JPH0772155B2 - Purification method of coenzyme Q - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for purifying coenzyme Q, and more specifically, to a large amount of a large amount by using a plurality of adsorption column chromatographs connected in series with each other. The present invention provides a novel method for purifying crude coenzyme Q.

Coenzyme Q is a useful substance that participates in the electron transport system of the terminal respiratory system in vivo and exhibits an excellent pharmacological effect against various diseases such as myasthenia gravis and emphysema.

[Problems to be Solved by Prior Art and Invention]

Coenzyme Q is obtained by a method such as synthesis or extraction from microbial cells or natural products. The crude coenzyme Q obtained by these methods contains a lot of impurities together with coenzyme Q and is extremely low in purity. is there. When coenzyme Q is used as a medicine or the like, it is required that coenzyme Q has an extremely high purity, and therefore an industrially excellent purification method is required.

Here, the industrially excellent purification method is purified coenzyme Q.
The purity of (hereinafter referred to as purified coenzyme Q) is high, and the amount of filler used per unit purified coenzyme Q for adsorption column chromatography (g-filler / g-purified coenzyme Q) and the amount of solvent used This is a purification method with less (-solvent / g-purified coenzyme Q).

To obtain purified coenzyme Q from crude coenzyme Q by adsorption column chromatography, many methods are known as follows. That is, there is a method of using an inorganic adsorption carrier as a filler, and methods of using silica gel as a carrier include, for example, JP-A-54-52790 and JP-A-57-18988.
JP-A-59-33354, JP-B-58-53917, JP-B-59
-26273, JP-B-57-21309 and JP-A-56-30941
The method described in the No. etc. is known. On the other hand, methods using florisil as a carrier include, for example, JP-A-54-110389, JP-B-58-12266 and JP-A-54-12.
The method described in JP-A-122795 and the method using alumina as a carrier include an extremely large number of methods such as the method described in JP-A-59-45894.

Purification methods using these inorganic adsorption carriers are all chromatographies using one column.

In addition, when trying to obtain highly purified purified coenzyme Q by a method using an inorganic adsorption carrier, the recovery rate (purified coenzyme Q /
Crude coenzyme Q) is extremely low, and it is general that the amount of filler and the amount of solvent used per unit of purified coenzyme Q must be increased. Therefore, it is necessary to make the purification apparatus large-scale for purification on an industrial scale, but in the case of large-scale purification, local solvent drift, mixing, and diffusion of crude coenzyme Q in the column are required. There are problems, etc., and it is very difficult to scale up due to the nature of adsorption column chromatography.

There is also a method of using a porous synthetic resin as a filler. That is, as an adsorption column chromatography using a non-polar porous synthetic resin and a polar solvent as a solvent used,
For example, JP-A-55-39701, JP-A-56-121490,
JP 57-2686, JP 57-6910, JP 59-5064
No. 8 and the like, and as an adsorption column chromatography using a polar porous synthetic resin and a non-polar solvent, for example, Japanese Examined Patent Publication No. 57-11302 is known, but when the inorganic adsorption simple substance is used. For the same reason as above, it is difficult to obtain highly purified purified coenzyme Q on an industrial scale. In addition, due to the nature of adsorption column chromatography using a porous synthetic resin, highly purified purified coenzyme Q can be obtained. In order to obtain a high recovery rate, it is necessary to carry out adsorption column chromatography and further purification such as adsorption column chromatography using an inorganic adsorption carrier and recrystallization.

The object of the present invention is that purification of crude coenzyme Q by conventional adsorption column chromatography is unsuitable for large-scale production, and that this purification alone usually yields coenzyme Q of sufficiently high purity. It is intended to provide a method for purifying coenzyme Q which can solve the problem of not being produced.

[Means and actions for solving problems]

The present inventors have conducted diligent research in order to obtain coenzyme Q which is capable of purification on an industrial scale even though it is a method by adsorption column chromatography, and has a sufficiently high purity without further post-treatment. As a result of the repetition, the present invention has been reached.

