JPH0327349A - Separation of protein, peptide and amino acid - Google Patents

Abstract

PURPOSE:To separate proteins, peptides and amino acids with a high accuracy in a short time by carrying out liquid chromatography separation thereof using a specified spherical porous calcium phosphate-based compound particle as the stationary phase and a phosphate buffer solution as the mobile phase. CONSTITUTION:In separation of proteins, peptides and amino acids by the liquid chromatography, the liquid chromatography separation, especially the gradient method or the isocratic method is carried out using a spherical porous calcium phosphate-based compound particle composed of a calcium phosphate-based compound with 1.5-1.80Ca/P ratio and having 1-40mum average particle size as the stationary phase and a phosphate buffer solution as the mobile phase. In case of a substance having >=10000 molecular weight among the above mentioned substances, liquid chromatography separation is preferably carried out by the isocratic method using a phosphate buffer solution of >=10mM as the single mobile phase and in the case of a substance having <=10000 molecular weight the liquid chromatography separation is preferably carried out also by the isocratic method using a phosphate buffer solution of <=10mM as the single mobile phase.

Description

DETAILED DESCRIPTION OF THE INVENTION "Used Separation" The present invention relates to a method for separating proteins, peptides, and amino acids. "Prior art and its problems" Calcium phosphate compounds are used as biomaterials due to their excellent biocompatibility, and are also often used as packing materials for liquid chromatography due to their ability to adsorb proteins. ．． JP-A No. 62-91410 discloses that gel-like hydroxyapatite is granulated and the resulting granules are
A method for producing macrogonal chromatographic separation particles having a specific unit cell constant by annealing at a temperature of 0°C is disclosed. However, in this method, since the incineration temperature is 700°C or less, the particle strength is not sufficient, and further improvement is desired for use as a packing material for high performance liquid chromatography. Therefore, increasing the firing temperature increases the strength, but the particles become denser and the sample loading amount decreases. Furthermore, with conventional apatite-based packing materials, it was possible to separate proteins, but it was not possible to separate peptides and ξ-anoic acids, so improvements in separation performance are desired. ``Object of the Invention'' The object of the present invention is to provide a method that can separate proteins, peptides, and amino acids by liquid chromatography in a short time with high separation accuracy. "Structure of the Invention" The method for separating proteins, peptides, and amino acids according to the present invention uses a spherical porous calcium phosphate compound with an average particle size of 1 to 40 μm, which is prepared from a calcium phosphate compound with a Ca/P ratio of 1.5 to 1.80. It is characterized by subjecting it to liquid chromatography using compound particles as a stationary phase and a phosphate buffer as a mobile phase. In the method of the present invention, porous calcium phosphate compound particles as described above are used as the stationary phase. As a calcium phosphate compound, a Ca/P ratio of 1. 5-1.8
Any calcium phosphate of 0 can be used, such as tricalcium phosphate, hydroxyapatite, fluoroapatite, etc. Furthermore, the porous particles need to be spherical particles with an average particle size of 1 to 40 μm, preferably 1 to 20 μm.
As such, the fine spherical particles function as a high-performance packing material for liquid chromatography, and if the particles fall outside the above range, the strength, degree of separation, etc. will not be improved. Any porous particles used as the stationary phase can be used as long as they meet the above conditions, but porous particles with an average pore diameter of 100
~4,000, especially 500-2,000 continuous pores, with 90% of the total pore volume being 0.50% of the average pore size. occupied by pores with pore sizes ranging from 5 to 2 times, and 1 g of particles
Preferably, the particles have a pore volume of 0.05d or more. If the average pore diameter is less than 100, the function of the porous body 1 will be insufficient and the adsorption capacity will be reduced.

Moreover, when the average pore diameter exceeds 4000, the strength tends to decrease. In addition, particles with such a narrow pore size distribution have a significantly uniform particle structure, resulting in improved strength and separation accuracy in chromatography. Furthermore, the pores as described above have a pore volume of 0.00% per 1 g of particles. 0 5 d or more is preferable. If the pore volume is less than 0.05 d, the adsorption capacity tends to be insufficient.

The porous calcium phosphate compound particles as described above are
A high-purity calcium compound and a high-purity phosphoric acid compound are reacted in water, and the Ca/P ratio is 1. 0.5 to 1.80 of calcium phosphate compounds. A 1-10% by weight aqueous suspension is prepared, and the viscosity of the aqueous suspension is lOcp=
Stirring was continued until it reached 100 cp, and then granulated by spray drying into particles with a particle size of 1 to 40 μm, and
It can be produced by burning at a temperature of 1200℃. More specifically, in order to produce the porous particles used in the present invention, a calcium phosphate compound with a Ca/P ratio of 1.5 to 1.80 is first prepared using high purity raw materials as described above. .1 to 10% by weight aqueous suspension is prepared. This concentration is 0. If it is less than 1% by weight, the productivity will drop significantly, and if it exceeds 10% by weight, the viscosity of the reaction solution will increase, and the reaction may become non-uniform. When the resulting aqueous suspension is continuously stirred, the viscosity initially increases;
The viscosity decreases again. Viscosity decreases to 10-100cP
When this is reached, stop stirring and spray dry. Viscosity is l0
If it does not decrease to 0 cP or less, the ripeness will be insufficient and particles with the above characteristics will not be obtained.
There is no need to lower it further. Achieving a viscosity in this range typically requires continued stirring for about 24 hours.

Spray drying can be carried out in various ways, such as by spraying from a nozzle or by using an atomizer. It is granulated into spherical particles with a particle size of 1 to 10 μm by spray drying. The particle size can be adjusted by appropriately setting various conditions during spray drying.

The spherical particles thus granulated are further heated at 700°C to 120°C.
By calcining at a temperature of 0° C., porous particles having the above-mentioned properties are obtained. The firing temperature can be appropriately selected within the above range depending on the type of calcium phosphate compound used. However, if the firing temperature is less than 700°C, the strength will be insufficient and 1
If the temperature exceeds 200°C, the material becomes dense.

The porous particles obtained as described above weighed 150 kg/r.
It has a high intensity exceeding that of RF, exhibits high separation ability in liquid chromatography, has high water retention, and does not require incubation, allowing immediate separation operations. In the method of the present invention, porous particles as described above are packed into a separator such as a column as a stationary phase, and subjected to liquid chromatography using a phosphate buffer as a mobile phase. The phosphate buffer used as the mobile phase may be one commonly used in liquid chromatography, such as sodium phosphate buffer (pH 6.8).

Chromatography according to the present invention can be performed using either a gradient method or an isocratic method. When applying the isocratic method, molecules! For separation of substances with a molecular weight of 10,000 or more, use a phosphate buffer of 10mM or more as the single mobile phase, and for separation of substances with a molecular weight of 10,000 or less, use a phosphate buffer of 10nM or less as the single mobile phase. It is preferable to use it as "Examples of the Invention" Next, the present invention will be explained in more detail based on Examples, but the present invention is not limited thereto. Reference example (manufacture of filler) Calcium hydroxide suspension (obtained by calcining JIS special grade calcium carbonate at 1000°C and hydrating it with pure water) and phosphoric acid aqueous solution (JIS special grade reagent) /P ratio is 1.
67, and produced hydroxyapatite by a wet method, and 8% by weight of hydroxyapatite.
An aqueous suspension was obtained.

This suspension is further stirred until the viscosity reaches 20 cP, after which stirring is stopped and spray-dried. Furthermore, the grain size was 2 μm, 10 μm, 2 μm, and
Porous spherical particles of 0 μm and 40 μm (hereinafter referred to as HP-02, HP-10, }IP-20 and HP-4, respectively)
) was obtained. The specific surface area, average pore diameter, and pore volume of each particle were measured, and the results are shown in Table 1.

The specific surface area was measured by the BET method, and the average pore diameter was measured by a mercury porosimeter. (Left below) Table 1 Furthermore, the X-ray diffraction diagram of the obtained particles (CuKα, 40K
V, 100 mA) is shown in Figure 1, and the infrared absorption spectrum is shown in Figure 2. In Figures 1 and 2, A is HP-
10, B is HP-02, C is the above spray-dried particles (
unfired particles).

From FIGS. 1 and 2, it was proved that the obtained particles were nohydroxyapatite.

Further, FIG. 3 shows the relationship between the pore diameter of the hydroxyapatite particles produced in the above reference example and the pore volume measured with a mercury pore volume meter. From FIG. 3, it was proved that 90% of the total pore volume of the obtained hydroxyapatite particles was occupied by pores having a pore diameter in the range of 0.5 to 2 times the average pore diameter.

Example 1 The hydroxyapatite particles produced in the above reference example were packed into a column with an inner diameter of 7.51III1 and a height of 100 am, and a sample containing bovine serum albumin, lysozyme and cytochrome C was chromatographically separated. From 1+*M to 40% in 30 min at a flow rate of 1M/min using sodium phosphate buffer (pH 6.8) as eluent.
Separation was performed using a linear gradient method down to 0mM. Figure 4 shows the chromatogram obtained using HP-02, Figure 5 shows the chromatogram obtained using HP-10, Figure 6 shows the chromatogram obtained using HP-20, and Figure 6 shows the chromatogram obtained using HP-20. Figure 7 shows the chromatogram obtained using -40. In Figures 4 to 7, a indicates the peak of bovine serum albumin, b indicates the peak of lysozyme, and C indicates the peak of cytochrome C. The chromatograms shown in Figures 4 and 5, which are examples of the present invention, have significantly sharper peaks than the chromatograms shown in Figures 6 and 7, which use a stationary phase with a large particle size. , it can be seen that the separation performance is high.

