CN105223281A - A kind of chromatographic process for acetic acid copaxone concentration in test card handkerchief pine preparation - Google Patents
A kind of chromatographic process for acetic acid copaxone concentration in test card handkerchief pine preparation Download PDFInfo
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- CN105223281A CN105223281A CN201410289379.2A CN201410289379A CN105223281A CN 105223281 A CN105223281 A CN 105223281A CN 201410289379 A CN201410289379 A CN 201410289379A CN 105223281 A CN105223281 A CN 105223281A
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Abstract
A kind of chromatographic process for acetic acid copaxone concentration in test card handkerchief pine preparation.Do you the present invention relates to a kind of employing Thermo? scientific? the Corona of Dionex liquid chromatograph? CAD electron spray detecting device, and be specific to the HPLC chromatographic condition of this model checkout equipment, for detecting the acetic acid copaxone concentration in the acetic acid copaxone sample (i.e. commercially available Ka Pasong preparation) containing sweet mellow wine auxiliary material.
Description
Technical field
The present invention relates to a kind of high performance liquid chromatography (HPLC) method for detecting acetic acid copaxone sample and internal standard peptide group matching used with it.
Background technology
Autoimmunity class disease (autoimmunediseases) refers to, the tissue of some bodies itself is used as the phenomenon that " exotic " carries out attacking by the immune system of living organism.Usual this disease by hindering living organism T cell and B cell to the reaction of body autologous tissue, can be eased.These early stage immune responses are attached on MHC molecule by antigen and promote, and are expressed by T cell.Autoimmunity class disease is exactly that the tissue of body itself and protein are taken as " from antigen ", is attacked by body immune system.Such as: multiple sclerosis, be exactly that the myelin of immune system to isolation and protection nerve is attacked and the disease caused, this disease progression, to losing myelin, will bring neuron and kinesitherapy nerve afunction; Other such as systemic loupus erythematosus, rheumatoid arthritis, from immune hemolytic anemia etc., also belong to this type of disease.
A lot of drug development is out used for the treatment of autoimmunity class disease, comprises multiple sclerosis.Acetic acid copaxone (being copolymer-1 again) is the potpourri of the different molecular weight polymers produced by polymerization by alanine, glutamic acid, lysine and tyrosine.Its four seed amino acid mol ratios are approximately 0.392 ~ 0.462:0.129 ~ 0.153:0.300 ~ 0.374:0.086 ~ 0.100 (Ala:Glu:Lys:Tyr), about 5000 ~ 9000 dalton of mean molecular weight of the multipolymer in potpourri.
The earliest; Teitelbaum etc. are at Eur.J.Immun.; 1; 242; the method describing synthetic copolymer-1 in 1971 prepares by the N-carboxylic acid anhydrides of the N-carboxylic acid anhydrides of the N-carboxylic acid anhydrides of the N-carboxylic acid anhydrides of the ALANINE being dissolved in Isosorbide-5-Nitrae-dioxane, TYR, gamma-glutamic acid benzyl ester and trifluoroacetyl lysine the copolymer-1 protected.Then with the γ-benzyl protecting group on the acetic acid solution removing glutaminic acid residue of 33% hydrogen bromide, the peptide chain of the cut-out polypeptide of part simultaneously, to trifluoroacetyl group copolymer-1.Then by the trifluoroacetyl group protecting group on aqueous piperidine solution removing lysine residue, thick copolymer-1 is obtained.Finally, through dialysis, by solution freeze-drying, copolymer-1 bulk drug had both been obtained.
Because copolymer-1 is a kind of polyblend, its structure between batches and amino acid composition can not be just the same, so can not prepare to detect its content with common UV detecting device, all the different of its response signal can be caused, so its measurement result also has comparatively big error from the subtle change of batch structural little difference and amino acid composition because criticize.Therefore, in order to its content of Accurate Determining, matter of utmost importance is just to provide a kind of mass flow rate sensitive detector irrelevant with structure.
In addition, in the R&D process of polymkeric substance-1, at initial period owing to there is no suitable reference substance, the preparation adopting TEVA is so just needed to make (Copaxone) for reference substance use, but it often props up the sweet mellow wine containing 40mg in preparation, owing to have employed mass flow rate sensitive detector, thus its raw material and sweet mellow wine fast and concentrate be separated into the another difficult problem needing to solve.If adopt gel chromatographic columns or anti-phase C18, because this product is a kind of potpourri, go out peak and do not concentrate, signal reproducibility will be caused bad, so just need a kind ofly raw material to be concentrated out peak and chromatographic condition sweet mellow wine can separated again.
