Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor
Raven, N. ; Rasche, F. ; Kuehn, C. ; Anderlei, T. ; Klöckner, W. ; Schuster, F. ; Henquet, M.G.L. ; Bosch, H.J. ; Büchs, J. ; Fischer, R. ; Schillberg, S. - \ 2015
Biotechnology and Bioengineering 112 (2015)2. - ISSN 0006-3592 - p. 308 - 321.
expanded-bed adsorption - enzyme replacement therapy - high-yield production - full-size antibody - pharmaceutical proteins - monoclonal-antibody - suspension-cultures - foreign proteins - cho-cells - purification
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.
Micropropagation of dahlia in static liquid medium using slow-release tools of medium ingredients
Klerk, G.J.M. de; Brugge, J. ter - \ 2011
Scientia Horticulturae 127 (2011)4. - ISSN 0304-4238 - p. 542 - 547.
tissue-cultures - suspension-cultures - growth - calcium - formulations - nutrition - segments - necrosis - invitro - apple
Growth of dahlia shoots in vitro was ca. 4 times faster in liquid medium than on solidified medium. In liquid standard medium (3% sucrose, macroelements according to Driver–Kuniyuki Walnut medium, microelements according to Murashige–Skoog medium, 0.44 µM benzylaminopurine), the major medium ingredients were consumed for 75–80% during the first 6 weeks. Addition of extra ingredients increased growth, demonstrating that the amount of ingredients added at the start of culture was suboptimal. When the extra ingredients were given at the start of the culture, concentrations became too high and therefore inhibitory. When the ingredients were added during the subculture cycle by means of small aliquots of a concentrated solution or by means of slow-release tools, growth was strongly increased. Osmocote gave satisfactory results as a slow-release tool for inorganics. For organic ingredients (sucrose and benzylaminopurine), a novel slow-release tool was developed.
Involvement of ethylene and nitric oxide in cell death in mastoparan-treated unicellular alga Chlamydomonas reinhardtii
Yordanova, Z.P. ; Iakimova, E.T. ; Cristescu, S.M. ; Harren, F.J.M. ; Kapchina-Toteva, V.M. ; Woltering, E.J. - \ 2010
Cell Biology International 34 (2010)3. - ISSN 1065-6995 - p. 301 - 308.
plant-pathogen interactions - tomato cells - suspension-cultures - phosphatidic-acid - hydrogen-peroxide - oxidative stress - no - model - activation - mechanisms
This work demonstrates a contribution of ethylene and NO in mastoparan (MP)-induced cell death in the green algae C. reinhardtii. Following MP treatment, C. reinhardtii showed massive cell death, expressing morphological features of programmed cell death (PCD). A pharmacological approach involving combined treatments with MP and ethylene and NO interacting compounds indicated the requirement of trace amounts of both ethylene and NO in MP-induced cell death. By employing a carbon dioxide laser-based photoacoustic detector to measure ethylene and a Quantum Cascade Laser (QCL) - based spectrometer for NO detection, simultaneous increases in the production of both ethylene and NO were observed following MP application. Our results show a tight regulation of the levels of both signalling molecules in which ethylene stimulates NO production and NO stimulates ethylene production. This suggests that, in conjunction with the elicitor, NO and ethylene cooperate and act synchronously in the mediation of MP-induced PCD in C. reinhardtii. To the best of our knowledge this is the first report on the functional significance of ethylene and NO in MP-induced cell death
The pectic polysaccharide rhamnogalacturonan II is present as a dimer in pectic populations of bilberries and black currants in muro and in juice
Hilz, H. ; Williams, P. ; Doco, T. ; Schols, H.A. ; Voragen, A.G.J. - \ 2006
Carbohydrate Polymers 65 (2006)4. - ISSN 0144-8617 - p. 521 - 528.
plant-cell-walls - structural-characterization - suspension-cultures - borate ester - acid - oligosaccharides - component - phenols - complex - roots
Rhamnogalacturonan II (RG II) can play an important role during processing of berries due to its enzyme resistance and its possible role as a pectic cross-linker. This article describes the presence of RG II in cell walls, in juice and in press cake of bilberries and black currants. RG II was identified and quantified via its diagnostic sugar residues. RG II, which was released from homogalacturonan, was probably present in its dimeric form in muro. Juice contained the free RG II dimer, while from press cake dimeric RG II was released by enzymatic degradation of homogalacturonan. A higher amount of RG II was present in juice than in press cake. During juice processing a cross-linker RG II might improve gel formation, which hinders the processability of berries. In addition, enzymes used during juice processing release dimeric RG II from pectin molecules and accumulate RG II in the juice.
Agrobacterium tumefaciens mediated transformation of Allium cepa L.: the production of transgenic onions and shallots
Zheng, S.J. ; Khrustaleva, L.I. ; Henken, G. ; Sofiari, E. ; Jacobsen, E. ; Kik, C. ; Krens, F.A. - \ 2001
Molecular Breeding 7 (2001)2. - ISSN 1380-3743 - p. 101 - 115.
manihot-esculenta crantz - oryza-sativa-l - gene-transfer - indica rice - t-dna - transient expression - suspension-cultures - plant-regeneration - immature embryos - reporter gene
This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology