Chromatographic method for purifying FC-containing proteins
AssigneeBoehringer Ingelheim International GmbH
InventorsChristian Eckermann, Dorothee Ambrosius, Franz Nothelfer, Thomas Rathjen
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Abstract
The present invention relates to methods of depleting impurities, in particular host cell proteins (HCP) and DNA from cell culture supernatants by means of protein A chromatography using a novel washing buffer.
Claims12
A method of depleting impurities from a composition that contains a protein which comprises the Fc domain of an immunoglobulin (target protein), by protein-A chromatography, comprising the steps of:
- a. applying a mobile phase which contains the target protein to a stationary phase which contains protein A, under conditions in which the target protein binds to the stationary phase;
- b. applying a washing buffer with a pH of between 4 and 8 as mobile phase, containing
- i. arginine in a concentration of 0.1-1 mol/l,
- ii. sodium chloride in a concentration of 0.2 to 2 mol/l,
- iii. an alcohol selected from among isopropanol, n-propanol and ethanol, in a concentration of 5-30% (w/v) and
- iv. polyvinylpyrrolidone in a concentration of 0.05-2% (w/v);
- c. using an elution buffer as mobile phase under conditions in which the target protein is eluted from the stationary phase.
A method of depleting impurities from a composition that contains a protein which comprises the Fc domain of an immunoglobulin (target protein), by protein-A chromatography, comprising the steps of:
- a. applying a mobile phase which contains the target protein to a stationary phase which contains protein A, under conditions in which the target protein binds to the stationary phase;
- b. applying a washing buffer with a pH of between 5 and 8 as mobile phase, containing
- i. arginine in a concentration of 0.1—1 mol/l,
- ii. sodium chloride in a concentration of 0.2 to 2 mol/l,
- iii. an alcohol selected from among isopropanol, n-propanol and ethanol, in a concentration of 5—30% (w/v) and
- iv. polyvinylpyrrolidone in a concentration of 0.05—2% (w/v);
- c. using an elution buffer as mobile phase under conditions in which the target protein is eluted from the stationary phase.
Dependencies are inferred from the claim text.