JPH10265499A - Purification of human growth hormone - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for inexpensively purifying a large amount of the subject hormone, useful as a therapeutic agent for pituitary dwarfism, chronic renal insufficiency, fracture, burn, etc., by subjecting human growth hormone solution to selective adsorption to and elution from a blue pigment- bound carrier. SOLUTION: This method purifies a large amount of the objective human growth hormone easily and in high purity, which is expected for expansion of applicable targets as a therapeutic agent for various disorders, such as chronic renal insufficiency, fracture and burn as well as for pituitary dwarfism, by subjecting a human growth hormone solution to affinity chromatography using its affinity for a blue pigment for selective adsorption to and selective elution from a blue pigment-bound carrier using eluting solution containing a protein- denaturing agent, such as 2-9 M urea, to remove contaminants in the human growth hormone solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for purifying human growth hormone.

[0002]

2. Description of the Related Art Growth hormone is a typical hormone secreted from the anterior pituitary gland, and is a hormone exhibiting growth promoting activity through production of somatomedin in the liver or binding to a receptor for growth hormone. In addition, it has recently been clarified that other effects are deeply involved in sugar / lipid metabolism, protein assimilation, cell growth and differentiation. It is known that the growth hormone produced from the human pituitary gland includes human growth hormone having a molecular weight of about 22,000 (hereinafter referred to as 22 KhGH) and human growth hormone having a molecular weight of about 20,000 (hereinafter referred to as 20 KhGH). 22KhGH and 20KhGH are on the same hGH gene,
Is thought to produce 20KhGH by alternative splicing against 22KhGH is a single-chain polypeptide consisting of 191 amino acid residues and accounts for 70-75% of the growth hormone in the pituitary gland of human adults. On the other hand, 20KhGH is a single-chain polypeptide consisting of 176 amino acid residues, in which the 15th amino acid residues from the 32nd to the 46th, counted from the N-terminal amino acid residue of 22KhGH, are deleted. 20K hGH has a 5-10% abundance in the pituitary gland of a human adult,
The ratio is lower than hGH.

[0003] In the future, human growth hormone is expected to be used as a therapeutic agent for pituitary dwarfism, as well as a therapeutic agent for diseases such as chronic renal failure, bone fractures and burns. Therefore, in order to stably supply human growth hormone as a therapeutic agent for these diseases, it is expected that an inexpensive mass production method that maintains high quality as a pharmaceutical and is inexpensive will become increasingly important in the future.

[0004] There have been many reports on the purification of human growth hormone, but most of them are based on the classical purification of proteins, and these methods utilize the precipitation method using ammonium sulfate and the properties of proteins. This is a method combining liquid chromatography methods such as gel filtration and ion exchange described above (Showa 56-21596, Showa 61-9312).
7). In addition, a hydrophobic chromatography method is known (Showa 56-30925). However, since the purification method described above has a large number of purification stages, it is considered that the final recovery rate may be extremely low or the purification cost may be high, and it is difficult to say that the purification method is an efficient purification method.

[0005] In contrast to the above-mentioned purification methods, affinity chromatography is the only adsorption chromatography method for purifying based on biological functions or individual chemical structures, and has a very high selectivity for adsorbed substances. Therefore, it is significantly more efficient than a multi-stage method with low selectivity, and is a useful purification method when high-purity and large-scale production is required, such as pharmaceuticals. In general, affinity chromatography is often applied to substances whose interactions are known, such as glycoproteins and lectins, immunoglobulin G and protein A, and enzymes and substrate substances. However, in the case of human growth hormone, no substance having an affinity other than proteins such as growth hormone binding protein and anti-growth hormone antibody is known, and a large amount of human growth hormone is obtained using a commercially available affinity chromatography gel. There are no reports on purification methods.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to establish a method for purifying human growth hormone which can be applied to the production of pharmaceuticals in a large amount, simply and with high purity.

[0007]

Means for Solving the Problems The present inventors have made intensive studies for the above purpose, and as a result, have found that a blue dye compound has a high affinity for human growth hormone and that a blue dye-binding carrier was used. It has been found that human growth hormone can be efficiently purified by affinity chromatography, and that human growth hormone can be selectively eluted by adding a protein denaturant to the eluate.

That is, an object of the present invention is to provide a method for purifying human growth hormone, which comprises selectively adsorbing and eluting a human growth hormone solution onto a blue dye-bound carrier.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As the blue dye used in the blue dye-bound carrier chromatography used in the method of the present invention, commercially available Cibacron Blue 3GA (Ciba
Geigy) is commonly used. For example, Japanese Patent Laid-Open No. 1-2
No. 2,2782 discloses a method for purifying a collagenase inhibitor using this blue dye. Formula 1 shows the structural formula of Cibacron Blue 3GA.

