METHOD FOR DETERMINING IMPURITIES IN POLYALKYLENETHERS AND ITS USE

Description

[0001] The invention relates to the determination of impurities in aliphatic polyalkylene ethers, in particular the determination of polyol impurities in polyalkylene glycols.

[0002] Polyalkylene glycols are used in many technical fields. Typical examples of polyalkylene glycols are polyethylene glycols (PEGs). Depending on their chain length, these are liquid or solid, water-soluble, and non-toxic polymers with the general molecular formula C 2n H 4n+2 O n+1 . Due to their properties, they are used in a variety of ways, for example as active ingredient carriers in pharmaceuticals, as components of cosmetic products, in a wide variety of industrial applications, or in cell biology research.

[0003] Another example of polyalkylene glycols is polypropylene glycols. These are used, for example, in non-ionic detergents and cleaning agents, in synthetic resins, as antifreeze agents, as flotation agents, or as chemical intermediates.

[0004] Polyalkylene glycols often contain polyol impurities. These impurities are undesirable in some applications.

[0005] One of the most important pharmaceutically relevant polymers is the well-established polyethylene glycol monomethyl ether (mPEG), which has also been approved by the Federal Drug Administration (FDA) for a variety of applications. A known problem of utmost importance is the existence of PEG diol impurities (PEG diols), which can lead to cross-linking of proteins or other pharmaceutical components of interest, thus causing complications.

[0006] PEG-diols and mPEG have very similar physicochemical properties (see Figures 1a and 1b) and therefore PEG-diol impurities are difficult to identify.

[0007] Chromatographic methods are generally suitable for the analytical determination of impurities and for the preparative processing of polyalkylene glycols.

[0008] Various chromatographic methods are available for the separation or purification of polymers using chromatographic techniques. These include, in particular, size exclusion chromatography (SEC), chromatography under critical conditions (LCCC), and liquid adsorption chromatography (LAC).

[0009] LCCC as an elution mode can offer elution that is independent of the macromolecule's chain length, i.e., determined only by the end group. However, the influence of the alpha-methoxy end in mPEG, in contrast to the hydroxyl group in unmodified PEG, implies only very limited differences in the hydrophilic / hydrophobic properties of PEG macromolecules (see Figures 1a and 1b), making simple identification methods based on liquid chromatography a challenging and as yet unsolved endeavor.

[0010] To date, there is no comprehensive approach that allows for the simple identification of PEG-diol impurities in mPEG with a broad range of molecular weights. Chromatographic methods do, in principle, offer possibilities. These are always models that are used for very limited ranges of molar masses, e.g., for molar masses well below 1000 g mol -1 or for individual molar masses. The patent literature on this topic suggests that identification is possible by modifying the terminal hydroxyl groups at the chain ends with groups of pronounced hydrophobic character, resulting in functional omega-chain ends or in functional alpha- and omega-chain ends that interact with the specifically selected chromatography sorbent. The efficiency of such labeling reactions is also an issue that must be considered.

[0011] Current methods for determining impurities in polyalkylene glycols therefore still leave much to be desired in many respects. Such methods include, for example, the analytical detection of impurities using SEC combined with a suitable detection method. This analytical method can, if necessary, be coupled with other analytical methods, such as magnetic resonance spectroscopy or mass spectrometry.

[0012] Monolithic molded bodies have proven effective as stationary phases in the purification and separation of biopolymers. EP 1 629 887 A1, WO 2006 / 013043 A1, DE 198 01 575 A1, and DE 197 26 151 A1 describe such processes.

[0013] The production of monolithic or particulate sorbents with uniform pore size is described in DE 102 58 491 A1.

[0014] DE 197 26 164 A1 discloses coated monolithic sorbents.

[0015] WO 2005 / 075976 A2 describes a separation process in which a capillary separation process is coupled with mass spectrometry. The process is preferably carried out using a monolithic sorbent.

[0016] A chromatographic method has now been discovered that is excellently suited for determining polyol impurities in polyalkylene glycols. The method according to the invention allows for the straightforward determination of polyol impurities in commercial products. The method can also be performed on polyalkylene glycols whose end groups have been fully or partially functionalized, or which contain functionalized polyol impurities, for example, etherified hydroxyl end groups. The method according to the invention also allows for the quantitative determination of very small amounts of polyol impurities, for example, amounts of less than 1 wt. % thereof.

[0017] The method according to the invention allows the quantitative determination of the amount of impurities in polyalkylene glycols. Impurities with a lower or higher molecular weight than the desired product can be determined and / or separated. Impurities with the same molecular weight as the desired product can also be determined and / or separated, provided the molecular weight does not exceed excessive values, for example, values of more than 20,000 g / mol.

