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Evaluation of a New Cation Exchanger for Purification of Plasma Proteins, Lactoferrin and Interferon

IP.com Disclosure Number: IPCOM000237475D
Publication Date: 2014-Jun-18
Document File: 8 page(s) / 1M

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Abstract

The present study concerns an evaluation of a new cation exchanger for purification of albumin, IgG and Factor VIII from plasma, lactoferrin from milk and a recombinant interferon-α2a from E Coli fermentation. 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 plasma proteins, lactoferrin and interferon

Background

Cation exchangers are generally useful in separations of proteins (11). They can for example be used in plasma processing for purification of IgG (1-4), albumin (5) and Factor VIII (6). It is also known to use cation exchangers for purification of recombinant proteins such as e.g. interferons (7,8) and of milk proteins such as e.g. lactoferrin (9). In all such processes there is a need for high selectivities to obtain high purities and for high capacities to allow high process throughput.

Outline

The present study concerns an evaluation of a new cation exchanger for purification of albumin, IgG and Factor VIII from plasma, lactoferrin from milk and a recombinant interferon-α2a from E Coli fermentation. 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 (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 purification of albumin

A 22 g/L albumin solution in acetate buffer at pH 4.5 was loaded onto a column packed with the new cation exchanger. The column was then eluted with acetate buffer at pH 5.5. The resulting chromatogram is shown in Fig. 1, which demonstrates the possibility of bind-elute purification of albumin. The yield of albumin was 70%.


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Fig. 1. Bind-elute purification of albumin.


2. Bind-elute purification of IgG/flowthrough purification of albumin

A solution of 5.2 g/L polyclonal IgG (Gammanorm) and 20.8 g/L albumin in 50 mM Na-acetate pH 5.5 was loaded (70 g IgG per mL resin) on a column packed with the new cation exchanger. The column was then eluted with 50 mM Na-acetate pH 5.5 + 500 mM NaCl. The resulting chromatogram is shown in Fig. 2. SEC chromatograms of the start material, pooled flowthrough and pooled eluate are shown in Fig. 3, which demonstrates that it is possible to fractionate the mixture such that albumin is recovered in the flowthrough and IgG in the eluate.


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Fig. 2. Separation of IgG and albumin.

Fig. 3. SEC chromatograms of pooled fractions from the chromatography experiment of Fig. 2.


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3. Aggregate removal from plasma IgG

10 g/L polyclonal plasma IgG (Gammanorm) in 50mM Na-acetate pH 5.5 + 100 mM Na Cl (A-buffer) was pumped through a column packed with the new cation exchanger until 50 g IgG/L resin had been loaded. The flowthrough was collected and the column was then eluted with A-buffer + 500 mM NaCl and cleaned in place with 1 M NaOH. The resulting chromatogram is shown in Fig. 3.

Fig. 3. F...