That is, in the present invention, when purifying crude coenzyme Q by adsorption column chromatography, a plurality of columns connected in series to each other are used, and the crude coenzyme Q solution is sequentially passed through the columns to remove the crude coenzyme Q from each column. A method for purifying coenzyme Q, which comprises discharging the precut fraction out of the system and recovering the fraction whose main component is coenzyme Q from the final column.

The crude coenzyme Q of the present invention refers to a coenzyme Q-containing substance itself containing a large amount of one or more kinds of impurities together with at least one of coenzymes Q _{4 to} Q ₁₂ , or previously extracted, concentrated and / or crystallized. It is a product that has undergone purification known per se such as chemical conversion. The coenzyme Q-containing product is not particularly limited in its production method and origin, and specifically, for example, an extract from an organism such as a microbial cell, an animal organ and blood mixture, and a reaction product obtained by synthesis The thing or its concentrate etc. are mentioned.

In the present invention, the crude coenzyme Q is made into a solution and sequentially passed through a plurality of columns connected in series, whereby the content of impurities therein is gradually reduced.
As a means therefor, there is a means for sequentially discharging impurities (precuts) from the bottom of each column.

The present invention will be specifically described with reference to the drawings. The crude coenzyme Q used in this description is assumed to have an elution pattern as shown in FIG. That is, this crude coenzyme Q
Is the main component of coenzyme Q and contains impurities x ₁ , x ₂ and x ₃ (impurities x ₁ , x ₂ and x ₃ respectively) that are sequentially eluted earlier than coenzyme Q over time. The category is referred to as the “precut category.” The same shall apply hereinafter), and impurities x ₄ , x ₅ and x that are sequentially eluted later than coenzyme Q over time.
₆ (the category containing each of impurities x ₄ , x ₅ and x ₆ is referred to as the post-cut category. The same applies hereinafter). However, the coenzyme Q and the respective impurities have long long curves, and the respective components are not clearly distinguished from each other and always contain a small amount or a trace amount of other components.

An example of the flow sheet of the refining device used for the present invention is shown in FIG. That is, this device is the first column 1,
It has three columns, a second column 2 and a third column 3. At the bottom of each column are a first bottom tube 5, a second bottom tube 6 and a third bottom tube 7, respectively. Each bottom pipe is provided with a first bottom valve 8, a second bottom valve 9 and a third bottom valve 10, respectively. In addition, each of the bottom tubes has a first detector 11 and a second detector between the bottom of each column and each bottom valve.
A detector 12 and a third detector 13 are provided. A portion of the first bottom pipe 5 between the first bottom valve 8 and the first detector 11,
The top of the second column 2 is communicated with the first communication pipe 14, so that the first column 1 and the second column 2 are connected in series with each other. Similarly, the second column 2 and the third column 3 are connected to each other in series by the second connecting pipe 15. Each communication pipe is provided with a first communication valve 16 and a second communication valve 17, respectively. A supply pipe 18 is provided at the top of the first column 1. As the detector at the bottom of each column, for example, an ultraviolet absorption measuring instrument (UV monitor) and an infrared absorption measuring instrument can be used. Further, instead of these measuring instruments, the valve can be opened and closed by a time schedule which is experimentally obtained in advance.

An operation for sequentially discharging impurities (precut) in crude coenzyme Q from the bottom of each column using this apparatus will be described. That is, a solution in which crude coenzyme Q is dissolved in a solvent is supplied from the supply pipe 18 to the first column 1, and each component is developed and adsorbed on the packing material in the first column 1. Next, while discharging the liquid from the first bottom valve 8 to the outside of the system,
The solvent is poured from the top pipe 4, and the precut section whose main component is the impurity x ₁ is discharged from the first column 1 through the first bottom valve 8 to the outside of the system.

