JPS5817594B2 - Cephalosporin C - Google Patents

Description

[Detailed description of the invention] The present invention relates to a novel method for purifying cephalosporin C.

More specifically, the present invention provides a solution containing cephalosporin C before using a weakly acidic carboxylic acid type cation exchange resin and a weakly basic or medium basic anion exchange resin, either separately or in combination. The pretreated liquid obtained from this pretreatment is treated with an anion exchange resin or a synthetic adsorbent or activated carbon to adsorb cephalosporin C, and the eluent removes the cephalosporin from the anion exchange resin or adsorbent. The gist of this invention is a method for recovering and purifying cephalosporin C, which is characterized by eluting phosphorus C and isolating cephalosporin C or a salt thereof from the active elution fraction.

In carrying out the method of the present invention, a solution containing cephalosporin C, especially a crude solution containing a large amount of dissolved impurities such as a culture solution, is treated with an H-type weakly acidic carboxylic acid type cation exchange resin. , a salt form such as an acetate form, or a free form of a weakly basic or medium basic anion exchange resin.

In this pretreatment step, the above-mentioned yin and yang ion exchange resins are used separately, for example in a continuous bed or in a mixed bed format, and the cephalosporin C solution is passed through the resin bed for pretreatment.

The active liquid obtained after passing through the resin bed is collected as a pretreatment liquid.

This is treated with a salt type strong, medium or weakly basic anion exchange resin such as acetate or formate to adsorb cephalosporin C.

Cephalosporin C is eluted using an inorganic or organic salt solution adjusted to pH 4 to 7, such as sodium acetate, potassium acetate, sodium formate, ammonium acetate, or pyridine-acetic acid buffer, as an eluent.

In order to isolate cephalosporin C from the active elution fraction containing eluted cephalosporin C, for example, the active elution fraction is concentrated under reduced pressure to crystallize cephalosporin C. Sporin C or its salt is purified and recovered.

Further, in the method of the present invention, as described above, after pre-treating a solution containing heptaph-1 allosporin C with a carboxylic acid type weakly acidic cation exchange resin and a weakly basic or medium basic anion exchange resin, Instead of the above-mentioned strong, medium or weak basic anion exchange resin, a synthetic adsorbent such as a styrene-divinylbenzene copolymer, e.g. Amberlite XAD series resin (Rohm Cephalosporin C can be adsorbed by adsorbing the pretreatment liquid using &Haas CO-), Diaion HP resin (Mitsubishi Chemical Industries, Ltd.), or activated carbon.

Cephalosporin C is eluted from this adsorbent using a water-containing organic solvent or dilute alkaline water as an eluent, and from the active elution fraction containing eluted cephalosporin C, monomers of previously known substances are extracted. Cephalosporin C or a salt thereof can be isolated by a separation method.

Conventionally, it has not always been easy to efficiently collect purified cephalosporin C on an industrial scale from an aqueous solution containing cephalosporin C, especially a solution containing a large amount of impurities such as a culture solution. 0 Particularly when adsorption/elution methods using ion exchange resins are employed, as cephalosporin C is a type of peptide that is a monoaminodicarboxylic acid, inorganic ions and other impurities in the solution may cause significant damage. easily susceptible to interference.

For this reason, as stated in Japanese Patent Publication No. 37-11695, cephalosporin C is
When recovering and purifying , the first stage pretreatment is performed by an adsorption/elution method using H-type strongly acidic cation exchange resin, and the second stage pretreatment is performed by an adsorption/elution method using a strongly basic anion exchange resin. Two steps of preprocessing are required.

In this case, it is possible that cephalosporin C is partially inactivated and adsorbed to the resin in the first step of pretreatment with a strongly acidic cation exchange resin, and the time required for these two steps of pretreatment is It takes a long time and the degree of deactivation is correspondingly large, which is disadvantageous in terms of yield and purity.

Therefore, it is industrially very difficult to obtain cephalosporin C by such a method.

Therefore, the current common method is to protect the amine group of cephalosporin C and then extract it with a solvent to obtain a cephalosporin C derivative.

The weakly acidic cation exchange resin used in the pretreatment step of the present invention hardly adsorbs cephalosporin C, and when the culture P solution is pretreated with it, the resulting pretreatment solution has a
H is 3 to 5, which is the stable pH range of cephalosporin C.

