HU195522B - Process for cleaning raw insulin by exclusion chromatography - Google Patents

Abstract

According to the invention, a hydrophobic alkylated silica gel stationary phase, which is equilibrated with an aqueous carrier phase containing 2 to 10% by weight of methanol and a buffer system of 1 to 3 pH, dissolved in the said carrier phase, is then applied to the crude insulin to be purified, followed by the main weight of pure insulin at a concentration of 10-100 mmol / l with the above carrier phase containing cetyl triinetylammonium bromide and optionally removing the residue from the stationary phase by displacement chromatography with a cetyltrimethylammonium bromide concentration above that used to remove the major mass. -1-

Description

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The present invention relates to a method for purifying insulin by displacement chromatography.

Insulin is an essential ingredient for the cure of people with diabetes. The pharmaceutical compositions generally contain porcine or bovine insulin. Animal insulins are processed during processing into a variety of substances such as insulin degradation products, insulin derivatives, insulin precursors and other polypeptide-like substances. These are detrimental because, to achieve the same therapeutic effect, they need to be given more and more insulin to patients: so-called increasing insulin starvation. In this respect, the pro-insulin precursor, insulin, has the most unfavorable effect. Gel chromatography methods, ion exchange methods, and combinations thereof are commonly used to remove impurities in insulin formulations. Of the first group, noteworthy was a crude insulin on a Sephadex G-50 (Pharmacia, Sweden) dextran gel with a 0.01 M hydrochloric acid eluent [J. Agr. Food Chem., 19 (4), 581 (1971)] and purification of Sephadex LH-60 (Pharmacia, Sweden) on a hydrophobized dextran gel with 65% ethyl alcohol, 35% acetate buffer, pH 7.3 (European 0439). patent application). A common disadvantage of gel chromatographic purifications is that they do not remove impurities of molecular weight close to insulin.

Effective ion-exchange purification is disclosed in German Patent Publication No. 2,629,568, which discloses an eluent containing 1% nonionic detergent, 0.1 M Tris buffer, pH 7, and 0.5 M sodium chloride. , DEAE Sephadex A-25 ion exchange (Pharmacia, Sweden).

It is well-suited to use a SP Sephadex C-25 ion exchanger (Pharmacia, Sweden) filled in a basket centrifuge and a 0.1 M Tris buffer pH 8.2. It is also a process using 65% ethyl alcohol (German Patent Publication No. 2,757,377).

A combination of ion exchange and gel chromatography purification steps is used in Diabetes, 17, 725, (1968), after pre-purification on a Biogel P-30 gel with pH 7.4 containing 7 U / L urea and 0.01 M citrate buffer DEAE-Cellulose (Pharmacia, Sweden) purifies insulin by ion exchange.

Column chromatography is usually used to separate the components of complex mixtures. The columns can be used in frontal, elution and displacement modes.

The most widespread mode of operation, even in the case of preparatory separations, is elution. During the formation, the components bound on the column, occupying only a small proportion thereof, are washed off the column with a solution which is less bound than the component, the so-called eluent.

However, the eluted sample is diluted during elution, which is disadvantageous for preparative separations.

In the case of displacement chromatography, which was first described by Ark. Kern. Mineral Geol. 16 (A), 1, (1943) described the component to be bound on the column with a more adsorbent component than the column. In a multicomponent pattern, the components are located in successive zones, which are filled at the same velocity by the displacement material front. Ideally, the concentration of the components leaving the column varies in a rectangular wave. The advantage of this method is that during separation, the column occupies a significant portion of the column and the components do not dilute during separation. It has been shown [J. Chromatogr., 218, 365 (1981) and J. Chromatogr., 215, 295 (1981)]. that a successful series of displacement chromatographic separations requires the formation of a series of equilibrium and constant velocities. In this case, the concentration of the components depends only on their material quality and the position of the working line, while the width of the zones depends on the amount of material applied to the column.