This effluent from the first column 1 is detected by the coenzyme Q as the first detector.
The first bottom valve 8 is closed and the first
Open the communication valve 16 and connect the liquid discharged from the first column 1 to the first communication pipe.
It is transferred to the second column 2 via 14 and almost all of the residual liquid in the first column 1 is transferred to the second column 2. Second
In column 2, as in the first column 1, each component is developed and adsorbed, and then the main component is an impurity.
The front cut section of x ₂ is discharged from the second column bottom pipe 6 to the outside of the system by opening the second column bottom valve 9. When coenzyme Q in the effluent from the second column 2 is detected by the second detector 12, this effluent is transferred to the third column 3 as described above, and almost all the residual liquid in the second column is removed. Transfer all to third column 3.

In the third column 3 as well, as in the first column 1 and the second column 2, each component is developed, separated and eluted, and the impurities x ₃ as the main component are extracted from the third bottom valve 10 of the third column 3. The pre-cut category containing, the category containing coenzyme Q as a main component, and each post-cut category containing each of impurities x ₄ , x ₅ , and x ₆ as main components are sequentially discharged. Coenzyme Q in this effluent is detected by the third detector 13,
The section whose main component is coenzyme Q is collected.

The column containing impurities remaining in each column is washed out with a solvent to regenerate the packing material in each column, and each column is again subjected to adsorption column chromatography. Properly combine the time of developing and adsorbing each component in each column, elution and washing out of impurities-for example, in parallel with the washing out of impurities in the first column 1, in the second column 2, developing, adsorption and By washing out a small amount of residual impurities in the 3 column 3, the play time of each column can be reduced and each column can be effectively used.

The filler used in the present invention is not particularly limited, and conventionally,
Generally used fillers can be used, but typical examples are as follows.

That is, as the inorganic filler, silica gel, alumina,
Examples include florisil, activated carbon, and hydroxyapatite. Examples of commercially available silica gel include Wakogel C-200 and Wakogel C-300 (both manufactured by Wako Pure Chemical Industries), silica gel art (Art) 9385 and silica gel art 7734 (both manufactured by Merck), and silica gel. Examples include KT-3063 and silica gel 4B (both manufactured by Fujigel Co., Ltd.).

In addition, a porous synthetic resin is generally used as the organic filler, and as a representative example of this, apolar synthetic resins such as Amberlite XAD-2 (manufactured by Rohm and Haas) and Amberlite XAD-4 (Rohm) are used. And styrene-divinyl copolymers such as High Porous Polymer HP (manufactured by Mitsubishi Kasei Co., Ltd.), and polar synthetic resins such as Amberlite XAD-7 (Rohm and Haas). Amberlite XAD-9, polyacrylic ester such as Amberlite XAD-8 (produced by Rohm and Haas)
(Rohm and Haas) and Amberlite XAD-11 (Rohm and Haas)
A polar synthetic resin such as that manufactured by Haas Co., Ltd. may be used.

The packing material packed in each column may be the same as or different from each other. It is preferably the same.
Particular preference is given to inorganic fillers of the same type.

In the present invention, the solvent for dissolving the crude coenzyme Q and the solvent added to each column may be any solvent capable of dissolving the crude coenzyme, and either a single solvent or a mixed solvent can be used.

Usually, when an inorganic filler is used, a non-polar solvent is preferable as the single solvent, and examples thereof include n-hexane, n-pentane, n-heptane, isooctane, cyclohexane and mixtures thereof, hydrocarbons such as petroleum ether. Is particularly preferable for practical use. As the mixed solvent, a mixed solvent obtained by adding an ether-based organic solvent such as ethyl ether, an ester-based solvent such as ethyl acetate or butyl acetate, or a ketone-based organic solvent such as acetone or methyl ethyl ketone to the above nonpolar solvent is particularly preferable. Further, in the case of using a porous synthetic resin as the filler, when using a non-polar porous synthetic resin, methanol, ethanol, isopropanol, n-propanol, acetone, methyl ethyl ketone, isopropyl ether, tetrahydrofuran,
Polar solvents such as dioxane, methyl cellosolve, dimethylformamide, acetonitrile and water can be used alone or as a mixture with one another, mixed solvents being preferred. Mixed solvents such as methanol and water, methanol and acetone, methanol and n-hexane, acetone and water are particularly preferably used. On the other hand, when a polar porous resin is used, nonpolar solvents such as benzene, toluene, n-hexane, petroleum ether, petroleum benzene, isopentane, carbon tetrachloride and chloroform should be used alone or as a mixture with each other. You can Further, a mixed solvent obtained by mixing each of these solvents with acetone, ether and ethyl acetate can also be used. Mixed solvents are generally preferred. A mixed solvent of n-hexane and each of acetone, ether and ethyl acetate is particularly preferable.

