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Evaluation of a New Cation Exchanger for Purification of Antibodies, Antibody Fragments, Domain Antibodies and Fc-Fusion Proteins

IP.com Disclosure Number: IPCOM000237474D
Publication Date: 2014-Jun-18
Document File: 11 page(s) / 2M

Publishing Venue

The IP.com Prior Art Database

Abstract

The present study concerns an evaluation of a new cation exchanger for purification of antibodies, antibody Fab and fragments, domain antibodies (Dab) and an Fc-fusion protein. The new cation exchanger is based on highly crosslinked agarose beads of 50 micrometer average diameter (d50v), onto which a sulfonate- and pyrrolidone-functional copolymer has been tethered by grafting, producing a strong cation exchanger with an ionic capacity of 37-63 micromol H+/ml.

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Evaluation of a new cation exchanger for purification of antibodies, antibody fragments, domain antibodies and Fc-fusion proteins

Background

Monoclonal antibodies are usually expressed in mammalian cells, e.g. CHO cells, and are recovered and further purified in processes including a series of chromatography steps. The first step is typically a capture step on a Protein A column, which is then followed by one or two steps involving ion exchange chromatography, hydrophobic interaction chromatography and/or multimodal chromatography (1). It is common practice to include a strong cation exchange step in the process, typically with the purpose of removing antibody aggregates, host cell proteins, leached Protein A, viruses, undesirable antibody variants etc. (2-6). The cation exchange step is often performed in bind- elute mode, but may also be performed in flow-through or weak partitioning mode (3).

In similar ways, cation exchange media are also commonly used in processes for purification of antibody fragments (7,8), Fc-fusion proteins (9) and other proteins containing antibody-derived moieties.

Outline

The present study concerns an evaluation of a new cation exchanger for purification of antibodies, antibody Fab and fragments, domain antibodies (Dab) and an Fc-fusion protein. The new cation exchanger is based on highly crosslinked agarose beads of 50 micrometer average diameter (d50v), onto which a sulfonate- and pyrrolidone-functional copolymer has been tethered by grafting, producing a strong cation exchanger with an ionic capacity of 37-63 micromol H+/ml. Similar constructions, including methods of manufacturing, are described in (10).

The performance of the new cation exchanger is illustrated by the experimental results below. The data are retrieved at an early stage and the experiments can be further optimized.

Experiments


1. Bind-elute removal of host cell proteins, fragments and aggregates

A monoclonal antibody having an isoelectric point at pH 8.4 was initially purified on a MabSelect SuRe LX column (GE Healthcare Bio-Sciences AB). The eluate was adjusted to pH 5.0 with NaOH and NaCl added to an end concentration of 50 mM and it was then applied on a column packed with the new cation exchanger and equilibrated in 50 mM Na acetate buffer pH 5.0 + 50 mM NaCl (A-buffer). After loading of 76 g mAb per L resin and washing with A-buffer, the column was eluted with a 20 column volume gradient from 50 to 400 mM NaCl concentration. After the elution, the column was cleaned in place with 1 M NaOH. The resulting chromatogram is shown in Fig. 1, with the content of fragments (green) and aggregates (red) in the fractions overlaid.


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Fig 1. Bind-elute removal of fragments and aggregates from a mAb.

The start material contained 2 % fragments, 2.1 % aggregates, 280 ppm host cell proteins (HCP) and 3 ppm Protein A, while the eluate pool contained 1.6 % fragments, 0.9 % aggregates, 170 ppm HCP and <1 ppm Protein A. The pool yiel...