JPS58126813A - Purification method of crude secretin - Google Patents

Abstract

PURPOSE:To purify crude secretin useful for treating peptic ulcer, etc. in high purity and yield, by using the change in the concentration of a hydrophilic organic solvent in a mixed solvent used as a mobile phase in the reversed-phase liquid chromatography. CONSTITUTION:In purifying crude secretin by a reversed-phase liquid chromatography using a reversed-phase silica gel as a fixed phase and a mixed solvent of a hydrophilic organic solvent, e.g. isopropyl alcohol, with water as a mobile phase, the change in the concentration of the hydrophilic organic solvent is set within 20-90% range to carry out the adsorption, separation and elution of the aimed secretin. If the concentration of the isopropyl alcohol is <=20%, the secretin is strongly adsorbed in th fixed phase and washed with the mixed solvent of the above-mentioned concentration. If the concentration of the isopropyl alcohol is >=25%, the secretin is desorbed from the fixed phase and can be eluted with the mixed solvent of the above-mentioned concentration.

Description

[Detailed description of the invention] The present invention relates to a method for purifying crude secretin.

Secretin is a type of gastrointestinal hormone, ie, a pancreatic secretion hormone, and is a useful substance used in the diagnosis of visceral and gallbladder diseases, and in the treatment of bleeding, ulcers, etc. in the gastrointestinal tract. Initially, the use of this substance was limited to very narrow areas, but since then its usefulness has been recognized in a variety of areas, and its applications are rapidly expanding. As a result, it is expected that there will be many opportunities to administer the substance as a so-called intravenous injection.
Therefore, in the medical field, highly purified substances are required.

In general, what are known methods for purifying polypeptides such as secretin?

For example, a salting out method using an inorganic salt such as common salt or ammonium sulfate, an organic solvent precipitation method using an organic solvent such as acetone, methanol, or ethanol, a purification method using selective adsorption/desorption using an ion exchange resin, Alternatively, industrially, these methods may be further combined as appropriate to purify the target product. - For example, when tryingpsin or chymotrypsin is manufactured industrially using bovine pancreas as a raw material, it is first extracted together with various other components contained in the pancreas under acidic sulfuric acid, and then concentrated by salting out with ammonium sulfate. After crystallization, desalting, and finally freeze-drying or jet drying, the desired specimen is obtained.

However, when manufacturing by conventional techniques, the yield is generally poor, and it is not possible to completely remove contaminant foreign components, so it is usually not possible to obtain a product of high purity. The same is true for the crude secretin according to the present invention, which is 5,000 units (C,
The situation is such that secretin of the same level as H, R, and single tl is finally being obtained.

In view of these circumstances, the present inventor investigated a method for industrially producing secretin of high purity using a simple and simple operation, and found that using reversed-phase silica gel as a stationary phase and a hydrophilic organic solvent and water. I learned that the intended purpose could be achieved by performing reversed-phase liquid chromatography using a mixed solvent as the mobile phase and changing the concentration of the hydrophilic organic solvent in the mixed solvent.

The invention has been completed.

The present invention will be explained below.

The gist of the present invention is to perform the following reverse phase liquid chromatography in purifying crude secretin. Specifically, in the chromatography, for example, octadecylated silica gel is used as a stationary phase, a mixed solvent of a hydrophilic organic solvent and water is used as a mobile phase, and the concentration of the hydrophilic organic solvent in the solvent is varied. 9 separations of secretin by adsorption.

It is to carry out elution.

The octadecyl silica gel serving as the stationary phase is a filler in which octadecyl groups are chemically bonded to silica gel, and the trade names and manufacturer names of available products are as follows.

μmBondapak C1g (Waters) Nuc 1eos i l C@@ (
Nagel) PoLygos mouth C1, (Nagel) Zorbax C16 (DuPont) jlnisil C16 (Gascro Industries) Unisil
Q (,1g Of Sukuo Kogyo) ODS-
) IYPER8IL (Chaton) 8PHELE I8U BOD8 (PS) In addition, the hydrophilic organic solvent in the mixed solvent of a hydrophilic organic solvent and water that is the mobile phase may be any organic solvent that is miscible with water in any proportion. Well 9 for example.

Mention may be made of isopropyl alcohol, ethyl alcohol, methyl alcohol and acetonitrile.