The solvent injected from the top of the first column is suitable for the conditions depending on the type of packing material packed in the column, the type, number and amount of impurities contained in the crude coenzyme Q, and the treatment temperature. It is common to use different solvents. The solvent supplied from the top of the first column may be changed to another solvent during the supply.

When changing the solvent midway, it is preferable to sequentially increase the polarity of the solvent when each column is filled with an inorganic filler and when each column is filled with a polar porous resin. On the other hand, when each column is filled with a non-polar porous resin, it is preferable to sequentially decrease the polarity of the solvent. In addition, in order to adjust the polarity of the solvent,
A plurality of solvents having different polarities may be mixed as in a conventional method.

For example, hexane / acetone (volume ratio) 100/0, 98 /
If mixed at a ratio of 2, 95/5, the polarity will increase in this order. In addition, methanol / acetone (volume ratio) 7 /
If mixed at 3, 5/5 and 3/7, the polarity becomes smaller in this order.

The temperature of the column, the temperature of the crude coenzyme Q solution supplied to the column, the feed rate, the amount of solvent poured into the first column, the feed rate, etc., depend on the type of packing used, the amount of crude coenzyme Q in the crude coenzyme Q, and the like. It varies depending on the number, type and amount of impurities and the type of solvent, and cannot be specified unconditionally. These conditions may be selected as appropriate conditions, but the conditions for each column may be changed, and the conditions for each column may be the same. However, in practice, the following conditions are usually adopted. That is, for example, the temperature of the crude coenzyme Q solution supplied to the column and the temperature of the column do not lose the adsorptive capacity of the packing material, respectively, and the coenzyme Q in the crude coenzyme Q is dissolved in the solvent and the boiling point of the solvent is increased. The temperature may be lower, usually 15 to 50 ° C., preferably room temperature to room temperature. Heating and cooling are not particularly required, but heating and cooling are not hindered. The temperature of the column is preferably set higher in the later columns.

The supply amount of the crude coenzyme Q solution is usually the total weight (g) of the packing material used in each column when the inorganic packing material is used.
Of 50% by weight or less, preferably 30% by weight or less for practical use.

When a porous synthetic resin is used as the filler,
Usually 50 weight percent of the total packing volume (ml) used in each column (g-crude coenzyme Q / ml-total packing volume)
The amount is below, and practically, the amount is preferably 35% by weight or less. Here, the total volume (ml) of the filler is not an apparent volume but an actual volume.

The amount of the solvent that dissolves the crude coenzyme Q is not particularly limited, but is usually 0.5 to 5 ml with respect to 1 g of the crude coenzyme Q, and preferably 1.3 to 2.5 ml.

The ratio of the packing material weight (g) between the columns used in the present invention is not particularly limited, but the ratio of the maximum packing amount (weight) to the minimum packing amount (weight) is usually 0.1 to It is assumed to be 1.

Apparent column empty velocity of the solvent poured into the first column in the present invention Is not particularly limited, but in practice, it is usually 0.001 to 0.3 cm / s.
ec, preferably 0.01 to 0.1 cm / sec. Further, the upper pressure in the column may be normal pressure, or increased pressure or reduced pressure as long as the apparent empty velocity of the feed solvent is maintained for separation.

〔Example〕

Next, the present invention will be described more specifically by way of examples. However, the invention is not limited by these examples.