[0018] Within certain dispersity limits, which are a prerequisite for the applicability of the polymers, the identification and quantitative determination of impurities is possible. The identity of the impurities, for example, the diol, can be confirmed by MALDI-MS measurements.

[0019] The invention relates to a method for determining impurities in polyalkylene ethers comprising the steps i) Introducing polyalkylene ethers as analyte into a chromatography column containing monolithic silica gel as stationary phase, ii) Eluting the analyte with a liquid eluent having a polarity such that the analyte is in adsorptive equilibrium with the stationary phase during chromatography, iii) Detecting the components of the analyte at the outlet end of the chromatography column to obtain a chromatogram which identifies different components of the analyte and their qualitative amount as a function of the elution time of the individual components, and iv) Determining bands in the chromatogram which have a small height or area compared to the band with the largest height or area as an indication of the presence of impurities in the analyte.

[0020] The polyalkylene glycols used in the invention are polyhydric alcohols or the corresponding derivatives partially or fully etherified or esterified at the end groups. Hydroxyl groups preferably form the end groups of the polymer chains.

[0021] Examples of polyalkylene glycols used according to the invention are polyethylene glycols, polypropyl glycols, or polytetrahydrofurans. Examples of these are: with m ≥ 1. Furthermore, branched polyalkylene glycols with several arms, for example with 3, 4, 6 or 8 arms, are also possible.

[0022] Other polyalkylene glycols which are preferably used are those in which some or all of the hydroxyl end groups are etherified, in particular alkyl ether groups or especially methoxy groups.

[0023] Particularly preferred are polyethylene glycols with a molecular weight range of 1000 to 50000 g*mol -1<, and in particular those with the typical pharmaceutically relevant molecular weight range of 1000 to 20000 g*mol -1<. For the purposes of this description, the molecular weight is determined by size exclusion chromatography.

[0024] Preferred processes are those in which the polyalkylene ether is a polyethylene glycol or a polypropylene glycol having hydroxyl end groups which may be partially or fully etherified.

[0025] Also preferred are processes in which the polyalkylene ether is a polyethylene glycol or a polypropylene glycol containing alkoxy end groups, in particular methoxy end groups.

[0026] The impurities to be analyzed or separated from the polyalkylene glycols can generally be organic compounds, such as protic organic compounds whose protic groups may be fully or partially functionalized. Typical impurities are polyols, such as monohydric alcohols or diols, whose hydroxyl groups may be partially or fully etherified, for example, as alkoxy groups, particularly as methoxy groups.

[0027] The amount of impurities in the initial sample is generally less than or equal to 20 wt. %, preferably less than 5 wt. % and in particular less than 1 wt. %, based on the total amount of the initial sample.

[0028] Preference is given to processes in which the impurities in the polyalkylene ethers are polyols whose average molecular weight is in the same range as or below the average molecular weight of the polyalkylene ethers to be analyzed, preferably alkylenediols and / or di- or trialkylene glycols.

[0029] In detail, the process according to the invention is characterized by the establishment of chromatographic conditions that determine the onset of the adsorption regime. Under these conditions, end-group-specific adsorption behavior begins. In the chromatography of polymers, three modi operandi are distinguished: size exclusion chromatography, chain-length-independent elution, and partition or adsorption chromatography, in which an interaction of the eluent with the stationary phase occurs. The process according to the invention is characterized by the presence of conditions under which adsorption chromatography occurs.

[0030] The present invention addresses the inherent problem of a defined approach to the analysis of protic and / or hydroxyl-containing impurities. The method according to the invention thus offers the possibility of qualitatively and quantitatively determining impurities within a defined molecular weight range. In particular, it is possible to perform the analysis without chemical modifications of the product and within a few minutes.

[0031] Furthermore, advantages of the method according to the invention arise from the obvious simplicity of the determination and from the rapid availability of the result, which can be obtained, for example, in a few minutes.

[0032] In addition, quantitative and reproducible purity analyses from batch to batch can be performed within minutes using rapid liquid chromatography.

[0033] A monolithic silica gel is used as the stationary phase in the process according to the invention. Such products are known and have already been used in other chromatographic processes.

[0034] The highly porous silica-based monolithic materials used in the invention are characterized by a macroporous and interconnected flow-through pore structure enclosed by a continuous and durable mesopore skeleton. Such stationary phases prove to be highly efficient and exhibit retention-stable performance in the separation of small analytes.

