Tailor-made expression hosts depleted in protease activity for recombinant protein production

acronym: PRODuCE

RESULTS of PRODuCE: Presentation can be found here.

Project coordinator
- Dr. rer. nat. Andreas Schiermeyer - Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen - Germany
Project leaders

- PhD Renier Adrianus Leonardus Van der Hoorn - Max Planck Institute for Plant Breeding Research (MPIPZ), Cologne - Germany
- PhD Christopher Mark Smales - Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury - UK
- PhD Rita Abranches - Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Oeiras - Portugal
- Christoph Heinrich - Teutocell AG, Bielefeld - Germany

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

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.