JPS6191132A - Separation and purification of human growth hormone releasing factor - Google Patents

Abstract

PURPOSE:To carry out the separation and purification of human growth hormone-releasing factor, by the high-performance liquid chromatography using a specific silica gel as a reverse-phase partition stationary phase, and using a mixture of an aqueous solution of acetic acid and acetonitrile as an eluting solution. CONSTITUTION:Human growth hormone-releasing factor is separated and purified by high-performance chromatography using a reverse-phase partition stationary phase consisting of a silica gel wherein 3-18C alkyl or cyanopropyl groups are bonded through chemical bonds and the remaining silanol groups are etherified with trimethylsilyl groups (having an average particle diameter of 5-20mum and narrow particle size distribution, and preferably having a pore diameter of 10-30nm), and using an eluting solution consisting of a mixture of an aqueous solution of acetic acid and acetonitrile, or a mixture of ammonium acetate buffer solution and acetonitrile, or their combination. The separated factor is useful as a diagnostic agent for the examination of the growth hormone productivity of the human anterior pituitary or remedy for dwarfism.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing human growth hormone releasing factor (hereinafter abbreviated as Gl?F).

薇Ju group GRF is a polypeptide consisting of 44 amino acids that was extracted from human pancreatic tumor, structurally determined, and chemically synthesized by Guillemin et al. in 1982 (Scie
nce 218. 585 (1982), Na
ture, 300, 276 (1982)), and is a physiologically active substance that has human growth hormone-releasing activity and can be used as a test agent for the growth hormone-producing ability of the human anterior pituitary gland or as a therapeutic agent for dwarfism.

GRF can be chemically synthesized by a liquid phase method or a solid phase method, but in this case, the synthesis process is long because amino acids are condensed one by one, and there is a high probability of contamination with impurities.

Therefore, purification of the final product requires a combination of gel filtration, ion exchange chromatography, partition chromatography or other methods. In some cases, it may be necessary to repeat the purification operation.

Conventionally, open-column gel filtration, ion-exchange chromatography, partition chromatography, or countercurrent partition have been used to produce chemically synthesized peptides. There are many problems such as it is time consuming, the recovery rate is low when purifying to high purity because the separation power is not high, and adsorption phenomena are observed in the column etc. during purification when dealing with trace amounts of peptides. had.

On the other hand, high performance liquid chromatography (HPLC) is a method that has rapidly developed in the field of chromatography in recent years.
(abbreviated as ), which has recently come to be used for the analysis of proteins and peptides. This HPLC method has an extremely high separation ability between peptides (1K) compared to conventional methods, can also handle at FJ components, and is simple, rapid, and has good reproducibility, making it suitable for analysis. Its applications are increasing steadily.

Using a reversed phase column, Riv i cr et al.
We report on the preparative purification of synthetic peptides containing F by HPLC (J, Chromatogr.
288 (1984) 303).

They mainly use triethylamine-phosphate buffer/acetonitrile-based /8 syneresis, but the inventors' test δ
- According to the results of 1, this system had low GRF purity and recovery rate, and required post-treatment such as desalting, making the operation complicated. Furthermore, although the purity of the peptides obtained is good with the eluent of water, acetonitrile, and trifluoroacetic acid, which is the most commonly used analytical condition for peptides, in the case of basic peptides such as GRF, trifluoroacetic acid is bound as a salt. It was recognized that it has drawbacks such as difficulty in desalting and salt exchange and requiring complicated operations. Therefore, the present inventors wish to develop a purification method that takes advantage of the characteristics of the HPLC method, such as good separation ability, simple and rapid method, and also has good reproducibility and recovery rate that can be used for the production of peptides such as GRF. , we have been considering it because we believe that its utility value is extremely large. That is, column size, column packing material, and 8 We focused on examining syneresis. In particular, packing materials are closely related to separation performance, sample load, recovery rate, etc., so the shape of the silica gel used as a carrier, particle size, pore size, functional groups chemically bonded to it, residual silanol treatment, etc. We also considered various points.

