Hoffmann-la Roche Inc.

Stefan Hepbildikler

Gerhard Winter

Wolfgang Kuhne

Eva Rosenberg

Adelbert Grossmann

Monika Baehner

Herein is reported a method for obtaining a polypeptide in monomeric form comprising the steps of a) providing a solution comprising the polypeptide in monomeric form and in aggregated form, wherein the ratio of monomeric to aggregated form is 4:1 or less as determined by size exclusion chromatography, b) performing a mixed-mode chromatography in bind-and-elute mode, or a hydrophobic interaction chromatography in flow-through mode, or a size-exclusion chromatography, and c) performing a weak cation exchange chromatography in bind-and-elute mode or flow-through mode, and thereby obtaining the polypeptide in monomeric form.

Method for separation of monomeric polypeptides from aggregated polypeptides

For the removal of high molecular weight compounds from recombinantly produced polypeptides generally chromatographic methods are employed. It has been found that underivatized controlled pore glass (uCPG) selectively binds high molecular weight compounds present in a solution. The purified polypeptide can be recovered e.g. from the flow through of a chromatography column containing uCPG as chromatography material. It has been found that this effect is pronounced at a pH value of about 4 to 6 in buffered solutions. With approximately 100 mto 150 muCPG surface per g of polypeptide almost 80% to 95% of the high molecular weight compounds are removed with a yield of 80% to 90% of polypeptide.

Immunoglobulin aggregate removal

Herein is reported a method for obtaining a polypeptide in monomeric form comprising the steps of a) providing a solution comprising the polypeptide m monomeric form and m aggregated form, wherein the ratio of monomeric to aggregated form is 4:1 or less as determined by size exclusion chromatography, b) performing a mixed-mode chromatography in bind-and-elute mode, or a hydrophobic interaction chromatography in flow-through mode, or a size-exclusion chromatography, and c) performing a weak cation exchange chromatography in bind-and-elute mode or flow-through mode, and thereby obtaining the polypeptide m monomeric form.

Herein is reported a tangential flow filtration method with a pre-filtration solute concentration adjustment in order to ensure a defined concentration of the components of the solution after tangential flow filtration.

Pre-filtration adjustment of buffer solutes

The current invention reports a method for concentrating an immunoglobulin solution by tangential flow filtration wherein the transmembrane pressure and the cross-flow are variable.

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Munich, Germany

Stefan Hepbildikler