Patent Application
20210324139
The invention discloses a method for preparation of polyoxyethylene 1,4-sorbitan fatty acid ester, such as Polysorbate 80, by a reaction of polyoxyethylene 1,4-sorbitan with a fatty acid chloride, and polyoxyethylene 1,4-sorbitan fatty acid esters obtainable by this method.
Polysorbate 80 is a hydrophilic non-ionic surfactant. Due to a good hydrotropic effect, it usually serves as a cosolvent, as an emulsifier, and as a stabilizer during preparation of a formulation of an API or drug for parenteral application, such as injection.
Chang Li et al., International Journal of Nanomedicine, 2014, 9, 2089-2100, in the following also abbreviated with “Li et al.”, describes polysorbates, such as Polysorbate 80, as a class of PNS (polyoxyethylene nonionic surfactants) and their use among others as a nanocarrier system with applications in tablets, emulsions and especially in the preparation of injections. It is used as a facilitator to improve delivery of drugs, especially hydrophobic drugs, such as hydrophobic anticancer drugs, to target tissues. However, due to the different synthetic processes used by different manufacturers to obtain polysorbates, their structure and composition are not identical from batch to batch. For example, in the United States Pharmacopeia 35-National Formulary 30, Polysorbate 80 was defined as a mixture of partial esters of fatty acids, mainly oleic acid, with sorbitol and its anhydrides ethoxylated with approximately 20 moles of ethylene oxide for each mole of sorbitol and sorbitol anhydrides, One method for preparation of Polysorbate 80 involves first the dehydration of sorbitol to a dehydrated derivative, and an esterification with oleic acid providing a sorbitan fatty acid ester, then a polyreaction of ethylene oxide with the sorbitan fatty acid ester.
Another method is to first to do the polyreaction of ethylene oxide with the dehydrated derivative of sorbitan, followed by esterification.
The widely accepted structure of Polysorbate 80 is compound of formula (PS80) with w+x+y+z=20, that is with an average content of EO (ethylene oxide) units of 20.
For various reasons Polysorbate 80 is a mixture of many compounds. One source of the diversity is the fatty acid moiety which is oleic acid in case of Polysorbate 80. However, oleic acid is used as a natural product from natural sources, it comprised other fatty acids such as myristic acid, palmitic acid, palmitoleic acid, stearic acid, linoleic acid, or linolenic acid. Thereby the product Polysorbate 80 comprises fatty acid esters not only derived from oleic acid, but also from those other fatty acids which are present in the natural product oleic acid.
As a further source for diversity Li et al. points out that the first step in the synthesis of polysorbate usually is dehydration of sorbitol to sorbitan, suggesting that the final product is a mixture of sorbitol, with general formula of compound of formula (SORBITOL), and sorbitol-derived cyclic ethers with different structures, such as
1,4-sorbitan, with general formula of compound of formula (1,4),
1,5-sorbitan, with general formula of compound of formula (1,5),
2,5-sorbitan, with general formula of compound of formula (2,5), and
1,4:3,6 isosorbide, with general formula of compound of formula (1,4:3,6).
1,4-sorbitan, 1,5-sorbitan, and 2,5-sorbitan are isomers of each other within the meaning of this invention, of not explicitly stated otherwise.
Further byproducts of the dehydration reaction can be sorbitol polymers.
So already the dehydrated product provided by the dehydration of sorbitol is a mixture of different compounds, and when this mixture is then converted with oleic acid and with ethylene oxide to Polysorbate 80, obviously the product called Polysorbate 80 will comprise compounds derived from any of the compounds found in the mixture provided by the dehydration of sorbitol.
Obviously the polyreaction of ethylene oxide again will introduce further diversity as the ethylene oxide can react with each hydroxyl residue of the product from the dehydration reaction of sorbitol, thereby building up a polyoxyethylene chain on the hydroxyl residue, and again varying numbers of ethylene oxide units can be introduced into the various polyoxyethylene chains that build upon the various hydroxyl residues.
Li et al. furthermore points out, that the potential of PNS to trigger pseudoallergy is well known. Pseudo allergy is the official term used by the World Allergy Organization. It is a reaction similar to an immune allergic reaction that is observed following the first administration of the offending agent. Unlike common allergies, pseudoallergic reactions can directly induce release of histamine from mast cells and activate the complement system, with abnormal synthesis of eicosanoids and inhibition of bradykinin degradation, which are not initiated or mediated by pre-existing immunoglobulin E antibodies. Although the exact mechanisms of pseudo allergy in response to PNS remain unclear, it is believed that activation of the complement system and degranulation of mast cells initiate the reactions that result in pseudo allergy, i.e., the initial step of the pseudo allergic reaction is the key step.
There is always the need for polysorbates, especially for Polysorbate 80, that has higher quality and better performance than the known products.
The polysorbate, that is the polyoxyethylene 1,4-sorbitan fatty acid ester, that can be prepared with the method of the invention, shows high purity;
A technical feature of the method of the invention is the use of fatty acid chlorides instead of free fatty acids for the esterification reaction.
The following terms and abbreviations are used throughout the specification, if not explicitly stated otherwise:
R1 is linear or branched C10-22 alkyl or linear or branched C10-22 alkenyl.
Preferably, R1 is linear C10-22 alkyl or linear C10-22 alkenyl.
even more preferably, ACIDCHLOR is oleic acid chloride;
REAC-A can be done at atmospheric pressure or at a pressure above atmospheric pressure;
preferably, REAC-A is done at atmospheric pressure.
Preferably, no solvent is present in or charged for or used for REAC-A.
Preferably, no water is charged for or used for REAC-A.
Preferably, no catalyst is charged for or used for REAC-A.
After REAC-A, the polyoxyethylene 1,4-sorbitan fatty acid ester can be isolated by standard methods known to the skilled person in the art. A steam distillation can be done after REAC-A.
Preferably, the polyoxyethylene 1,4-sorbitan is prepared by a reaction REAC-B,
wherein 1,4-sorbitan is reacted with ethylene oxide.
Preferably, REAC-B is done in the presence of a base BASE-B.
Preferably, the alkali metal of the alkali metal C1-4 alkoxide is Na or K;
Preferably, the alkyl metal hydroxide is preferably NaOH or KOH.
even more especially, BASE-B is NaOH or KOH;
in particular, BASE-B is KOH.
REAC-B can be done at atmospheric pressure or at a pressure above atmospheric pressure;
Preferably, REAC-B is done under inert atmosphere, such as nitrogen or argon atmosphere.
in STEP2 ethanol is mixed with MIX1, STEP2 provides a mixture MIX2;
in STEP3 isopropanol is mixed with MIX2, STEP3 provides a mixture MIX3;
in STEP4 1,4-sorbitan is isolated from MIX3.
Preferably, no solvent is present in or used for DEHYDREAC.
Preferably, no water is charged for DEHYDREAC.
Preferably, STEP2, STEP3 and STEP4 are done at atmospheric pressure.
Preferably, water is removed during DEHYDREAC.
Preferably, STIRR2 is done at TEMP2.
Preferably, COOL2 is done after STIRR2.
Preferably, COOL2 is done from TEMP2 to TEMP3.
Preferably, STEP2 comprises STIRR2 and COOL2, and COOL2 is done after STIRR2.
Preferably, STIRR3 is done after COOL3.
Preferably, STIRR3 is done at TEMP3-2.
More preferably, STIRR3 is done after COOL3 and STIRR3 is done at TEMP3-2.
In one embodiment,
Preferably, in STEP2 ethanol is charged to MIX1 providing MIX2.
Preferably, in STEP3 isopropanol is charged to MIX2 providing MIX3.
Preferably, STEP1, STEP2 and STEP3 are done consecutively in one and the same reactor.
Preferably, TBAB is used for STEP1AQU as a mixture of TBAB with water;
The mixture of TBAB with water can be a solution or a suspension of TBAB in water.
Preferably, the amount of ethanol is such that crystallization starts during COOL2AQU; more preferably, the amount of ethanol is such that
even more preferably, the amount of ethanol is such that
Preferably, MIX2AQU after COOL2AQU is a suspension.
Preferably, MIX3AQU is a suspension.
In one embodiment,
preferably,
more preferably,
wherein ACIDCHLOR and R1 are defined as herein, also with all their embodiments.
preferably, said polyoxyethylene 1,4-sorbitan fatty acid ester has an average content of ethylene oxide units of from 19 to 23, preferably from 20 to 22, more preferably 20 or 22.
as an excipient in the formulation of drug formulations;
with the method and REAC-A as defined herein, also with all its embodiments.
The descriptions in the figures means the following, if not otherwise stated:
The following materials were, if not stated otherwise:
Density of thionyl chloride: 1.683 kg/L
NOF Polysorbate 80 (HX2)™, Lot 704352, NOF Corporation, Tokyo, Japan
HPLC-ELSD is a reversed phase HPLC using Evaporative Light Scattering Detection.
Column: Agilent Zorbax Eclipse XDB-C18 (150 mm×3 mm; 3.5 micrometer)
Pump:
Injection:
Autoinjektor:
Detector
Column oven
SAT/IN
Typical Integration Parameters
Sample preparation:
50 mg+/−5 mg sample were dissolved in 50 ml of acetonitrile.
The percentage determined by an HPLC chromatogram are the area percentage of the respective signal.
The LOD (Limit of Detection) with a Signal-to-noise ratio of 3 was 0.06 area-%.
The LOQ (Limit of Quantification) with a Signal-to-noise ratio of 10 was 0.20 area-%.