Based on the results of these 5 experiments, a filler in which an alkyl group or cyanopropyl group having 3 to 18 carbon atoms was chemically bonded to silica gel was used as the filler, and a mixed solvent of acetic acid aqueous solution/acetonitrile and ammonium acetate were used as the eluent. Reversed-phase HP using a mixed solvent of buffer and acetonitrile
We have completed the present invention by discovering a method that allows GRF to be purified quickly, easily, with good reproducibility and recovery rate, and to a high degree of purity using the LC method.

! '! ＋ (7) 3Rusei, -man "Liyu U This invention is,
In high-performance liquid chromatography, silica gel is used as a reversed-phase partitioning stationary phase to which alkyl groups or cyanopropyl groups having 3 to 18 carbon atoms are chemically bonded, and the remaining silanol is etherified with trinotylsilyl groups. Is the liquid a mixed solvent of acetic acid/acetonitrile or ammonium acetate? (This invention provides a method for purifying human growth hormone-releasing factor, which is characterized by using a mixed solvent of acetonitrile or a combination of these two types of solvent systems.)

In the method of the present invention, ordinary chemically bonded HPLC fillers for reverse phase distribution can be used as the filler, but
Particularly preferred is a silica gel for HPLC to which an alkyl group such as an octadecyl group or an octyl group or a cyanopropyl group is chemically bonded.

The silica gel preferably has a spherical shape, an average particle diameter of 5 to 20 μm, and a narrow particle size distribution, and a pore diameter of 10 to 3 Q nmO. Furthermore, after chemically bonding the above alkyl group or cyanopropyl group to the silica gel, the remaining silanol groups on the silica gel surface are almost completely treated with a suitable silane cuffing agent, such as trimethylchlorosilane or hexamethyldisilazane. Etherification is more preferred. If this treatment is not performed, GRF will be adsorbed onto the silica gel surface due to interaction with silanol groups, resulting in a decrease in recovery rate.

As a packing material for chemically bonded HPLC for reverse phase distribution under such conditions, for example, Nucleosil C18 (
Nagel), YMC-GEL 00S (Yamamura Chemical), Develosil ODS (Nomura Chemical)
YMC-GEL C8 (Yamamura chemical type), 5ynchr
opak RP-P (manufactured by Zinchrome), YMC
-GEL CN (Yamamura chemical type) and the like.

The column used in the present invention has a normal internal diameter of 1 lfl~
A column having a diameter of about 50 mm and a length of about 250 to 500 mm is used, and an appropriate combination with a packing material is selected depending on the objective such as the amount of sample to be loaded, the purity to be obtained, and the price of the column.

For GRF analysis, a column packed with the packing material of the present invention with a particle size of 5 μm in a stainless steel tube with an inner diameter of 4.6 mm and a length of 250 mm is used, but for preparative purification of GRF, a single sample loading is required. When the amount is several mg to 10 mg, a column is prepared by filling a stainless steel tube with an inner diameter of IQ mm and a length of 25 Q mm with the packing material of the present invention having a particle size of 5 to 7 μm,
Or, if the sample loading amount is 100 mg or more,
A column made of stainless steel with an inner diameter of 30 to 50 mm and a length of 250 to 500 mm filled with the packing material of the present invention having a particle size of 7 to 20 μm is recommended.