Summary of the invention
In order to solve the problem run in the above-mentioned research and development at polymkeric substance-1 and preparation process, inventor is through screening, determine the CoronaCAD electron spray detecting device adopting ThermoscientificDionex liquid chromatograph, and devise the chromatographic condition being specific to this model checkout equipment.This detecting device testing result is relevant with analyte particles, marking current is directly proportional to the quality analyzing thing in sample, therefore no matter which kind of compound, as long as sample introduction response identical in quality is all basically identical, so CoronaUltra detecting device can detect all non-volatile matters, comprise not containing chromophoric material, no matter measured object molecular structure.
First the present invention relates to a kind of concentration special color spectrum detection method detecting the acetic acid copaxone sample (commercially available Ka Pasong preparation) containing sweet mellow wine auxiliary material, the method uses the CoronaCAD electron spray detecting device of ThermoscientificDionex liquid chromatograph, chromatographic column is: Kromasil60-5CN, 4.6 × 250mm, 5um; In described chromatographic process, the compound method of mobile phase is that buffer salt solution and organic phase are according to buffer salt solution: organic phase solvent=4:1 ~ 16 (v/v) mixes; The pH value of described mobile phase is 2.0-5.0, is preferably 3.0; Described organic phase solvent is acetonitrile or methyl alcohol, is preferably acetonitrile; Described buffer salt composition is ammonium formate, ammonium acetate, is preferably ammonium formate; The concentration of described buffer salt solution is 0.1-0.5mol/l, is preferably 0.2mol/l.
In chromatographic detection method of the present invention, chromatographic condition is, column temperature 20-60 DEG C, is preferably 25 DEG C.
In chromatographic detection method of the present invention, the sample introduction concentration of sample is 0.01-0.5mg/ml, is preferably 0.1mg/ml.
In chromatographic detection method of the present invention, the detecting device used is electron spray detecting device (CAD) or evaporative light-scattering detector (ELSD).
Accompanying drawing explanation
The linear regression curves of Fig. 1, the logarithm of acetic acid copaxone concentration and the logarithm of relative detector response.
The linear regression curves of the logarithm of Fig. 2, mannitol concentration and the logarithm of relative detector response.
The chromatogram that Fig. 3, Ka Pasong (Copaxone) preparation is curved, leading peak is that acetic acid lattice take over thunder, and postpeak is sweet mellow wine.
Fig. 4,4a-4c, the chromatogram of Ka Pasong (Copaxone) preparation under different chromatographic condition.
Embodiment
The screening of embodiment 1. chromatographic condition and checking
Chromatographic column Kromasil60-5CN, 4.6 × 250mm, 5um; Mobile phase A is the ammonium formate (adjusting pH3.0 with formic acid) of 0.1M, and Mobile phase B is acetonitrile, and A:B=50:50 carries out isocratic elution; Adopt UV-detector connect with electron spray detecting device, its medium ultraviolet at front electron spray detecting device rear.The determined wavelength of UV-detector is 275nm, and electron spray detecting device atomization gas adopts high pure nitrogen, and atomization temperature is 25 DEG C; Flow velocity 1.0ml/min, column temperature: 25 DEG C, the degree of separation of the chromatographic peak of acetic acid copaxone and the chromatographic peak of sweet mellow wine should be greater than 4; Acetic acid copaxone tailing factor should be less than 2.5.
