Establishment of long-term perfusion cultures of recombinant moss in a pilot tubular photobioreactor

The moss Physcomitrella patens has been successfully used for the expression of interesting heterologous proteins. Humanization of native glycosylation patterns in Physcomitrella has opened the possibility to express complex glycoproteins in cultures with simplified down stream processing. In order to characterize this expression system, the transgenic moss tWT11.51VEGF that produces the vascular endothelial growth factor VEGF121 has been cultured in a pilot 30-l tubular photoreactor. Long-term perfusion cultures with total cell recovery were performed by means of cross-flow filtration (CFF). Morphology, concentration and debris of moss showed to be determinant for the performance of CFF. Controlled cell disruption by means of rotor–stator devices and medium perfusion of 0.2 day−1, helped to delay cell differentiation, maintain low the proportion of leafy gametophores and postpone the decay of rhVEGF121 secretion that has been observed in standard batches. However, only through continuous cultures (D = 0.2–0.3 day−1), it was possible to stabilize the concentration of recombinant protein in the biosuspension. Here, perfusion of 18 and 30 day−1 showed to increase the specific production rate of rhVEGF121.