Purification of antibodies using simulated moving bed chromatography

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The present invention relates to compositions and methods for the chromatographic purification of antibodies, such as monoclonal antibodies, employing improved simulated moving bed separation strategies and, in certain embodiments, Raman spectroscopy.

1. INTRODUCTION

The present invention relates to compositions and methods for the chromatographic purification of antibodies, such as monoclonal antibodies ("mAbs"), employing improved simulated moving bed ("SMB") separation strategies and, in certain embodiments, Raman spectroscopy.

2. BACKGROUND OF THE INVENTION

Protein purification strategies commonly employ one or more chromatographic separation steps in order to exclude host cell proteins ("HCPs") from final purified protein preparations. Such chromatographic separation steps are traditionally performed in "batch mode", where a single column packed with a particular chromatographic support is sequentially equilibrated, loaded, washed, eluted, and then regenerated. Because batch mode chromatography relies on loading the column only to the column's dynamic capacity rather than loading the column to its saturation capacity, each cycle of loading and separation makes use of only 30% to 50% of the column's actual binding capacity. Thus, batch mode separation requires the use of columns having two to three times more volume than would be needed if the columns were operated at their saturation capacity. By utilizing only 30%-50% of the column's actual binding capacity, batch mode chromatography therefore involves the use of significantly higher quantities of chromatographic separation supports and extends the time necessary to complete each cycle of loading and separation, which substantially raises the costs associated with protein purification. Furthermore, the use of columns having two to three times the volume that would be necessary if the separation was performed at saturation, leads to significant increases in the amount of equilibration, wash, and elution buffers employed in a single separation cycle, resulting in additional cost and time inefficiencies. In light of the foregoing, there exists a need in the art for improved methods to more efficiently purify proteins, including therapeutic antibodies. The present invention addresses this need by incorporating improved simulated moving bed separation strategies into the purification of proteins.

3. SUMMARY OF THE INVENTION

In certain embodiments the present invention is directed towards methods for producing a host cell-protein (HCP) reduced target protein preparation from a sample mixture comprising a target protein and at least one HCP. In certain embodiments, the methods of the instant invention comprise contacting a target protein-containing sample mixture to a chromatography resin such that the resin is loaded to about 50%-100%, including greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, and greater than about 90%, of its saturated binding capacity, and collecting a chromatographic sample, wherein said chromatographic sample comprises said HCP-reduced target protein preparation. In certain of such embodiments, Raman spectroscopy is employed in order to monitor and/or determine the composition of one or more of the multi-component mixtures involved in the production of such HCP-reduced target protein preparations.

Certain embodiments of the present invention are directed to the production of HCP-reduced target protein preparations that comprise contacting a target protein-containing sample mixture to a chromatography resin such that the resin is loaded to between about 50%-100%, including greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, and greater than about 90%, of its saturated binding capacity, and collecting a chromatographic sample, wherein said chromatographic sample comprises said HCP-reduced target protein preparation and the chromatographic resin selected from the group consisting of affinity chromatographic resin, ion exchange chromatographic resin, and hydrophobic interaction chromatographic resin. In certain of such embodiments, Raman spectroscopy is employed in order to monitor and/or determine the composition of one or more of the multi-component mixtures involved in the production of such HCP-reduced target protein preparations.

Certain embodiments of the present invention are directed to the production of HCP-reduced target protein preparations that comprise contacting a target protein-containing sample mixture to a chromatography resin such that the resin is loaded to about 50%- 100%, including greater than about 50%, greater than about 60%, greater than about 70%», greater than about 80%, and greater than about 90%, of its saturated binding capacity, and collecting a chromatographic sample, wherein said chromatographic sample comprises said HCP -reduced target protein preparation and the target protein is selected from the group consisting of: enzymes; peptide hormones; polyclonal antibodies; human monoclonal antibodies; humanized monoclonal antibodies; chimeric monoclonal antibodies; single chain antibodies; Fab antibody fragments; F(ab')2 antibody fragments; Fd antibody fragments; Fv antibody fragments; isolated CDRs; diabodies; DVDs, and immunoadhesions. In certain of such embodiments, Raman spectroscopy is employed in order to monitor and/or determine the composition of one or more of the multi-component mixtures involved in the production of such HCP-reduced target protein preparations.

Certain embodiments of the present invention are directed to the production of HCP-reduced target protein preparations that comprise contacting a target protein-containing sample mixture to a chromatography resin such that the resin is loaded to about 50%-100%, including greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, and greater than about 90%, of its saturated binding capacity, and collecting a chromatographic sample, wherein said chromatographic sample comprises said HCP-reduced target protein preparation and the chromatography resin is packed into a series of fluidly-connected columns separated by fluid conduits, wherein the number of fiuidly connected columns is selected from the group consisting of: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 individual columns. In certain of such embodiments, Raman spectroscopy is employed in order to monitor and/or determine the composition of one or more of the multi-component mixtures involved in the production of such HCP-reduced target protein preparations.

Certain embodiments of the present invention are directed to the production of HCP-reduced target protein preparations that comprise contacting a target protein-containing sample mixture to a chromatography resin such that the resin is loaded to about 50%- 100%, including greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, and greater than about 90%, of its saturated binding capacity, and collecting a chromatographic sample, wherein said chromatographic sample comprises said HCP-reduced target protein preparation and the chromatography resin is packed into a series of at least 2 fluidly-connected columns separated by fluid conduits, wherein the columns are separated by fluid conduits that permit the introduction buffers, such as equilibration, wash, and elution buffers, as well as the withdrawal of eluates. In certain of such embodiments, Raman spectroscopy is employed in order to monitor and/or determine the composition of one or more of the multi-component mixtures involved in the production of such HCP-reduced target protein preparations.