[0010]

Embedded image (Here, R ₁ and R ₂ are a hydrogen atom or a —SO ₂ ONa group.) Examples of the blue dye-binding carrier include Blue Sepharose CL-6B, Blue Sepharose 6FF (both Pharmacia) and Matrek Gel Blue A (Amicon Co., Ltd.), Affigel Blue (Bio-Rad), Blue Cellulofine (Seikagaku), TSKgel Blu
e-5PW (Tosoh Corporation) and the like. Further, a gel obtained by binding a blue dye to an activated gel can also be used. The blue dye-bound carrier according to the present invention is not particularly limited to the above blue dye-bound carrier.

In general, human growth hormone can be obtained by a method of producing it by a genetic recombination method or a method of extracting it from human pituitary gland. The human growth hormone solution as the purified material of the present invention is obtained by either method. May be. That is, the human growth hormone solution according to the present invention is an aqueous solution containing human growth hormone secreted or accumulated inside or outside cells by bacteria or animal cells into which the human growth hormone gene has been introduced, and a crude purified solution thereof, or human pituitary tissue. It is a human growth hormone solution partially homogenized and then partially purified by a known technique such as a precipitation method.

In practicing the present invention, a blue dye-bound carrier is first packed in a column, washed with distilled water, and then equilibrated with a buffer. Phosphoric acid, sulfuric acid acetate, citric acid, Tris, HEPES, boric acid or the like is used as a buffer. The concentration of the buffer is 0.005M to 0.1M.
5M, preferably 0.01M to 0.1M, more preferably 0.02M to 0.05M. The pH of the buffer is between 5 and 8.5, preferably between 6 and 7.5. The above human growth hormone solution is added to the column thus prepared. Thereafter, by washing with a buffer containing 0 to 0.3 M sodium chloride or potassium chloride, contaminants adsorbed on the blue dye-bound carrier in a non-specific manner can be removed.

Elution of human growth hormone from the blue dye-bound carrier column can be performed by increasing the ionic strength of the eluate. To increase the ionic strength of the eluate, simply increase the concentration of the buffer solution or add a neutral salt such as sodium chloride or potassium chloride to the eluate. For example, human growth hormone adsorbed on a blue dye-bound carrier can be eluted by adding 1 M sodium chloride to the eluate. Human growth hormone is selectively eluted by adding a protein denaturant to the eluate. In the present invention, the protein denaturant refers to urea and a chaotropic reagent, and examples of the chaotropic reagent include potassium thiocyanate, sodium thiocyanate, sodium perchlorate, guanidine hydrochloride, sodium iodide and the like.

The selective elution according to the present invention is considered to differ in principle from the elution method using high ionic strength in that human growth hormone can be selectively eluted. When urea is used as a denaturant, the urea concentration in the eluate is 2
９9M, preferably 4-6M.

In the column from which the human growth hormone has been eluted in this way, the contaminants may still be strongly adsorbed to the blue dye-bound carrier. In this case, the column can be regenerated by washing the column with a buffer containing 2 M sodium chloride or a 0.2 M aqueous sodium hydroxide solution to remove contaminants.

[0016]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited thereto.

Example 1 A crude purified solution for studying a method for purifying 20 KhGH was prepared in the following manner according to the description in JP-A-6-269292. E. coli transformant MT-10765
(This strain is deposited under Accession No. FERM BP-5020 under the Budapest Treaty with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry) in a medium containing polypeptone, yeast extract and the like. After completion of the culture, the cells are collected by centrifugation, the outer membrane of the cells is ruptured by an osmotic shock method, the disrupted liquid is centrifuged to remove the cells, and only the periplasm fraction is recovered. In order to remove nucleic acids and the like from the obtained periplasm fraction, an anion exchange chromatography, for example, a product passed through a column of Q Sepharose FF (Pharmacia) was used as a crude 20 KhGH solution. 2400 ml of this 20 KhGH roughly purified liquid
Was purified using Blue Sepharose 6FF (Pharmacia) as follows. Blue Sepharose 6F
F98ml (5cmφ × 5cm) is 300ml 20m
After equilibration with an M phosphate buffer (pH 6.5), 20 KhGH was adsorbed to Blue Sepharose 6FF through the above-described 20 KhGH crude purified solution. After sufficiently washing the column with 20 mM phosphate buffer (pH 6.5) containing 0.3 M sodium chloride, 550 ml of 20 M containing 6 M urea was added.
The eluate was collected by flowing in mM phosphate buffer (pH 6.5). The amount of 20K hGH before and after purification on a Blue Sepharose column was quantified by enzyme immunochemistry. As a result, the recovery rate of 20KhGH in the purification was 96.5%, which was a high yield. In addition, the quantified amount of 20 KhGH was 28
When the specific activity was calculated by dividing by the absorbance at 0 nm, the specific activity was increased about 80 times before and after purification. Further, the value obtained by multiplying the absorbance at 280 nm of the sample added to the column by the amount of the solution was taken as 100%, and the recovery of each fraction was calculated.
%, 3.8% and 1.2% of eluate. A portion corresponding to 5 μg of 20 KhGH in the obtained eluate was subjected to SDS polyacrylamide electrophoresis, and the gel after electrophoresis was confirmed for protein by Coomassie brilliant blue staining. As a result, only one band corresponding to 20 khHG was confirmed. From the above, it is apparent that the method of the present invention was capable of selectively adsorbing and separating 20 KhGH from a partially purified solution containing a large amount of contaminants.