Isopropyl alcohol is a particularly preferred example.

The feature of the present invention is that it has been found that when the concentration of the hydrophilic organic solvent in the mixed solvent changes, secretin is adsorbed to and desorbed from the stationary phase accordingly. In other words, it has been found that secretin can be easily separated from other impurities during this adsorption/desorption process. For example, as shown in the Examples below, when an isopropyl alcohol water mixed solvent is used as the mobile phase, when the isopropyl alcohol concentration is 20% or less, secretin is strongly (adsorbed) to the stationary phase, so the concentration Secretin is clearly separated from other impurities by washing with a mixed solvent of Therefore, as a concrete procedure, crude secretin is first loaded onto the stationary phase, and isopropyl alcohol and water mixed solvent is used.

It is sufficient to wash thoroughly with isopropyl alcohol having a concentration of 20%, and then to elute with E2796. After cleaning with a 24% concentration solution, wash with a 24% concentration solution.
Finally, it may be eluted with 27% of the same. In short, 2 for each solution used for washing and elution.
The chromatography can be carried out by monitoring the adsorption or desorption of secretin by keeping the absorbance value of 10 fins constant and changing the alcohol concentration as appropriate. , is extremely simple and convenient.

In addition, in order to ensure the stability of secretin in the process of carrying out the present invention, it is desirable to maintain the liquid nature of the mixed solvent at an acidic level, particularly at a pH of 5 or less. Therefore, it is a preferred embodiment to use a mixed solvent of, for example, alcohol and N/100 hydrochloric acid. Specifically, it will be shown in the examples below.

A mixed solvent of isopropyl alcohol and N/100 hydrochloric acid with a concentration of 11% isopropyl alcohol and 20% or 27% may be used.

Next, there are no particular requirements to limit the crude secretin to which the present invention is applied. In short, any source secretin raw material that is contaminated with other components and has low purity is sufficient. In general, most secretin is obtained by extraction from pig duodenum, and therefore gels are not obtained from extracts from pig duodenum.

and ion exchange chromatography.

If the present invention is applied to crude secretin in which contaminant foreign proteins are not completely removed even by these treatments and the purity remains low, the clear results of the present invention can be obtained.
・Also, the secretin-containing eluate obtained by carrying out the present invention may be used as it is or diluted with water and freeze-dried to obtain secretin as dried rice. It may also be obtained as a highly pure dried product by performing lithography and finally freeze-drying. The present invention is not limited to these post-treatments.

The effects of the method of the present invention are clearly demonstrated by comparing the number of active units of secretin and the peaks of high performance liquid chromatography before and after implementing the method of the present invention. For example, as shown in the Examples below, the number of active units increased nine times, and the liquid chromatography peak was only for secretin.

The present invention will be explained in more detail with reference to the following examples.

Example 1 Extracts from pig duodenum were exposed to organic solvents.

Furthermore, gel filtration (5 ephadex G-25) and ion exchange chromatography (CM-cellulose and QAE-8 ephadex) were performed and lyophilized secretin was used as a raw material.

Nucleosil C, @ diameter 5.7ca * height 3
The column was loaded with crude secretin 4f dissolved in water, washed with methanol and water, and loaded with isopropyl alcohol and N/N.
150 ml of 41, a mixed solvent with 100 hydrochloric acid and an isopropyl alcohol concentration of 2096
It was drained and washed at a rate of 100 min.

Next, a mixed solvent of isopropyl alcohol and N/100 hydrochloric acid with an isopropyl alcohol concentration of 27
96, 31, was eluted by flowing out at a speed of 150 n/min, and was used as an eluent. After washing and elution, the absorbance at 210 nm of each fluxin solution was measured, and a chromatogram was obtained, as shown in FIG. 1. In the figure.

Continue until Blution Volume reaches 41.
The madogram is obtained when a mixed solvent with an isopropyl alcohol concentration of 20% is flowed out.

The chromatogram for 41 or above is the one obtained when a mixed solvent of 2796 was eluted.

In the figure, the non-shaded peaks are peaks due to contaminant components,
In addition, the peak in the shaded area is a peak due to secretin, as shown in the measurement results of the number of active units shown later and in Figure 21 and Figure 3.
This is clear from the high performance liquid chromatography results shown in .