All measurements were measured in an identical way, the samples were used as such, if not otherwise stated, with sample weights ranging from 2 to 12 mg for the different products. If not otherwise stated the samples were dried in a vacuum pistol over night at room temperature, then they were immediately sealed in a glove bag into 40 microliter aluminum pans with pins, Mettler Toledo, in order to avoid and minimize any uptake of humidity from the atmosphere, and then the pans were subjected to DCS measurements with a DSC 1 STARe system from Mettler Toledo. The samples were run from 25 to −80° C., equilibrated for 5 min at −80° C., then heated from −80 to +80° C., equilibrated for 5 min at +80° C. (denoted 1st cycle). Then this thermal cycle was repeated, +80 to −80° C., equilibration at −80° C., −80 to +80° C., equilibration at +80° C. and then back to 25° C., with all heating and cooling segments at 10° C./min. If not stated otherwise, the heating segments from the first thermal cycle are displayed. If nothing else is reported, the measurement of the second heating cycle produced the same signal as the measurement of the first heating cycle, thereby it was confirmed that the samples did not show any thermal history.
(C) MALDI and DSC from a Preparative HPLC and from Non-Fractionated Samples
All samples were used as such, if not otherwise stated. The samples were dissolved in ACN to provide a solution with a concentration of 300 mg/ml. 300 microliter of this solution were injected (Waters sample manager 2700) and loaded onto a C18 column (Xterra Prep MS C18 OBD, 5 micrometer, 19×100 mm, Waters). The polysorbate species were separated using an ACN:H2O gradient starting at 45% ACN and increasing to 100% in 30 min with a flow rate at 10 ml/min and a column temperature of 50° C. (Thermostated column compartment TCC-100, Dionex). The separation continued at 100% ACN until reaching 120 min and no more species could be detected. The species were detected with a UV detector (Waters 2487 dual absorbance detector) at 195 nm (lamda max with epsilon=11000 for C═C bonds present in oleic acid). The MassLynx V4.0 software was used for data acquisition. 10 ml fractions were manually collected in 20 ml glass tubes. From each tube 10 microliter were taken out for MALDI analysis prior to evaporation until dryness under vacuum (GeneVac centrifugal evaporator EZ-2, SP Scientific). The evaporated fractions were then used for DSC analysis.
(C2) MALDI of Samples from Preparative HPLC (C1) and of Non-Fractionated Samples
2.5-Dihydroxybenzoic acid (super-DHB>=99.0%, Sigma Aldrich) was used as matrix and prepared as a 5 mg/ml solution in EtOH with 10 mM NaCl added in order to exclusively detect sodiated adducts. Prior to use, the matrix was sonicated for 10 min in a bath in order to obtain a solution. Non-fractionated samples were dissolved in ethanol to provide a solution with a concentration of 5 mg/ml, and for the HPLC fractions the 10 microliter samples were used without further preparation. All samples were mixed 1:1 (vol:vol) with the matrix and vortexed before spotting 1 microliter of each sample onto a target plate (MPT 384 polished steel, Bruker) in triplicates. All sample spots were allowed to dry and crystallize on the plate before MALDI measurements were performed. Positive ion MALDI-TOF mass spectrometry was carried out on an Ultraflex TOF/TOF, Bruker Daltonics instrument equipped with a 337 nm N2 laser operated at a frequency of 5 Hz in reflection mode. Spectra were recorded at an accelerating voltage of 25 kV and with matrix suppression until 450 Da with 1000 summed acquisitions per measurement. The laser power was kept slightly above the threshold for detection (usually ca. 40%) in order to get optimal peak resolution. All mass spectra were acquired with FlexControl 3.4 and analyzed with the FlexAnalysis 3.4 software.
(C3) DSC of Samples from Preparative HPLC (C1)
The evaporated samples from the preparative HPLC separation were extracted from the 20 ml tubes by dissolving in acetone and transfer (with three washes) to 1.5 ml glass vials equipped with 0.1 ml micro-inserts (Sigma Aldrich). The samples were then evaporated to dryness under vacuum (GeneVac centrifugal evaporator EZ-2, SP Scientific). All samples were afterwards dried overnight in a vacuum pistol before they were transferred to DSC pans (40 microliter, aluminum pans with pins, Mettler Toledo) and sealed in a vacuum bag at controlled humidity (ca. 7% or lower) to avoid uptake of moisture from the atmosphere. The DSC measurements were done as described under the method description (B) DCS
Add 2 g sample to 5 ml pyridine and 10 ml acetic anhydride in a screw-cap bottle (25 mL) and heat up to 120° C. for 2 hours under stirring.
0.5 ml of Sample stock solution is added into an auto sampler vial with 1 ml of dichloromethane and mixed
1,4-Sorbitan is detected at ca. 12.3 min.
1H NMR is a routine analytical method for the skilled person, so only one exemplary set of parameters is given in the following which can be used:
Solvent: DMSO-d6
5 to 10 mg of sample are dissolved in 0.6 ml of DMSO-d6 and mixed.
13C NMR is a routine analytical method for the skilled person, so only one exemplary set of parameters is given in the following which can be used:
Solvent: DMSO-d6
20 to 50 mg of sample are dissolved in 0.6 ml of DMSO-d6 and mixed well.