[0035] Preferred monolithic silica gels are characterized by the presence of a communicating network of pores, i.e., by the convective and / or diffusive accessibility of the eluent to these pores. Preferred sizes of the pores convectively accessible to the eluent range from 0.2 to 3 µm. In addition, the monolithic silica gels may also have pores that are accessible to the eluent by diffusion. The sizes of these pores preferably range from 3 to 200 nm.

[0036] Preferred monolithic silica gels are modified with functional groups on the surface, including the inner surface. These can be hydrophilic or hydrophobic groups. Examples of hydrophilic groups are hydroxyl groups. Examples of hydrophobic groups are alkyl groups, for example, alkyl groups with 4, 8, 12, or 18 carbon atoms.

[0037] Alternatively, the monolithic silica gel can be used in unmodified form, i.e. as a polar material.

[0038] The monolithic silica gel can therefore be used as a polar material or in a hydrophobic form, i.e. as a non-polar material.

[0039] Monolithic silica gel rods derivatized with long alkyl groups, e.g. with C 18 alkyl, are preferably used.

[0040] In the process according to the invention, an eluent with a polarity such that the analyte is in adsorptive equilibrium with the stationary phase during chromatography is used. The elution strength can be adjusted, for example, by mixing solvents of different polarities. The elution strength is adjusted such that the chromatography proceeds as adsorption / partition chromatography. The adjustment can be carried out by a person skilled in the art through dilution series using simple serial experiments.

[0041] When using polar monolithic silica gel, eluents with low polarity are suitable. Examples include nonpolar solvents, which are preferably anhydrous. Preferred eluents are aliphatic or aromatic hydrocarbons that are liquid at 25°C, such as hexane, heptane, octane, decane, benzene, toluene, or xylene, as well as halogenated aliphatic hydrocarbons that are liquid at 25°C, such as dichloromethane, chloroform, or carbon tetrachloride, or carboxylic acid esters that are liquid at 25°C, such as methyl acetate or ethyl acetate, or ethers that are liquid at 25°C, such as diethyl ether, or carbon disulfide. These eluents can also be used as mixtures to adjust the required elution strength.

[0042] Nonpolar solvents can be mixed with small amounts of water, alcohols or aprotic polar solvents to adjust a specific polarity of the eluent.

[0043] When using non-polar monolithic silica gel (corresponding to a stationary phase designed as a reversed phase), eluents with high polarity can be considered.

[0044] Examples of polar eluents are water, water-soluble alcohols or aprotic polar solvents.

[0045] Examples of water-soluble alcohols are aliphatic alcohols with one to four carbon atoms.

[0046] Examples of aprotic polar solvents are ketones such as acetone, lactones such as gamma-butyrolactone, lactams such as N-methyl-2-pyrrolidone, nitriles such as acetonitrile, nitro compounds such as nitromethane, tertiary carboxylic acid amides such as dimethylformamide or dimethylacetamide, urea derivatives such as tetramethylurea or dimethylpropyleneurea (DMPU), sulfoxides such as dimethyl sulfoxide (DMSO), sulfones such as sulfolane, carbonic acid esters such as dimethyl carbonate or ethylene carbonate.

[0047] Particular preference is given to using mixtures of water with aprotic polar solvents. Examples of these mixtures are mixtures of methanol and water, ethanol and water, or acetone and water, or especially mixtures of water with acetonitrile.

[0048] The required polarity of the eluent, corresponding to a volume ratio of water to aprotic polar solvent, such as water to acetonitrile, of 6:4, is an excellent starting point. Fine-tuning of the separation parameters is achieved through the series of experiments mentioned above in infinitesimal steps. For the water / acetonitrile system, a volume ratio of 6.5:3.5 to 5.5:4.5 is preferred.

[0049] A method is preferred in which the monolithic silica gel is designed as a reversed phase and in which the eluent contains water and an organic solvent in such an amount that the elution of the analyte takes place as adsorption / partition chromatography.

[0050] The process according to the invention can be carried out at atmospheric pressure or, preferably, at superatmospheric pressure. The pressure in the chromatography column is preferably 10 to 200 bar.

[0051] The process according to the invention is carried out at temperatures at which the eluent in the column is liquid. The temperature in the chromatography column is preferably 4 to 50 °C.

[0052] Column lengths and diameters can be selected within a wide range. Typical column lengths range from 1 cm to 1 m. Typical column diameters range from 10 µm to 10 cm.

[0053] The detection of the analyte components at the outlet end of the chromatography column can be performed using any method known to those skilled in the art. Examples include the detection of physical properties of the mobile phase, such as light absorption, fluorescence, light scattering, or thermal conductivity, or the detection of chemical properties of the mobile phase, such as the use of indicator substances.

[0054] Preferred methods are those in which the components of the analyte are detected at the outlet end of the chromatography column by light scattering detection (ELSD) or by UV / VIS spectroscopy.

[0055] The result of the detection of the components of the analyte at the outlet end of the chromatography column is a chromatogram in which species of the sample with different elution times are plotted as a function of time.

[0056] The qualitative amount of the respective component can be determined from the height or area of the bands in the chromatogram. Quantitative determinations can also be performed using appropriate calibration standards.

[0057] The impurities in the sample are present only in small amounts compared to the polymers, which make up the main part of the sample.

[0058] If bands appear in the chromatogram that have a small height or area compared to the band with the largest height or area, it can be concluded that impurities are present in the sample.

[0059] A method in which the height of the low-level bands is less than 10%, in particular less than 5%, of the height of the highest-level band is preferred. For example, with PEG-diol, detection is possible down to a band height of 0.8% of the height of the highest-level band.

[0060] The process according to the invention can be carried out batchwise or continuously. Standard chromatography equipment, such as conventional HPLC systems equipped with the specific column material, can be used for this purpose.

[0061] The method according to the invention is suitable for the rapid testing of polyalkylene glycols for impurities. It can therefore be used advantageously for quality control of such products.

[0062] The process according to the invention can also be used to remove impurities from polyalkylene glycols. It can therefore also be advantageously used in the preparative purification of these compounds.

[0063] These uses are also the subject of the present invention.

[0064] The following examples illustrate the invention without limiting it. Examples

[0065] Chromatographic measurements were performed using a chromatography system (Agilent Technologies 1200 Series, Polymer Standards Service GmbH (PSS, Mainz)). The system consisted of a column oven and a light scattering detector (ELSD) operated with nitrogen as the carrier gas. Measurements were performed on commercially available polyethylene glycol diols (PEG-diols; PSS, Mainz, Germany and PL, Shropshire, UK) and on synthesized polyethylene glycol monomethyl ethers (mPEG). A high-resolution Chromolith column (monolithic silica gel end-capped with RP-18) was used. The Chromolith column was obtained from Merck KGaA (Darmstadt, Germany). The column was 100 mm long and had an inner diameter of 4.6 mm. The column material had a high porosity of more than 80% and featured macropores of approximately 1.1 µm and mesopores of approximately 15 nm. The inner surface area of the column material was 250 m 2< g -1< .The internal surface was determined by mercury porosimetry and nitrogen adsorption / desorption isotherms.

[0066] Molar masses (number average M n and weight average M w ) of the synthesized mPEG samples were determined by size exclusion chromatography and showed narrow dispersities (Ð ≤ 1.1). Details can be found in Table 1 below. Table 1: Overview of the properties of the polyethylene glycol diols (PEG) and polyethylene glycol monomethyl ether (mPEG) used product M n (g mol -1< ) M w (g mol -1< ) Ð PEG 1 375 400 1,07 PEG 2 1840 2010 1,09 PEG3 2800 3060 1,09 PEG4 7500 11200 1,51 PEG 5 22100 25800 1,17 PEG6 34000 42700 1,26 mPEG 1 2300 2400 1,04 mPEG 2 5600 5800 1,04 mPEG 3 7500 7700 1,03 mPEG 4 120000 13200 1,10 mPEG 5 21800 22700 1,04 mPEG 6 34900 37200 1,07 mPEG 7 46200 50100 1,08 Chemicals and materials

[0067] The reagents and solvents used were commercially available products purchased from Aldrich or Linde. Ethylene oxide (EO) was stirred over sodium in burettes prior to distillation. Prior to use, 2-methoxyethanol and diphenylmethane were stirred over calcium hydride and then distilled under vacuum. The purified reactants were purged with argon, stored in Schlenk tubes in a glove box, and used within three days. Tetrahydrofuran was dried by refluxing over freshly prepared sodium benzophenone until a deep blue color appeared. The dried tetrahydrofuran was subsequently stored in a Schlenk tube under inert gas and used within a short time.

[0068] HPLC-grade acetonitrile was purchased from Sigma (Taufkirchen, Germany), and ultrapure water was freshly prepared in a Thermo Scientific™ Barnstaedt™ GenPure™ -xCAD water purification system (Thermo Electron LED GmbH, Langenselbold, Germany). The methoxypolyethylene glycol (mPEG) samples used were synthesized. The PEG samples were purchased as SEC standards from PSS (Polymer Standards Service GmbH, Mainz, Germany) and PL (Polymer Laboratories, Shropshire, United Kingdom). Preparation of mPEG samples

[0069] The initiator potassium 2-methoxyethanolate was prepared under inert conditions. 2-Methoxyethanol was dissolved in tetrahydrofuran, and diphenylmethyl potassium was added dropwise until a precipitate of the product was observed, forming a light orange mixture. The product was washed four times with tetrahydrofuran until the orange color had completely disappeared. The product was then dried under vacuum and obtained as a gray powder.

[0070] The preparation of the initiator solutions for the polymerization of EO to mPEG by living anionic ring-opening polymerization (AROP) was carried out under the exclusion of water and air (inert). First, tetrahydrofuran and potassium 2-methoxyethanolate were added to a GL45 bottle under inert conditions. To ensure the entire process was carried out under inert conditions, the fine suspension was then transferred via PTFE tubing into a PicoClave glass autoclave reactor (BüchiGlasUster, Uster, Switzerland) and cooled to -20 °C with stirring. The appropriate amount of EO was then added to the reaction mixture using a Mini-CORI-FLOW mass flow control apparatus (Bronkhorst High-Tech BB, Ruurlo, Netherlands). The reaction mixture was then heated to 45 °C within 120 min and stirred for a further 48 hours. The polymerization was terminated by adding a mixture of ethanol / acetic acid (95 / 5%, v / v).For isolation and purification, the polymer was filtered through cold diethyl ether and dried under vacuum. The product was obtained as a white powder. Determination of the molecular mass of the synthesized mPEG samples

[0071] Size exclusion chromatography (SEC) measurements were performed on a Shimadzu SEC system (control unit: CBM-20A VP, degasser: DGU-20A5, pump: LC-10ADVP, autosampler: SIL-10AD VP, oven: TechLab, RI detector: RID-10A) using a ternary mobile phase consisting of chloroform / isopropanol / triethylamine (94 / 2 / 4%, v / v / v) as eluent. The PS5 SDV linear S column (5 µm particle size) was operated at a flow rate of 1 mL min -1 at a temperature of 40 °C. The system was calibrated in the range from 194 g mol -1< to 106000 g mol -1< using a PEG / PEO standard obtained from (i) Polymer Standards Service GmbH (PSS, Mainz, Germany) (PEO 106000, 55800, 42700, 26100 g mol -1< ) and from (ii) Polymer Laboratories (PL, Shropshire, UK) (PEG 12600, 7100, 4100, 1470, 960, 600, 440, 194 g mol -1<). Liquid chromatography

[0072] Chromatographic measurements were performed using a modified Agilent Technologies 1200 series system from PSS (Polymer Standards Service GmbH, Mainz). Column dead volumes were reduced by using 130 µm ID tubing running from the injector to the column head and from the column outlet to the detector. The injection volume was set to 10 µl in all experiments, i.e., 0.6% of the column volume. The column was housed in a TCC 6000 column oven from PSS (Polymer Standards Service GmbH, Mainz) and annealed at 30 °C. A light scattering detector (ELSD) (Softa Model 400) from PSS (Polymer Standards Service GmbH, Mainz) was connected to the column outlet line. Nitrogen was used as the carrier gas for the ELSD detector. The chamber and drift tube temperatures were set to 45 and 70 °C, respectively. The detector was operated at the maximum data rate of 10 Hz.Elutions were performed on a high-resolution Chromolith column (monolithic silica gel end-capped with RP-18). The Chromolith column was obtained from Merck KGaA (Darmstadt, Germany). The column length was 100 mm and the diameter was 4.6 mm. The total dead volume of the system was determined by replacing the column with a tubing connector with a dead volume of zero and injecting the smallest and largest PEG samples. The dead volume was calculated to be approximately 2% of the total column volume.

[0073] All samples were prepared at concentrations between 0.1 and 2.0 mg mL by dissolving them in the respective mobile phase used for the chromatography experiments. Before analysis, samples were filtered through a 0.45 µm pore-size PTFE filter. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF-MS) experiments

[0074] MALDI-TOF-MS experiments were performed on the collected elution fractions using an UltraFlex TOF / TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an Nd-YAG laser. All spectra were measured in positive reflector mode. The instrument was calibrated before each measurement using an external PMMA standard from PSS (Polymer Standards Services GmbH, Mainz) within the required mass measurement range. MS data were analyzed using Flex Analysis 3.4 software, and isotope patterns were generated using the Isotope Pattern Calculator software from Bruker Daltonics. For sample preparation of the MALDI-MS samples, the collected elution fractions, a solution of trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malonic acid dinitrile (DCTB, Sigma-Aldrich) in chloroform at a concentration of 30 mg mL -1< and the doping salt sodium iodide dissolved in chloroform at a concentration of 60 mg mL -1< were used.The elution fraction containing the sample was first placed on the sample plate, and the sample was then allowed to dry. After drying, the matrix and salt solutions were applied to the appropriate location on the sample plate. For each sample, 0.5 µl of the sample solution was used, followed by 0.5 µl of the matrix-salt mixture solutions. Example 1: Determination of elution times depending on the composition of the eluent.