Example 2 Inner diameter 7. filled with hydroxyapatite particles produced in Reference Example. Using a column with a height of 5 rtm and 100 nn, the sample containing lysozyme was mixed from 1 mM to 400+++M.
The particles were chromatographically separated using a 30-minute linear gradient method using sodium phosphate buffer (pH 6.8), and the relationship between the particle size and the number of theoretical plates was investigated. The results are shown in Figure 8. Furthermore, using the same column, 4 samples containing cytidine were added.
Chromatographic separation was performed using an isocratic method using 00 mM sodium phosphate buffer (pH 6.8) as a mobile phase, and the relationship between particle size and number of theoretical plates was investigated. The results are shown in Figure 8. From FIG. 8, it can be seen that the smaller the particle size, the higher the number of theoretical plates and the extremely high separation performance in both the gradient method and the isocratic method.

Actual 1N example 3 Column with inner diameter 7.5 m and height 100 mm filled with HP-02, inner diameter 7.5 ms and height 10 mm filled with HP-10
0 column, inner diameter 10.7 column filled with HP-10,
Column with a height of 100 m, inner diameter 2 filled with HP-2 0
4 types of columns (1.5m, 100mm height)
Column A, Column B, Column C and Column D, respectively.
) at a flow rate of 1rII/min using sodium phosphate buffer (pH 6.8) as the eluent.
A mixture of lysozyme and cytochrome C was separated by a linear gradient method from 1 mM to 400 mM in 0 minutes. The amount of sample and the degree of separation were measured and the results are shown in FIG.

These results demonstrated that high sample loading was possible.

Example 4 Inner diameter 7.5 filled with particles HP-02 manufactured in Reference Example
Using a column with a height of 100 am and a sodium phosphate buffer (pH 6.8) as a mobile phase eluent,
Various proteins were separated by passing the solution from 1n+M to 400mM in 30 minutes using a linear gradient method at a flow rate of ld/min, and the retention times are shown in Table 2 below. Furthermore, Table 2 also shows the molecular weight and isoelectric point of the separated substances.

Similarly, peptides were separated and Table 3 shows their molecular weights, isoelectric points and retention times.

(Left below) Example 5 Inner diameter 7.0mm filled with particles HP-02 manufactured in Reference Example.
Using a column with a diameter of 5 mm and a height loOan, a sodium phosphate buffer (pH 6.8) was used as the mobile phase solution M for 30 minutes at a flow rate of 1 d/min. A mixture of protein and bebutide was separated by a linear gradient method from 0 O 1 mM to 0.4 mM. The pressure is 37kg/c
It was ii. The obtained chromatogram is shown in Figure 1O. In Figure 10, 1 is (D1^1a", D
-LeuS) 1 peak of enkephalin, 2 peak of angiotensin H, 3 peak of angiotensin ■, 4 peak of ovalbumin, 5 peak of bovine serum albumin, 6 peak of myoglobin, 7 peak of lysozyme, 8 peak 9 shows the peak of α-chymotrypsinogen-A and the peak of cytochrome C.

Example 6 Inner diameter 7.5 filled with particles HP-02 manufactured in Reference Example
Using a column with a height of 100 m, 1+M was used as the eluent.
l using sodium phosphate buffer (pll6.8)
Various amino acids were separated in an isocratic manner at a flow rate of Id/min. The isoelectric point and retention time of each ξ-anoic acid are shown in Table 4 below. Table 4 11e Pro Val Phe Glu nyp Tyr Met Ala Thr Asn Ser cty Asp His 6.02 6.30 5.96 5.48 3.22 5.83 5.66 5.74 6.00 6.16 5. 41 5.68 5.97 2.77 7.59 2.71 2.73 2.73 2.73 2.79 2.86 2.87 2.89 3.01 3.35 3.67 3.78 3 .90 4.08 4.78 Arg 1 0. 7 6
7. 9 0Cys-Cys 4.
6 0 8. 4 0Ls
9. 7 4 8. 9
0 Example 7 Inner diameter 7.0 filled with particles HP-0 2 produced in Reference Example.
Using a 5M, 100m high column, 1m as eluent.
1 using M sodium phosphate buffer (pH 6.8).
The mixture of amino acids was separated in an isocratic manner at a flow rate of -/min. Detection was performed using UV at 1951m. The obtained chromatogram is shown in FIG. 11th
In the figure, d is the isoleucine peak, e is the alanine peak, f is the threonine peak, g is the aspartic acid peak, and h is the arginine peak.

