CN105223281B - A kind of chromatographic process for being used to detect acetic acid copaxone concentration in the loose preparation of Kappa - Google Patents
A kind of chromatographic process for being used to detect acetic acid copaxone concentration in the loose preparation of Kappa Download PDFInfo
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- CN105223281B CN105223281B CN201410289379.2A CN201410289379A CN105223281B CN 105223281 B CN105223281 B CN 105223281B CN 201410289379 A CN201410289379 A CN 201410289379A CN 105223281 B CN105223281 B CN 105223281B
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Abstract
A kind of chromatographic process for being used to detect acetic acid copaxone concentration in the loose preparation of Kappa.The present invention relates to a kind of Corona CAD EFI fog detectors of use Thermo scientific Dionex liquid chromatographs, and the HPLC chromatogram condition of the model detection device is specific to, for detecting the acetic acid copaxone concentration in the acetic acid copaxone sample (the i.e. commercially available loose preparation of Kappa) containing mannitol auxiliary material.
Description
Technical field
The present invention relates to it is a kind of be used for detect acetic acid copaxone sample high performance liquid chromatography (HPLC) method and and its
Matching used internal standard peptide group.
Background technology
Autoimmunity class disease (autoimmune diseases) refers to, the immune system of living organism is by some bodies
The phenomenon that the tissue of itself is attacked as " exotic ".Usual this disease can be by hindering living organism T cell and B
Reaction of the cell to body autologous tissue, is eased.The immune response of these early stages is by antigen binding to MHC molecule
Promote, and expressed by T cell.Autoimmunity class disease is exactly body tissue in itself and protein be taken as " from
Antigen ", is attacked by body immune system.For example：Multiple sclerosis is exactly immune system to isolating and protecting neural myelin
Disease caused by being attacked, this disease, which is developed into, loses myelin, can bring about neuron and kinesitherapy nerve function is lost；
Other fall within such disease such as systemic loupus erythematosus, rheumatoid arthritis, from immune hemolytic anemia.
Many drug developments are out used to treat autoimmunity class disease, including multiple sclerosis.Acetic acid copaxone
(being called copolymer-1) is by polymerizeing the different molecular weight polymers produced by alanine, glutamic acid, lysine and tyrosine
Mixture.Its four kinds of amino acid molar ratios are about 0.392~0.462:0.129~0.153:0.300~0.374:
0.086~0.100 (Ala：Glu：Lys：Tyr), the dongle of the mean molecule quantity of the copolymer in mixture about 5000~9000
.
Earliest, the method that Teitelbaum etc. describes synthetic copolymer -1 in Eur.J.Immun., 1,242,1971
Be by be dissolved in the N- carboxylic acid anhydrides of the ALANINE of 1,4- dioxane, the N- carboxylic acid anhydrides of TYR, gamma-glutamic acid benzyl ester N-
The N- carboxylic acid anhydrides of carboxylic acid anhydrides and trifluoroacetyl lysine prepares the copolymer-1 of protection.Then the acetic acid with 33% hydrogen bromide is molten
Liquid removes γ-benzyl protecting group on glutaminic acid residue, while the peptide chain of partial cut-out polypeptide, to trifluoroacetyl group copolymerization
Thing -1.Then the trifluoroacetyl group protection group on lysine residue is removed with aqueous piperidine solution, obtains thick copolymer-1.Finally,
By dialysis, solution is freezed, copolymer-1 bulk drug has both been obtained.
Because copolymer-1 is a kind of polyblend, its between batches structure and amino acid composition can not possibly completely
Equally, so can not be prepared to detect its content with common UV detectors, because criticizing and the small difference and ammonia in batch structure
The minor variations of base acid constituents can cause the difference of its response signal, so its measurement result also has larger error.Therefore,
For its content of Accurate Determining, matter of utmost importance is just to provide a kind of mass flow rate sensitive detector unrelated with structure.
In addition, in the R＆D process of polymer -1, in initial period due to unsuitable reference substance, then be accomplished by
Use TEVA preparation to make (Copaxone) to use for reference substance, but the mannitol containing 40mg in its every preparation, due to adopting
With mass flow rate sensitive detector, so its raw material and the quick and concentration of mannitol are separated into the another problem for needing to solve.
If using gel chromatographic columnses or anti-phase C18, because this product is a kind of mixture, appearance is not concentrated, and may result in signal reproduction
Property it is bad, so be accomplished by a kind of raw material being concentrated into appearance and the chromatographic condition that can separate mannitol again.
The content of the invention
The problem of in order to solve encountered in the above-mentioned research and development in polymer -1 and preparation process, inventor passes through screening,
The Corona CAD EFI fog detectors using Thermo scientific Dionex liquid chromatographs are determined, and devise
It is specific to the chromatographic condition of the model detection device.The detector testing result is relevant with analyte particles, signal code and sample
The quality of analyte is directly proportional in product, thus no matter which kind of compound, as long as sample introduction it is identical in quality response it is all basically identical, so
Corona Ultra detectors can detect all non-volatile matters, including the material without chromophore, no matter measured object molecular structure
How.
Present invention firstly relates to a kind of acetic acid copaxone sample (commercially available Kappa pine system detected containing mannitol auxiliary material
Agent) the special chromatographic detection method of concentration, this method is using Thermo scientific Dionex liquid chromatographs
Corona CAD EFI fog detectors, chromatographic column is：Kromasil 60-5CN, 4.6 × 250mm, 5um；Described chromatographic process
In, the compound method of mobile phase is that buffer salt solution is with organic phase according to buffer salt solution：Organic phase solvent=4：1~16 (v/
V) mix；The pH value of the mobile phase is 2.0-5.0, preferably 3.0；Described organic phase solvent be acetonitrile or methanol, preferably
For acetonitrile；Described buffering salt component is ammonium formate, ammonium acetate, preferably ammonium formate；The concentration of the buffer salt solution is
0.1-0.5mol/l, preferably 0.2mol/l.
In chromatographic detection method of the present invention, chromatographic condition is, 20-60 DEG C of column temperature, preferably 25 DEG C.
In chromatographic detection method of the present invention, the sample introduction concentration of sample is 0.01-0.5mg/ml, preferably 0.1mg/
ml。
In chromatographic detection method of the present invention, used detector is EFI fog detector (CAD) or evaporative light
Scatter detector (ELSD).
Brief description of the drawings
The linear regression curves of Fig. 1, the logarithm of acetic acid copaxone concentration and relative detector response logarithm.
The linear regression curves of Fig. 2, the logarithm of mannitol concentration and relative detector response logarithm.
The curved chromatogram of loose (Copaxone) preparation of Fig. 3, Kappa, leading peak is that acetic acid lattice take over thunder, and postpeak is mannitol.
The chromatogram of loose (Copaxone) preparation of Kappa under Fig. 4,4a-4c, different chromatographic conditions.
Embodiment
The screening and checking of the chromatographic condition of embodiment 1.
Chromatographic column Kromasil 60-5CN, 4.6 × 250mm, 5um；Mobile phase A (is adjusted for 0.1M ammonium formate with formic acid
PH3.0), Mobile phase B is acetonitrile, A:B=50:50, carry out isocratic elution；Using UV-detector and EFI fog detector string
Connection, wherein it is ultraviolet in preceding EFI fog detector rear.The Detection wavelength of UV-detector is 275nm, the atomization of EFI fog detector
Gas uses high pure nitrogen, and atomization temperature is 25 DEG C；Flow velocity 1.0ml/min, column temperature：25 DEG C, the chromatographic peak of acetic acid copaxone
4 are should be greater than with the separating degree of the chromatographic peak of mannitol；Acetic acid copaxone tailing factor should be less than 2.5.