Pure water
300±3 mg of 1,4-Sorbitan was added into a 100 ml volumetric flask, then dissolved with water and diluted to volume.
The samples were dissolved in deuterated chloroform prior to the measurements.
Approximately 90 to 120 mg of material were mixed with 0.55 ml of d1-chloroform. 0.5 ml of solution was filled in 5 mm NMR tube. The 1H-decoupled 13C-NMR, 13C(1H)-NMR, were performed with proton decoupling and nuclear Overhauser effect (NOE). The measurements were carried out at 25° C., on a 400 MHz spectrometer at a resonance frequency of 100.61 MHz. The samples were run with 8192 scans using a pulse length of 14.5 micro-sec (90°), a 20 Hz spin, an acquisition time of 1.301 s, and a relaxation delay of 5 s. 32768 complex data points were collected, using a spectral width of 25188.9 Hz (250 ppm). All spectra were Fourier transformed with a line broadening of 1 Hz and zero filling to 128 k data points. The spectra were phase and baseline corrected, and the chloroform peak was used as a reference peak, determined to 77.23 ppm relative to TMS for the 13C-NMR.
A two-neck round bottom flask equipped with a stir bar was charged with oleic acid (12.62 g, 40.9 mmol, 1.0 equiv) and the flask was purged with N2. After heating to 40° C., thionyl chloride (12.5 ml, 172.0 mmol, 4.2 equiv) was added dropwise over 10 min by an addition funnel while stirring, gas evolution was observed. Then the temperature was increased to 65° C. and the reaction mixture was stirred for 1 hour. Then the reaction mixture was cooled to room temperature. Excess SOCl2 was removed by a rotary evaporator followed by drying under vacuum providing oleoyl chloride. The yield of oleoyl chloride was assumed to be 100%.
PEO sorbitan (47.1 g, 42.9 mmol, 1.0 equiv), prepared according to Example 10, were weighed into a single-neck round bottom flask and the atmosphere in the flask was exchanged for N2. Oleoyl chloride, the whole amount that was prepared according to Example 1, was added at room temperature and the reaction mixture was stirred for 15 min at room temperature.
The mixture steam distilled under reduced pressure of ca. 80 mbar for ca. 10 min. The pH was raised by this steam distillation from ca. 1.5 to ca. 4.5.
The product from the steam distillation was used as is for analysis.
A two-neck round bottom flask equipped with a stir bar was charged with oleic acid (30.0 g, 97.3 mmol, 1.0 equiv) and the flask was purged with N2. After heating to 40° C., thionyl chloride (30 ml, 413.0 mmol, 4.2 equiv) was added dropwise over 10 min by an addition funnel while stirring, gas evolution was observed. Then the temperature was increased to 65° C. and the reaction mixture was stirred for 1 hour. Then the reaction mixture was cooled to room temperature. The excess SOCl2 was removed by a rotary evaporator followed by drying under vacuum providing oleoyl chloride. The yield of oleoyl chloride was assumed to be 100%.
PEO sorbitan (22.4 g, 21.4 mmol, 1.0 equiv), prepared according to Example 10, were weighed into a single-neck round bottom flask and the atmosphere in the flask was exchanged for N2. Oleoyl chloride (7.74 g, 25.7 mmol, 1.2 equiv, prepared according to example 3) was added at room temperature and the reaction mixture was stirred for 15 min at room temperature.
The mixture steam distilled under reduced pressure of ca. 80 mbar for ca. 10 min. The pH was raised by this steam distillation from ca. 1.5 to ca. 4.5.
The product from the steam distillation was used as is for analysis.
Example 4 was repeated with the difference that 1.4 equiv oleoyl chloride were added instead of 1.2 equiv.
Example 4 was repeated with the difference that 1.6 equiv oleoyl chloride were added instead of 1.2 equiv.
A two-neck round bottom flask equipped with a stir bar was charged with oleic acid (2.0 g, 7.1 mmol, 1.0 equiv) and the flask was purged with N2. DCM (6.5 mL) was added, a clear solution formed. Then oxalyl chloride (1.21 ml, 14.2 mmol, 2.0 equiv) was added dropwise at room temperature over 10 min by an addition funnel while stirring, then the reaction mixture was stirred at room temperature for 2 hour. The DCM and excess oxalyl chloride were removed at the rotary evaporator followed by drying under vacuum. The yield of oleoyl chloride was assumed to be 100%.
PEO sorbitan (7.4 g, 7.1 mmol, 1.0 equiv), prepared according to Example 10, were weighed into a single-neck round bottom flask and the atmosphere in the flask was exchanged for N2. Oleoyl chloride, the whole amount that was prepared according to example 7, was added at room temperature and the reaction mixture was stirred for 15 min at room temperature. The product was used as is for analysis.
PEO sorbitan (5.96 g, 5.7 mmol, 1.0 equiv), prepared according to Example 10, were weighed into a single-neck round bottom flask and the atmosphere in the flask was exchanged for N2. Oleoyl chloride (1.885 mL, 5.7 mmol, 1.0 equiv, Sigma Aldrich) was added at room temperature and it was stirred for 15 min at this temperature.
The product was used as is for analysis.
Table 1 shows the HPLC-ELSD results of Examples 1, 4, 5, 6, 8 and 9, reported are the area % of the elution peaks of the Mono-, Di- and Tri-ester (denoted with “Mono”, “Di” and “Tri” in the Table 1) in the respective HPLC chromatogram; the first value is the absolute percentage of the area of the respective peak (“abs %”) based on the total peak area of the chromatogram, the second value is the percentage of the area of the respective peak based on the sum of the areas of the three peaks (“rel”).
The maximum of the elution peak is observed:
The elution peaks of the three esters are well separated from each other.
Results from (B) DSC
Table 2 shows the DSC results, values of T(peak) and for delta H are an average of 3 DCS analysis per sample in case of Croda HP and NOF, whereas they are values of one DSC analysis in case of Example 2, 4, 5 and 6.
The DSC of Example 8, 9 and 13 look similar to the DSC of the four Examples 2, 4, 5 and 6. So Examples 2, 4, 5, 6, 8, 9 and 13 do not show at all or at least not clearly a melting of crystallization behavior.
Neither NOF nor Examples 2, 4, 5, 6, 8, 9 and 13 show a peak in any of the two cooling cycles (
Results from (C) MALDI and DSC from a Preparative HPLC
The samples have been tested by MALDI. Example 5 and Croda HP were examined in detail by separation on a preparative HPLC and fractionation into 100 individual fractions, which were consecutively collected between 0 and 100 min, so each fraction was collected for 1 min (10 ml fractions), and that were analyzed by MALDI. The actual weight of all fractions was determined and a weight distribution was created and overlaid with the UV chromatogram:
From this separation pure fractions of PEO sorbitan mono oleate were tested by DSC: Fractionation of Example 5 yielded pure PEO sorbitan monoester fractions, which also did not show any melting peaks in DSC analysis.
In a MALDI mass spectrum all ethoxylated distributions are separated by 44 Da, which is equal to one EO unit. In order to calculate the average EO content of a mass distribution the mass peak list was exported and fitted to a Gaussian distribution function:
Where a is the height of the Gaussian distribution function, b is the position of the Gaussian distribution function center and c can be used as an estimate of the EO spread or dispersity of the Gaussian distribution function around the center mass. The Gaussian distribution function center position thus indicates the mass of the molecule present in the mixture, which gives the highest MALDI peak.
In the case of Example 10, the PEO sorbitan, this value corresponded to 1146 Da. A sodiated PEO sorbitan with 21 EO units has a molecular mass of 1112 Da and a sodiated PEO sorbitan with 22 EO units has a mass of 1156 Da. The average integer EO number for this Gaussian distribution function will thus be estimated to be 22 (
The same methodology can be used for analysis of the pure PEO sorbitan monooleate fractions. Non-fractionated products, though, contain PEO sorbitan mono- di and tri-esters with overlapping distributions due to the fact that one oleate, having 264 Da, is isobaric with six EO units. The mass peak of a sodiated PEO sorbitan monooleate with 20 EO units has 1332 Da and therefore falls on top of a PEO sorbitan diester with 14 EO units and a PEO sorbitan triester with 8 EO units. It is therefore not possible to calculate the average EO content from a MALDI mass spectrum of non-fractionated samples alone, even with the knowledge of the weights of the HPLC fractions. However, with the knowledge gained from fractionated samples, the average EO content of the non-fractionated samples can be estimated.
In case of the monoester species, which elute between ca. 16 and 27 min:
In case of the diester species, which elute between ca. 30 and 46 min:
The isosorbide based species and the PEO ester species elute noticeably later than the sorbitan species, and this both in case of the mono- and of the diester species, even though there is an overlap due to the distribution of the molecular weight which is caused by the distribution of the number of EO units.
The isosorbide based species and the PEO ester species actually more or less coelute.
Also in case of Example 5 also PEO esters were observed, but only in trace or small amounts in comparison to the major peaks of the PEO sorbitan esters.
In case of Example 10 also PEO isosorbides were observed, but only in trace amounts just above the noise level in comparison to the major peaks of the PEO sorbitan.
Isosorbide species have not been observed in Example 5.
In general any of the ethoxylated species in Example 5 shows lower number of EO units in comparison the respective species in Croda HP and in Croda SR, the difference is always roughly between 5 and 10 EO units.
Obviously the MALDI peaks in case of Example 5 have been shifted to lower m/z values compared to NOF, Croda HP and Croda SR.
The MALDI mass distribution of pure 1,4-sorbitan monoester fractions fits well to a Gaussian distribution function. In the case of non-fractionated material, that is Example 5, NOF, Croda HP and Croda SR, the main mass distribution contains overlapping mass distributions due to the presence of PEO sorbitan mono- di- and tri-oleate, which are all isobaric molecules. The polyester species are present in a lower amount than the monoesters but will shift the total mass spectrum slightly towards higher masses. A MALDI mass distribution from a non-fractionated sample will thus deviate from a Gaussian distribution function. If, however, a Gaussian distribution function is fitted to the left side of the mass distribution as illustrated in
The MALDI of Example 5 shows a distribution of signals with only one maximum, whereas the MALDI of NOF, Croda HP and Crode SR show in the signal distribution in addition to a main maximum two additional maxima; one of the additional maxima has a b value at a lower m/z value relative to the b value of the main maximum, the other additional maximum has a b value at a higher m/z value relative to the b value of the main maximum. Both additional maxima have a lower intensity than the main maximum.
Table 4 shows the b values of the three Gaussian curves fitted to each the respective maximum, as well as the b value of the Gaussian curve fitted to the one maximum in the MALDI spectrum of Example 5. These fitted curves are illustrated in
The MALDI spectrum of Examples 2, 4, 5, 6, 8, 9 and 13 show a signal distribution with only one maximum.
This difference of the products according to instant invention versus the known polysorbates products can also be illustrated when only one Gaussian curve is fitted to all the signals, that is to the whole distribution, in a MALDI spectrum. The c value of the Gaussian distribution function can be used as an estimate of the spread of the m/z values of the signals, that is of the dispersity of the Gaussian distribution function around the center m/z value of the Gaussian distribution function, which is expressed by the b value. Table 5 shows these c values for Example 5, NOF, Croda HP and Croda SR.
This fit of one Gaussian curve to all the signals in the MALDI spectrum is illustrated in
200 g Naphtha (petroleum), heavy alkylate, CAS 64741-65-7, 100 g (0.61 mol, 1 equiv) 1,4-sorbitan, prepared according to Example 11, and 0.6 g KOH were charged into a 4 L autoclave. The autoclave was rendered inert by evacuating first and then applying afterwards 0.5 bar pressure with N2, this was done for four times in total.
The mixture was heated to 150° C., 553 g (12.6 mol, 20.7 equiv) ethylene oxide were added in such speed that the temperature did not raise above 160° C. and the pressure did not raise above 3.8 bar; the addition was done in 4 h. Then the mixture was stirred for 2 h at 150° C.
After cooling to 60° C. 2.3 g AcOH were added. Two phases formed, one with solvent, the other with product, and were separated. Residual solvent was removed by steam distillation at a rotary evacuator. 625 g product was obtained.
Yield: 95% based on the assumption that a PEO sorbitan with an average of 20 EO was obtained. This assumption was also applied when this product was used in further reactions. 1H-NMR and 13C-NMR confirmed the structure.
DSC analysis showed no sign of crystallization or melting, neither in both heating cycles nor in both cooling cycles.
D-sorbitol (300 g, 1.647 mol, 1 equiv) was charged into a 1.5 L reactor. p-Toluenesulfonic acid monohydrate (2.665 g, 0.014 mol, 0.0085 (0.85%) equiv) was charged, followed by charging of TBAB (9.6 g, 0.03 mol, 0.0182 (1.81%) equiv). Vacuum of reactor 4 to 6 mbar was applied. Then the mixture was heated to 110° C. (the mixture melted at around 90° C.) and stirred at 110° C. for 6 hours. The mixture was cooled to 70 to 75° C. in 30 min. Ethanol (150 mL) was charged. The resulting mixture was stirred at 70 to 75° C. for 2 hours and formed a clear solution. Then the solution was cooled to 20° C. in 3 hours. A yellow suspension was formed. Isopropanol (150 mL) was charged. The mixture was cooled to 0° C. in 1 hour. The mixture was slurry at 0° C. for 4 hours. The mixture was filtered, and the cake was washed with isopropanol (150 mL). The cake was dried at 50° C. for 16 hours under vacuum to provide 142.2 g of product as white solid.
Yield 52.6%
1H NMR and 13C NMR confirmed the structure.
GC area-%:
Specific Rotation: −22.26°, c=3.1 (water)
From Nov. 4 to 7, 2018, on the Walter E Washington Convention Center, Washington, D.C., the conference “aaps 2018 PharmSci 360” was held with a Move-In on Friday, Nov. 2, 2018 and Pre-Conference Activities on Saturday, Nov. 3, 2018.
From 9:00 am to 5:00 pm of these Pre-Conference Activities Workshops and Short Courses took place. One of these Workshops took place between 9.45 AM and 10:15 AM with the title: “SC1-Synthesis and Control of Polysobates for Bioüharmaceuticel Applications”, which was held by Sreejit R. Menon, representing the company CRODA, www.crodahealthcare.com, Croda, Inc., Edison, N.J., USA.
The presentation showed on slide 12 the Polysorbate Synthesis of Croda, see
Sorbitol-(Dehydration)->Sorbitan-(Esterification with Fatty Acid)->Sorbitan Fatty Ester-(Ethoxylation)->Polysorbate-(Finishing)->High quality Polysorbate.