Further, even if the pretreatment is performed with a weakly basic or medium basic anion exchange resin that is simultaneously used in this pretreatment step, there is almost no loss of cephalosporin C.

As in the method of the present invention, the method of pretreating cultured lachrymal fluid using these two types of resins, i.e., a weakly acidic cation exchange resin and a weakly or moderately basic anion exchange resin, is conventionally carried out using a car bed or a mixed bed. There are no examples of either being used.

Of course, the above two types of resins are effective even when used separately in a continuous bed, but when used in a mixed bed type, the effect is even better and pretreatment can be performed in a short period of time.

In order to adsorb and elute cephalosphorin C, it is necessary to remove inorganic anions such as chloride ions, but in this case, the cations that form a pair with the anions must be removed first. .

In this case, it goes without saying that strongly acidic cation exchange resins are superior to weakly acidic cation exchange resins in terms of simply removing cations, and in fact, if the amount of resin is selected appropriately, it is possible to A pretreatment effect can be obtained.

However, as mentioned above, in the case of a strongly acidic resin, cephalosporin C is more likely to be deactivated than in the case of a weakly acidic resin.

Therefore, it is preferable to use a weakly acidic resin as long as it does not adversely affect the adsorption and elution of cephalosporin C, which is the next step.

In particular, even carboxylic acid type weakly acidic cation exchange resins have a pH of 4.
Decomposable neutral salts (e.g. Amberlite)
If IRC-84 (Rohm & Haas Co., Imac Z-5 (Imac-ti Co.), etc.) is used, there is no problem in practice.

In addition, other weakly acidic resins that can be used include Am
berlite I RC75, IRC-50 (Ro
tun &; Haas Co-), Diaion WK
-10, WK-11, WK-20 (Sanjomori Koriki, DOw
ex CCR-2 (The Dow Chemical
Co-)+10NACONN, CC(Ionac
chemicalCo・), Duol ite CC
-3(Diamond-8hamro-ck Chem
ical Co-), Lewatit CNP--80
(Farbenfabriken Bayer A
-G-) etc.

Further, the weak or medium basic anion exchange resin used in the present invention is, for example, Amberlite I RA 94.

I RA93, I RA68 (Rohm &;
Haas Co-Dowex MWA-1 (The
Dow Chemical-co, entered 10NACAF
P-329 (IonacC-bemical Co.)
, Imac A-2OR, A-205.

A-24 (Imacti Co.), Duolite
A-43.

A-6(Diamond-8hamrock Che
mical-Co, diamond ion WA-20, WA-
21.

WA-30 (Mitsubishi Chemical Industries), Lewatit MP-
64 (Farbenfabriken Bayer A
-G), etc., and the pretreated solution obtained by pretreating a crude solution containing cephalosporin C with the weakly acidic cation exchange resin or strongly acidic cation exchange resin described above is passed through these anion exchange resins. In this case, the adsorption rate of cephalosporin C is very low, and it adsorbs coexisting organic acids, fatty acids, and pigments well, and is also excellent in removing inorganic anions such as chloride ions.

The basic feature of the present invention is that the culture p solution containing cephalosporin C is pretreated with the above two types of ion exchange resins, but when both types of resins are used as a mixed bed, a better pretreatment effect can be obtained. Since this is recognized, the mixed bed pretreatment method will be mainly explained below.

According to the method of the present invention, when the culture filtrate containing cephalosporin C is passed through a mixed bed containing 1/10 to 1/20 of resin in an appropriate ratio in a descending method or an ascending method, the initial The part contains almost no cephalosporin C,
In the middle to latter part, a cephalosporin solution with 10 to 30 fb higher activity than the original solution is eluted as an active solution.
Finally, by washing with water in an amount of 2 to 5 times the amount of resin, cephalosporin C is recovered with almost no loss.

In this case, organic acids and fatty acids are hardly contained in the passing liquid and are eluted from the latter half of the washing liquid.

One cycle of cephalosporin C in this pretreatment step is usually 20 or more times, and the liquid amount is reduced to 80 to 90%.

Some chlorine ions may be detected in the pretreated liquid obtained by pretreatment using the above mixed bed, but even in that case,
There is no problem with adsorption by basic anion exchange resin in the next step.

As the resin used for adsorbing the pretreatment liquid, ordinary strong basic, medium basic, or weak basic anion exchange resins can be used, but particularly desirable resins include Amberli
teIRA401, IRA402, IRA411
.

IRA47, IRA68 (Rohm & HaasCo-
Diamond Aeon PA408, PA308, SAI
I.A.

8A21A, WAI O, WA20 (Mitsubishi Shichiyu Dingyo)
, Dowex I X 4 t 2 X 4 r 1
1 + 21 K t W G R (The Dow
Chemical Co, entered 10NACA-540,
A-305 (Ionac Chemical Co.)
, Asmlt 259 N, Imac S-5
-42, ImacA-27 (Imacti Co), [
) uo I ite A-102-D, A-10
] D, A-57 (Diamond -8ha-m
rock Chemical Co.), Lewa t
it M-504.

MK-70 (Parbenfabriken Baye
rA-G-) and XT5007 (organo).

In any case, these resins are used in the form of a salt such as acetic acid or formic acid.

The resin adsorbed with cephalosporin C may be washed with water and then pre-washed with a dilute acetic acid solution, a dilute alcohol solution, etc., but even if it is eluted directly with a salt solution after washing with water, it will yield high yield and high purity. of cephalosporin C eluate can be obtained.

In this case, the salt solution used as the eluent is suitably an aqueous solution of sodium acetate, potassium acetate, sodium formate or the like adjusted to pH 4 to 7, or a salt solution containing an organic solvent such as lower alcohol.

The eluate containing cephalosporin C obtained here can be extracted and isolated as a cephalosporin C derivative by protecting the amino group as it is, but it can also be concentrated under reduced pressure,
It can also be crystallized and isolated as cephalosporin C crystals.

That is, when the eluate is concentrated to a solid content of 40 to 70%, crystals of cephalosporin C are precipitated as salts of sodium, potassium, etc., and the crystals can be obtained by filter washing.

Instead of the adsorption and elution using the strongly basic anion exchange resin described above, the pretreatment liquid is used with various adsorbents, such as activated carbon, Am
It is also possible to obtain highly pure cephalosporin C crystals by adsorbing it onto a berIlteXAD-based resin, a Diaion HP-based resin, or the like, and then eluting it.

According to the method of the present invention, the decomposition and deactivation of cephalosporin C can be minimized, and the adsorption and elution and crystallization of cephalosporin C by ion exchange resins, which are usually extremely difficult, can be suppressed. It is an industrially excellent and useful method that can be carried out with high yield.

In addition, as shown in the comparative example, when adsorption with a resin is performed after pretreatment using an existing method, for example, the method of Japanese Patent Publication No. 11695/1983, which uses a strongly acidic cation exchange resin and a strongly basic anion exchange resin, Compared to the examples of the present invention, results were obtained that were significantly inferior in both yield and purity.

Comparative Example 1 Culture filtrate 51 containing cephalosporin C (pH 5,9,
3700 u/ml) according to the method of Japanese Patent Publication No. 37-11695.

That is, a strongly acidic cation exchange resin Ambe is added to the culture slurry.
rl ite IR120 (H type, Rohm & Haa
After adjusting the pH to 2.8 by adding s Co-), it was filtered, and the solution was left at room temperature for 3 hours, and the precipitate formed again was filtered, washed with a small amount of water, and acidic lachrymal fluid 5.111 (pH2
,8,2830u/rul! .

A yield of 78% was obtained.

This is mixed with strongly basic anion exchange resin Amber l i
teIRA400 (acetic acid type, Rohm & Haas Co
) The amount of resin passed through the 300ml column was determined by separately measuring the penetration point of chlorine ions on a small scale), 500ml
Wash with water and add pretreatment solution 5.21 (pH 4.0, 228
0 u/ml, yield 82%; pretreatment yield 64).

The pretreatment liquid was prepared using a strong basic anion exchange resin Asn1t 252 N (acetic acid type) 50 in the same manner as in Example 1 described later.
After adsorbing through 0rrLl and washing with water, add sodium acetate solution (
0.5M, adjusted to pH 6.0 with acetic acid).
Active eluate 800TLl (11800u/ml.

80o; b yield) was obtained.

This was concentrated under reduced pressure, but since no crystals precipitated, cephalosporin C sodium salt crystals were added as seeds and cooled overnight to allow crystallization.