[0075] Figures 1 a) and 1 b) show the elution times for various mPEG samples (closed symbols) and PEG-diol samples (open symbols) as a function of the eluent composition. Mixtures of acetonitrile with water were used as the eluent.

[0076] Figure 1a) shows the results for mPEG samples with different molecular weights (M n = 2300 g / mol; M n = 5600 g / mol; M n = 7500 g / mol; M n = 12000 g / mol; M n = 21800 g / mol; M n = 34900 g / mol; M n = 46200 g / mol).

[0077] Figure 1b ) shows the results for PEG-diol samples with different molecular weights (M n = 1840 g / mol; M n = 2800 g / mol; M n = 7500 g / mol; M n = 22100 g / mol; M n = 34000 g / mol).

[0078] Figure 2 shows gradient elution results of a PEG-diol sample, an mPEG sample, and a mixture of both samples. The upper curve shows the elution of a PEG-diol sample with an average molecular weight M n = 1840 g / mol. The middle curve shows the elution of an mPEG sample with an average molecular weight M n = 2300 g / mol, and the lower curve shows the elution of a 50 / 50 (%, v / v) mixture of these two samples.

[0079] Conditions: Binary composition of the mobile phase with 10% acetonitrile in water, which was kept isocratic for 3 minutes and then increased linearly within 50 minutes to a content of 50% acetonitrile in water (%, v / v) Figure 3 shows the elugrams of an mPEG sample with an average molecular weight of M n = 12,000 g / mol (middle curve) and 50 / 50 (%, v / v) mixtures of this mPEG sample with PEG-diol with an average molecular weight of M n = 2,800 g / mol (upper curve) or with PEG-diol with an average molecular weight of M n = 22,100 g / mol (lower curve). The mobile phase composition was 60 / 40 (%, v / v) water / acetonitrile. The mobile phase flow rate was 1 mL / min.

[0080] Figure 4 a) shows the elugrams of mixtures of m-PEG with different PEG-diol contents. The m-PEG content with an average molecular weight of M n = 2300 g / mol was kept constant at 1 mg / mL, and increasing contents of PEG-diol with an average molecular weight of M n = 1840 g / mol were used.

[0081] Figure 4 b ) shows a double-logarithmic plot of the band height of the elution signal in relation to the PEG-diol content in the mixture (with error bars obtained from three replicate injections and chromatographic analysis). The measurements were repeated the next day with the same samples (open symbols).

[0082] Figures 5a and 5b show the results of the elution of mPEG and PEG-diols. The average molecular weights M n of the mPEG and PEG-diols used range from 1500 to 50000 g / mol. The upper figure shows results obtained using 50 / 50 to 40 / 60 acetonitrile / water (%, v / v) as eluent. The lower figure shows a significantly enlarged section of the Figure 5a .

[0083] In the upper Figure 5aThe molecular weights (M n ) of PEG-diols (open symbols) and mPEG (closed symbols) are plotted against elution time at 50 / 50 acetonitrile / water (%, v / v), indicating the transition between size exclusion mode (squares) and adsorption / partition mode (diamonds and pentagons). The latter is reached first for the larger molecular weights. The lower panel (5b) with a lower concentration of acetonitrile as eluent shows that at 40% acetonitrile in the eluent, the lower molecular weight mPEGs elute significantly later than the PEG-diols. The mobile phase composition was 40 / 60 acetonitrile / water (%, v / v). The mobile phase flow rate was 1 mL min -1 for both Figures 5a and 5b.

[0084] From Figures 5a and 5b, it is clear that a mobile phase composition of 50 / 50 acetonitrile / water (%, v / v) results in elution patterns influenced by size exclusion effects, i.e., the largest mPEG and PEG-diols elute first. Increasing the polarity of the mobile phase eluent to 42% acetonitrile shows increased elution times for the PEGs with larger molar masses, such that all PEGs elute at similar elution times, with virtually no selectivity (circles) across all molar masses. These results demonstrate the limited ability of liquid chromatography under critical conditions (LCCC elution mode) to differentiate between mPEG and PEG-diol, as the difference between alpha-hydroxyl compared to alpha-methoxy is simply too small to allow clear critical adsorption conditions based on end-group character ( Fig. 1 ).

