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

Abstract

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.

In‐solution antibody harvesting with a plant‐produced hydrophobin–Protein A fusion

“…We have previously reported 10‐fold increase in productivity with a stable model protein GFP–HFBI in BY‐2 suspension cells (Reuter et al ., ). Several means for improving the productivity of BY‐2 suspension cells have been published recently, including improved culture media (Holland et al ., ), FACS‐based clone screening (Kirchhoff et al ., ), protease knockout lines (Mandal et al ., ) and development of culture systems (Raven et al ., ). However, improving the yield in the BY‐2 suspension cells was not the aim of this study.…”

“…In comparison with N. benthamiana‐ based transient production systems, plant suspension cells may prove to be a useful alternative. As demonstrated here and in previous studies, BY‐2 cell lines can be propagated in conventional industrial‐scale bioreactors and the downstream processing is readily scalable (Raven et al ., ; Reuter et al ., ). Low productivity is nevertheless an issue.…”

Recombinant protein production from stable mammalian cell lines and pools

“…In addition, TubeSpin 1 bioreactor 50 and 600 tubes, 50-mL and 600-mL centrifugation tubes with a vented cap, have been developed for working volumes of 2-20 mL and 100-500 mL, respectively [17,18]. For cultures of 10-200 L, disposable orbitally shaken bioreactors of 50-L and 200-L have recently been introduced [19,20]. Disposable WAVE bioreactor bags for culture volumes starting from 100 mL remain a popular alternative choice [21].…”

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

“…The molecular toolbox for plant cells is still limited, however, the transformation of the gene of interest is done by Agrobacterium infiltration in a well-established process (Leuzinger et al, 2013). In plants, the performance of PTMs, correct assembly, and folding of complex therapeutics, as well as their secretion into the cell culture medium is possible (Raven et al, 2015). In plants, the performance of PTMs, correct assembly, and folding of complex therapeutics, as well as their secretion into the cell culture medium is possible (Raven et al, 2015).…”

“…So far, only one biopharmaceutical produced recombinantly in carrot-derived cells was approved by the FDA (Marsian & Lomonossoff, 2016;Zimran et al, 2011). In terms of N-glycosylation, biopharmaceuticals generated from the plant-based system N. tabacum show plant-specific glycan structures and lack mammalian-specific sialic acids, which can result in immunogenicity and poor pharmacokinetics, respectively (Mercx et al, 2017;Raven et al, 2015). In terms of N-glycosylation, biopharmaceuticals generated from the plant-based system N. tabacum show plant-specific glycan structures and lack mammalian-specific sialic acids, which can result in immunogenicity and poor pharmacokinetics, respectively (Mercx et al, 2017;Raven et al, 2015).…”