JPH0479964B2 - - Google Patents

Description

[Detailed Description of the Invention] (1) Purpose of the Invention [Field of Industrial Application] The present invention is a method for separating and purifying biopolymer substances such as chemical components in aqueous solutions, especially proteins and nucleic acids. This invention relates to a method for producing hydroxylapatite Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ .

[Prior Art] As a method for isolating biopolymer substances such as proteins and nucleic acids from a mixed aqueous solution, a method of adsorbing and desorbing them onto solid particles has been studied, and solid particles for separation with various performances have been devised and provided. ing.

Ciselius and his collaborators prepared hydroxylapatite in 1956 by boiling the calcium phosphate precipitate obtained by mixing calcium chloride solution and dibasic sodium phosphate solution at room temperature (around 25°C) in alkali. It was shown that this method is useful for separating and purifying each component from a biopolymer mixture.
[A. Ciselius, S. Hierten, E.
Levin; Archives of Biochemistry and Biophysics (A.
Tiselius, S. Hjerten and O. Levin; Archives
of Biochemistry and Biophysics) Volume 65, 132~
155 pages, 1956] [Problems to be solved by the invention] Hydroxylapatite for separation by the method of Chiselius et al. has been used for the separation and purification of biopolymers, but it is difficult to fill it into a glass cylinder or other column. When subjected to chromatography, the problem is that the flow rate is extremely slow due to the small particle size. To solve this problem, conventional methods have been to mix cellulose particles, use silica as a core when producing intermediate calcium phosphate, or maintain a constant pH during alkaline boiling. It has been done. However, all of these methods are complicated to operate, do not always produce the results as reported, and have not been sufficiently improved.

(2) Structure of the Invention [Means for Solving the Problems] With the aim of solving the above-mentioned difficulties, the present inventor has reexamined in detail each step of the method of Ciselius et al. Discovered special conditions for preparing large hydroxylapatite,
The present invention has now been completed.

That is, in the method of Chiselius et al., calcium chloride (CaCl ₂ ) is used to prepare calcium phosphate precipitate as a precursor leading to hydroxylapatite.
Aqueous solutions of and dibasic sodium phosphate (Na ₂ HPO ₄ ) were directly mixed at room temperature (around 25°C), but the inventors changed the temperature over a wide range from 25°C to 100°C. As a result of experimenting and examining the effects, we found that by injecting both solutions into a 0.5M NaCl aqueous solution and allowing them to react, we found that
When calcium chloride solution and dibasic sodium phosphate solution are mixed under the conditions of two temperature ranges, the particle size of the resulting calcium phosphate precipitate is 60 microns or more in diameter, which is much larger than in other temperature ranges. Hydroxylapatite, which will be derived by heating in an alkali, will maintain a similar large particle size, and in column chromatography, the flow rate of the developing eluate that is 10 times higher than that possible for hydroxylapatite using conventional methods. became possible.

The above phenomenon was completely unexpected by the inventors at first, but based on these results, the formation mechanism is considered to be as follows. That is, the crystal growth of calcium phosphate precipitate, which is a precursor of hydroxylapatite, It is strongly influenced by the actual temperature.

Calcium phosphate precipitate is formed at a temperature of 25
As the temperature rises above ℃, brushite CaHPO ₄ 2H ₂ O crystals grow gradually larger, but after reaching a peak of 45 ℃, the brushite crystals gradually become smaller. Then, at temperatures above 80°C, minute brassyite is dehydrated and becomes minute monetite.
It becomes a crystal of _CaHPO4 . Furthermore, the temperature rose to 90
When the temperature exceeds .degree. C., the particle size of the formed calcium phosphate precipitate suddenly increases and hardens. The discontinuous change at temperatures above 90°C is thought to be due to the fact that at this high temperature, the nascent, ultra-fine brassiaite crystals are immediately dehydrated and become monetite, which at the same time coagulates to form strong particles. Monetite crystals produced at 80 to 90°C or commercially available monetite do not have this ability to coagulate and remain in the form of fine crystals.

Next, the calcium phosphate precipitate thus generated is transformed into hydroxylapatite by heating in an alkali. Conventionally, at this time, 1 mole or more of calcium chloride (CaCl ₂ ), which is the raw material, is converted into hydroxylapatite. A large amount of alkali was added in a proportion of .

As a result of careful consideration of this matter, the inventor found that
It is possible to reduce the amount of alkali added, at a ratio of 0.4 to 0.6 mol per 1 mol of CaCl ₂ of the raw material,
It has been found that it is most preferable to add caustic soda (NaOH) in a proportion of 0.5 mole.

Conventional methods required washing with large amounts of water or phosphate buffer to remove excess alkali, but the method proposed by the present inventors greatly simplifies cleaning and finishing of products. It is something that will be done.

The product hydroxylapatite has also traditionally been stored in phosphate buffers. Of course, this is also utilized in the embodiments of the present invention, but the inventors have also added more convenient embodiments.

In other words, even if the produced hydroxylapatite is heated and dried at temperatures above the boiling temperature and up to 220°C,
It has been found that dry powder can be obtained while maintaining good performance as a separating agent.

Dry products offer great storage and transportation convenience.

[Examples and Comparative Examples] Hereinafter, embodiments of implementation and effects thereof will be specifically shown by Examples and Comparative Examples.

Comparative example 0.5M (molar concentration) CaCl ₂ aqueous solution and 0.5M
Prepare 2 parts each of Na ₂ HPO ₄ aqueous solutions and add 1 part of 0.5M NaCl to each at a rate of 2.5 ml per minute while keeping the desired constant temperature and stirring as slowly as possible.
Inject into aqueous solution. The temperature of the mixed solution is maintained at the above constant value throughout the injection.