Example 54 g of crude coenzyme obtained from microbial cells (coenzyme Q ₁₀ content
37.8 g) was purified using the apparatus shown in FIG.
Wakogel C-300 suspended in n-hexane in each of three columns (22 mm in diameter, 450 mm in length)
Each column was packed with 210 g of 70 g.

A solution prepared by dissolving 54 g of the above-mentioned crude coenzyme in 108 ml of n-hexane at 25 ° C. was supplied to the upper part of the first column 1.

Then, while the liquid was extracted from the bottom of the first column 1 to the outside of the system, a solvent (a mixed solvent of acetone and n-hexane with an acetone content of 0.5% by volume) was poured from the top of the system. The solvent supply rate was 684 ml / hr, the apparent column empty velocity was 0.05 cm / sec, and the column temperature was maintained at 25 ° C.

By this operation, when 200 ml of the impurity solution was taken out of the system from the first bottom valve 14 of the first column 1, the first detector 17 (U
Coenzyme Q was detected at the V monitor wavelength of 254 nm and below. At this time, the first bottom valve 8 is closed, the first communication valve 16 and the second bottom valve 9 are opened, and the section containing coenzyme Q ₁₀ is set to the second section.
Transferred to column 2.

At this time, the solvent supplied to the first column 1 was changed to a solvent (a mixed solvent of acetone and n-hexane having an acetone content of 1.0% by volume, the same applies hereinafter). The supply rate is 684 ml / hr as with the solvent.

The temperature of the second column 2 was also 25 ° C. Also, the first column 1
And the top pressure of each of the second column 2 is 0.
It was 3 Kg / cm ² -G and 0.1 Kg / cm ² -G.

Here, the apparent empty space velocity of the solvent in both the first column 1 and the second column 2 was 0.044 cm / sec.

The eluate from the second bottom valve 9 of the second column 2 is used as a second detector.
Two fractions were collected in the order of elution, and were taken out of the system as two fractions, an elution fraction 560 ml containing only impurities detected in 12, and a fraction 190 ml containing impurities and a trace amount of coenzyme Q ₁₀ .

Next, the second bottom valve 9 is closed, the second communication valve 17 and the third bottom valve 10 are opened, and a section mainly containing only coenzyme Q ₁₀ and impurities (postcut) and a trace amount of coenzyme Q ₁₀ are added. The containing section was moved to the third column 3.

The temperatures of the first column 1 and the second column 2 were not changed, and the temperature of the third column 3 was set to 35 ° C.

The top pressure of each column is 0.4Kg / cm ² -G, 0.2Kg / c
Atsuta in m ² -G and 0.1Kg / cm ² -G.

From the start of adsorption column chromatography, the total amount of eluate was 20
When it reached 60 ml (200 ml from the bottom of the first column 1, 750 ml from the bottom of the second column 2, 1110 ml from the bottom of the third column 3), the solvent poured into the first column 1 was used as a solvent (acetone content rate). It was changed to a mixed solvent of 3.0% by volume of acetone and n-hexane, and so on). The supply rate was 684 ml / hr, similar to the solvent.

Next, the eluate from the third column bottom valve 10 of the third column 3 is separated into an elution section containing only impurities, a section containing impurities and a trace amount of coenzyme Q ₁₀ , a section mainly containing only coenzyme Q ₁₀ and impurities ( (Post-cut) and trace amount of coenzyme Q ₁₀
The samples were collected in the order of elution as one category. The amounts of these eluates were 2370 ml, 620 ml, 1750 ml and 410 ml, respectively.
The total amount of solvent used from the start of the adsorption column chromatography was 6100 ml (200 ml from the bottom of the first column 1, 750 ml from the bottom of the second column 2 and 5150 m from the bottom of the third column 3).
l)

To give coenzyme Q ₁₀ of 26.27g was mainly removing the solvent from the division 1750ml containing only coenzyme Q _10. The melting point of this coenzyme Q ₁₀ is
At 48 ° C., reverse phase and normal phase high performance liquid chromatography, mass spectrum and infrared absorption spectrum confirmed that the enzyme was coenzyme Q ₁₀ having a purity of 100%.