Example 2 A crude liquid for studying a method for purifying 22 KhGH was prepared in the following manner according to the description in JP-A-6-269292. E. coli transformant MT-10773
Has been deposited under the Budapest Treaty with the Accession No. FERM BP-5019 under the Budapest Treaty under the Ministry of International Trade and Industry under the Industrial Technology Research Institute of the Ministry of International Trade and Industry in the same manner as in Example 1.
2KhGH crude semen was obtained. This 22 KhGH crude purified liquid 1
200 ml was purified using Blue Sepharose 6FF (Pharmacia) as follows. Blue Sepharose 6FF 98ml (5cmφ × 5cm) 300m
After equilibrating with 1 l of a 20 mM phosphate buffer (pH 6.5), the solution was passed through the above-mentioned crudely purified solution of 22 KhGH to give 22 KhGH.
Was adsorbed on Blue Sepharose 6FF. 0.3M
20 mM phosphate buffer containing sodium chloride (pH 6.
After sufficiently washing the column in 5), 550 ml of a 20 mM phosphate buffer (pH 6.5) containing 6 M urea was introduced, and the eluate was collected. The amount of 22 KhGH before and after purification with a Blue Sepharose column was quantified by enzyme immunochemistry. As a result, the recovery rate of 22KhGH in the purification was 9
The yield was as high as 7.2%. In addition, the determined 22KhG
When the specific activity was calculated by dividing the H content by the absorbance at 280 nm, the specific activity increased about 6.5-fold before and after purification. Further, when the value obtained by multiplying the absorbance at 280 nm of the sample added to the column by the amount of the solution was set to 100%, the recovery rate of each fraction was calculated.
The eluate was 0.9%, 4.0%, and 14.9%. A portion corresponding to 5 μg of 22 KhGH in the obtained eluate was subjected to SDS polyacrylamide electrophoresis, and the gel after electrophoresis was confirmed for proteins by Coomassie brilliant blue staining. As a result, only one band corresponding to 20 khHG was confirmed. From the above, it is clear that the method of the present invention was capable of selectively adsorbing and separating 22KhGH from a partially purified solution containing a large amount of contaminants.

[0019]

According to the present invention, the human growth hormone is mixed with the crudely purified human growth hormone solution by utilizing the affinity between the human growth hormone and the blue dye-binding carrier and the selective elution method of the human growth hormone using a protein denaturant. Remove contaminants,
It is possible to purify human growth hormone in a large amount, simply and with high purity. Production of high-purity human growth hormone in large quantities is important for stable supply of human growth hormone as a pharmaceutical. In addition, in order to expand human growth hormone not only to pituitary dwarfism but also to new indications, the utility value of 20KhGH with few side effects is great.
Therefore, not only 22KhGH which has been marketed until now, but also 20KhGH which has been difficult with the prior art.
The present invention, which also enables mass production of, is very useful.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Nobuumi Kusuhara 1144 Togo, Mobara-shi, Chiba Mitsui Toatsu Chemicals Co., Ltd.

Claims (4)

[Claims] 1. A method for purifying human growth hormone, which comprises selectively adsorbing and eluting a human growth hormone solution to a blue dye-bound carrier. 2. The purification method according to claim 1, wherein the human growth hormone adsorbed on the blue dye-bound carrier is selectively eluted using a protein denaturing agent. 3. The purification method according to claim 2, wherein the elution is carried out using an eluate containing 2 to 9 M urea. 4. A human growth hormone-containing composition obtained by the purification method according to any one of claims 1 to 3.