The expression of recombinant polypeptides in host cells is a standard technology widely used in biotechnology and the pharmaceutical industry. Especially microbial hosts, like for example E. coli, are commonly used, since relatively simple expression systems and cell culture conditions are available for these host cells. In general the cultivation process is therefore comparably economic.
It is, however, often difficult to obtain a polypeptide of interest in soluble and active form when it is expressed in microbial cells. Often, expression of a recombinant polypeptide leads to the production of poorly soluble intracellular aggregates of the polypeptide in denatured form, the so-called inclusion bodies [Baneyx, F. and Mujacic, M. (2004) Nat. Biotechnol. 22, 1399-1408 and Sorensen, H. P. and Mortensen, K. K. (2005) Microb. Cell Fact. 4, 1], also referred to as classical inclusion bodies.
Classical inclusion bodies are generally easy to isolate, typically by centrifugation at moderate speed. To recover the active, i.e. correctly folded, polypeptide from the inclusion bodies, the inclusion bodies have to be solubilized and the protein renaturated after isolation. Several patent applications and patents deal with the aspect of solubilizing the inclusion bodies and renaturing the proteins obtained from inclusion bodies. For example EP0512097, EP0364926, EP0219874, WO01/87925, Rudolph 1996, Rudolph 1990, Marston 1986 and Dietrich 2003 describe general techniques relating to the solubilization and renaturing of denatured proteins. For example, EP0219874 discloses generic methods for refolding of recombinant proteins from E. coli inclusion bodies. For the solubilisation the chaotropic agents GuHCl and arginine were used at high pH. EP0219874 describes the formation of disulfide bridges under redox conditions provided by GSH/GSSG.
Despite the fact that numerous processes for the isolation and solubilization of inclusion bodies are known, the results are not always satisfactory. One major problem is that the structure of the inclusion bodies can vary. It is known that the formation and the structure of the inclusion bodies can be influenced by parameters of the cell culture process, including for example media composition, growth temperature and production rate. WO2004/015124 describes the formation of “non-classical” inclusion bodies by modulating culture conditions.
Obtaining recombinant proteins from inclusion bodies, in particular in active form and sufficient amounts, can be problematic. Sometimes the structure of the inclusion bodies is too “soft” which leads to the situation that the isolation of the inclusion bodies by centrifugation is difficult. On the other hand it is also possible that the inclusion bodies are too compact. This results in inclusion bodies which cannot be solubilized even under rough conditions.
To overcome these disadvantages the present invention provides new cell culture processes which lead to large amounts of properly folded protein in inclusion bodies that can easily be isolated and solubilised, resulting in increased yield of the recombinant protein.