[0085] With eluents containing only 40 to 41% acetonitrile, larger PEGs elute significantly later than the smaller ones, ie at molar masses of more than 10000 g mol -1< (diamonds and pentagons in Fig. 5a ) the distribution / adsorption of the polymer backbone is entered, ie at molecular weights above 10000 g mol -1< the distribution / adsorption mode is clearly entered (diamonds and pentagons in the upper part of Figure 5a ). Above 10000 g mol -1< the result is determined exclusively by the molecular weight dependent adsorption / distribution of the polymer backbone with a gradually decreasing contribution resulting from the identity of the alpha groups. This is shown in the lower part of the Fig. 5a which concerns the smaller molecular masses (< 10000 g mol-1, pentagons).

[0086] At this point, partitioning and adsorption begin to dominate the elution. It is also worth noting that small but noticeable differences in the elution time for the populations of the mPEG and PEG-diol species begin to develop. Figures 5a and 5b thus describe a unique possibility for the separation of mPEG and PEG-diol species with very similar molecular weights, i.e., a separation in partition / adsorption mode at an acetonitrile content of 40%. While PEG-diols still elute quite similarly between 1000 and 10000 g mol -1<, mPEG show a delayed elution (pentagons in Fig. 5a and 5b ). This is demonstrated below, for example, by separations of mixtures of an mPEG / PEG-diol pair with similar molecular weights ( Figure 6a ).

[0087] In Figure 6aelugrams of PEG-diol (M n = 1840 g mol -1< , upper trace), of mPEG (M n = 2300 g mol -1< , middle trace) and of their mixture 50 / 50 (%, v / v} (lower trace) are shown.

[0088] In Figure 6b Elugrams of mPEG (M n = 2300 g mol -1< , middle lane), of mixtures of this m-PEG with PEG-diol (M n = 375 g mol -1< , upper lane) and of mixtures of this m-PEG with PEG-diol (M n = 2800 g mol -1< , lower lane) are shown.

[0089] In the experiments shown in Figures 6a and 6b, the mobile phase flow rate was 1 mL min -1< and the mobile phase had a composition of 60 / 40 water / acetonitrile (%, v / v).

[0090] These results show that the mPEG of the example of Figure 6a elutes as a narrow band that differs from that of a PEG-diol of similar molecular weight. Larger mPEGs with molecular weights of 20000 g mol -1< elute similarly to model diols of comparable molecular weight ( Fig. 1 This situation is not surprising and arises from a loss of selectivity towards the end group character at molecular weights greater than 20000 g mol -1, where adsorption of the polymer chain strongly dominates the elution.

[0091] It is found that when using a diol with significantly different molecular weight and molecular weight distribution, separation of mPEG and PEG-diol is possible even at larger molecular weights.

[0092] To support the pronounced selectivity between mPEG and PEG-diol, the Fig. 6a and 6b mPEG used was analyzed by gradient liquid chromatography ( Fig. 2 ). The experiments demonstrated the expected dispersity of the PEG standards and the mPEG samples by the appearance of numerous bands in both the PEG-diol and the mPEG sample. Gradient elution does not improve the discriminability between mPEG and PEG-diol ( Fig. 2 ).

[0093] Due to different initiation scenarios and reaction kinetics, as well as the polymerization procedure, it is unlikely that PEG-diol impurities will have the same molecular weight values as the mPEG samples. The different initiation probabilities and kinetics of growing polymer chains with two possible attachment points for monomers require consideration. Therefore, it is desirable to demonstrate whether smaller and, in particular, larger amounts of PEG-diol impurities are present in mPEG products. To mimic such conditions, mPEG samples were investigated that contained a controlled amount of PEG-diol with lower and higher molecular weights at the lower and upper ends of the molecular weight range, respectively. Selected elugrams are shown in Figures 6b and 3.

[0094] It is clear that diols with lower molecular weight differ significantly from the respective mPEG (Figures 6b and 3, top lanes), but become increasingly difficult to identify with higher molecular weight of the diol (Figures 6b and 3, bottom lane), although two species can be recognized in the elugrams. The example for the PEG-diol with the higher molecular weight ( Figure 3 , bottom lane) elutes in two distinguishable fractions originating from a bimodal molecular weight distribution. However, these elute later than the mPEG fraction. This is an inherent result of the elution of the PEG diols, which is based on the adsorption / distribution of the polymer chains of the macromolecules with higher molecular weight ( Figure 5). The smaller elution fraction also shows some overlap with the mPEG fraction. Regardless, and in contrast to pure mPEG (Figures 6a and 6b, middle lane), the PEG-diol is indicated by a clear elution pattern as shown in the chromatograms (Fig. S3, bottom lane opposite the middle lane).

[0095] To demonstrate the general suitability of the method for estimating the amount of PEG-diols present, experiments were carried out with mixtures of different concentrations of PEG-diol (M n = 1840 g mol -1< ) with a fixed concentration of mPEG (M n = 2300 g mol -1< ) ( Figure 4a ).

[0096] Although the typical nonlinear dependence of the ELSD is evident even in the double logarithmic plot of the band height against the solution concentration ( Figure 4), the repeatability of the measurements on different days allows an estimation of the concentrations and the content of PEG-diols of less than 1% at a fixed concentration of mPEG 1 mg mL -1< ( Figure 4 ).

[0097] To demonstrate the performance of the approach described here, a sample of mPEG prepared in the presence of a protic impurity, e.g., water, was analyzed. The water was added at the beginning of the living anionic ring-opening polymerization. In this case, a diol impurity is indicated by a distinct shoulder in the band in the chromatogram ( Figure 7a ).

[0098] In Figure 7a An example of the application of the described method is shown. The same chromatographic conditions were used as in Figure 6 described.

[0099] Figure 7ashows the elution trace of 1 mg mL -1< of an anionic polymerization product containing a diol impurity. In the representation on the right side of Figure 7a This is an enlargement of the band on the left side of this figure.

[0100] Figure 7b The left half shows MALDI-TOF MS spectra of the collected small elution fraction identified as PEG-diol (shown in black) and the larger elution fraction identified as mPEG (shown in gray). The right half of Figure 7b The isotopic fragmentation pattern of PEG-diol and mPEG is shown. A section of the mass spectrum is shown, with the dashed line representing the calculated isotopic fragmentation pattern for PEG-diols.

[0101] The elution fraction collected from the chromatogram ( Figure 7a) was identified by MALDI-TOF-MS as a PEG-diol impurity with a broad molecular weight distribution from less than 1000 m / z up to 3000 m / z ( Figure 7b , left part, black mass spectrum). The amount of PEG-diol was estimated to be about 8%.

[0102] From the data of these examples it can be deduced that under experimental conditions which do not lead to a selective elution of species based on a molecular weight distribution (e.g. Figure 3 ), but the populations of mPEG and PEG-diols occur in rather narrow elution bands (e.g. Figure 3 ), a rapid identification of PEG-diol impurities in mPEG in the pharmaceutically relevant molar range is possible (e.g. Figure 5 ).

Claims

1. A method for the determination of impurities in polyalkylene ethers comprising the steps of i) introducing polyalkylene ethers as analyte into a chromatography column containing monolithic silica gel as stationary phase ii) eluting the analyte with a liquid eluent having a polarity such that the analyte is in adsorptive equilibrium with the stationary phase during chromatography, iii) detecting the components of the analyte at the outlet end of the chromatography column to obtain a chromatogram which shows different components of the analyte and their qualitative quantity as a function of the elution time of the individual components, and iv) determining bands in the chromatogram which have a small height or area compared to the band with the largest height or area as an indication of the presence of impurities in the analyte.

2. The method according to claim 1, characterized in that the polyalkylene ether is a polyethylene glycol or a polypropylene glycol which has hydroxyl end groups which can be partially or completely etherified.

3. The method according to claim 2, characterized in that the polyalkylene ether is a polyethylene glycol or a polypropylene glycol which contains alkoxy end groups, in particular methoxy end groups.

4. The method according to at least one of claims 1 to 3, characterized in that the impurities in the polyalkylene ethers are polyols whose average molecular weight is in the same range or below the average molecular weight of the polyalkylene ethers to be analyzed, preferably alkylene diols and / or di- or trialkylene glycols.

5. The method according to at least one of claims 1 to 4, characterized in that the monolithic silica gel is designed as a reversed phase and that the eluent contains water and an organic solvent in such an amount that the elution of the analyte takes place as adsorption chromatography.

6. The method according to claim 5, characterized in that the eluent contains water and acetonitrile.

7. The method according to at least one of claims 1 to 6, characterized in that the chromatographic process is a high-pressure liquid chromatography.

8. The method according to at least one of claims 1 to 7, characterized in that the detection of the components of the analyte at the outlet end of the chromatography column is carried out by light scattering detection (ELSD) or by UV / VIS spectroscopy.

9. The method according to at least one of claims 1 to 8, characterized in that the height of the bands with low height is less than 10 %, in particular less than 5 %, of the height of the band with the greatest height.

10. Use of the method according to at least one of claims 1 to 9 for quality control of polyalkylene glycols or for preparative purification of polyalkylene glycols.