Separation of High-Activity Heparin by Affinity Chromatography on Supported Protamine

This invention relates generally to the isolation of high-activity heparin and, more particularly, to the separation of heparin into fractions of relatively high specific activity and relatively low specific activity by affinity chromatography.

Heparin is a highly sulphated dextrorotatory mucopolysaccharide which is commonly employed in various biomedical applications for its anticoagulant properties in prolonging the clotting time of whole blood. It is typically isolated from mammalian tissue, such as animal livers, lungs or intestines, and thereafter purified by methods well known in the art. The anticoagulant potency of the isolated and purified heparin, is generally referred to as its "specific activity" and expressed as IU/mg.

Commercially available heparin typically has a specific activity of approximately 150 IU/mg, and has previously been shown to be composed of a mixture of different heparin species varying in their specific activities. Recent use of heparin as a coating for biomaterials exposed to blood has prompted a search for methods capable of separating the most active fractions. Several different techniques have previously been proposed for effecting a separation of the heparin of relatively high specific activity out of its mixture with heparin of relatively low specific activity, including gel filtration, sucrose density gradient centrifugation after binding with antithrombin III, affinity chromatography on antithrombin III-agarose, ion-exchange chromatography on DEAE-cellulose, and electrofocusing. However, all of these procedures permit fractionation of only small quantities of heparin, and have not proved to be practical for the large scale isolation of high-activity heparin in high yield.

It is, accordingly, a primary object of this invention to provide an improved procedure for the isolation of high-activity heparin in relatively high yields practical for large commercial production.

Another object of the invention is to provide an improved procedure for the separation of high-activity heparin from a mixture thereof with low-activity heparin employing affinity chromatography techniques.

A further object of the invention is to provide an improved procedure for the isolation of high-activity heparin in accordance with the preceding objects, which is relatively simple and economical to perform.

The above and other objects are achieved in accordance with the present invention by providing an affinity chromatography method by which commercially available heparin may be readily separated into fractions of relatively high specific activity and relatively low specific activity. The method utilizes an affinity column composed of protamine coupled to a water-insoluble solid support material such as agarose. The mixture of high-activity heparin and low-activity heparin (e.g., commercially available heparin) is dissolved in a solvent of about 0.6 molar sodium chloride buffered with about 0.01-0.03 molar imidazole to pH 6.5-7.5, and the resulting heparin solution is then applied onto the affinity column. Thereafter, a plurality of heparin fractions are successively eluted from the affinity column with a series of elution buffers of successively increasing molarity of sodium chloride buffered with about 0.01-0.03 molar imidazole to pH 6.5-7.5. The elution buffers employed have a sodium chloride molarity ranging from about 1.3 to about 2.0. In this manner, there is effected a stepwise removal from the affinity column of first the heparin of relatively low specific activity and finally the heparin of relatively high specific activity.

The affinity chromatography method of the present invention employs an affinity column composed of protamine coupled to a water-insoluble solid support material. Protamine is a simple protein material which is obtained from the sperm of certain fish, yields basic amino acids on hydrolysis, and which is used in medicine to neutralize heparin and to control hemorrhage.

Suitable water-insoluble solid support materials include, for example, agarose, cross-linked dextrans such as Sephadex, hydroxyl-modified polyacrylates such as hydroxyl ethyl methacrylate polymer, agaropectin, collagen, glass, silica clays, cellulose derivatives, and the like. The preferred support material is agarose, for example, Sepharose gel manufactured by Pharmacia Fine Chemicals, Uppsala, Sweden.

Preparation of the affinity column material may be carried out by the standard coupling techniques well known in the art, for example, the cyanogen bromide coupling technique (Cuatrocasas, J. Biol. Chem. 245, 3059, 1970), wherein the support material is first activated with cyanogen bromide, and the protamine thereafter coupled to the activated support material.

The heparin starting material employed in the method of the present invention is composed of a mixture of heparin of relatively high specific activity and heparin of relatively low specific activity, such as heparin commercially available from various sources, for example, Abbott Laboratories, North Chicago, Ill., and Sigma Chemical Company, St. Louis, Mo. A loading solution of the heparin mixture is prepared by dissolving the heparin mixture in a solvent of about 0.6 molar sodium chloride buffered with about 0.01-0.03 molar imidazole to pH 6.5-7.5. Preferably, such solvent is buffered with 0.02 molar imidazole to pH 7.35.

