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Evaluation of a New Cation Exchanger for Purification of Insulin

IP.com Disclosure Number: IPCOM000237473D
Publication Date: 2014-Jun-18

Publishing Venue

The IP.com Prior Art Database

Abstract

The present study concerns an evaluation of a new cation exchanger for further purification of insulin previously purified on a conventional SP Sepharose FF column (GE Healthcare Bio-Sciences AB, Sweden). The new cation exchanger is based on highly crosslinked agarose beads of 50 micrometer average diameter, 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 insulin

Background

The industrial purification of recombinant insulin is usually performed in a complex multistep process involving several subsequent chromatography steps, such as adsorption chromatography, ion exchange chromatography and reversed phase chromatography (RPC). The requirements on purity of the final product are high and it must e.g. contain <2% insulin-like polypeptides, <2% desamidoinsulin and <10 ppm immunogenic polypeptides (1). RPC is often used as the final polishing step and can provide a high purity insulin (2-6). The use of flammable solvents and in many cases highly toxic solvents such as acetonitrile in the RPC step is however a general issue and it would be advantageous if solvent-free methods could be used to a higher extent. There is also a general need for improving the efficiency of insulin processes.

Outline

The present study concerns an evaluation of a new cation exchanger for further purification of insulin previously purified on a conventional SP Sepharose FF column (GE Healthcare Bio-Sciences AB, Sweden). The new cation exchanger is based on highly crosslinked agarose beads of 50 micrometer average diameter, 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 (7).

The insulin preparation has been purified by bind-elute chromatography on columns packed with the new cation exchanger and the purity of the main peak has been assessed by RPC-HPLC. A comparative experiment with a conventional sulphopropyl cation exchanger (Capto SP ImpRes) has been made.

Materials

Cation exchange


Stock solution: 100 mM Acetate buffer, pH 4 (2.178 g Na acetate 3.H2O + 4.804 ml acetic acid dissolved in 1L MQ water).

Buffer A: 47.5% Ethanol in 50 mM Na acetate pH 4 prepared from 525 ml stock solution +475 ml ethanol. Buffer B: 47.5% Ethanol in 50 mM Na acetate pH 4 + 0.5 M NaCl prepared from 300 ml buffer A +8.76 g NaCl (Merck).

Column: A Tricorn 5/50 (GE Healthcare Bio-Sciences AB) column packed with the new cation exchanger. Sample: Partially purified insulin, derived as the main peak from a previous SP Sepharose FF column . 4 mg was dispersed in 400 microliters buffer A and 10 microliters HAc was added to solubilize. Conc = 10 mg/ml
Äkta avant 25 chromatography system

RPC analysis
Äkte Explorer chromatography system
Column: Kromasil 100-3.5 C4 4.6x150mm (3.5 µm, 4.6 x 150 mm) Phosphate/perchlorate pH 2.5 buffer system used containing ACN as: Buffer A: Phosphate/perchlorate pH 2.5/CAN 70/30 % (v/v)

Buffer B: Phosphate/perchlorate pH 2.5/CAN 50/50 % (v/v)


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Gradient:
0-2 min 26%B 2-22min 26-43%B 22-23 min 43-100%B 23-28min 100%B 28-29min 100.26%B 29-34min 26%B

Experiment no. 1. 20 ul Insulin was injected on the new cation exchanger column...