In order to ensure the feasibility of this analytical approach, we verify the linear of this analytical approach and precision, and result is as follows:
1. concentration linear relationship is analyzed:
Because the detecting device that this analysis method is used is mass flow rate sensitive detector, the relation between its concentration and peak response value is not direct linear relationship, is that both logarithm value are linear.Therefore, under first detecting this chromatographic condition, the concentration linear relationship of polymkeric substance-1 (acetic acid copaxone) sample, the results are shown in Table 1.Further with the logarithm of concentration in above-mentioned data with carry out linear regression, linear equation is y=0.8054x+7.171, its related coefficient square be 0.9995, illustrate that in the method, the logarithm of concentration and the logarithm of relative detector response are good linear relationship.(Fig. 1)
Table 1. acetic acid copaxone is linearly investigated
| Weigh (mg) | Area | Log concentration | Area logarithm |
| 16.65 | 142476801 | 1.221414238 | 8.1537442 |
| 18.36 | 155042181 | 1.263872677 | 8.1904499 |
| 19.90 | 164595532 | 1.298853076 | 8.216418 |
| 20.70 | 170294711 | 1.315970345 | 8.2312012 |
| 22.55 | 182710424 | 1.353146546 | 8.2617633 |
| 24.55 | 194854847 | 1.390051496 | 8.2897112 |
Next, under detecting this chromatographic condition, the concentration linear relationship of mannitol sample, the results are shown in Table 2.Further with the logarithm of concentration in above-mentioned data with carry out linear regression, linear equation is y=0.5649x+7.4936, its related coefficient square be 0.9996, illustrate that in the method, the logarithm of mannitol concentration and the logarithm of relative detector response are good linear relationship.(Fig. 2)
Table 2. sweet mellow wine is linearly investigated
| Weigh (mg) | Area | Log concentration | Area logarithm |
| 33.99 | 228688342 | 1.531351165 | 8.359244 |
| 36.35 | 236804547 | 1.560504415 | 8.37439 |
| 42.15 | 257792429 | 1.624797579 | 8.4112702 |
| 43.63 | 263634289 | 1.639785213 | 8.4210019 |
| 46.88 | 273557161 | 1.670987603 | 8.4370481 |
| 48.02 | 277732914 | 1.681422156 | 8.4436274 |
2. detect precision to investigate:
In order to determine the steadiness of this mass flow rate sensitive detector to this product response, the acetic acid copaxone sample of sweet mellow wine is specifically contained for one, the precision situation of this method investigated by continuous sample introduction 6 pin, and concrete data are as shown in table 3 below, and wherein the testing result of sequence number 3 is as shown in Figure 3:
The precision situation of table 3. acetic acid copaxone peak area and sweet mellow wine peak area
| Sequence number | Acetic acid copaxone peak area | Sweet mellow wine peak area |
| 1 | 183016783 | 246887661 |
| 2 | 183023567 | 246832189 |
| 3 | 183026356 | 246897541 |
| 4 | 183012567 | 246886543 |
| 5 | 183024526 | 246865321 |
| 6 | 183009875 | 246867536 |
| SD | 6856.404189 | 23472.25985 |
| Average | 183018945.7 | 246872798.5 |
| RSD | 0.37％ | 0.95％ |
Can find out that these detecting device Dichlorodiphenyl Acetate lattice draw and have stable response signal for thunder and sweet mellow wine, have reliable sample introduction precision in this way from above-mentioned data.
The screening of embodiment 2. chromatographic condition and checking
Chromatographic column Kromasil60-5CN, 4.6 × 250mm, 5um; Mobile phase A is the ammonium formate (adjusting pH5.0 with formic acid) of 0.1M, and Mobile phase B is acetonitrile, and A:B=80:20 carries out isocratic elution; Adopt UV-detector connect with electron spray detecting device, its medium ultraviolet at front electron spray detecting device rear.The determined wavelength of UV-detector is 275nm, and electron spray detecting device atomization gas adopts high pure nitrogen, and atomization temperature is 25 DEG C; Flow velocity 1.0ml/min, column temperature: 25 DEG C, the degree of separation of the chromatographic peak of acetic acid copaxone and the chromatographic peak of sweet mellow wine is 4.5; Acetic acid copaxone tailing factor 2.1.(Fig. 4 a)
The screening of embodiment 3. chromatographic condition and checking
Chromatographic column Kromasil60-5CN, 4.6 × 250mm, 5um; Mobile phase A is the ammonium formate (adjusting pH3.5 with formic acid) of 0.5M, and Mobile phase B is acetonitrile, and A:B=35:65 carries out isocratic elution; Adopt UV-detector connect with electron spray detecting device, its medium ultraviolet at front electron spray detecting device rear.The determined wavelength of UV-detector is 275nm, and electron spray detecting device atomization gas adopts high pure nitrogen, and atomization temperature is 25 DEG C; Flow velocity 1.0ml/min, column temperature: 50 DEG C, the degree of separation of the chromatographic peak of acetic acid copaxone and the chromatographic peak of sweet mellow wine is 4.6; Acetic acid copaxone tailing factor 1.9.(Fig. 4 b)
The screening of embodiment 4. chromatographic condition and checking
Chromatographic column Kromasil60-5CN, 4.6 × 250mm, 5um; Mobile phase A is the ammonium acetate (adjusting pH5.0 with formic acid) of 0.1M, and Mobile phase B is acetonitrile, and A:B=50:50 carries out isocratic elution; Adopt UV-detector connect with electron spray detecting device, its medium ultraviolet at front electron spray detecting device rear.The determined wavelength of UV-detector is 275nm, and electron spray detecting device atomization gas adopts high pure nitrogen, and atomization temperature is 25 DEG C; Flow velocity 1.5ml/min, column temperature: 25 DEG C, the degree of separation of the chromatographic peak of acetic acid copaxone and the chromatographic peak of sweet mellow wine is 4.6; Acetic acid copaxone tailing factor 2.2.(Fig. 4 c)
Embodiment 5. with chromatographic process of the present invention, measurement card handkerchief pine sample in acetic acid copaxone
Ka Pasong (Copaxone) the analysis of pharmaceutical dosage forms result of 6 lot numbers adopting chromatographic of the present invention to produce at different regions different time TEVA company as following table 4,
The Ka Pasong preparation (Copaxone) of table 4. five crowdes of TEVA detects data summarization
From the analysis of 5 wholesale bright business's preparations essence condition, its acetic acid lattice take over the content of thunder or suitable consistent, the RSD%=2.46 of the relative detector response of 6 batches; 6 crowdes of RSD%=0.42 of its sweet mellow wine.Method of the present invention is described, can be good at the detection and the demarcation that are applicable to acetic acid copaxone sample.