Cephalosporin C is pale yellow after being filtered and washed with methanol.
8.1 g of sodium salt crystals (720 u/~, yield: 62%, total yield: I).

260 nm (D.1 ratio - 4°) and 1 crr
did.

A comparison of this with the method of the present invention obtained in Example 1 is as follows.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Culture solution 501 containing cephalosporin C (pH 5,9
, 3700 u/ml), the furnace solution was treated with carboxylic acid-type weakly acidic cation exchange resin "berlite".
I RC84 (H type) and weakly basic anion exchange resin A
amberlite I RA 94 (acetic acid type) 51 each
(diameter 8cm, height 250cIrL)
After passing through the column, the column was washed with water 151, extruded, and the fractions showing an activity of 1000 u/ml or more were combined to obtain active fraction 431) (pH 3, 8, 4080 u/IrLl,
(yield: 94.8%) was obtained as a pretreatment liquid.

This is strongly basic anion exchange resin Asmlt 259
A column packed with N (acetic acid type) 51 (diameter 5.8 cwt,
Cephalosporin C was adsorbed.

The column was then washed with 101 of water and then eluted with a sodium acetate solution (0.5M, adjusted to pH 6.0 with acetic acid) as eluent, resulting in an active eluent of 7.511 (20000 u/ml).

A yield of 85.5%) was obtained.

This eluate was concentrated under reduced pressure to precipitate crystals of cephalosporin C sodium salt, and an equal amount of methanol was added thereto and filtered.

After filtration and washing with cold methanol, the crystals were dried and
．． 9.9 (880u/”&, yield 85.0%, total yield 1%
A white crrL-colored cephalosporin C sodium salt was obtained with a yield of 68.9% and an E 178 at 260 nm.

Example 2 Culture p solution 501 (pH 5, 7, 4) was prepared in the same manner as in Example 1.
200u/ml) and 42 liters of pretreatment liquid (pH
3,9,4550 u/ml (yield 92.1%) was obtained.

This is mixed with strongly basic anion exchange resin Amber ite.
The mixture was applied to a column packed with 51 ml of IRA 401 (acetic acid type), washed with 101 ml of water, and eluted with an aqueous solution of sodium acetate (0.5 M, adjusted to pH 6.0 with acetic acid) as an eluent.

The obtained active eluate 6.61 (23200 u/ml, yield so, 1%) was concentrated under reduced pressure to precipitate crystals, filtered by adding an equal amount of methanol, and washed with methanol to obtain cephalosporin C. Sodium salt 151.0g (830u/
7Q, yield 8□, 8%, total yield.

. , 3rd section, 260 nm (7) E""crrL =168) was obtained.

1 Example 3 Pretreatment liquid 251 (pH 3, 9) obtained in the same manner as Example 2
, 4550 u (yield 92.1 from 7ml culture p solution) was adjusted to pH 2.5 with ION sulfuric acid, and the resulting precipitate was removed by filtration.

The furnace liquid is synthesized and adsorbent resin Diamond I-IP-2
0 (Mitsubishi Chemical Industries) 81 (diameter 8 crr)
t, height 200crrL) and cephalosporin C
was adsorbed.

After washing the column with 161 of water, 0.05 N-
Elute with NaOH aqueous solution, and the active eluent was 7.51 nm (pH
4,7,11800 u/ml, yield 77.8%).

This was adjusted to pH 6.0 with 2N-N a OH,
After concentrating under reduced pressure, ethanol was added to crystallize.73.
Cephalosporin C sodium salt of 99 (874 u/hi yield 72.9 mm, total yield 52.2 mm, 260 nm (
1) E”%=□76) was obtained.

ICrrL

Claims (1)

[Claims] 1 A solution containing cephalosporin C is subjected to pretreatment using a weakly acidic carboxylic acid type cation exchange resin and a weakly basic or medium basic anion exchange resin, either separately or in a mixture, and the resulting product is obtained from this pretreatment. The pretreated solution obtained is treated with an anion exchange resin, a synthetic adsorbent, or activated carbon to adsorb cephalosporin C, and the cephalosporin C is eluted from the anion exchange resin or adsorbent using an eluent to determine its activity. A method for recovering and purifying cephalosporin C, which comprises isolating cephalosporin C or a salt thereof from an elution fraction.