The eluent used in the method of the present invention is a mixed solvent system of an acetic acid aqueous solution and acetonitrile, or a mixed solvent system of an ammonium acetate buffer solution and acetonitrile, and an isocratic elution method is used in which the mixing ratio is appropriately set and elution is carried out at a constant composition. can be successfully preparatively purified. Furthermore, a gradient elution method in which the acetonitrile concentration is continuously increased has excellent separation performance and reproducibility, and allows effective preparative purification of GRF. In the case of the isocratic elution method, when using a mixed solvent system of aqueous acetic acid and acetonitrile, the mixing ratio of the solvents is acetonitrile concentration of 10 to 30% (% means V/V%, hereinafter the same), ammonium acetate buffer In the case of liquid acetonitrile, the concentration of acetonitrile is preferably in the range of about 20 to 40%.
In the case of the gradient elution method, when using an acetic acid aqueous solution/acetonitrile mixture, the acetonitrile concentration should be
In the case of ammonium acetate/acetonitrile, it is preferable to increase the acetonitrile concentration in the range of 20 to 40% over time (for example, 30 minutes to 1.5 hours). It is preferable that the acetic acid aqueous solution used in the method of the present invention has a concentration of 1 to 5%, and that the #acid ammonium buffer has a concentration of 0, an O of 1 to 0.2 molar concentration, and a pH of 3 to 5. These eluents are volatile, and highly pure GRF can be easily obtained from the eluate separated by conventional vacuum distillation or freeze-drying.

The present invention will be explained in more detail with reference to Examples below. The embodiments of the present invention are not limited only to the following examples.

Purification was carried out under the conditions shown below using silica gel into which an octadecyl group had been introduced.

GRF crude product 300 with purity of 76.0% by wood refining method
With a recovery rate of 51.8% and 72.8%, the purities were able to be purified to 98.0% and 94.9%, respectively.

GRFO preparative separation conditions (1) are shown below.

Column: Octadecylated HPLC silica gel IC-GEL ODS Type A; manufactured by Yamamura Kagaku Kenkyusho.

Spherical, particle size 1um, pore size 10nm) with an inner diameter of 30mm,
Filled in a 250mm long stainless steel tube.

4i? &: 2s % acet
Nitrile 10.05M Ammonium acetate (p
H3, 6) j Dispense method near isocratic method? *Fit: 5.85m6/min Temperature: Room temperature Detector: Ultraviolet absorption meter (measurement wavelength 280nm) GRF
Chromatograms of the crude product and the purified product obtained are shown in FIGS. 1 and 2.

Note that the purity test was performed using HPLC conditions not shown below.

The analysis conditions (purity assay method) for GRF are shown below.

Column: Octadecylated HPLC gel (YMCGel, 005 Type A M; manufactured by Hakuichi Kagaku Kenkyusho, r, Y shape, particle size 5 μm, pore size 10n)
m) was filled into a stainless steel tube with an inner diameter of 4.6 mm and a length of 250 mm.

one? ? :δ1: 8; & 0.1% trifluoroacetic acid in acetonitrile/water (20%: 80%) solution B & 0.1% trifluoroacetic acid in acetonitrile/water (50%: 50%) Gent condition: B? Pi, is increased linearly from 30% to 80% in 50 minutes.

Flowffi: 1. Oml/min Temperature: Room temperature detection "g'jI, UV absorption meter (measurement wavelength 220r
1m) Final Example 2 Purification was carried out under the conditions shown below using silica gel into which an octadecyl group had been introduced.

GRF crude product 100 with purity of 76.0% by wood refining method
It was possible to refine the product to a purity of 96.2% with a recovery rate of 78.1%.

GRF preparative separation conditions (2) are shown below.

1L-yA: Octadecylated silica gel for HPLC C-GEL ODS Type A; manufactured by Yamamura Kagaku Kenkyushin, spherical, particle size 1O ~ 20 μm, pore size 10
) filled in a stainless steel tube with an inner diameter of 50 mm and a length of 500IIIffl.

filFIi't;t: 28% acetonitrile 10.05M ammonium acetate (p)t3.6)? J-Arashi 1 Rebellion Flow rate: 22
．． 5 mi! /min Temperature: Room temperature Detector: Ultraviolet absorption meter (measurement wavelength 280 nm) Example 3 Purification was performed under the conditions shown below using silica gel into which an octadecyl group was introduced.