Recovery of Shutoku was purified coenzyme Q ₁₀ 69.5 a, Shutoku purified coenzyme Q solvents used total amount per ₁₀ 1 g and fillers used total amount was found to be respectively 232ml and 8.0 g.

A large amount of impurities remained in the first column 1 and the second column 2.

Comparative Example The procedure was carried out according to the example except that the liquid was not discharged from the bottom of each of the first column 1 and the second column 2 to the outside of the system. That is, a solvent prepared by dissolving 54 g of crude coenzyme Q _{10 5} similar to that in Example in 108 ml of n-hexane at 25 ° C. was supplied to the first column 1. The third tower bottom valve 7 was opened to allow the crude coenzyme described above.
Each component was developed and adsorbed by sequentially passing Q ₁₀ through each column.

The solvent is supplied from the bottom of the first column 1 at a feed rate of 684 ml / h.
r, apparent empty cylinder speed was 0.05 cm / sec, and 200 ml was poured.

The temperature of each column was maintained at 25 ° C.

Then, the solvent was changed to a solvent, and 3240 ml of the solvent was poured without changing the supply rate.

The temperature of each column was changed to 35 ° C. First
Column 1, column top pressure of the second column 2 and the third column 3 respectively ^{0.4Kg / cm 2 -G, 0.2Kg /} cm 2 -G and 0.1 Kg /
cm ² -G.

The eluate from the third column bottom valve 16 is an elution section containing only impurities, a section containing impurities and coenzyme Q ₁₀ , mainly coenzyme Q ₁₀
The four groups were separated in the order of elution, namely, a group containing only and a group containing impurities and coenzyme Q ₁₀ . The volume of each eluate was 3200 ml, 1190 ml, 1050 ml and 800 ml. The solvent was removed from the 1050 ml section containing mainly coenzyme Q ₁₀ alone to obtain 16.9 g of 99% pure coenzyme Q ₁₀ .

Recovery 44.7 percent Shutoku was purified coenzyme Q _10, Shutoku purified coenzyme Q ₁₀ (purity 99%) solvent using the total amount and fillers used total amount per 1g was found to be 384ml and 12.4g, respectively.
The recovery obtained in Comparative Example 2 was significantly inferior to that in Examples, and the purity of purified coenzyme Q ₁₀ was inferior to that in Examples.

Further, the total amount of the solvent used and the total amount of the filler used per 1 g of the purified coenzyme were extremely large as compared with the examples.

〔The invention's effect〕

In the present invention, the recovery rate of coenzyme Q is remarkably improved, and the amount of filler used and the amount of solvent used per unit weight of purified coenzyme are greatly reduced. Therefore, while the present invention is a method by adsorption column chromatography, coenzyme Q can be purified on an industrial scale. Moreover, the purity of the coenzyme Q obtained according to the present invention is high.

[Brief description of drawings]

FIG. 1 shows an example of the elution pattern of crude coenzyme Q, and FIG. 2 shows an example of the flow sheet of the purification apparatus used for the present invention. In the drawings, 1 ... First column, 2 ... Second column, 3 ... Third column, 4 ... First tower top pipe, 5 ... First tower bottom pipe, 6 ... Second column
Tower bottom tube, 7 ... Third tower bottom tube, 8 ... First tower bottom valve, 9 ...
Second bottom valve, 10 ... Third bottom valve, 11 ... First detector, 12
...... Second detector, 13 ...... Third detector, 14 ...... First connecting pipe, 15 ...... Second connecting pipe, 16 ...... First connecting valve, 17 ...... Second
Connection valve and 18 ... Supply pipe

Claims (1)

[Claims] 1. When purifying crude coenzyme Q by adsorption column chromatography, a plurality of columns connected in series to each other are used, and the crude coenzyme Q solution is sequentially passed through the columns to remove the crude coenzyme Q from each column. Eject the front cut category out of the system,
A method for purifying coenzyme Q, which comprises recovering a fraction whose main component is coenzyme Q from the final column