JP5618248B2 - Method for purifying carboxyl group-containing polyoxyethylene derivatives - Google Patents

Description

  The present invention relates to a method for purifying a high molecular weight carboxyl group-containing polyoxyethylene derivative.

  In recent years, polyoxyethylene has been used as a material to add blood retention, targeting function to target sites, etc. to nucleic acid compounds including nucleic acids, including polypeptides, enzymes, antibodies, genes, oligonucleic acids, nucleic acid drugs and other physiologically active substances. Derivatives are very often used. This is because of its low steric repulsion effect, the interaction with other biological components is low, and a bioactive substance modified with a polyoxyethylene derivative or a drug carrier modified with a polyoxyethylene derivative such as a liposome is not It is known to exhibit longer blood retention in the body than modified ones. In addition, it has been reported that this effect is larger as the polyoxyethylene derivative has a higher molecular weight. Furthermore, by attaching an active group or antibody to the terminal of this polyoxyethylene derivative, it is possible to add a targeting function. Polyoxyethylene derivatives are extremely useful and indispensable materials in the field of drug delivery systems. It has become. Among them, polyoxyethylene derivatives having a carboxyl group are very important raw materials as intermediates for various active ester groups. From the viewpoint of the performance and safety of a drug produced using this, a polyoxyethylene derivative having a carboxyl group is required to have higher purity and less impurities. Currently, polyoxyethylene derivatives having a carboxyl group have been developed having various skeletons or other functional groups, and impurities produced as a by-product vary depending on the production method. An example is shown below.

  In general, a polyoxyethylene derivative having a carboxyl group can be synthesized by using a polyoxyethylene derivative having a hydroxyl group at a terminal as a raw material and oxidizing the terminal hydroxyl group to a carboxyl group. Usually, in the case of a terminal modification reaction of a polyoxyethylene derivative, since it is a reaction at the end of a polymer, the concentration of reaction sites in the molecule decreases, the reaction rate deteriorates, and a hydroxyl group remains. Further, as the molecular weight increases, the viscosity of the reaction solution increases, the reaction rate deteriorates, and unreacted hydroxyl groups remain. In the case of the polyoxyethylene derivative having a large number of hydroxyl groups in the multi-arm type, impurities having different numbers of unreacted hydroxyl groups are generated during terminal modification. In general, the terminal hydroxyl group and the terminal carboxyl form are the same polyoxyethylene derivative and are difficult to purify. In particular, the larger the molecular weight, the closer the properties are, and therefore the separation and purification is difficult. Further, in the case of such a polyoxyethylene derivative in which a terminal unreacted hydroxyl group remains, dimerization proceeds in the next active esterification, and the purity of the active ester is significantly reduced. In addition, when impurities such as a hydroxyl group and a dimer remain, they remain as polymer impurities even during a drug modification reaction in a later step, resulting in a non-uniform drug and a problem as a preparation.

  As a method for purifying a polyoxyethylene derivative having a carboxyl group at the terminal, column chromatography purification using an ion exchange resin shown below is known (Patent Document 1 (US 5,298,410): Patent Document 2 (special Table 2008-514693)). In these patent documents, for the purpose of removing diols contained in methoxypolyethylene glycol, after the hydroxyl group is modified to a carboxyl group, the monocarboxylate and dicarboxylate are separated and separated by ion exchange chromatography. A method of taking is disclosed. As a specific purification method, a terminal carboxyl group methoxypolyethyleneglycol derivative (molecular weight 20,000) is dissolved in distilled water to make an about 2% aqueous solution, charged to an ion exchange resin, and then distilled water is poured into the impurities and the purpose. Things are separated. The obtained fraction is further diluted to an aqueous solution having a concentration of about 1%. After pH adjustment, purification is performed by extraction with dichloromethane, dehydration, and crystallization, but in the case of column chromatography purification using an ion exchange resin as described above, the sample is highly diluted (PEG concentration 1 to 2% aqueous solution). This is because the target product is a carboxyl compound having only a polymer terminal, and the interaction with the ion exchange resin is extremely low. In addition, a large-capacity extraction device is required to remove water from the diluted aqueous solution after fractionation to obtain the target product. In addition, the efficiency is poor even when considering the time required from the purification to the recovery of the target product, and the purification by ion exchange chromatography has a problem as an industrial production method.

  As another example of a polyoxyethylene derivative having a carboxyl group, there is a method in which a methoxypolyoxyethylene derivative is chemically bonded to an amino group or a thiol group of a compound containing a carboxyl group such as an amino acid for synthesis.

  For example, in Patent Document 3 (US05932462), a method for synthesizing a polyoxyethylene derivative (molecular weight 40,000) in which two methoxypolyoxyethylene derivatives (molecular weight 20,000) are introduced into two amino groups of lysine and its A purification method is described. The impurities in this case are an unreacted methoxypolyoxyethylene derivative having a molecular weight of 20,000 and one introduced into lysine (molecular weight 20,000), and a diol which is an impurity in the methoxypolyoxyethylene derivative (molecular weight 20,000). Isomers (molecular weight 40,000) introduced into lysine (molecular weight 40,000 or more), dimers that are impurities in lysine with three polyoxyethylene derivatives introduced (molecular weight 40,000 or more), etc. It is done.