Previously, the displacement chromatography procedure had been carried out only on a small scale. used primarily for the separation and purification of components with a relative molecular weight of less than 500 (Chromatogr., 218, 365 (1981)). This is due to the rapid diffusion of the low molecular low bandwidth setting.

Using silica gel alkylated with lower alkyl groups and ion-pair chromatography, it was possible to extend the molecular weight limit of the components separable by displacement chromatography to 1200 [J. Chromatogr., 215, 295 (1981)]. In this way, the isolation of Polymixin B-type antibiotics was achieved under laboratory conditions using an aqueous solution of octyldodecyldimethylammonium clone displacement agent.

Chromatographic eluates report a two-dimensional gel electrophoresis assay in aClin. Chem., 28 (4), 998-999, (1982). Methods for assaying proteins for ion exchange chromatography are described in Methods Enzymol, 104, 113-13 (1984).

Surprisingly, it has now been found that displacement chromatography can also be used to purify high molecular weight crude insulin by equilibrating with a hydrophobic aqueous phase containing 2 to 10% v / v methanol and a pH 1 to 3 buffer system. dissolving the crude insulin to be purified into said stationary phase of an alkylated silica gel in said carrier phase, followed by the above carrier phase containing cetyltrimethylammonium bromide at a concentration of 10-100 mmol / l of pure insulin and, if desired, is removed from the stationary phase by displacement chromatography with the aforementioned carrier phase containing ammonium bromide.

The present invention is based on the discovery that very high molecular weight polypeptide insulin is bound to the column by strong silanophilic and hydrophobic interactions in reverse phase liquid chromatography systems [J. Chromatogr., 236, 51 (1982)], can be used to purify weakly bound impurities using a suitable displacement agent capable of both silanophilic and hydrophobic interactions. According to the present invention, displacement chromatography can reduce the time required for separation, increase the concentration of the more strongly bound components, improve the productivity of the preparative displacement separation, and reduce the amount of inactivated prior to re-separation of the completely separated components. (This kind of separation method is hereafter called gradient

Another advantage of the present invention is that, using a gradient displacement, insulin formulations containing impurities can be purified to a preparative size in a single operation. According to the present invention, the extent of the overlap between adjacent component zones is achieved by the fact that the degree of overlap between the adjacent component zones is primarily dependent on the column pack grain size, bed structure and flow rate, but not on the actual displacement concentration. If the purpose of the separation according to the invention is to purify the weakly bound components of the sample 10 purely and to prepare the more strongly bound components purely, the separation begins with a low concentration of displacement agent, and after purifying the desired components has left the column 15, concentration or feed! speed. This results in the removal of residual material from the column at a higher concentration than the original with minimal volume of solution.

The buffer of the present invention is preferably a phosphoric acid / dihydrogen phosphate buffer system. It is particularly advantageous if the carrier phase contains 210% by volume, for example 5% by volume of methanol, and the concentration of phosphate buffer is 10-100 mmol / l. preferably 50 mmol / l. 25

The raw crude insulin used in our exemplary embodiment was commercially available crude bovine insulin. The crude bovine insulin content was 76.7%.

Further details of the present invention are illustrated by the following non-limiting examples.

Example 1

A column of 4.6 mm internal diameter, 250 mm long, 5 μπι grain size (Nucleosil C8. Alkylated silica gel, Macherey-Nagel, Federal Republic of Germany) filled with a hydrophobic stationary column containing 5% v / v methanol, pH 2.2, It is equilibrated with 50 mM aqueous phosphate buffer (carrier phase). The column is injected with 1400 μΐ of the above carrier phase and injected with 140 mg of contaminated crude bovine insulin at a flow rate of 0.1 ml / min. After sample application, a displacement medium of 10 mM cetyltrimethylammonium bromide at a flow rate of 0.1 ml / min is applied to the column. Measure the absorbance of the solution exiting the 45 columns at 295 nm using i. The displacement chromatogram shown in FIG.