25, 40, 45, 55, 70, as the desired constant temperature.
Experiments were conducted using temperatures of 85 and 95°C. Calcium phosphate precipitates produced by the operation at each experimental temperature were collected from the reaction solution, each thoroughly washed with water, suspended in 0.25M NaOH (5), and boiled for 1 hour with stirring. The obtained hydroxylapatite was thoroughly washed with warm water at 70℃, and then
After washing with 10mM phosphate buffer (PH6.9), store in the same buffer.

1 ml of static precipitate of hydroxylapatite derived from calcium phosphate produced at each temperature was filled into a glass cylinder with an inner diameter of 8.5 mm with a nylon mesh placed on the bottom.
A 10 mM phosphate buffer solution was passed under a pressure of 80 cm of water column, and the flow rate was measured, the settling height of the hydroxylapatite layer was observed, and the filled bed volume V (ml) occupied by the hydroxylapatite layer was calculated.

In addition, from the above flow rate measurement value, calculate the value per 1 cm of water column pressure and 1 cm of packed bed height, and use this as the normalized flow rate U.
It is expressed in (ml/min/cm/cmHH).

Additionally, calf thymus DNA was added to 10mM phosphate buffer.
A solution containing a concentration of 100 μg/ml was flowed under a pressure of 80 cm of water column, and the DNA adsorption amount C (mg) was calculated from the amount of flow until DNA was detected in the liquid discharged from the column.

The temperature at which calcium phosphate precipitate, which is the precursor of each hydroxylapatite, is formed is taken on the horizontal axis.
The settled packed bed volume V, the normalized flow rate U, and the corresponding hydroxylapatite sample
FIG. 1 is a graph plotting the amount of DNA adsorption C on the vertical axis.

As shown in the figure, it has become clear that the settled packed bed volume V and the normalized flow rate U vary greatly depending on the formation temperature of the calcium phosphate precursor.

The maximum is seen in hydroxylapatite from calcium phosphate (hereinafter abbreviated as H45 and H95, respectively) produced at 45°C and 95°C.

In particular, there is a large difference in flow rate, which is 10 times that of hydroxylapatite (hereinafter abbreviated as H25) from calcium phosphate produced at 25℃, which is around room temperature, as proposed by Chiselius. It has also reached

This suggests that H45 and H95 have large particle sizes and are hardly compressed when packed in a column. On the other hand, the amount of DMA adsorption is lower in hydroxylapatite produced from calcium phosphate produced at 70 to 85°C, but other than that there is almost no difference.Especially at 45°C and 95°C, which are the focus of this invention, it is equivalent to the case at 25°C. It is clear that he possesses the above abilities.

Furthermore, Figure 2 shows the results of developing calf serum albumin as protein, yeast ribonucleic acid as RNA, and calf thymus deoxyribonucleic acid as DNA for columns packed with H25, H45, and H95 hydroxylapatite. H45, H95
It is confirmed that both methods achieve the same separation and fractionation accuracy as the conventional method H25.

Example 1 Two aqueous solutions of 0.5M (molar concentration) CaCl ₂ and 0.5M Na ₂ HPO ₄ were prepared, and each was added to one 0.5M NaCl aqueous solution kept at 45°C and stirred as slowly as possible. Inject at a rate of 2.5 ml per minute, mix, and react. Calcium phosphate (brassiaite) produced
Wash the large crystals thoroughly with water and suspend them in water of 1.
Add 100ml of 10M NaOH and boil for 1 hour.
The obtained hydroxylapatite is thoroughly washed with water and phosphate buffer and stored as is until use.

Example 2 Two aqueous solutions of 0.5M (molar concentration) CaCl ₂ and 0.5M Na ₂ HPO ₄ were prepared and poured into one 0.5M NaCl aqueous solution kept at 95-100°C and stirred as slowly as possible. Inject each at a rate of 2.5 ml per minute to mix and react. Calcium phosphate (monetite) produced
Wash the large, hard particles thoroughly with water, suspend them in 1 water, add 100 ml of 5M NaOH, and boil for 1 hour. The obtained hydroxylapatite is washed with water and stored.

Example 3 Wet hydroxylapatite obtained in the same manner as in Example 2 is heated and dried.

The final temperature is reached at 220°C to obtain a dry powder.

(3) Effects of the invention According to the method described in claims 1 and 2 of the present invention, it has excellent separation and fractionation accuracy as a chromatographic packing material for separating biopolymer substances, and also has high Hydroxylapatite for separation can be prepared quickly due to the high flow rate of the developing solution.

[Brief explanation of the drawing]

FIG. 1 shows the relationship between the temperature at which calcium phosphate, which is a hydroxylapatite precursor, is produced and the performance of hydroxylapatite derived therefrom. In the figure, V and C indicate the volume after column filling (ml) and adsorbed DNA amount (mg) per 1 ml of hydroxylapatite static precipitation, respectively, and U indicates the developing solution standardized flow rate (ml/min/cm/cmHH). show. FIG. 2 shows chromatographic fractionation patterns for calf serum albumin (P), yeast ribonucleic acid (R), and calf thymus deoxyribonucleic acid (D) using columns packed with H45, H95, and H25 for comparison.

Claims (1)

[Claims] 1. Calcium chloride solution and dibasic sodium phosphate solution are heated in a temperature range of 40°C to 50°C, most preferably
A method for producing hydroxylapatite for separation, which comprises mixing at a temperature of 45°C and heating the resulting calcium phosphate precipitate in an alkali to produce hydroxylapatite. 2. Mixing a calcium chloride solution and a dibasic sodium phosphate solution at a temperature range of 90°C to 100°C, most preferably at a temperature of 95°C, and heating the resulting calcium phosphate precipitate in an alkali to form hydroxylapatite. A method for producing hydroxylapatite for separation, which is characterized by: