title: Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei.
abstract: The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.
authors: VTT Technical Research Centre of Finland, Espoo, Finland.
authors: Christopher P
authors: CP
authors: Landowski
authors: VTT Technical Research Centre of Finland, Espoo, Finland.
authors: Anne
authors: A
authors: Huuskonen
authors: Novartis Pharma AG, Basel, Switzerland.
authors: Ramon
authors: R
authors: Wahl
authors: VTT Technical Research Centre of Finland, Espoo, Finland.
authors: Ann
authors: A
authors: Westerholm-Parvinen
authors: Glykos Finland Oy, Helsinki, Finland.
authors: Anne
authors: A
authors: Kanerva
authors: Glykos Finland Oy, Helsinki, Finland.
authors: Anna-Liisa
authors: AL
authors: Hänninen
authors: Glykos Finland Oy, Helsinki, Finland.
authors: Noora
authors: N
authors: Salovuori
authors: VTT Technical Research Centre of Finland, Espoo, Finland.
authors: Merja
authors: M
authors: Penttilä
authors: Glykos Finland Oy, Helsinki, Finland.
authors: Jari
authors: J
authors: Natunen
authors: Novartis Pharma AG, Basel, Switzerland.
authors: Christian
authors: C
authors: Ostermeier
authors: Novartis Pharma AG, Basel, Switzerland.
authors: Bernhard
authors: B
authors: Helk
authors: Glykos Finland Oy, Helsinki, Finland.
authors: Juhani
authors: J
authors: Saarinen
authors: VTT Technical Research Centre of Finland, Espoo, Finland.
authors: Markku
authors: M
authors: Saloheimo
mesh_terms: D001688:Biological Products / Q000191:economics* / Q000378:metabolism*; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D010447:Peptide Hydrolases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D059748:Proteolysis; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
chemical_list: D001688:Biological Products; D007074:Immunoglobulin G; D011480:Protease Inhibitors; D010447:Peptide Hydrolases