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Separating PEGylated monoclonal antibodies on MacroCap SP

IP.com Disclosure Number: IPCOM000132615D
Publication Date: 2005-Dec-26
Document File: 2 page(s) / 70K

Publishing Venue

The IP.com Prior Art Database

Abstract

Disclosed in this publication is a method using a highly porous ion exchange medium for separation of large native and functionalized or conjugated bio-molecules. In this study it is shown, that proteins in a PEGylation reaction mixture as large as PEGylated antibodies can be separated from nonPEGylated by using a highly porous ion exchanger like Macro Cap SP.

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Separating PEGylated monoclonal antibodies on MacroCap SP

Summary

Disclosed in this publication is a method using a highly porous ion exchange medium for separation of large native and functionalized or conjugated bio-molecules.

In this study it is shown, that proteins in a PEGylation reaction mixture as large as PEGylated antibodies can be separated from nonPEGylated by using a highly porous ion exchanger like Macro Cap SP.

Introduction

PEGylated pharmaceutical have gained a great interest and use in resent years.

PEG

polymers have, compared to proteins, 7-10 times higher hydration radius and since both the molecular weight of used Peg’s and proteins has become higher, the demand for chromatography media with suitable high porosities is rising.  The pores of a media should be wide enough to allow sample molecules to penetrate in to the beads in order to give an optimal available surface for adsorption, which is larger than the outer surface of the beads of the media.

The ion exchanger used in this study was MacroCap SP which is a large pore medium intended for separation of large molecules such as PEGylated proteins, and other large proteins such as IgM, with a hydrodynamic radius >4 nm.

A reaction mixture from PEGylation of an antibody has been loaded on a MacroCap SP column and eluted with a salt-gradient at pH 5. The result shows that it is possible to absorb and separate proteins as large as PEGylated and nonPEGylated antibodies by using the highly porous ion exchanger Macro Cap SP.

Materials and Methods

PEGylation reaction           

200 µl monoclonal IgG ( 6mg/ml)

5 mg

PEG

SPA

20k

1000 µl 200 mM Sodium Borate pH 7.8

20 minutes reaction at room temperature

Reaction stopped with 48 µl 1 M Acetic acid and cooling on ice bath.

Ion...