  As a method for purifying impurities as described above, Patent Document 3 (US05932462) proposes column chromatography of an ion exchange resin. Specifically, an aqueous solution adjusted to have a polyoxyethylene derivative concentration of 0.2% is charged into an ion exchange resin column and fractionated using ion exchange water as an eluent. At this time, an impurity with a molecular weight of 20,000 flows out before the target product, and then the target product elutes. After the target product, one impurity is added to impurities with a molecular weight of 40,000 or more and lysine with a molecular weight of 20,000. Impurities introduced with methoxypolyoxyethylene derivatives are eluted. In order to fractionate the impurities and the target product, it is necessary to finely fractionate and analyze the eluted solution during the purification, and recover an aqueous solution containing only the target product. Further, this method is a method for separating a high molecular weight polyoxyethylene derivative, and it is necessary to dilute the charge sample to the column more than the above method (solution concentration 0.2%). Further, for purification in an aqueous solution, subsequent steps of extraction, dehydration, concentration and crystallization are necessary, and the operation is very complicated. Considering the time required for the amount to be purified, the equipment used, and the treatment of distilled water and organic solvent used for purification, purification by column chromatography is industrially disadvantageous as in the above method.

US 5,298,410 Special table 2008-514693 US 05932462

  An object of the present invention is a method for producing a polyoxyethylene derivative having a carboxyl group having a high purity and a high molecular weight by reducing impurities contained in a main component.

  As a result of intensive studies to solve the above problems, the present inventors selected a high molecular weight polyoxyethylene derivative from an organic solvent such as toluene or ethyl acetate from the group consisting of magnesium, silicon and aluminum. By adsorbing using an inorganic adsorbent containing at least one of oxides and hydroxides of one or more elements and adsorbing and filtering the target polyoxyethylene derivative having a carboxyl group Then, the polyoxyethylene derivative having no carboxyl group as an impurity is separated to the filtrate side. Furthermore, the adsorbent on the filter paper is again adsorbed with an alcohol solvent such as ethanol, and the polyoxyethylene derivative having the target carboxyl group is desorbed from the adsorbent and filtered to adsorb impurities having multiple carboxyl groups. It is a method of isolation by leaving it in the agent, and it is a purification method capable of obtaining a polyoxyethylene derivative having a high purity and a high molecular weight by separating regardless of the impurities and the molecular weight of the target product.

  According to the present invention, without performing column chromatography, the adsorbent is mixed with a solution in which a polyoxyethylene derivative is dissolved from the beginning, like a filter aid, and is simply filtered. can do.

That is, the present invention relates to the following method.
A purification method for separating impurities from a carboxyl group-containing polyoxyethylene derivative having a molecular weight of 2,000 to 100,000 represented by the general formula [1], comprising the following steps (A) to (E).

（式中、Ｚが２〜８個の活性水素を持つ多価アルコール、アミノ酸またはペプチドの残基であり、Y¹及びY²がエーテル結合、アミド結合、エステル結合、ウレタン結合、カーボネート結合、２級アミノ基、チオエーテル結合、ジスルフィド結合、チオエステル結合、またはこれらを含むアルキレン基であり、Polymer¹及びPolymer²が直鎖のポリオキシエチレン鎖または分岐のポリオキシエチレン鎖であり、Ｒが炭素数1〜７の炭化水素基または炭素数３〜９のアセタール基であり、Xはアミノ基、保護アミノ基、カルボキシル基、保護カルボキシル基、アルデヒド基、保護アルデヒド基、水酸基、保護水酸基、チオール基、保護チオール基、シアノ基またはこれらを含むアルキレン基であり、ｍ及びｎが、それぞれ、m＝1または０、ｎ＝1または0、かつ1≦ｍ＋ｎであり、i及びｋが、それぞれ、１≦i≦５、ｋ＝１または０であり、ａ、ｂ及びｃが、それぞれ、０≦ａ、０≦ｂ、１≦ｃかつ１≦ａ＋ｂ、２≦ａ＋ｂ＋ｃ≦８を満たす整数であり、m＝０かつｎ＝1の場合には１≦ｂであり、m＝1かつｎ＝0の場合には１≦ａであり、a、ｂ及びｃが２以上の整数の場合、Y¹及びY²、Polymer¹、Polymer²、Ｒ、ｍ、ｎ、i、ｋは分子中で同一または異なっても良い）
(A) 前記ポリオキシエチレン誘導体の5質量倍以上のトルエン、キシレン、ベンゼン、酢酸エチルおよび酢酸ブチルからなる群より選択される有機溶剤を使用し、前記ポリオキシエチレン誘導体を溶解して溶液とする工程、
(B) マグネシウム、ケイ素およびアルミニウムからなる群より選ばれた一種または二種以上の元素の酸化物と水酸化物の少なくとも一方を含む無機系吸着剤を前記溶液に対して前記ポリオキシエチレン誘導体の0.5〜１０質量倍添加することで、スラリーを生成させる工程、
(C) 25℃以上の温度で前記スラリーを攪拌する工程、
(D) このスラリーをろ過して得られたろ過ケーキに、トルエン、キシレン、ベンゼン、酢酸エチルおよび酢酸ブチルからなる群より選択される有機溶剤を添加し、さらにろ過を行う工程、および
(E) 前記ポリオキシエチレン誘導体の5質量倍以上のメタノール、エタノールおよび2-プロパノールからなる群より選択される有機溶剤を前記ろ過ケーキに添加してろ過し、得られたろ液から前記ポリオキシエチレン誘導体を回収する工程

(Wherein Z is a residue of a polyhydric alcohol, amino acid or peptide having 2 to 8 active hydrogens, and Y ¹ and Y ² are ether bonds, amide bonds, ester bonds, urethane bonds, carbonate bonds, 2 A ^primary amino group, a thioether bond, a disulfide bond, a thioester bond, or an alkylene group containing these, Polymer ¹ and Polymer ² are linear polyoxyethylene chains or branched polyoxyethylene chains, and R is carbon number 1 -7 hydrocarbon group or acetal group having 3 to 9 carbon atoms, X is amino group, protected amino group, carboxyl group, protected carboxyl group, aldehyde group, protected aldehyde group, hydroxyl group, protected hydroxyl group, thiol group, protection A thiol group, a cyano group or an alkylene group containing these, and m and n are each m = 1 or 0, n = 1 or 0, 1 ≦ m + n, i and k are 1 ≦ i ≦ 5, k = 1 or 0, and a, b and c are 0 ≦ a, 0 ≦ b, 1 ≦ c and 1 ≦, respectively. a + b, 2 ≦ a + b + c ≦ 8, 1 ≦ b when m = 0 and n = 1, 1 ≦ a when m = 1 and n = 0, a, b And c is an integer of 2 or more, Y ¹ and Y ² , Polymer ¹ , Polymer ² , R, m, n, i, and k may be the same or different in the molecule)
(A) Using an organic solvent selected from the group consisting of toluene, xylene, benzene, ethyl acetate and butyl acetate at least 5 times the mass of the polyoxyethylene derivative, dissolve the polyoxyethylene derivative to obtain a solution. Process,
(B) An inorganic adsorbent containing at least one of an oxide and a hydroxide of one or more elements selected from the group consisting of magnesium, silicon and aluminum is added to the polyoxyethylene derivative with respect to the solution. A step of generating a slurry by adding 0.5 to 10 times by mass,
(C) stirring the slurry at a temperature of 25 ° C or higher,
(D) a step of adding an organic solvent selected from the group consisting of toluene, xylene, benzene, ethyl acetate and butyl acetate to the filter cake obtained by filtering this slurry, and further filtering; and
(E) An organic solvent selected from the group consisting of methanol, ethanol and 2-propanol at least 5 times by mass of the polyoxyethylene derivative is added to the filter cake and filtered, and the polyoxyethylene is obtained from the obtained filtrate. Process for recovering derivatives

In the polyoxyethylene derivative represented by the general formula [1], Y ¹ is a urethane bond or an ether bond, Polymer ¹ is a linear polyoxyethylene chain, R is a methyl group, and m = 1 , A = 1 or 2, b = 0, c = 1, i = 1 or 5.

In the polyoxyethylene derivative represented by the general formula [1], Z is a residue of lysine, Y ¹ is a urethane bond, and k = 0 and a = 2.

  A method in which the molecular weight of the polyoxyethylene derivative represented by the general formula [1] is 10,000 to 100,000.

  The method whose quantity of the said adsorption agent is 2-6 mass times of a polyoxyethylene derivative.

  A method in which the solvent in the step (A) is selected from toluene and ethyl acetate.

  A method in which the solvent in step (E) is selected from methanol and ethanol.

  A method in which the amount of the solvent in the step (A) is 10 to 20 times by mass that of the polyoxyethylene derivative.

  A method in which the amount of the solvent in the step (E) is 10 to 20 times by mass that of the polyoxyethylene derivative.

  According to the present invention, regardless of the molecular weight of the target product, the molecular weight of impurities, the structure and the functional group contained in the target, it can be separated and purified with high purity by the presence or absence of the carboxyl group or the number thereof. Furthermore, since the present invention is easy to operate, it can be said to be a highly suitable purification method with good reproducibility by scaling up to industrial production. The high molecular weight polyoxyethylene derivative obtained by the purification method of the present invention has a low impurity content and a high purity. Moreover, a high purity thing is easily obtained by manufacturing with the manufacturing method of this invention. The bio-related substance modified with the high molecular weight polyoxyethylene derivative of the present invention has few impurities, is excellent in safety and stability, is suitable for the pharmaceutical and cosmetic fields, and is useful.

  The details of the present invention are a method for purifying a polyoxyethylene derivative having a molecular weight of 2,000 to 100,000 represented by the general formula [1] by an adsorption treatment step including the operations (A) to (E).

  Z is a residue of polyhydric alcohol, amino acid or peptide having 2 to 8 active hydrogens, and specific compounds include ethylene glycol, propylene glycol, trimethylene glycol, isopropylene glycol, butylene glycol, tetramethylene Glycols, trimethylolpropane, glycerin, diglycerin, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, erythritol, pentaerythritol, dipentaerythritol, polyhydric alcohols such as sorbitol, mannitol, xylitol, or lysine, glutamic acid, etc. Examples include amino acids having amino groups, carboxyl groups, or thiol groups, and dimers and trimers thereof. Preferred compounds are ethylene glycol, glycerin, pentaerythritol, xylitol, lysine and glutamic acid, more preferred are ethylene glycol, glycerin and lysine, and even more preferred is lysine.

  Here, the residue is a residue obtained by removing a functional group having an active hydrogen group such as a hydroxyl group, an amino group, a carboxyl group, or a thiol group.

Y ¹ and Y ² are bonding groups between Z and the polyoxyethylene chain, and are not particularly limited as long as they are covalent bonds, but preferably ether bonds, thioether bonds, ester bonds, urethane bonds, amide bonds , Carbonate bond, secondary amino group, disulfide bond and alkylene group containing them. Preferred linking groups are shown below.

When Z is a residue of a polyhydric alcohol having 2 to 8 active hydrogens, a polyoxyethylene derivative is bonded to the hydroxyl group of the polyhydric alcohol
-O-, -OCONH (CH ₂ ) _p O-, -O (CH ₂ ) _p NHCO (CH ₂ ) _q O-, -O (CH ₂ ) _p NHCOO-

When Z is a residue of an amino acid or peptide and a polyoxyethylene derivative is bound to the amino group of the amino acid or peptide
-NHCO (CH ₂ ) _q O-, -NHCOO-, -NHCO-
When Z is an amino acid or peptide residue, and a polyoxyethylene derivative is bound to the carboxyl group of the amino acid or peptide
-COO-, -CONH (CH ₂ ) _p O-
When Z is an amino acid or peptide residue, and a polyoxyethylene derivative is bound to the thiol group of the amino acid or peptide
-S-, -SS-
(Where p and q are integers of p = 2 or 3, q = 1-5)

Polymer ¹ and Polymer ² are linear or branched polyoxyethylene chains. A branched polyoxyethylene chain is a polyoxyethylene chain branched into two or more chains via a branch point in the middle, and there may be a plurality of branch points. As an example, it is a polyoxyethylene chain branched from two or more chains with a polyhydric alcohol such as glycerin as shown in the following formula (i) as a branch point.

  R is a hydrocarbon group having 1 to 7 carbon atoms or an acetal group having 3 to 9 carbon atoms. Specific hydrocarbon groups and acetal groups include methyl, ethyl, propyl, isopropyl, and butyl groups. , T-butyl group, pentyl group, isopentyl group, cyclopentyl group, hexyl group, 2-hexyl group, 3-hexyl group, cyclohexyl group, phenyl group, benzyl group, diethyl acetal group, dimethyl acetal group, diisopropyl acetal group, 1 1,3-dioxolane and the like. Preferred hydrocarbon groups are methyl, t-butyl and benzyl groups, and preferred acetal groups are diethyl acetal groups.

  X is an amino group, protected amino group, protected carboxyl group, aldehyde group, protected aldehyde group, hydroxyl group, protected hydroxyl group, thiol group, protected thiol group, sulfonyl group, vinyl group, allyl group, cyano group, and alkylene groups containing them It is. Specific examples of the alkylene group include a methylene group, an ethylene group, a propylene group, an isopropylene group, a butylene group, an isobutylene group, a pentylene group, an isopentylene group, and a hexylene group, preferably a methylene group, an ethylene group, A propylene group, more preferably an ethylene group.

m and n are m = 1 or 0, n = 1 or 0, and 1 ≦ m + n, i and k are 1 ≦ i ≦ 5, k = 1 or 0, and a, b, and c are respectively , 0 ≦ a, 0 ≦ b, 1 ≦ c and 1 ≦ a + b, 2 ≦ a + b + c ≦ 8, and when m = 0 and n = 1, 1 ≦ b, m = 1 and n When 0 = 0, 1 ≦ a. When a, b, and c are integers of 2 or more, Y ¹ and Y ² , Polymer ¹ and Polymer ² , R, m, n, i, and k are in the molecule. It may be the same or different.

  The molecular weight of the polyoxyethylene derivative is 2,000 to 100,000. However, in the preparation using a polyoxyethylene derivative in recent medical applications (PEGylated preparation), the higher the molecular weight, the higher the blood retention. Therefore, the molecular weight of the desired polyoxyethylene derivative tends to increase. Therefore, the upper limit is not particularly limited, but the molecular weight of the preferred polyoxyethylene derivative is 6,000 to 100,000, more preferably 10,000 to 100,000, still more preferably 20,000 to 80,000, and most preferably 30,000 to 50,000.

Hereinafter, each process will be described in detail.
“(A) Using an organic solvent selected from toluene, xylene, benzene, ethyl acetate, and butyl acetate at least 5 times the mass of the polyoxyethylene derivative represented by the general formula [1], Process of dissolving into solution "

 The solvent is an organic solvent selected from toluene, xylene, benzene, ethyl acetate, and butyl acetate, and is preferably a solvent having low polarity and high solubility of the polyoxyethylene derivative. Preferred organic solvents are toluene and ethyl acetate, and more preferably toluene. A highly polar solvent is not preferable because it interferes with the interaction between the polyoxyethylene derivative and the adsorbent.