According to this sequence Crode produces two product ranges:
Slide 11, see
On Slide 17, see
In Slide 15, see
Clearly the MALDI spectrum shows even for the SR grade, which is the grade with the highest purity that is currently available on the market not only the one desired peak area of Sorbitan ester ethoxylates, but also significant peak areas caused by the presence of isosorbide and sorbitol derivatives, which are present in the SR grade.
A two-neck round bottom flask was charged with oleic acid (469.3 g, 1.64 mol, 1.0 equiv) and the flask was purged with N2. Dichloromethane (DCM) (1520 mL) was added, a clear, colorless solution formed. Then oxalyl chloride (288 ml, 3.3 mol, 2.0 equiv) was added dropwise at room temperature over 50 min while stirring, then the reaction mixture was stirred at room temperature for 2 h. The DCM and excess oxalyl chloride were removed at the rotary evaporator at ca. 35° C. and ca. 450 to 8 mbar followed by drying under vacuum. The yield of oleoyl chloride was assumed to be 100%.
PEO sorbitan (1001.9 g, 0.96 mol, 1.0 equiv, prepared according to Example 10) were weighed into a 21 reactor and the atmosphere in the flask was exchanged for N2. Oleoyl chloride (435.5 g, 1.34 mol, 1.4 equiv, prepared according to Example 12) was added at room temperature during ca. 40 min and the reaction mixture was stirred for 1 h at room temperature. Then the reaction mixture was heat up to 60° C. and vacuum was applied under stirring (200 mbar) for 1 day.
The formed HCl could be removed and the pH increased to 5.9. The pH was measured preparing a solution of a sample of the product in water with a content of 5 wt % of the sample.
The prep-HPLC method as described under (C1) Sample Preparation and preparative HPLC (except for the last sentence “The evaporated fractions were then used for DSC analysis.”) was used to separate PEO isosorbide oleate, prepared according to example 15. The material eluted, in time similar to the second peak, the peak B (see
PEO isosorbide oleate with an average of 12 EO units, which is needed for standardization purpose, can be synthesized according to known procedures, in this example the PEO isosorbide oleate prepared according to example 15, was used.
The isosorbide calibration material, the PEO isosorbide oleate, prepared according to example 15, was dissolved into three separate solutions: at 0.001 mg/ml, 0.002 mg/ml, 0.006 mg/ml. 10 microliter of each of the three solutions was injected into the LC (Water 2795 Alliance HT, Waters AG, 5405 Baden-Dättwil, Switzerland) and loaded onto a C18 column (Luna C18(2), 3 micrometer, 75×4.6 mm, Phenomenex, 63741 Aschaffenburg, Germany). The analyte species were separated using an can (Acetonitrile): H2O gradient starting at 45 vol % of ACN and increasing to 100 vol % of ACN in 45 min with a flow rate at 0.8 ml/min and a column temperature of 50° C. The separation continued at 100% ACN until reaching 60 min. The species were detected with a mass spectrometer (Waters Micromass Quattro Micro™) equipped with an electrospray ionization source (ESI). The MassLynx V4.0 software was used for data acquisition. Full scan mass spectra were acquired between m/z 200 and 2000 at a speed of 1 scan per second. The parameters for the MS scans were as follows: a desolvation gas temperature of 300° C., ion source temperature of 100° C., a nitrogen gas flow rate of 500 L/hour, nebulizing (N2) gas pressure was 6 bar, capillary voltage was 3000 V, and the cone voltage was 30 V.
Mass spectra were collected and combined over the peak of interest using the MassLynx V4.0 software. The mass spectra were combined, ranging from the time when PEO isosorbide monooleate species were detected, (elution times between 28 to 34 min depending on sample). Each calibration concentration corresponds to one mass spectrum, used for the calibration curve. Four different distributions were detected in each spectrum, corresponding to four different adducts: Na+, K+, H+ and H2O. Each adduct distribution displayed a range of peaks, separated by 44 Da, corresponding to one EO unit. The intensity of all peaks of each distribution was added together, given four intensities, one for each adduct (see figure, circle: sum of all adducts, square: H2O adduct, triangle: H+ adduct, star: Na+ adduct, diamond (visible in the
Two calibration curves were used, one for the H2O adduct (dashed line) and one for the K+ adduct (continuous line), to determine to PEO isosorbide content as these peaks do not overlap with PEO monooleates in the polysorbate samples.
Two polysorbate samples, Croda HP and a polysorbate prepared according to Example 13, were dissolved in H2O to provide a solution with concentration of 0.05 mg/ml. One combined mass spectrum for each sample was collected, using the same method as for the isosorbide calibration material, the PEO isosorbide oleate, for the peak eluting between 28 to 34 min (sample dependent). The intensities for each adduct distribution was calculated, and the calibration curves were used to calculate the amount of PEO isosorbide monooleate species (in wt % based on the weight of the sample) for each sample. The polysorbate prepared according to Example 17 contained 1 wt % PEO isosorbide monooleate. The Croda HP contained more than 12 wt % PEO isosorbide monooleate, a specific concentration could not be determined as it was outside the scope of the calibration range.