As is clear from FIG. 11, it was possible to clearly separate amino acids that had low molecular weights and could not be separated by conventional liquid chromatography. Separation of such low molecular weight substances has conventionally been carried out by chromatography using ion exchange resins, which usually takes about 30 minutes.

However, according to the present invention, separation can be performed in an extremely short time.

Example 8 Inner diameter 7.0 filled with particles HP-02 produced in Reference Example.
Using a 5 m, 100 m high column and 400 mM sodium phosphate buffer (pH 6.8) as the eluent, lad! A mixture of bovine serum albumin, lysozyme and cytochrome C was separated in an isocratic manner at a flow rate of /min, and the resulting chromatogram is shown in Figure 12A. Separately, using the same column, the same sample as above was separated using a linear gradient method from 1 mM to 400 min at a flow rate of 1-/min for 30 min using sodium phosphate buffer (pH 7.8) as the eluent. The obtained chromatogram is shown in FIG. 12B. Furthermore, Figure 12A and Figure 1
In Figure 2B, a is the peak of bovine serum albumin, b
indicates the peak of lysozyme, and C indicates the peak of cytochrome C.

Furthermore, a correlation diagram between the retention time by the gradient method and the retention time by the isocratic method was created from the above chromatogram, and is shown in Figure 13. As is clear from this correlation diagram, the two methods show an almost linear correlation, and the isocrate ink method can perform separation in an extremely short time. It is extremely suitable for

"Effects of the Invention" According to the separation method of the present invention, since a stationary phase with extremely high separation ability is used, it is possible to not only separate proteins, but also to
Low molecular weight peptides and amino acids, which are difficult to separate using conventional liquid chromatography, can be separated with high separation accuracy. In addition, high sample loading is possible. The separation method of the present invention can also be performed by an isocratic method, and separation can be performed in an extremely short time.

[Brief explanation of drawings]

Figure 1 is an X-ray diffraction diagram of the particles produced in the reference example, Figure 2 is an infrared absorption spectrum, Figure 3 is a diagram of the relationship between pore diameter and pore volume, and Figure 4 is a graph obtained using HP-02. Figure 5 is the chromatogram obtained using HP-10, Figure 6 is the chromatogram obtained using HP-20, and Figure 7 is the chromatogram obtained using HP-40. Chromatogram, Figure 8 is a diagram of the relationship between the average particle diameter and the number of theoretical plates measured in Example 2, Figure 9 is a diagram of the relationship between the sample loading amount and degree of separation measured in Example 3, and Figure 1O is a diagram of the relationship between the sample load and the degree of separation measured in Example 3. Chromatogram obtained in Example 5, FIG. 11 is the chromatogram obtained in Example 7, FIG. 12A is the isocratic chromatogram obtained in Example 8, and FIG. 12B is the chromatogram obtained in Example 8.
FIG. 13 is a correlation diagram between the retention time obtained in Example 8 using the gradient method and the retention time obtained using the isocratic method. Explanation of symbols A...HP-10, B...HP-02, C...unburned particles, a...peak of bovine serum albumen, b...
...Lysozyme peak, C...Cytochrome C peak, ■... (D Ala", D-Leus) - Enkephalin peak, 2... Angiotensin ■
peak, 3... peak of angiotensin ■, 4...
・・Peak of Oval ξ, 5 ・・Bovine serum Alp ξ
6. Myoglobin peak, 7.・・・
-Lysozyme peak, 8...α-chymotrypsinogen-A peak, 9...cytochrome C peak,
d...Isoleucine peak, e...Alanine peak, f...Threonine peak, g...Aspartic acid peak, h...Arginine peak.

Claims (1)

[Claims] 1. Spherical porous calcium phosphate compound particles with an average particle size of 1 to 40 μm made of a calcium phosphate compound with a Ca/P ratio of 1.5 to 1.80 are used as the stationary phase, and phosphorus is used as the mobile phase. A method for separating proteins, peptides, and amino acids, which comprises subjecting them to liquid chromatography using an acid salt buffer. 2. The separation method according to claim 1, wherein the liquid chromatography is performed by a gradient method or an isocratic method. 3. For the separation of substances with a molecular weight of 10,000 or more, use a phosphate buffer of 10mM or more as a single mobile phase, and for the separation of substances with a molecular weight of 10,000 or less, use a phosphate buffer of 10mM or less as a single mobile phase. 2. The separation method according to claim 1, wherein liquid chromatography is carried out using an isocratic method using one of the mobile phases.