Removal of host cell proteins

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The present invention relates to a method for the removal of host cell protein from a cell broth containing cells, culture medium and a secreted desired biological substance having an overall positive charge in the cell broth by contacting the cell broth with a particulate anion exchanger, allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, separating the resulting cell pellet from the supernatant layer, and determining the reduction of the HCP content in the supernatant layer as compared to the cell broth. The present invention further relates to a method for the recovery of a secreted desired biological substance from a cell broth containing cells producing the secreted desired biological substance which has an overall positive charge in the cell broth by contacting the cell broth with particulate anion exchanger, allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, separating the resulting cell pellet from the supernatant layer, determining the reduction of the HCP content in the supernatant layer as compared to the cell broth and extracting the secreted desired biological substance from the supernatant layer.

The present invention relates to a method for the reduction of host cell protein (HCP) in a cell broth and to a method for the recovery of secreted desired biological substances from a cell broth containing cells producing the secreted desired biological substance.

Fermentative production of biological substances, such as pharmaceuticals and in particular monoclonal antibodies, delivers a complex cell broth from which the biological substances should be isolated and purified by a great number of steps.

As a first step solid material such as the cells and cell debris is to be separated from the cell broth fluid - a step called clarification. In many instances the biological substances are present extracellularly and will thus be present in the cell broth fluid. Examples of clarification methods used to-date include centrifugation, filtration (such as microfiltration, depth filtration and filtration through absolute pore size membranes) and expanded bed chromatography. Flocculation may be employed in order to enhance any of these clarification methods, in particular in combination with filtration. Known flocculation agents for this purpose can range from simple electrolytes to synthetic poly-electrolytes (such as DEAE dextran, acryl-based polymers, polyethylene amine) or inorganic materials (such as diatomaceous earth or perlites). A recent development is the use of chitosan for this purpose.

An objective of the present invention is to reduce the HCP content of the secreted desired biological substance from a cell culture prior to further downstream processing.

In a specific embodiment the present invention relates to a method for the reduction of host cell protein in a cell broth containing mammalian cells, culture medium and a secreted desired biological substance having an overall positive charge in the cell broth by: a. contacting the cell broth with a particulate anion exchange material, b. allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer, and d. determining the reduction of the HCP content in the supernatant layer as compared to the cell broth.

The international patent application published as WO2007/108955 relates to a protein purification process, in particular to purification of antibodies or antibody-like proteins, from the supernatant of a cell culture, by using cation exchange followed by anion exchange chromatography. This encompasses the removal of HCP. Also, it is described in the document that these chromatography steps may be carried out using a batch process followed by a separation step (separation of supernatant from solid material). The essential difference between the instant invention and the disclosure of WO2007/108955 is that the latter relates to purification from a clarified protein-containing medium, whereas the instant invention relates to a process carried out on a unclarified material containing cells.

The international patent application which is published as WO2008/079280 relates to the purification of biomolecules from a mixture, by adding soluble polymers (e.g. soluble ion exchange polymers) to the mixture and changing the conditions so as to precipitate these and the biomolecules bound to it, thus separating the biomolecules into fractions. The essential difference between the instant invention and the disclosure of WO2008/079280 is that in the latter the ion-exchange polymer is soluble, whereas in the instant the anion exchange material is particulate. Neither of the two documents suggests a purification process carried out on the complete cell broth. The problem which can be solved by the present invention is that there is a desire in the industry to reduce the number of purification steps in a protein purification process. The present invention solves the problem by combining the removal of HCP with the clarification step of the cell broth.