A PROCESS FOR PURIFICATION OF POLYETHER BLOCK COPOLYMERS

Abstract

Process for providing purified polyether block copolymers comprising polyoxyethylene (PEO) and polyoxypropylene (PPO) moieties wherein the purified product is obtained by an ultrafiltration step of a solution of the polyether block copolymers and wherein the block copolymers are depleted in lower molecular impurities.

Claims

1. A process for providing purified polyether block copolymers comprising polyoxyethylene (PEO) and polyoxypropylene (PPO) moieties wherein the purified polyether block copolymer is obtained by an ultrafiltration step of a solution of the polyether block copolymer.

2. The process according to claim 1, wherein the polyether block copolymers comprising polyoxyethylene and polyoxypropylene moieties are selected from the group consisting of polyethylene oxide-block-polypropylene oxide copolymers and polyethylene oxide-polypropylene oxide random copolymers.

3. The process according to claim 1, wherein the polyether block copolymers comprising polyoxyethylene and polyoxypropylene moieties are triblock (PEO-PPO-PEO)-copolymers

4. The process according to claim 1 wherein the triblock (PEO-PPO-PEO)-copolymers are poloxamer 188 or poloxamer 407.

5. The process according to claim 1, wherein the solution of the polyether block copolymers treated by ultrafiltration is an aqueous solution or an organic solution or an aqueous-organic solvent mixture.

6. The process according to claim 1, wherein the solution of the polyether block copolymers treated by ultrafiltration is an aqueous solution.

7. The process according to claim 1, wherein a concentration of the polymer in the solution is 2-20% b.w.

8. The process according to claim 1, wherein a concentration of the polymer in the solution is 5-15% b.w.

9. The process according to claim 1, wherein the ultrafiltration is carried out using a membrane separating layer which is a ceramic or polymer or carbon material.

10. The process according to claim 1, wherein the ultrafiltration is carried out using a membrane separating layer with a cut-off in the range of from 1 to 50 kD (kiloDalton).

11. The process according to claim 1, wherein the ultrafiltration step is combined with an acid treatment step, wherein the acid treatment step is carried out prior to or simultaneously with the ultrafiltration step.

12. The process according to claim 11, wherein the acid treatment step is carried out in a solution of the polyether block copolymer in water or an organic solvent or aqueous-organic solvent mixtures

13. The process according to claim 11, wherein the acid treatment is carried out at a pH in the range of from pH 1 to pH 5

14. The process according to claim 11, wherein the acid is selected from the group of inorganic acids consisting of sulfuric acid, nitric acid, hydrochloric acid, sulfonic acid, and phosphoric acid,

15. The process according to claim 11, wherein the acid is hydrochloric or sulfuric acid.

16. The process according to claim 11, wherein the acid is selected from the group of organic acids consisting of formic acid, acetic acid, propionic acid, fumaric acid, tartaric acid, butyric acid, benzoic acid, succinic acid, oxalic acid, malic acid, lactic acid, adipic acid, and citric acid

17. The process according to claim 11 wherein the acid treatment is carried out at temperatures in the range of from 20 to 60 C.

18. The process according to claim 17, wherein the acid treatment is carried out at temperatures in the range of from 25 to 40 C.

19. The process according to claim 1, wherein the purified polyether block copolymer obtained by the ultrafiltration step is further subjected to sequential multi-column size exclusion chromatography in a simulated moving bed apparatus.

20. The process according to claim 19, wherein a process cycle comprises (vi) providing a feed mixture comprising the block copolymers dissolved in an eluent in a feed vessel, (vii) subjecting the feed mixture to a chromatographic separation by introducing the feed mixture into an apparatus comprising a plurality of chromatographic columns sequentially linked together, each column comprising a stationary phase, (viii) after separation collecting a first eluent portion enriched in the purified target block copolymer and a second eluent portion depleted of the purified target block copolymer, (ix) collecting the purified block copolymer from the first eluent portion, and (x) recovery of the depleted eluent and recycling the depleted eluent into the process.

Description

[0038] The process is further illustrated by FIG. 1:

[0039] FIG. 1 depicts a test unit with a feed/retentate circulation loop consisting of a feed/retentate circulation vessel B1, a circulation pump P1, a heat exchanger W1, a membrane module M1 and a pressure control valve V1. Between the heat exchanger W1 and the module entrance a temperature TM, a flow FM and a pressure PH and between the module outlet and the pressure control valve V1 an additional pressure P12 measurement is sited. The permeate pressure is measured by P13 and adjusted by the permeate pressure control valve V2. Via the three-way valve V3 the permeate can be led to the permeate vessel B2 or back to the circulation vessel B1 (the permeate is recycled). The permeate flux is measured by the weight increase over time in the permeate vessel B2.

[0040] Two operation modes are possible:

[0041] Concentration mode: In this case the permeate is gathered in B2 and the hold-up of the circulation loop and B1 is reduced respectively. The mass concentration factor CF is defined by CF=mret,0/mret,t=mret,0 (mret,0mperm,t) mret,0 =initial feed mass in B1 and the circulation loop, trial time=0 mret,t=mass in B1 and the circulation loop at trial time t mperm,t=permeate mass in B2 at trial time t

[0042] Diafiltration mode: In this case the gathered permeate in B2 is balanced by the permeate medium which is fed from B3 by the pump P2. The mass diafiltration coefficient DF is defined DF=mperm,t/m0

[0043] mret,0=initial feed mass in B1 and the circulation loop, constant over trial time mret,0=mret,t

[0044] mperm,t=permeate mass in B2 at trial time t

[0045] permeate mass=diafiltration medium mass at each trial time

[0046] Further definitions:

[0047] The selectivity of the membrane is defined by the Rejection R=1(cP/cR)

[0048] cR=concentration on the feed side of the membrane

[0049] cP=concentration on the permeate side of the membrane

[0050] The samples for the determination of both concentrations have to be taken at the same time, concentration factor or mass diafiltration coefficient.

[0051] The concentration can refer to the solid content, a specific molecule or a polymer fraction defined by the molecular weight.

[0052] The trans membrane pressure TMP is calculated from P11, P12 and P13 TMP=((P11+P12)/2)P13

[0053] The cross flow velocity is calculated from the feed flow and area of the feed flow channel.

[0054] According to another embodiment of the invention the ultrafiltration step can be combined with an acid treatment step in order to remove impurities that are prone to hydrolysis, particularly to remove aldehyde impurities. This acid treatment is carried out prior to the ultrafiltration step or simultaneously with the ultrafiltration step. The acid treatment can be carried out in a separate apparatus or in the ultrafiltration apparatus. The acid treatment is carried out by (i) dissolving the block copolymers in a solvent, preferably water, methanol, ethanol or aqueous mixtures thereof (ii) adding one or more acids and (iii) removal of the hydrolysed impurities. According to the embodiment wherein the acid treatment is carried out simultaneously with the ultrafiltration step, the acid is added to the feed solution for the ultrafiltration. The acid added to the block copolymer solution can be any acid that is strong enough to start the hydrolytic reaction and not too corrosive. The acid can be chosen from the group of inorganic acids consisting of sulfuric acid, nitric acid, hydrochloric acid and sulfonic acid, phosphoric acid, preferably hydrochloric or sulfuric acid. The acid can also be chosen from the group of organic acids consisting of formic acid, acetic acid, propionic acid, fumaric acid, tartaric acid, butyric acid, benzoic acid, succinic acid, oxalic acid, malic acid, lactic acid, adipic acid or citric acid. Pure acids or mixtures can be used and the amount of acid depends on the desired pH value. The pH for the acid treatment can be in the range of from 1 to 5, preferably 3. The concentration of the aqueous acid used to set the pH is preferably in the range of from 0.1 to 2 mol/ L.

[0055] Treatment time depends on the amounts of impurities to be hydrolysed, but is usually not less than one hour and up to two hours. The skilled artisan will know how to adapt the required time period for a given case.

[0056] The acid treatment can be carried out at temperatures in the range of from 20 to 60 C.,

[0057] preferably 25 to 40.

[0058] The pressure for the acid treatment is chosen to lie in the range of from 0.1 to 1 MPa, preferably 0.1 to 0.5 MPa.

[0059] The reaction can be carried out under ambient atmosphere or under protective atmosphere. Suitable inert gases for the treatment are nitrogen, helium, argon, carbon dioxide.

[0060] After the ultrafiltration step which optionally combined with the acid hydrolysis step, the solution comprising the purified target block copolymer can be converted to give a powderous polymer by known processes such as heating and distilling the water off until the polymers are molten. The molten polymers can be cooled to give a solid mass which can be converted to powders by convention methods such as for instance cryo-milling. Alternatively, the molten mass can be converted to powders by spray processes.

[0061] According to another embodiment of the invention the method of purification of poloxamers by ultrafiltration is combined with a process for purification of polyether block copolymers comprising polyoxyethylene and polyoxypropylene moieties using sequential mufti-column size exclusion chromatography in a simulated moving bed apparatus.

[0062] Before the solution obtained by ultrafiltration is subjected to the size exclusion chromatography in a simulated moving bed apparatus the solution can be concentrated by removing water and subsequently be diluted with methanol.

[0063] The process for purification of polyether block copolymers comprising polyoxyethylene and polyoxypropylene moieties using sequential mufti-column size exclusion chromatography in a simulated moving bed apparatus (SMB chromatography) wherein a process cycle comprises the steps of [0064] (i) providing a feed mixture comprising the block copolymers dissolved in an eluent in a feed vessel, [0065] (ii) subjecting the feed mixture to a chromatographic separation by introducing the feed mixture into an apparatus comprising a plurality of chromatographic columns sequentially linked together, each column comprising a stationary phase, [0066] (iii) after separation collecting a first eluent portion enriched in the purified target block copolymer and a second eluent portion depleted of the purified target block copolymer, [0067] (iv) collecting the purified block copolymer from the first eluent portion, and [0068] (v) recovery of the depleted eluent and recycling the depleted eluent into the process.

[0069] According to this embodiment impurities with a molecular weight higher than the molecular weight of the target block copolymer can be removed.

[0070] The respective equipment for carrying out the size exclusion chromatography is commercially available and can be adapted by the skilled expert to the specific needs of the separation process, operated under different pumps, valves and configuration and columns in static position or as actual moving bed (AMB, CSEP, ISEP apparatus) as described in U.S. Pat. No. 7,141,172.

[0071] The eluent can be an organic solvent or water or a mixture thereof.

[0072] According to a particularly preferred embodiment the eluent is methanol or a mixture of methanol with water or other organic solvents, particularly acetonitrile and/or acetone.

[0073] The stationary phase comprises a size exclusion chromatographic packing material. According to one preferred embodiment the stationary bed comprises as an inorganic adsorbent a silica based material, more preferably a silica diol. The silica diols are silica particles modified with 1,2-dihydroxypropane to cover the surface of the particles with diol groups. Such silica diol materials are commercially available at bulk quantities and different pore and particle sizes.

[0074] The number of columns used in each apparatus is not particularly limited. A skilled person would easily be able to determine an appropriate number of columns depending the amount of material to be purified.

[0075] The SMB separation can be operated as a high pressure process or as a low pressure process. The separation process is preferably carried out at high pressures >0.5 MPa up to an upper limit in the range of 10 MPa.

[0076] Typically, the temperature of the columns is limited from a lower level where the formation of crystals or particulates may be observed up to vaporization of solute or solvent. According to one embodiment the process is carried out at constant room temperature from 20 to 25 C. Optionally the process can be carried out at higher temperatures in the range of from 30 to 65. C.

[0077] After the block copolymers of the invention have been subjected to an ultrafiltration they are distinguished by very low residual contents of low molecular impurities. It is thus possible to provide block copolymers which are suitable in particular as excipients in the cell culture production of biologicals, as processing aids for other biotechnological applications such as cell engineering or as excipients in specific pharmaceutical dosage forms. In combination with a SMB chromatographic process higher molecular impurities can be removed as well so that poloxamers with a very defined molecular weight fraction can be achieved.

[0078] The successful removal of low molecular weight compounds or impuritites can be controlled by the following analytical methods:

[0079] The molecular weight distribution of poloxamers is determined by size exclusion chromatography (HPLC) The principle of this analytical technique is known to the expert.

[0080] This method is preferably carried out under the following conditions:

[0081] Stationary phase (columns): 3 columns sulfonated polystyrene/divinylbenzene resins (3008 mm SDV 1000A/100.000A/1.000 000A)

[0082] 1 analytical pre-column (sulfonated polystyrene divinylbenzene copolymer

[0083] Solvent: Tetrahydrofuran (THF)

[0084] Column temperature: 60 C.

[0085] Flow: 1 mL/min

[0086] Injection volume: 100 DL

[0087] Concentration: 2 mg/mL

[0088] Samples were filtered prior to analysis Macherey-Nagel PTFE-20/25 (0.2 Dm)

[0089] Calibration: Polyethylene glycol with narrow molar mass in the range of 106-1.522.000 g/mol)

[0090] Detector: RID Agilent 1100

[0091] However, other variations of this method will work as well and can be adapted by the skilled artisan to this specific problem.