  The amount of the solvent is preferably 5 times by mass or more with respect to the polyoxyethylene derivative represented by the general formula [1]. If it is less than 5 times by mass, the viscosity of the solution is high, the purification efficiency is deteriorated, and the yield is also deteriorated. Even if the amount of the solvent is 30 mass times or more, the purification efficiency is good without changing, but in the subsequent filtration operation, the treatment capacity increases, the man-hour increases and the yield decreases, which is disadvantageous in terms of cost. For these reasons, the preferred amount of solvent is 5 to 30 times, more preferably 10 to 20 times.

  The polyoxyethylene derivative is dissolved using the solvent. The order of charging into the processing vessel may be either a polyoxyethylene derivative or an organic solvent. Depending on the molecular weight of the polyoxyethylene derivative, heating may be necessary, and the method is not particularly limited, but in general, it can be dissolved by heating to 30 ° C. or higher.

“(B) an inorganic adsorbent containing at least one of oxides and hydroxides of one or more elements selected from the group consisting of magnesium, silicon and aluminum with respect to the solution; Step of generating slurry by adding 0.5 to 10 times the mass of

  In the adsorbent used in the present invention, magnesium, aluminum and silicon oxides and hydroxides are magnesium oxide, aluminum oxide, silicon oxide, or a composite oxide of these two or three kinds, or magnesium water. It is an oxide, an aluminum hydroxide, a silicon hydroxide, or a composite hydroxide of two or three of these.

  In the inorganic adsorbent, these oxides (including complex oxides) and hydroxides (including complex hydroxides) may be contained singly, and a mixture of two or more kinds is contained. Good. The inorganic adsorbent may further contain a hydrate.

Specific examples include magnesium oxide, activated alumina, silica gel, Kyoward series manufactured by Kyowa Chemical Industry Co., Ltd., Kyoward 200B (Al ₂ O ₃ .H ₂ O), Kyoward 200 (Al (OH) ₃ XH ₂ O) (x is 1 ≦ x ≦ 3), Kyoward 500 (Mg ₆ Al ₂ (OH) ₁₆ CO ₃ .4H ₂ O), Kyoword 700 (Al ₂ O ₃ .9SiO ₂ .H ₂ O ), Kyoward 1000 (Mg _4.5 Al ₂ (OH) ₁₃ CO ₃ .3.5H ₂ O), Kyoward 2000 (Mg _0.7 Al _0.3 O _1.15 ). Each of the above inorganic adsorbents may be used alone or in combination.

In order to separate a polyoxyethylene derivative having a carboxyl group from a polyoxyethylene derivative having no carboxyl group, an adsorbent having an interaction of carboxyl groups is advantageous. As the particularly preferred inorganic adsorbents in terms _{Al 2 O 3 · H 2 O} , Al (OH) 3 · xH 2 O (x is _{1 ≦ x ≦ 3), Mg} 0.7 Al 0.3 O 1. ₁₅ , more preferably Al ₂ O ₃ .H ₂ O.

  The amount of adsorbent is preferably in the range of 0.5 to 10 times the mass of the polyoxyethylene derivative represented by the general formula [1]. If it is less than 0.5 mass times, a sufficient purification effect cannot be obtained, and if it is more than 10 mass times, the amount of treatment increases and the yield decreases, and the number of steps increases. For these reasons, the adsorbent amount is preferably 1 to 6 times by mass, more preferably 2 to 6 times by mass.

"(C) Step of stirring the above solution at 25 ° C or higher"
In this step, an adsorbent is added to the solution in step (B) to form an adsorbent slurry, which is subjected to an adsorption treatment. The treatment temperature is preferably 25 to 80 ° C. At temperatures higher than 80 ° C, the polyoxyethylene derivative may be deteriorated by heat. If the temperature is lower than 25 ° C., the viscosity of the solution is high and the purification efficiency deteriorates. Moreover, since crystals may be deposited depending on the structure and molecular weight of the polyoxyethylene derivative, the temperature is preferably 25 ° C. or higher. A preferred temperature range is 40 to 80 ° C, more preferably 40 to 60 ° C. The slurry is agitated for 30 minutes or more in an inert gas atmosphere or while blowing the slurry in the above temperature range. The stirring time is preferably 30 minutes or more in order to perform sufficient adsorption, and there is no limit on the upper limit. By this operation, the carboxyl group is preferentially adsorbed because the interaction with the adsorbent is greater than the hydroxyl group.

“(D) A step of filtering this slurry, adding an organic solvent selected from toluene, xylene, benzene, ethyl acetate, and butyl acetate to the filter cake, and further filtering”

  This step is a step of separating the adsorbent and the solvent in which the impurities are dissolved from the adsorption treatment solution in the above step (C). The method for removing the adsorbent is not particularly limited, but in general, the removal is performed by vacuum filtration, centrifugal filtration, or pressure filtration. At this time, it is desirable to heat the filter in advance at the same processing temperature as in step (C) for the purpose of preventing the precipitation of crystals due to a decrease in temperature during filtration. In order to improve filterability, a filter aid may be placed on the filter paper in advance.