The wt % are based on the weight of the respective polysorbate sample, the Croda HP and the polysorbate prepared according to Example 17.
The saturation of the detector occurs with 10 microliter of a PEO isosorbide oleate solution with a concentration above 0.006 mg/ml, to be more specific, between 0.006 mg/ml and 0.01 mg/ml is injected, this is equal to an amount of between 0.06 microgram and 0.1 microgram of PEO isosorbide oleate. Since 10 microliters of sample solutions of a concentration of 0.05 mg/ml are injected, this injection is equal to an amount of 0.5 microgram of sample material injected. Therefore the detection limit is between 12 wt % and 20 wt %.
Oleic acid (204.1 g) and DCM (660 ml) were mixed, oxalyl chloride (185 g) were added at 20° C. during 40 min, after stirring for 2 h at 20° C. the reaction mixture was concentrated at 33° C. from 450 to 22 mbar, obtained was a yellow, clear liquid (216.6 g).
PEO isosorbide (254.5 g, prepared according to example 16) were weighed into a 21 reactor and the atmosphere in the flask was exchanged for N2. Oleoyl chloride (160.9 g of the 216.6 g) was added at room temperature during 30 min and the reaction mixture was stirred for 40 min at room temperature. Then the reaction mixture was heat to 60° C. and vacuum was applied under stirring (200 mbar) for 1.5 day.
The formed HCl could be removed and the pH increased to 3.8. The pH was measured preparing a solution of a sample of the product in water with a content of 5 wt % of the sample.
200 g Naphtha (petroleum), heavy alkylate, CAS 64741-65-7, 89.1 g (0.61 mol, 1 equiv) isosorbide (Sigma-Aldrich), and 0.6 g KOH were charged into a 4 L autoclave. The autoclave was rendered inert by evacuating first and then applying afterwards 0.5 bar pressure with N2, this was done for four times in total.
The mixture was heated to 150° C. 333 g (7.6 mol, 12.4 equiv.) ethylene oxide were added in such speed that the temperature did not raise above 160° C. and the pressure did not raise above 3.8 bar; the addition was done in 4 h. Then the mixture was stirred for 2 h at 150° C.
After cooling to 60° C. 1.4 g AcOH were added. Two phases formed, one with solvent, the other with product, and were separated. Residual solvent was removed by steam distillation at a rotary evacuator. ca. 376 g product was obtained.
Yield: 95% based on the assumption that a PEO sorbitan with an average of 120 EO was obtained. This assumption was also applied when this product was used in further reactions.
1H-NMR and 13C-NMR confirmed the structure.
DSC analysis showed no sign of crystallization or melting, neither in both heating cycles nor in both cooling cycles.
PEO sorbitan (502, 0.44 mol, 1.0 equiv, prepared according to Example 18) were weighed into a 21 reactor and the atmosphere in the flask was exchanged for N2. Oleoyl chloride (215.8 g, 0.7 mol, 1.5 equiv, prepared according to Example 12) was added at room temperature during ca. 40 min and the reaction mixture was stirred for 1 h at room temperature. Then the reaction mixture was heat up to 60° C. and vacuum was applied under stirring (200 mbar) for 3 days.
The formed HCl could be removed and the pH increased to 6.9. The pH was measured preparing a solution of a sample of the product in water with a content of 5 wt % of the sample.
200 g Naphtha (petroleum), heavy alkylate, CAS 64741-65-7, 100 g (0.61 mol, 1 equiv) 1,4-sorbitan, prepared according to Example 11, and 0.6 g KOH were charged into a 4 L autoclave. The autoclave was rendered inert by evacuating first and then applying afterwards 0.5 bar pressure with N2, this was done for four times in total.
The mixture was heated to 150° C. 612 g (13.92.6 mol, 22.8 equiv) ethylene oxide were added in such speed the temperature did not raise above 160° C. and the pressure did not raise above 3.8 bar; the addition was done in 4 h. Then the mixture was stirred for 2 h at 150° C.
After cooling to 60° C. 2.5 g AcOH were added. Two phases formed, one with solvent, the other with product, and were separated. Residual solvent was removed by steam distillation at a rotary evacuator. 688 g product was obtained.
Yield: 95% based on the assumption that a PEO sorbitan with an average of 22 EO was obtained. This assumption was also applied when this product was used in further reactions.
1H-NMR and 13C-NMR confirmed the structure.
DSC analysis showed no sign of crystallization or melting, neither in both heating cycles nor in both cooling cycles.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/104219 | Sep 2018 | CN | national |
| 18 200 298.0 | Oct 2018 | EP | regional |
| 19 157 032.4 | Feb 2019 | EP | regional |
| 19 157 068.8 | Feb 2019 | EP | regional |
| 19 157 297.3 | Feb 2019 | EP | regional |
| 19 194 776.1 | Aug 2019 | EP | regional |
| 19 195 046.8 | Sep 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/073509 | 9/4/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62745676 | Oct 2018 | US |
