Journal of Chromatography, 641 (1993) 71-79 Elsevier Science Publishers B.V., Amsterdam CHROM. 25 112 Analysis and purification of monomethoxy-polyethylene glycol by vesicle and gel permeation chromatography Barbara Selisko*, Cristina Delgado and Derek Fisher zyxwvutsrqponmlkjihgfedcbaZYXWVUTS M olecular Cell Pathology, Royal Free Hospital, School of Medicine, Rowland Hill Street, London NW 3 2PF (UK) Rudolf Ehwald Institute of Plant Physiology and Cell Biology, Department of Biology, Humboldt-University Berlin, Invalidenstrasse 42, 1040 Berlin (Germany) (First received December llth, 1992; revised manuscript received March 18th, 1993) ABSTRACT Vesicle chromatography (VC) and gel permeation chromatography (GPC) were used for characterisation and purifkation of monomethoxy-polyethylene glycol (M-PEG), a reagent for protein modification. Detection of low concentrations of contaminat- ing PEG was facilitated by a very sensitive colourimetric detection method with a detection limit of 1 &ml. For analytical purposes GPC on Superose 12 was superior to VC. Molecular masses, polydispersity and percentage of contaminating PEG were estimated. As a comparison ‘H NMR spectroscopy was carried out. The results were in good accordance with GPC. A two-step preparative purification with VC of M-PEG containing 22.9% PEG reduced the PEG content to 4.4%. INTRODUCTION Proteins can be modified by attaching macro- molecules. In general, the modification results in an alteration of the physiological properties and/ or an increase of the stability of proteins. The main objectives of the modification of proteins are to use them as therapeutic agents or as biocatalysts in biotechnological processes. Poly- ethylene glycol (PEG) has been applied to various proteins as an agent for modification [ 11. Various coupling methods have been developed so far using mainly monomethoxy-polyethylene glycol (M-PEG) as starting material. In this way a PEG molecule is provided which is activated at only one end of the polymeric chain, thus pre- venting cross-linking of two proteins or the formation of even larger aggregates. However, M-PEG preparations are often contaminated by PEG with free hydroxyl groups at each end (also called diol-PEG). This contaminant is reported to be formed as a result of simple hydrolysis of some of the ethylene oxide monomers in the starting period of the polymerisation due to the presence of free hydroxyl ions [2]. As a con- sequence these molecules grow at both ends of the chain and, thus, should have about double the molecular size of M-PEG. The use of narrow-range M-PEG with a high degree of purity is important to minimise the heterogeneity of the modification products and to prevent the above mentioned formation of aggregates. Thus, the characterisation of the purity and molecular mass distribution of the polymer is necessary. In this study we used gel permeation chromatography zyxwvutsrqponmlkjihgfe (GPC) , vesicle chromatography (VC) and ‘H NMR spectros- copy * * Corresponding author. VC is a type of permeation chromatography CKt21-9673/93/$06.00 @ 1993 Elsevier Science Publishers B.V. All rights reserved