[0092] The successful removal of impurities by acid treatment, specifically aldehyde or acetal impurities, can be controlled by the following analytical method:

[0093] The aldehydes are determined by reversed phase HPLC after reaction of the sample with 2,4-dinitrophenylhydrazine as the respective dinitrophenyl hydrazine derivatives. For quantification an external standard is applied using UV detection at 370 nm.

[0094] Sample derivatization: 60 mg of poloxamer 188 are weighted (accurate to 0.01 mg) into a 10 mL volumetric flask, dissolved in 1 mL acetonitrile, and derivatized by addition of 1-2 mL reagent solution followed by heating to 60 C. for 5 min. After cooling down to ambient temperature, the flask is filled up to the mark with acetonitrile/ water (1:1)

[0095] Reagent solution: 4 g with 2,4-dinitrophenylhydrazine (stabilized with 50% b.w. water) are weighed into a 1 L Erlenmeyer flask. 800 mL water and 200 mL concentrated hydrochloric acid are added. The mixture is stirred until it is clear.

[0096] Stationary phase: Symmetry Shield RP 18-5 m, Waters (2.1 mm diameter, stainless steel)

[0097] Calibration solutions: 20 mg of aldehyde dinitrophenylhydrazones are weighed, accurate to 0.01 mg and dissolved in acetonitrile. Dilutions are adjusted in such a way that the concentration of hydrazine is within the ranges listed below:

[0098] Formaldehyde derivative 0.0021-0.43 mg/10 mL injection solution

[0099] Acetaldehyde derivative 0.0024-0.47 mg/10 mL injection solution

[0100] Propionic aldehyde derivative . 0.0024-0.47 mg/10 mL injection solution

[0101] Mobile phase: water (A)/acetonitrile (B) gradient:

TABLE-US-00003 t/min A [%] B [%] 0 60 40 25 30 70 35 30 70 36 60 40 45 60 40

[0102] Flow: 0.4 mL/min

[0103] Injection volume: 5 L

[0104] Temperature: 45 C.

[0105] Detection: UV/VIS, lambda=370 nm

[0106] Thus, the present invention is characterized by the following specific embodiments.

Embodiment 1

[0107] Process for providing purified polyether block copolymers comprising polyoxyethylene (PEO) and polyoxypropylene (PPO) moieties wherein the purified product is obtained by an ultrafiltration step of a solution of the polyether block copolymers.

Embodiment 2

[0108] Process according to Embodiment 1, wherein the polyether block copolymers comprising polyoxyethylene and polyoxypropylene moieties are selected from the group consisting of polyethylene oxide- block- polypropylene oxide copolymers or polyethylene oxide-polypropylene oxide random copolymers.

Embodiment 3

[0109] Process according to Embodiment 1 or 2, wherein the polyether block copolymers comprising polyoxyethylene and polyoxypropylene moieties are triblock (PEO-PPO-PEO)-copolymers.

Embodiment 4

[0110] Process according to any of Embodiments 1 to 3 wherein the triblock (PEO-PPO-PEO)-copolymers are poloxamer 188 or poloxamer 407.

Embodiment 5

[0111] Process according to any of Embodiments 1 to 4 wherein the triblock (PEO-PPO-PEO)-copolymers is poloxamer 188.

Embodiment 6

[0112] Process according to any of Embodiments 1 to 5, wherein the solution of the polyether block copolymers treated by ultrafiltration is an aqueous solution or an organic solution or an aqueous-organic solvent mixture.

Embodiment 7

[0113] Process according to any of Embodiments 1 to 6, wherein the solution of the polyether block copolymers treated by ultrafiltration is an aqueous solution.

Embodiment 8

[0114] Process according to any of Embodiments 1 to 6, wherein the solution of the polyether block copolymers treated by ultrafiltration is an organic solution or an aqueous-organic solvent mixture.

Embodiment 9

[0115] Process according to any of Embodiments 1 to 6 and 8, wherein the organic solvent is selected from the group consisting of methanol, ethanol, acetonitril, acetone, ethyl acetate.

Embodiment 10

[0116] Process according to any of Embodiments 1 to 9, wherein the concentration of the polymer in the solution is 1-50% b.w.

Embodiment 11

[0117] Process according to any of Embodiments 1 to 10, wherein the concentration of the polymer in the solution is 2-20% b.w.

Embodiment 12

[0118] Process according to any of Embodiments 1 to 11, wherein the concentration of the polymer in the solution is 5-15% b.w.

Embodiment 13

[0119] Process according to any of Embodiments 1 to 12, wherein the block polymers to be purified by ultrafiltration to remove low molecular compounds are dissolved in the aqueous solution, and the aqueous solution is brought into contact with a membrane under pressure, and the permeate free of the target block copolymers (filtrate) comprising the dissolved impurities is drawn off on the reverse side of the membrane at a lower pressure than on the feed side.

Embodiment 14

[0120] Process according to any of Embodiments 1 to 13, wherein the ultrafiltration is carried out using a membrane separating layer which is a ceramic or polymer or carbon material.

Embodiment 15

[0121] Process according to any of Embodiments 1 to 14, wherein the ultrafiltration is carried out using a membrane separating layer which is a ceramic material.

Embodiment 16

[0122] Process according to any of Embodiments 1 to 15, wherein the ultrafiltration is carried out using a membrane separating layer which is a ceramic material selected from the group consisting of alpha-Al.sub.2O.sub.3, ZrO.sub.2, TiO.sub.2, SiC and/or mixed ceramic materials.

Embodiment 17

[0123] Process according to any of Embodiments 1 to 18, wherein the ultrafiltration is carried out using a membrane separating layer which is a ceramic material with ZrO.sub.2 layered on alpha-Al.sub.2O.sub.3.

Embodiment 18

[0124] Process according to any of Embodiments 1 to 17, wherein the ultrafiltration is carried out using a membrane separating layer with a cut-off in the range of from 1 to 150 kD (kiloDalton).

Embodiment 19

[0125] Process according to any of Embodiments 1 to 18, wherein the ultrafiltration is carried out using a membrane separating layer with a cut-off of 1, 5, 10, 20 or 100 kD (kiloDalton).

Embodiment 20

[0126] Process according to any of Embodiments 1 to 19, wherein the ultrafiltration is carried out using a membrane separating layer with a cut-off in the range of from 1 to 50 kD (kiloDalton).

Embodiment 21

[0127] Process according to any of Embodiments 1 to 20, wherein the ultrafiltration is carried out with a transmembrane pressure preferably in a range from 0.02 to 2 MPa, particularly preferably in a range from 0.03 to 0.66 MPa.

Embodiment 22

[0128] Process according to any of Embodiments 1 to 21, wherein the ultrafiltration step is combined with an acid treatment step wherein the acid treatment step is carried out prior to or simultaneously with the ultrafiltration step.

Embodiment 23

[0129] Process according to any of Embodiments 1 to 22, wherein the acid treatment step is carried out in a solution of the polyether block copolymer in water or an organic solvent or aqueous-organic solvent mixtures.

Embodiment 24

[0130] Process according to any of Embodiments 1 to 23, wherein the acid treatment is carried out at a pH in the range of from pH 1 to pH 5.

Embodiment 25

[0131] Process according to any of Embodiments 1 to 24, wherein the acid treatment is carried out at a pH in the range of pH 3.

Embodiment 26

[0132] Process according to any of Embodiments 1 to 25, wherein the acid is selected from the group of inorganic acids consisting of sulfuric acid, nitric acid, hydrochloric acid, sulfonic acid and phosphoric acid.

Embodiment 27

[0133] Process according to any of Embodiments 1 to 26, wherein the acid is hydrochloric or sulfuric acid.

Embodiment 28

[0134] Process according to any of Embodiments 1 to 27, wherein the acid is sulfuric acid.

Embodiment 29

[0135] Process according to any of Embodiments 1 to 25, wherein the acid is selected from the group of organic acids consisting of formic acid, acetic acid, propionic acid, fumaric acid, tartaric acid, butyric acid, benzoic acid, succinic acid, oxalic acid, malic acid, lactic acid, adipic acid or citric acid.

Embodiment 30

[0136] Process according to any of Embodiments 1 to 29, wherein the concentration of the aqueous acid used to set the pH is preferably in the range of from 0.1 to 2 mol/ L.

Embodiment 31

[0137] Process according to any of Embodiments 1 to 30 wherein the acid treatment is carried out at temperatures in the range of from 20 to 60 C.

Embodiment 32

[0138] Process according to any of Embodiments 1 to 31, wherein the acid treatment is carried out at temperatures in the range of from 25 to 40 C.

Embodiment 33

[0139] Process according to any of Embodiments 1 to 32, wherein the pressure for the acid treatment is chosen to lie in the range of from 0.1 to 1 MPa, preferably 0.1 to 0.5 MPa.

Embodiment 34

[0140] Process according to any of Embodiments 1 to 33, wherein the purified product obtained by the ultrafiltration step is further subjected to sequential mufti-column size exclusion chromatography in a simulated moving bed apparatus.

Embodiment 35

[0141] Process according to Embodiment 34, wherein a process cycle comprises the steps of providing a feed mixture comprising the block copolymers dissolved in an eluent in a feed vessel, subjecting the feed mixture to a chromatographic separation by introducing the feed mixture into an apparatus comprising a plurality of chromatographic columns sequentially linked together, each column comprising a stationary phase, after separation collecting a first eluent portion enriched in the purified target block copolymer and a second eluent portion depleted of the purified target block copolymer, collecting the purified block copolymer from the first eluent portion, and recovery of the depleted eluent and recycling the depleted eluent into the process.

Embodiment 36

[0142] Process according to any of embodiments 1 to 36, wherein the removal of low molecular weight compounds is determined by size exclusion chromatography. The method which can be applied for all embodiments is described in detail above.

Embodiment 37

[0143] Process according to any of embodiments 1 to 37, wherein the removal of impurities by acid treatment is be controlled by the following analytical method:

[0144] the aldehydes are determined by reversed phase HPLC after reaction of the sample with 2,4-dinitrophenylhydrazine as the respective dinitrophenyl hydrazones. For quantification an external standard is applied using UV detection at 370 nm. The method is described in detail above.

Embodiment 38

[0145] Process according to any of Embodiments 1 to 37, wherein a low molecular impurity is an impurity with a molecular weight below the average molecular weight of the target polyether block polymer to be purified.

Embodiment 39

[0146] Process according to any of Embodiments 1 to 38, wherein a low molecular impurity is an impurity with an average molecular weight at least 1500 g/mol below the average molecular weight of the target polyether block polymer to be purified.

Embodiment 40

[0147] Process according to any of Embodiments 1 to 38, wherein a low molecular impurity is an impurity with an average molecular weight at least 2000 g/mol below the average molecular weight of the target polyether block polymer to be purified and wherein the impurity is a polyether polymer.

Embodiment 41

[0148] Process according to any of Embodiments 1 to 38, wherein a low molecular impurity is an impurity with an average molecular weight at least 3000 g/mol below the average molecular weight of the target polyether block polymer to be purified and wherein the impurity is a polyether polymer

Embodiment 42

[0149] Process according to any of Embodiments 1 to 40, wherein a low molecular impurity is an impurity carrying aldehyde or acetal groups.

[0150] The following examples further describe the invention without limiting its scope.

EXAMPLES

[0151] In the following examples %, if not further specified. means % by weight

[0152] All ultrafiltration experiments in the following examples were carried out using a membrane with zircon dioxide layered on alpha-Al.sub.2O.sub.3.

[0153] The molecular weight distribution of poloxamers was determined by size exclusion chromatography (HPLC) under the followings conditions:

[0154] Stationary phase (columns): 3 columns sulfonated polystyrene/divinylbenzene resins (3008 mm SDV 1000A/100.000A/1.000 000A)

[0155] 1 analytical pre-column (sulfonated polystyrene divinylbenzene copolymer

[0156] Solvent: Tetrahydrofuran (THF)

[0157] Column temperature: 60 C.

[0158] Flow:1 mL/min

[0159] Injection volume: 100 DL

[0160] Concentration: 2 mg/mL

[0161] Samples were filtered prior to analysis Macherey-Nagel PTFE-20/25 (0.2 Dm)

[0162] Calibration: Polyethylene glycol with narrow molar mass in the range of 106-1.522.000 g/mol)

[0163] Detector: RID Agilent 1100

[0164] The aldehydes were determined by reversed phase HPLC after reaction of the sample with 2,4-dinitrophenylhydrazine as the respective dinitrophenyl hydrazones. For quantification an external standard was applied using UV detection at 370 nm.

[0165] Sample derivatization: 60 mg of poloxamer 188 were weighed (accurate to 0.01 mg) into a 10 mL volumetric flask, dissolved in 1 mL acetonitrile, and derivatized by addition of 1-2 mL reagent solution followed by heating to 60 C. for 5 min. After cooling down to ambient temperature, the flask is filled up to the mark with acetonitrile/water (1:1)

[0166] Reagent solution: Approx. 4 g with 2,4-dinitrophenylhydrazine (stabilized with 50% water) re weighed into a 1 L Erlenmeyer flask. 800 mL water and 200 mL concentrated hydrochloric acid were added. The mixture is stirred until it is clear.

[0167] Stationary phase: Symmetry Shield RP 18-5 m, Waters (2.1 mm diameter, stainless steel)

[0168] Calibration solutions: 20 mg of aldehyde dinitrophenylhydrazones were weighed, accurate to 0.01 mg, and dissolved in acetonitrile. Dilutions were adjusted in such a way that the concentration of hydrazine is within the following ranges:

[0169] Formaldehyde derivative 0.0021-0.43 mg/10 mL injection solution

[0170] Acetaldehyde derivative 0.0024-0.47 mg/10 mL injection solution

[0171] Propionic aldehyde derivative Approx. 0.0024-0.47 mg/10 mL injection solution

[0172] Mobile phase: water (A)/acetonitrile (B) gradient:

TABLE-US-00004 t/min A [%] B [%] 0 60 40 25 30 70 35 30 70 36 60 40 45 60 40

[0173] Flow: 0.4 mL/min

[0174] Injection volume: 5 L

[0175] Temperature: 45 C.

[0176] Detection: UV/VIS, lambda=370 nm

[0177] The total aldehyde contents listed in the tables for the starting materials are calculated on the basis of the hydrazone derivatives (comprising hydrazine derivatives of free aldehydes and the acetals, which are trapped in the polymer and are released after hydrolysis).

Example 1

[0178] The first experiment was performed using a 100 mm long 10/6 UF5 kDZ membrane (atech innovations Gmbh). The circulation vessel B1 was filled with 2829 g Poloxamer 188 solution, having a solid content of 10.26%. The experimental conditions were set to obtain a temperature of 60 C., a cross-flow velocity of 4 m/s and a trans-membrane pressure of 0.1MPa. Table 1 summarizes the results of this experiment. First, the experiment was run in a diafiltration mode until a diafiltration factor DF of 3.91 was reached. During this step, the solid contents in the retentate dropped from 10.26 to 6.66%. Because of the decreased polymer content in the retentate, an increase in the membrane flux was recorded. Subsequently, the polymer was concentrated by a factor 2 to a retentate concentration of 12.74% as shown in Table 2.

TABLE-US-00005 TABLE 1 DF Flux C.sub.Ret C.sub.Perm R kg/m.sup.2/h % % 0.00 22.4 10.26 1.34 86.9 0.48 25.4 9.75 1.05 89.2 0.96 29.6 9.04 0.75 91.7 2.05 37.3 7.88 0.59 92.5 2.91 42.3 7.30 0.48 93.5 3.91 47.6 6.66 0.38 94.3

TABLE-US-00006 TABLE 2 CF Flux C.sub.Ret C.sub.Perm R kg/m.sup.2/h % % 1.00 47.6 6.66 0.38 94.3 1.20 36.5 1.40 27.9 1.60 20.3 1.80 14.5 2.04 10.0 12.74 0.97 92.4

[0179] Results obtained by size exclusion chromatography according to Table 3 show that low molecular weight compounds can be removed effectively from a standard commercial grade Poloxamer 188:

TABLE-US-00007 TABLE 3 Area Mn Mw D Area % < 5000 % > 13000 Sample [g/mol] [g/mol] [Mn/Mw] g/mol g/mol Starting 7330 7870 1.07 5.99 1.04 material Example 7900 8140 1.03 1.22 1.25 #1

[0180] Total aldehyde levels obtained by using the HPLC method described above are summarized in the following Table 4:

TABLE-US-00008 TABLE 4 Formaldehyde Acetaldehyde Propionic aldehyde Sample [ppm] [ppm] [ppm] Starting material <10 163 257 Example #1 <10 87 29

[0181] The results show that aldehydes levels were significantly reduced by the acid treatment in comparison to the levels of the starting material.

Example 2

[0182] The second experiment was performed using a 100 mm long 10/6 UF5kDZ membrane (atech innovations gmbh). The circulation vessel B1 was filled with 3185 gram Poloxamer 188 solution, having a solids content of 10.44%. Prior to starting the filtration, the Poloxamer was recycled in the setup under permeate recycle into the feed vessel at 30 C. The pH was set to 3 using 1.81 g 10% H.sub.2SO.sub.4. Directly after setting the pH, the temperature was increased to 60 C. and the mixture was recirculated for 2h. Dosing of the acid did not result in a change in solid contents. Subsequently, the mixture was diafiltrated at 60 C., a cross-flow velocity of 4 m/s and a trans-membrane pressure of 0.1 MPa until a diafiltration factor of 3.76 was reached. Table 5 summarizes the results of this experiment. During this step, the solid contents dropped from 10.46% to 6.23%. Because of the decreased polymer content in the retentate, an increase in the membrane flux was recorded. The flux behavior was similar to that obtained in Example 1.