Finally, the eluent was concentrated under reduced pressure and 5ephadex (j
-25 gel filtration, freeze-drying, and secretin 4 made from Hinoki cypress.
001! I got it.

Analytical Samples and Methods Using secretin and purified secretin as samples, the number of secretin activity units (C, H, L units) was measured and high performance liquid chromatography was performed under the following conditions.

Stationary phase: Nucleosil C, 5IJm (φ4.
Mobile phase: 6596 methylaldiyl water mixed solvent (0,5
96-1 (Contains C10) Flow rate: 1.2-/min Detection: 210nm Sample 1: 20μ1 Analysis result The number of secretin activity units is 1.50 in pro-secretin
0u/'! and 14, in purified secretin.
000 u/”? Tea Tsuta.

Next, the results of high performance liquid chromatography were as shown in -2 for the original secretin, and as shown in FIG. 3 for the purified secretin. Note that the numerical values shown in FIGS. 2 and 3 represent the elapsed time (min) after sample injection.

As shown in Figure 3, purified secretin exhibits almost a single peak, and the activity and activity of purified secretin are nine times higher than that of crude secretin.
The single peak shown in is determined to be due to purified secretin itself.

On the other hand, crude secretin exhibits two peaks, large and small, in FIG. 2, and its activity is 1/9 that of purified secretin. In other words, in addition to secretin, secretin slave devices exist as contaminant components, so the large peak in Figure 3 that indicates outflow at an early stage is due to contaminant components, and the small peak that indicates outflow at a late stage is due to contaminant components. It turns out that the other peak is due to secretin.

Example 2 Nucleosil C1 plate with a diameter of 10 mm and a height of 3
001vLTIL column as a stationary phase, washed well with methanol and water, and added crude secretin 10
A solution of 0.0 cm dissolved in 1-1 water was loaded. After that, I drained it out at a meth speed and washed it. Next, methanol and N/100
120-, a mixed solvent with hydrochloric acid having a methanol concentration of 5896, was allowed to flow out at a rate of 4 m/m111 to elute secretin. The eluate was concentrated under reduced pressure, and the gel was over-desalted using 8ephadex (j-25) and further freeze-dried to obtain purified secretin ioq. The secretin activity of this product increased to 13,500 u/W.

Example 3 JA-Bondapak Cts with a diameter of 10 mm,
A column having a height of 300 rrLrIL was packed as a stationary phase, washed thoroughly with methanol and water, and loaded with a solution of secretin 100 in water. Thereafter, a mixed solvent of isopropyl alcohol and N/loo hydrochloric acid having an isopropyl alcohol concentration of 2096 ml was flowed out at a rate of 4 wd/min for washing. Next, a mixed solvent of isopropyl alcohol and N/100 hydrochloric acid with an isopropyl alcohol concentration of 2
120 ml of 7% solution was flowed out at a rate of 4 d/min to serve as an eluent. The absorbance peak at 210 nm of the eluent was collected, diluted 3 times with water, and lyophilized.
Purified secretin 1OWII was obtained.

The secretin activity of this product increased to 13,0 OOU/''v.

[Brief explanation of the drawing]

FIG. 1 corresponds to FIG. 1 described in Example 1 and shows the results of liquid chromatography obtained when the method described in Example 1 is carried out. FIG. 2 corresponds to FIG. 2 described in the analysis results section of Example 1, and shows the results of high performance liquid chromatography for crude secretin. FIG. 3 corresponds to FIG. 3 described in the analytical results section of Example 1, and shows the results of high performance liquid chromatography on loaded secretin. Patent application ElutJonVO1ame (11) 2 Zeromi ml, n

Claims (1)

[Claims] (1) Crude secretin is purified to a high purity by reversed-phase liquid chromatography using reversed-phase silica gel as a stationary phase and a mixed solvent of a hydrophilic organic solvent and water as a mobile phase. (2) Claims in which the hydrophilic organic solvent is alcohol The method for purifying crude secretin described in Section 1 (3) The alcohol is isopropyl alcohol, and the concentration changes from 20 to
(4) A method for purifying crude secretin according to claim 2, carried out within the scope of Patent No. 9096, wherein the pH of the mixed solvent is 5 or more. Purification method