Production of a recombinantly expressed laminin fragment by HEK293-EBNA cells cultured in suspension in a dialysis-based bioreactor

Laminin 5 is a multifunctional extracellular matrix protein, which supports epithelial cell adhesion through multiple cell binding sites. For elaborate studies, a 35 kDa fragment localized at the C-terminal extremity of the molecule, the LG4/5 fragment was recombinantly expressed in mammalian HEK293-EBNA cells. As the production of the LG4/5 fragment by adherent cell monolavers was very low (< 1 mu g/ml), we used the commercially available small scale bioreactor system rniniPERM (TM). The HEK293-EBNA transfectants were adapted to grow in suspension in defined medium containing low level of fetal calf serum and produced the recombinant LG4/5 protein with quality consistent with that produced in conventional static cell culture conditions. Cells grew without forming aggregates in the bioreactor and the resulting HEK293-EBNA-LG4/5 cell line was Suitable for unlimited passages in the bioreactor. 2.5 x 10(5) cells/ml were cultured for 25 days to reach the maximal cell density of 1.6 x 10(7) cells/ml. The quantification of protein synthesis revealed that the highest level of 2.4 mg of recombinant LG4/5 protein was harvested when 107 transfected cells/ml were injected in the bioreactor and allowed to grow for 2 days. The mean daily recombinant LG4/5 fragment product yield of 1.2 mg of protein per minifermenter shows that cultivation of HEK293-EBNA transfectants in suspension is highly convenient for the production of recombinant laminin fragments. (c) 2006 Elsevier Inc. All rights reserved.