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Improving Purity on Protein A Affinity Chromatography Media through use of an Arginine Intermediate Wash Step

IP.com Disclosure Number: IPCOM000127319D
Publication Date: 2005-Aug-22
Document File: 4 page(s) / 57K

Publishing Venue

The IP.com Prior Art Database

Abstract

The use of a 1M arginine pH 5.0 phosphate/acetate buffer as an intermediate wash step has been demonstrated to be effective in reducing non-specifically bound host cell protein co-eluting with IgG from ProSep-vA High Capacity media. The use of arginine provides an effective alternative to TMAC.

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Improving Purity on Protein A Affinity Chromatography Media through use of an Arginine Intermediate Wash Step

S. Barron1, C. Major1,

S. Cross1

, J. Madan
2, F. Mann1

Millipore Corporation: 1 Consett, UK; 2 Billerica, USA

SUMMARY

The use of a 1M arginine pH 5.0 phosphate/acetate buffer as an intermediate wash step has been demonstrated to be effective in reducing non-specifically bound host cell protein co-eluting with IgG from ProSep-vA High Capacity media. The use of arginine provides an effective alternative to TMAC.   

INTRODUCTION

Protein A affinity chromatography is the most commonly used initial capture chromatography step for production of therapeutic monoclonal antibodies, exhibiting high selectivity with purities typically 95% or greater. The remaining impurities contain residual host cell proteins(HCP), nucleic acids, product variants i.e. aggregates and (potentially) viruses, which are removed by subsequent chromatographic steps. Differences may be seen in terms of HCP carry over into the protein A eluate depending on the nature of the base matrix. Glass based matrices, for instance ProSep®-A, may exhibit relatively higher HCP levels compared to agarose based matrices. This is thought to be due to higher non-specific binding (NSB) inherent to the glass matrix compared to agarose.  The use of TMAC ( tetramethyl ammonium chloride) or TEAC ( tetraethyl ammonium chloride) as an intermediate wash have been shown to significantly reduce HCP carry over on such resins (ref 1).  Such compounds may however be undesirable due to corrosive properties.

There is a substantial body of literature showing that arginine containing buffers have interesting effects on protein folding, protein-protein interactions and protein solubility.  Arginine containing buffers have been widely used to increase the recovery of recombinant protein products from inclusion bodies and are thought to help prevent aggregation of partially folded intermediates (ref 2-4).  The precise mechanism by which arginine exerts these effects remains obscure.  There is some evidence to show that arginine containing buffers can promote antibody stability during purification by allowing elution from Protein A affinity media at less acidic pH than usually used. 

The aim of this study was to investigate the potential benefits of an arginine intermediate wash in improving purity by removal of non-specifically bound proteins from the protein A affinity media.

METHOD

Comparison of different intermediate wash buffers was performed using gravity fed disposable columns packed with ProSep-vA High Capacity media (Millipore Corporation) to a bed volume of 2ml+/- 10%.

Columns were equilibrated with PBS buffer pH 7.4 and then loaded with a model feedstock to a level of 15mg of IgG per ml of media.

The model feedstock was obtained by the addition of 1mg/ml human polyclonal IgG into a clarified non-expressing CHO (Chinese Hamster Ovary) cell culture supernatant. The feedstock was then filte...