TABLE-US-00009 TABLE 5 DF Flux C.sub.Ret C.sub.Perm R kg/m.sup.2/h % % 0.00 22.4 10.46 1.42 86.4 0.95 25.4 9.06 0.78 91.4 1.89 29.6 8.28 0.63 92.4 2.83 37.3 7.74 0.51 93.4 3.76 42.3 6.23 0.38 93.9

[0183] The resulting product was further characterized by size exclusion chromatography.

TABLE-US-00010 TABLE 6 Results obtained by size exclusion chromatography Area Mn Mw D Area % < 4667 % > 13280 Sample [g/mol] [g/mol] [Mn/Mw] g/mol g/mol Starting 7760 8410 1.08 5.0 1.3 material Example 8210 8610 1.05 1.3 1.5 #2

TABLE-US-00011 TABLE 7 Results of acid treatment Formaldehyde Acetaldehyde Propionic aldehyde Sample [ppm] [ppm] [ppm] Starting material <10 163 257 Example #2 <10 6 <1

[0184] The results according to Table 7 show that total aldehyde level in the purified product was below 20 ppm

Example 3

[0185] The third experiment was performed using a 100 mm long 10/6 UF10kDZ membrane (atech innovations gmbh). The circulation vessel B1 was filled with 3275 gram Poloxamer 188 solution, having a solids content of 9.89%. Prior to starting the filtration, the Poloxamer was recycled in the setup under permeate recycle into the feed vessel at 60 C. The pH was set to 3 using 1.84 g 10% H2SO4. The mixture was recirculated for 1 h 20 m. Dosing of the acid did not result in a change in solid contents. Subsequently, the mixture was diafiltrated at 60 C., a cross-flow velocity of 4 m/s and a trans-membrane pressure of 0.1 MPa until a diafiltration factor of 2.79 was reached. Table 8 summarizes the results of this experiment. During this step, the solid contents dropped from 9.96% to 4.30%. Because of the decreased polymer content in the retentate, an increase in the membrane flux was recorded. The retention using this membrane was significantly lower than that in Example 1 and 2.

TABLE-US-00012 TABLE 8 DF Flux C.sub.Ret C.sub.Perm R kg/m.sup.2/h % % 0.00 28.1 9.96 2.62 73.7 1.00 46.7 6.95 1.66 76.1 1.87 70.8 4.89 1.12 77.1 2.79 78.4 4.30 0.97 77.4

TABLE-US-00013 TABLE 9 Results obtained by size exclusion chromatography Area Mn Mw D Area % < 4667 % > 13280 Sample [g/mol] [g/mol] [Mn/Mw] g/mol g/mol Starting 7760 8410 1.08 5.0 1.3 material Example 8260 8750 1.06 1.8 1.3 #3

TABLE-US-00014 TABLE 10 Results of acid treatment Formaldehyde Acetaldehyde Propionic aldehyde Sample [ppm] [ppm] [ppm] Starting material <10 163 257 Example #3 <10 4 <1

[0186] The results according to Table 10 show that total aldehyde levels in the purified product were below 20 ppm.

Example 4

[0187] This example was performed using a 100 mm long mono-channel Type 1/6 UF5kD Z membrane (atech innovations gmbh). The circulation vessel B1 was filled with 4003 gram Poloxamer 188 solution, having a solids content of 10.37%. The Polaxomer solution was set to a pH of 3 before filling it into the setup. The mixture was diafiltrated at 60 C., a cross-flow velocity of 4 m/s and a trans-membrane pressure of 0.1 MPa until a diafiltration factor of 3.82 was reached. The fluxes and rejections were within the experimental error comparable to those obtained in Example 2. During the diafiltration step, the solid contents dropped from 10.37% to 4.86%. Because of the decreased polymer content in the retentate, an increase in the membrane flux was recorded.

TABLE-US-00015 Area Mn Mw D Area % < 4936 % > 11612 Sample [g/mol] [g/mol] [Mn/Mw] g/mol g/mol Starting 7330 7770 1.06 4.9 1.9 material Example 7790 8050 1.03 1.2 1.8 #4

TABLE-US-00016 Formaldehyde Acetaldehyde Propionic aldehyde Sample [ppm] [ppm] [pppm] Starting material <10 150 234 Example #4 <10 <1 6

[0188] The results show that total aldehyde levels in the purified product were below 20 ppm