Project: Tailor-made expression hosts depleted in protease activity for recombinant protein production
The demand for biopharmaceuticals is high and predicted to increase further and as such efficient production processes are required that yield quality protein. Despite the improvements in the production of complex biopharmaceuticals in Chinese Hamster Ovary (CHO) cells a number of challenges remain. A key challenge is the degradation of protein products during fermentation or downstream processing steps. Within the proposed project we will systematically evaluate the proteolytic activities that hamper the successful production and/or purification of selected target proteins (e. g. antibodies, erythropoietin). The project will include CHO cells as the current industry work horse for protein production but also plant suspension cells (tobacco, Medicago) as emerging alternative production platforms. Proteolysis will be blocked using selective protease inhibitors to classify proteases responsible for product degradation. Proteolytic activities will be determined in cells and spent culture medium by activitybased protein profiling (ABPP) using chemical probes for proteases. Libraries of these probes are available for cysteine-, serine- and metalloproteases and for the proteasome. Probes for aspartic proteases will be produced within the project. The knowledge gained about the nature of the proteases will be utilized to engineer cell lines with reduced endogenous protease activities. Different strategies will be used to suppress product degradation: Knock-out of protease genes by gene targeting Knock-down of protease genes by posttranscriptional gene silencing Co-expression of selective protease inhibitors together with the target protein Rational design of cell culture media to suppress proteolytic activities With the project we expect to establish new production cell lines (CHO and plant suspension cells) with reduced endogenous protease activities and to develop novel CHO cell culture medium formulations. The consortium will seek protection of intellectual property rights for the novel engineered cell lines because cell lines with reduced proteolytic activities are of great value for the industry to produce sensitive biopharmaceutical proteins (e.g. factor VIII). Patent applications for novel cell culture medium formulations are uncommon and therefore IP protection will only be sought for novel ingredient compounds.
| Acronym | PRODuCE |
| Duration | 01/01/2013 - 31/05/2016 |
| Website | visit project website |
| Network | ERA-IB-2 |
| Call | 3rd ERA-IB Joint Call |