By this purification method, GRF crude product 415 with a purity of 8164% was obtained.
It was possible to refine the product to a purity of 98.5% with a recovery rate of 43.4%.

G RFO preparative separation conditions (3) are shown below.

Column: Octadecylated silica gel for HPLC (YMCGEL 005 Type A; manufactured by Yamamura Kagaku Kenkyushin, spherical, particle size 7 pm, pore size 10 nm)
) filled in a stainless steel tube with an inner diameter of 30 mm and a length of 250 mm.

jfi L'lJ i (1: Solution A 3% acetic acid aqueous solution/acetonide 'J JLim?'11. (8
0/2 0) Solution B 3% acetic acid aqueous solution/acetonitrile Runn 11 (50150) Elution method Igradient conditions) 2 After flowing Solution B at 5% for 10 minutes,
% to 15% in 70 minutes.

Flow rate: 9 m+2/min Temperature: Room temperature Detector: Ultraviolet absorption meter (measurement wavelength 220 nm) J-Kei, (1- Purification was performed under the conditions shown below using silica gel into which an octyl group was introduced.

According to this purification method, 3 mg of GRF with a purity of 80.6% could be purified to a purity of 99.6% with a recovery rate of 94.4%.

GRF preparative separation conditions (4) are shown below.

Column: Octylated silica gel for HPLC <YMC-GEL Cs Type A; manufactured by Yamamura Kagaku Kenkyushin, spherical, particle size 5. p m, pore diameter 10n
m) filled in a stainless steel tube with an inner diameter of 10 mm and a length of 250+++ m.

? @: 1tUL: Helium 20% acetonitrile/
80% 0.1M ammonium acetate buffer (pH, 4,3> B solution 50% complete, tonitrile 150% 0.1M ammonium acetate buffer (pl! 4.31) Elution method (gradient conditions): B?li is increased linearly from 30% to 40% over 40 minutes.

Flow rate: 3. Omj! /min Temperature: Room temperature Detector: Purification was carried out under the conditions shown below using a comparative octadecyl group-introduced silica gel on an ultraviolet absorption meter (measurement wavelength: 220 nm).

GRF crude product 180 with purity 72.1% by wood refining method
It was possible to refine the product to a purity of 88.1% using 8.5% recovered weight.

However, the purity of the triethylamine-phosphoric acid-based dissolved Allium solution in this experimental example is low (to reach the purity level of Examples 1 to 4, the recovery rate is considerably reduced (40% or less).
Furthermore, since the triethylamine-phosphate used in the eluent remains, the operation is complicated, such as requiring desalting.

GRF preparative separation conditions (5) are shown below.

Column: Octadecylated HPLC silica gel C-GEL ODS Type A; manufactured by Yamamura Kagaku Kenkyushin, spherical, particle size 7 μm, pore size Ions) packed in a stainless steel tube with an inner diameter of 3' Omm and a length of 250 mm.

Elution 8: 28% acetonitrile 10.25M phosphoric acid-triethylamine (pH 2,3) Elution method: Isocratic polymethod Flow rate: 22.5ml 17ml Temperature: Room temperature Detector: Ultraviolet absorption meter (measurement wavelength 280nm)

[Brief explanation of drawings]

FIG. 1 shows a chromatogram of the GRF crude product. FIG. 2 is a diagram showing a chromatogram of a GRF purified product. Figure 1 Figure 2 above

Claims (1)

[Claims] (1) When purifying human growth hormone-releasing factor using high-performance liquid chromatography, alkyl groups or cyanopropyl groups having 3 to 18 carbon atoms are chemically bonded as a reversed-phase partitioned stationary phase to remove residual silanol. A human growth method using silica gel etherified with a trimethylsilyl group, and characterized in that the eluent is a mixed solvent of acetic acid aqueous solution and acetonitrile, a mixed solvent of ammonium acetate buffer and acetonitrile, or a combination of these two types of solvent systems. Purification method of hormone releasing factor