  After filtering the slurry solution, an organic solvent selected from toluene, xylene, benzene, ethyl acetate, and butyl acetate is added, followed by filtration to remove impurities remaining in the filter cake. The organic solvent used is preferably the same solvent as the adsorption treatment solution.

  The amount of the solvent is preferably 5 times by mass or more with respect to the polyoxyethylene derivative represented by the general formula [1]. If it is less than 5 mass times, sufficient impurities cannot be removed from the filter cake, so 5 mass times is advantageous. Even if the amount of the solvent is 30 mass times or more, the purification efficiency is good without changing, but in the subsequent filtration operation, the treatment capacity increases, the man-hour increases and the yield decreases, which is disadvantageous in terms of cost. For these reasons, the preferred amount of solvent is 5 to 30 times, more preferably 10 to 20 times. Furthermore, the solvent removal efficiency becomes high by preheating the solvent to the temperature at the time of treatment.

“(E) A step of adding an organic solvent selected from methanol, ethanol, 2-propanol 5 times or more times the polyoxyethylene derivative and recovering the polyoxyethylene derivative from the filter cake”

  The solvent is an organic solvent selected from methanol, ethanol and 2-propanol, and is preferably a solvent having high polarity and high solubility of the polyoxyethylene derivative. Preferred solvents are methanol and ethanol, more preferably ethanol.

  The amount of the solvent is preferably 5 times by mass or more with respect to the polyoxyethylene derivative represented by the general formula [1]. If it is less than 5 times by mass, the viscosity of the solution is high, the purification efficiency is deteriorated, and the yield is also deteriorated. Even if the amount of the solvent is 30 mass times or more, the purification efficiency does not change and is good. However, in the subsequent filtration operation, the processing capacity increases, man-hours increase, and this is disadvantageous in terms of cost. For these reasons, the preferred amount of solvent is 5 to 30 times, more preferably 10 to 20 times.

  Using the solvent, the target polyoxyethylene derivative having a carboxyl group is desorbed and recovered from the filtered cake after filtration. As a method for recovery, the above-mentioned solvent heated in a filter is added and filtered, and the filtrate is isolated from the filtrate. The filter cake is again slurried with the above-mentioned solvent, and then filtered. The method of releasing is mentioned.

  In the former method, the recovery efficiency is increased by preheating the solvent to the temperature at the time of the treatment in step (C).

  The latter method is the same operation as steps (C) and (D) in which the solvent to be used is the above solvent, and the latter has higher recovery efficiency than the former method because the filter cake is sufficiently mixed with the solvent. Become. In this case, a filter aid may be preliminarily placed on the filter paper in order to improve filterability. The filter aid mentioned here is a powder for preventing clogging of filter paper such as diatomaceous earth, for example, Oplite W-3050 (manufactured by Oplite Mining Co., Ltd.).

  After filtration, an organic solvent selected from methanol, ethanol, and 2-propanol is further added, followed by further filtration, and recovery of the target product remaining in the filter cake. The organic solvent used at this time is preferably the same solvent as the desorption treatment solution. The amount of the solvent is preferably 5 times by mass or more with respect to the polyoxyethylene derivative represented by the general formula [1]. If it is less than 5 mass times, sufficient impurities cannot be removed from the filter cake, so 5 mass times is advantageous. Even if the amount of the solvent is 30 mass times or more, the purification efficiency does not change and is good. However, in the subsequent filtration operation, the processing capacity increases, man-hours increase, and this is disadvantageous in terms of cost. For these reasons, the preferred amount of solvent is 5 to 30 times, more preferably 10 to 20 times. Furthermore, the solvent removal efficiency becomes high by preheating the solvent to the temperature at the time of treatment.

  A method for isolating the target product from the filtrate is not particularly limited, but a method using distillation under reduced pressure or a method using reprecipitation is preferable.

  The operations described above can be repeated after returning to the step (A) or the step (E) after the steps (A) to (E). Moreover, when it repeats, refinement | purification efficiency may go up by changing combinations, such as changing the kind or quantity of an adsorbent of the 1st time and the 2nd time, and the organic solvent to be used. This depends on the number of carboxyl groups in the polyoxyethylene derivative to be purified, and the number, type, and content of impurities contained, but impurities that have weaker interaction with the adsorbent than those of the target are mixed. This is particularly effective in some cases. In general, when removing impurities that weakly interact with the adsorbent (such as impurities of polyoxyethylene derivatives that do not have a carboxyl group), toluene is used as the solvent in step (A), and the target has strong adsorption power. Use of an adsorbent increases the purification efficiency, and conversely, when removing impurities that interact strongly with the adsorbent (such as impurities of polyoxyethylene derivatives having two or more carboxyl groups) By using a weak adsorbent and using ethanol as the solvent in the step (D), the purification efficiency is improved. As for the amount of adsorbent, an optimum amount may be used as appropriate depending on the impurity content. However, in the case of impurities having a smaller interaction than the target product, the removal efficiency increases by reducing the amount of adsorbent as the amount of impurities increases. Thus, in the case of an impurity having a larger interaction than that of the target product, the removal efficiency is increased by increasing the amount of the adsorbent as the amount of the impurity increases.