Finally, it should be noted that, above-described embodiment only understands the particular content of technical scheme of the present invention for those skilled in the art, and is not used as limiting the scope of the present invention.
Claims (6)
1. the concentration special color spectrum detection method of acetic acid copaxone in test card handkerchief pine formulation samples, it is characterized in that, use the CoronaCAD electron spray detecting device of ThermoscientificDionex liquid chromatograph, chromatographic column is: Kromasil60-5CN, 4.6 × 250mm, 5um detect the acetic acid copaxone concentration in sample.
2. method according to claim 1, is characterized in that, the compound method of mobile phase during detection sample is that buffer salt solution and organic phase are that 4:1 ~ 16 (v/v) mix according to buffer salt solution and the ratio of organic phase solvent; The pH value regulating mobile phase is 2.0-5.0; Described organic phase is acetonitrile or methyl alcohol; Described buffer salt is ammonium formate, ammonium acetate; The concentration of described buffer salt solution is 0.1-0.5mol/l.
3. method according to claim 2, is characterized in that, described mobile phase pH is 3.0; Organic phase is acetonitrile, and buffer salt is ammonium formate, and buffer concentration is 0.2mol/l.
4., according to the arbitrary described method of Claims 2 or 3, it is characterized in that the column temperature 20-60 DEG C when chromatogram detects is preferably 25 DEG C.
5., according to the arbitrary described method of claim 1-4, it is characterized in that, the sample introduction concentration detecting sample is 0.01-0.5mg/ml, is preferably 0.1mg/ml.
6., according to the arbitrary described method of claim 1-5, it is characterized in that, the detecting device used is electron spray detecting device (CAD) or evaporative light-scattering detector (ELSD).
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Citations (4)
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| CN101479607A (en) * | 2006-04-28 | 2009-07-08 | 莫门塔制药股份有限公司 | Methods of evaluating peptide mixtures |
| CN102271681A (en) * | 2008-11-04 | 2011-12-07 | 凯莫森特里克斯股份有限公司 | Modulators of cxcr7 |
| CN102844325A (en) * | 2010-04-27 | 2012-12-26 | 雷迪博士实验室有限公司 | Preparation of polypeptides and salts thereof |
| US20130210054A1 (en) * | 2012-02-09 | 2013-08-15 | Momenta Pharmaceuticals, Inc. | Amino Acid Copolymer Assay |
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- 2014-06-24 CN CN201410289379.2A patent/CN105223281B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101479607A (en) * | 2006-04-28 | 2009-07-08 | 莫门塔制药股份有限公司 | Methods of evaluating peptide mixtures |
| CN102271681A (en) * | 2008-11-04 | 2011-12-07 | 凯莫森特里克斯股份有限公司 | Modulators of cxcr7 |
| CN102844325A (en) * | 2010-04-27 | 2012-12-26 | 雷迪博士实验室有限公司 | Preparation of polypeptides and salts thereof |
| US20130210054A1 (en) * | 2012-02-09 | 2013-08-15 | Momenta Pharmaceuticals, Inc. | Amino Acid Copolymer Assay |
Non-Patent Citations (3)
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| CHRISTOPHER CRAFTS等: "Direct Measurement of Mass Balance in the Digestion of a Protein", 《LC GC NORTH AMERICA》 * |
| STEFANIE WERNISCH等: "Application of cinchona-sulfonate-based chiral zwitterionic ion exchangers for the separation of proline-containing dipeptide rotamers and determination of on-column isomerization parameters from dynamic elution profiles", 《ANALYTICA CHIMICA ACTA》 * |
| 朱晓囡等: "反相液相色谱在蛋白质及多肽分离分析中的应用", 《分析化学 》 * |
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