Prior to use, the affinity column should be equilibrated with the same solvent used in preparing the heparin loading solution, i.e., 0.6 molar sodium chloride buffered with about 0.01-0.03 molar imidazole to pH 6.5-7.5. Thereafter, the heparin loading solution is applied onto the affinity column.

The heparin is then eluted from the affinity column in a plurality of fractions by successive elution with a series of sodium chloride-imidazole elution buffers of successively increasing sodium chloride molarity ranging from about 1.3 to about 2.0. Each of the elution buffers is buffered with about 0.01-0.03, preferably 0.02, molar imidazole to pH 6.5-7.5, preferably 7.35. The stepwise elution is preferably carried out so as to obtain four separate heparin fractions, employing a first elution buffer having a sodium chloride molarity of from about 1.3 to about 1.4, a second elution buffer having a sodium chloride molarity of from about 1.4 to about 1.7, a third elution buffer having a sodium chloride molarity of from about 1.7 to about 1.9, and a fourth elution buffer having a sodium chloride molarity of from about 1.9 to about 2.0.

In this manner, there is effected a stepwise removal from the affinity column of first the heparin of relatively low specific activity and finally the heparin of relatively high specific activity. The penultimate heparin fraction eluted with the elution buffer having a sodium chloride molarity of from about 1.7 to about 1.9, will generally have a specific activity ranging from about 210 to about 260 IU/mg. The final heparin fraction eluted with the elution buffer having a sodium chloride molarity of from about 1.9 to about 2.0, will generally have a higher specific activity ranging from about 270 to about 305 IU/mg.

The invention is further illustrated by way of the following examples.

For the preparation of protamine-Sepharose gel, 500 ml of Sepharose 4B swollen in distilled water was equilibrated with 0.2 M Na.sub.2 CO.sub.3, then suspended in one liter of 0.2 M Na.sub.2 CO.sub.3, and 100 g cyanogen bromide in 100 ml dimethyl formamide added. The temperature was maintained between 15.degree. and 20.degree. C. by the addition of crushed ice, and the pH was maintained by the dropwise addition of 50% sodium hydroxide. After ten minutes, the gel was washed in a Buchner funnel under suction with 5 liters cold (4.degree. C.) 0.15 M sodium phosphate buffer, pH 7.0, and with one to two liters of distilled water, (4.degree. C.). Suction was continued until excess water was removed. 500 ml of 2% (w/v) protamine in 0.2 M sodium bicarbonate were then added to the wet gel, and the pH adjusted to 8.2 by the addition of 1 M NaOH. Incubation with gentle stirring for two and a half hours at room temperature was followed by washing sequentially with 10 liters of distilled water, 10 liters of normal saline and 5 liters of 1 M sodium chloride. Final equilibration was with 0.15 M sodium chloride, 0.02 M imidazole, pH 7.35. The resulting protamine-Sepharose gel was stored with 0.02% sodium azide at 4.degree. C.

A plastic affinity column (0.9.times.16 cm) packed with protamine-Sepharose gel as prepared in Example 1 above, was equilibrated with 0.6 molar sodium chloride-0.02 molar imidazole buffer solution, pH 7.35. A heparin loading solution was prepared by dissolving 100 mg commercial heparin (Sigma Chemical Co., specific anticoagulant activity 158.7 IU/mg) in 2 ml of 0.6 molar sodium chloride-0.02 molar imidazole buffer solution, pH 7.35. The loading solution was applied onto the equilibrated protamine-Sepharose affinity column. Gradient elution was then carried out with a series of elution buffers of successively increasing molarity of sodium chloride ranging from 1.3 to 2.0, buffered with 0.02 molar imidazole to pH 7.35. The eluates were pooled into four separate fractions over the eluant sodium chloride molarity ranges of 1.3-1.4, 1.4-1.7, 1.7-1.9, and 1.9-2.0, respectively. A 2.5.times.30 cm column of Sephadex G-25 equilibrated with distilled water was used for desalting the pooled fractions. Each of the pooled fractions was then analyzed for heparin specific activity by both the kaolin activated partial thromboplastin time assay of Proctor et al (AM. J. CLIN. PATH. 36:212-219, 1961) and the X.sub.a neutralization assay of Yin et al (J. LAB. CLIN. MED. 81:298-310, 1973).

The results obtained when carrying out the above-described elution procedure on five separate protamine-Sepharose affinity columns, are set forth in Table I, below, wherein the heparin specific activity values given represent the means values and the deviations therefrom over the five columns. PG,9