The insulin sample leaves the column between 76 and 114 ml in an eluent volume of 38 ml at an average concentration of 3.7 mg / ml. Calculated from the displacement chromatogram (Fig. 2), computed from the HPLC analysis of the fractions, the amount and purity of the insulin in the sample, depending on the volumes of eluate selected for use, are as follows: _cc

From the data in the table it can be seen that 50.6% (88 mg) of the 107.4 mg (100% purity) insulin used as starting material in crude bovine insulin (140 mg) was obtained in a purity of 90.7% as exemplified. The yield obtained at the expense of the purity of the final product may be increased.

Example 2

All of the procedures described in Example 1 were followed except that 150 mg of crude bovine insulin was used and eluted with a 25 mM cetylnonium bromide carrier phase. The displacement chromatogram at 295 nm is shown in Figure 3. Insulin leaves the column at a mean concentration of 8.8 mg / ml between 34 and 51 ml of eluent. The displacement chromatogram constructed from the analysis of the fractions is shown in Figure 4. From this it can be calculated that 15.3% of the 115 mg of pure insulin contained in 150 mg of crude insulin can be recovered in 99% purity using fractions between 34 and 36 µl.

Example 3

All procedures described in Example 1 were followed, except that elution was continued after passing through 92 ml of a 10 mM cetyltrimethylammonium bromide carrier phase with a 50 mM cetyltrimethylammonium bromide carrier phase. The displacement chromatogram at 295 nm is shown in Figure 5. The displacement chromatogram obtained from the analysis of the fractions is shown in Figure 6. This shows that after the 95 ml fraction, the concentration of insulin and impurities in the column solution increased significantly. The presence of cluted substances at 102 ml of eluate volume returns to baseline. By this time all the applied material had come down from the column. Thereafter, the column is rinsed again with the carrier phase, which does not contain cetyltrimethylammonium bromide, and becomes again suitable for purifying insulin.

Similar results to those of Examples 1 and 2 were obtained when the column packing was made from octadecylated silica gel (LiChrosorb RP-18 from Merck, Germany) instead of Nucleosil C8 above, or when the pH of the carrier phase was between 1 and 3. or by eluting the bulk mass of pure insulin with the above vehicle phase containing 10-50 mM cetyltrimethylammoniumumbonide. The methanol content of the solutions used for the loading and dual application of crude insulin was between 2 and 10% by volume.

Selected eluent fraction

Insulin content of selected fraction [mg] Purity [%]

77 to 82 ml 16 > 99 77 to 87 ml 38 96.6 77 to 92 ml 61 94.2 77 to 97 ml 83 91.3 77 to 98 ml 88 90.7

Claims (6)

A process for purifying crude insulin by displacement chromatography, comprising applying to said stationary phase a hydrophobic, alkylated silica gel, equilibrated with an aqueous carrier phase containing 2 to 10 volumes of methanol and an aqueous carrier phase having a pH of 1 to 3, crude insulin followed by the main weight of pure insulin at a concentration of 10-100 mmol / liter cetyltrimethylammonium3 The residue is eluted with the above carrier phase containing 195,522 bromides and, if desired, the remainder is eluted with the above carrier phase having a concentration of? -Trimethylaminonium bromide greater than that used for bulk removal. The process of claim 1, wherein the bulk of pure insulin is eluted with an aqueous carrier phase having a concentration of 10 mM cetyltrimethylammonium bromide. 3. The process according to claim 1, wherein the aqueous carrier phase is a mixture comprising a pH 2.2 phosphate buffer. A process according to claim 1, wherein the aqueous carrier phase comprises 5% by volume of methanol. 5 mixtures were used. The process according to any one of the preceding claims, wherein the aqueous carrier phase is a mixture of phosphate buffer at pH 2.2, 50 mM and 5% methanol. 6 drawings, 6 figures Published by the National Office for Inventions; Zoltán Hinier Head of Department Published by Technical Publisher COPYLUX Printing and Duplicating Small Cooperative