The polyoxyalkylene derivative was synthesized by the following method.
Dissolve 548 g of lysine monohydrochloride in 12 L of 0.1 M phosphate buffer adjusted to pH 8 and add 3 kg of NOF MENP-20T (α-methoxy-ω-pnitrophenylcarbonyl-polyoxyethylene with a molecular weight of 20,000) And stirred at room temperature overnight. 24 kg of sodium chloride was dissolved in the reaction solution, extracted with 30 kg of chloroform, and the chloroform layer was washed with 12.5 kg of 20% brine. After concentration, it was dissolved in 15 kg of toluene, dehydrated and filtered using 1 kg of magnesium sulfate. The filtrate was crystallized by adding 12 kg of hexane and dried to obtain 2.4 kg of a polyoxyethylene derivative in which methoxypolyoxyethylene was introduced into one of two amino groups of lysine.

  450 g of the obtained crystals are dissolved in 2.7 kg of dehydrated dimethylformamide, and 114 g of triethylamine and 475 g of NOF MENP-20T (α-methoxy-ω-pnitrophenylcarbonyl-polyoxyethylene with a molecular weight of 20,000) are added at 60 ° C. The reaction was performed for 8 hours. Crystallization was performed by adding 4.5 kg of ethyl acetate to the reaction solution and further adding 3.2 kg of hexane. The crystals were dissolved in 5.85 kg of ethyl acetate, recrystallized by adding 3.15 kg of hexane again, and then dried to obtain 940 g of a polyoxyethylene derivative in which two polyoxyethylenes were introduced into two amino groups of lysine. Obtained.

Regarding the purity of the obtained polyoxyethylene derivative, the molecular weight distribution of the polymer was measured by gel permeation chromatography (GPC). The measurement method and the result are described below.
As GPC system, developing solvent using SH-10ADV LC-10AVP: N, N-dimethylformamide (10 mM LiBr), flow rate: 0.7 ml / min,
Column: PL gel MIXED-D x2 (polymer laboratory),
Column temperature: 65 ° C, detector: R
I, Sample amount: 30mg / 30mL, Injection amount: 100ul

  For GPC measurement values, high molecular weight impurities and low molecular weight impurities are divided perpendicularly to the baseline from the inflection point of the elution curve with the main peak, and the area percentage of each peak is calculated from the area value of each peak obtained. Is calculated.

  Here, high molecular weight impurities are those in which lysine is bonded to both ends of polyoxyethylene having a molecular weight of 40,000 or more (derived from diol), and three polyoxyethylene derivatives are introduced into the dimer which is an impurity in lysine. The low molecular weight impurities are unreacted methoxypolyoxyethylene derivatives and monosubstituted lysine methoxypolyoxyethylene derivatives having a molecular weight of 20,000.

The present invention will be described in more detail with reference to the following examples using the LY-400 obtained by the above method. Evaluation at that time was performed according to the following criteria.
When the target peak after purification is 90% or more and the yield is 50% or more → ◎
When the target peak after purification is 90% or more and the yield is 20% or more → ○
When the target peak after purification is 90% or more and the yield is 10% or more → △
The target peak after purification is less than 90% → ×

(Example 1-1)
30 g of the above polyoxyethylene derivative and 450 g of toluene were charged into a 1 L four-necked flask, a three-one motor, a cooling tube, and a nitrogen blowing tube were attached, and dissolved at 55 ° C. using a mantle heater. 120 g of KYOWARD 200B (Kyowa Chemical Industry) was added thereto, and the mixture was stirred at 50 ° C. for 1 hour to carry out an adsorption operation. Nutsche with 5A filter paper and Oplite W-3050 (manufactured by Oplite Mining Co., Ltd.) was prepared, and suction filtration was performed. Thereafter, the filter cake was washed with 300 g of toluene heated to 50 ° C. in advance. Thereafter, ethanol was added to the filter cake, heated and stirred at 50 ° C., suction filtered again, and the filter cake was washed with 300 g of ethanol heated to 50 ° C., and the filtrate was collected. The filtrate was concentrated using an evaporator, dissolved in 150 g of ethyl acetate, crystallized by adding 100 g of hexane, the precipitated crystals were separated by filtration, dried, and a sample was collected (21 g).

  As a result, the methoxypolyethylene glycol impurity content with a molecular weight of 20,000 was reduced to 2.0%, the high molecular weight impurities with a molecular weight of 40,000 or more were reduced to 2.0%, and the GPC purity could be purified to 96.0%. Furthermore, as a result of processing the obtained target object again by the same operation, it was possible to improve the GPC purity to 98.5%.

(Examples 1-2 to 7)
The same operation as in Example 1-1 was performed under the other conditions using the adsorbents shown in the following table as the adsorbents used for the adsorption. The results are shown below.

  Here, the adsorbent species of Examples 1-1 to 5 are Kyoward series product names, which are inorganic adsorbents manufactured by Kyowa Chemical Industry.

(Comparative Examples 1-1 to 1-2)
The same operation was carried out using the following magnesium sulfate and calcium oxide in place of the adsorbent in the method of Example 1-1. The results are shown below.

  From the above results, no purification effect was observed.

