L of the stabilizing agent polyvinylpyrrolidone. Analysis of hairy root cross sections revealed that the optimized medium induced lateral root formation and morphological changes in the inner cortex and pericycle cells, indicating that the improved productivity was at least partially based on the enhanced efficiency of antibody secretion. We found that 57% of the antibody was secreted, yielding 5.9 mg of product per liter of induction medium. Both the secreted and intracellular forms of the antibody could be isolated by protein A affinity chromatography and their functionality was confirmed using vitronectin-binding assays. Glycan analysis revealed three major plant complex-type glycans on both forms of the antibody, although the secreted form was more homogeneous due to the predominance of a specific glycoform. Tobacco hairy root cultures therefore offer a practical solution for the production of homogeneous pharmaceutical antibodies in containment.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody

Häkkinen

Raven

Henquet³

et al. 2013

Biotech & Bioengineering

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“…Other groups have intensified their efforts to develop a new regulatory pathway for whole plants (Figure 2) resulting in updated guidelines [49]. There have been impressive efforts to incorporate the latest regulatory innovations in plant-based manufacturing processes, including quality by design through the use of statistical experimental models to optimize each production process [28,29 ], process analytical technologies to improve product quality and consistency [22], automated production platforms to increase process robustness and scalability [50] and single-use technologies to reduce the risk of contamination and product carry-over, including single use bioreactors [16 , 51,52], novel filter materials [42] and chromatography media [40].…”

Section: Regulatory Considerationsmentioning

confidence: 99%

High-value products from plants: the challenges of process optimization

Fischer¹,

Vasil'ev²,

Twyman³

et al. 2015

Current Opinion in Biotechnology

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“…Several types of stainless steel or disposable bioreactor systems have been used for the cultivation of plant cells, including stirred‐tank bioreactors (Holland et al, ; Huang, ; Reuter et al, ; Schmale et al, ; Trexler et al, ), bubble column reactors (Huang et al, ), single‐use bubble column bioreactors (Shaaltiel et al, ), wave‐mixed bioreactors (Eibl and Eibl, ; Huang and McDonald, ; Raven et al, ), and miniature bioreactors for high‐throughput screening (Eibl et al, ; Huang and McDonald, ). Among those systems, wave‐mixed bioreactors have been widely used for the cultivation of hairy roots and plant cells that benefit from constant illumination, such as mosses (Eibl and Eibl, ; Weathers et al, ).…”

Section: Introductionmentioning

confidence: 99%

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

Raven

Rasche

Kuehn

et al. 2014

Biotech & Bioengineering

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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.

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Growth of BY‐2 Suspension Cells and Plantibody Production in Single‐Use Bioreactors

Cited by 18 publications

References 44 publications

Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody

Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody

High-value products from plants: the challenges of process optimization

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

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