(Examples 1-8 to 1-9)
The same procedure as in Example 1-1 was performed under the other conditions using the amounts of adsorbent used for adsorption in the amounts shown in the following table. The results are shown below.

 From the above results, increasing the amount of adsorbent tends to improve both the effect and yield, but there is almost no difference between 3 times and 4 times the mass. Although it affects the purity of the polyoxyethylene derivative before purification, a sufficient purification effect was observed even in double amount.

(Example 1-10)
The same operation as in Example 1 was performed except that the solvent used in the step (A) was the solvent shown in the following table. The results are shown below.

  From the above results, ethyl acetate is effective, but toluene has better purity and yield.

(Comparative Examples 1-3 to 1-4)
The same operation as in Example 1 was performed except that the solvent used in the step (A) was the solvent shown in the following table. The results are shown below.

以上の結果より、上記の溶剤では効果があまり見られなかった。

From the above results, the above solvent was not very effective.

(Examples 1-11 to 12)
The same operation as in Example 1 was performed under the other conditions using the amount of solvent in the step (A) shown in the following table. The results are shown below.

  From the above results, the effect can be seen even at 10 mass times, but the purification effect is higher when the amount of the solvent is larger. However, the purification effect is almost unchanged at 15 mass times or more.

Claims (9)

A purification method for separating impurities from a carboxyl group-containing polyoxyethylene derivative having a molecular weight of 2,000 to 100,000 represented by the general formula [1], comprising the following steps (A) to (E).

(Wherein Z is a residue of a polyhydric alcohol, amino acid or peptide having 2 to 8 active hydrogens, and Y ¹ and Y ² are ether bonds, amide bonds, ester bonds, urethane bonds, carbonate bonds, 2 A ^primary amino group, a thioether bond, a disulfide bond, a thioester bond, or an alkylene group containing these, Polymer ¹ and Polymer ² are linear polyoxyethylene chains or branched polyoxyethylene chains, and R is carbon number 1 -7 hydrocarbon group or acetal group having 3 to 9 carbon atoms, X is amino group, protected amino group, carboxyl group, protected carboxyl group, aldehyde group, protected aldehyde group, hydroxyl group, protected hydroxyl group, thiol group, protection A thiol group, a cyano group or an alkylene group containing these, and m and n are each m = 1 or 0, n = 1 or 0, 1 ≦ m + n, i and k are 1 ≦ i ≦ 5, k = 1 or 0, and a, b and c are 0 ≦ a, 0 ≦ b, 1 ≦ c and 1 ≦, respectively. a + b, 2 ≦ a + b + c ≦ 8, 1 ≦ b when m = 0 and n = 1, 1 ≦ a when m = 1 and n = 0, a, b And c is an integer of 2 or more, Y ¹ and Y ² , Polymer ¹ , Polymer ² , R, m, n, i, and k may be the same or different in the molecule)

(A) Using an organic solvent selected from the group consisting of toluene, xylene, benzene, ethyl acetate and butyl acetate at least 5 times the mass of the polyoxyethylene derivative, dissolve the polyoxyethylene derivative to obtain a solution. Process,
(B) An inorganic adsorbent containing at least one of an oxide and a hydroxide of one or more elements selected from the group consisting of magnesium, silicon and aluminum is added to the polyoxyethylene derivative with respect to the solution. A step of generating a slurry by adding 0.5 to 10 times by mass,
(C) stirring the slurry at a temperature of 25 ° C or higher,
(D) a step of adding an organic solvent selected from the group consisting of toluene, xylene, benzene, ethyl acetate and butyl acetate to the filter cake obtained by filtering this slurry, and further filtering; and
(E) An organic solvent selected from the group consisting of methanol, ethanol and 2-propanol at least 5 times by mass of the polyoxyethylene derivative is added to the filter cake and filtered, and the polyoxyethylene is obtained from the obtained filtrate. Process for recovering derivatives
In the polyoxyethylene derivative represented by the general formula [1], Y ¹ is a urethane bond or an ether bond, Polymer ¹ is a linear polyoxyethylene chain, R is a methyl group, and m = 1 The method of claim 1, wherein a = 1 or 2, b = 0, c = 1, i = 1 or 5. The method according to claim 2, wherein in the polyoxyethylene derivative represented by the general formula [1], Z is a residue of lysine, Y ¹ is a urethane bond, and k = 0 and a = 2.   The method according to any one of claims 1 to 3, wherein the molecular weight of the polyoxyethylene derivative represented by the general formula [1] is 10,000 to 100,000.   The method according to claim 1, wherein the amount of the adsorbent is 2 to 6 times by mass of the polyoxyethylene derivative.   The method according to claim 1, wherein the organic solvent in the step (A) is selected from the group consisting of toluene and ethyl acetate.   The method according to claim 1, wherein the organic solvent in the step (E) is selected from the group consisting of methanol and ethanol.   The method according to any one of claims 1 to 7, wherein the amount of the organic solvent in the step (A) is 10 to 20 times by mass that of the polyoxyethylene derivative.   The method according to any one of claims 1 to 8, wherein the amount of the organic solvent in the step (E) is 10 to 20 times the mass of the polyoxyethylene derivative.