In order to ensure the feasibility of the analysis method, we are verified the linear and precision to the analysis method,
As a result it is as follows：
1. concentration linear relationship is analyzed：
Because the detector used in this analysis method is mass flow rate sensitive detector, the relation between its concentration and peak response value is simultaneously
It is not direct linear relationship, is that both logarithm values are linear.Therefore, detect first under the chromatographic condition, polymer-
The concentration linear relationship of 1 (acetic acid copaxone) sample, the results are shown in Table 1.Further with the logarithm of concentration in above-mentioned data with entering
Row linear regression, linear equation is y=0.8054x+7.171, and its coefficient correlation square is 0.9995, illustrates dense in this method
The logarithm of degree and the logarithm of relative detector response are in good linear relationship.(Fig. 1)
The acetic acid copaxone of table 1. 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, detecting under the chromatographic condition that the concentration linear relationship of mannitol sample the results are shown in Table 2.Further use
The logarithm of concentration and progress linear regression in data are stated, linear equation is y=0.5649x+7.4936, square of its coefficient correlation
For 0.9996, the logarithm for illustrating the logarithm of mannitol concentration and relative detector response in this method is in good linear relationship.(figure
2)
The mannitol of table 2. 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 that precision is investigated：
In order to determine steadiness of the mass flow rate sensitive detector to this product response, specifically contain sweet dew for one
The acetic acid copaxone sample of alcohol, the pin of continuous sample introduction 6 investigates the precision situation of this method, and specific data are as shown in table 3 below,
The testing result of wherein sequence number 3 is as shown in Figure 3：
The precision situation of the acetic acid copaxone peak area of table 3. and mannitol peak area
| Sequence number | Acetic acid copaxone peak area | Mannitol 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% |
It can be seen that the detector Dichlorodiphenyl Acetate copaxone and mannitol have stable response signal from above-mentioned data, so should
Method has reliable sample introduction precision.
The screening and checking of the chromatographic condition of embodiment 2.
Chromatographic column Kromasil 60-5CN, 4.6 × 250mm, 5um；Mobile phase A (is adjusted for 0.1M ammonium formate with formic acid
PH5.0), Mobile phase B is acetonitrile, A:B=80:20, carry out isocratic elution；Using UV-detector and EFI fog detector string
Connection, wherein it is ultraviolet in preceding EFI fog detector rear.The Detection wavelength of UV-detector is 275nm, the atomization of EFI fog detector
Gas uses high pure nitrogen, and atomization temperature is 25 DEG C；Flow velocity 1.0ml/min, column temperature：25 DEG C, the chromatographic peak of acetic acid copaxone
Separating degree with the chromatographic peak of mannitol is 4.5；Acetic acid copaxone tailing factor 2.1.(Fig. 4 a)
The screening and checking of the chromatographic condition of embodiment 3.
Chromatographic column Kromasil 60-5CN, 4.6 × 250mm, 5um；Mobile phase A (is adjusted for 0.5M ammonium formate with formic acid
PH3.5), Mobile phase B is acetonitrile, A:B=35:65, carry out isocratic elution；Using UV-detector and EFI fog detector string
Connection, wherein it is ultraviolet in preceding EFI fog detector rear.The Detection wavelength of UV-detector is 275nm, the atomization of EFI fog detector
Gas uses high pure nitrogen, and atomization temperature is 25 DEG C；Flow velocity 1.0ml/min, column temperature：50 DEG C, the chromatographic peak of acetic acid copaxone
Separating degree with the chromatographic peak of mannitol is 4.6；Acetic acid copaxone tailing factor 1.9.(Fig. 4 b)
The screening and checking of the chromatographic condition of embodiment 4.
Chromatographic column Kromasil 60-5CN, 4.6 × 250mm, 5um；Mobile phase A (is adjusted for 0.1M ammonium acetate with formic acid
PH5.0), Mobile phase B is acetonitrile, A:B=50:50, carry out isocratic elution；Using UV-detector and EFI fog detector string
Connection, wherein it is ultraviolet in preceding EFI fog detector rear.The Detection wavelength of UV-detector is 275nm, the atomization of EFI fog detector
Gas uses high pure nitrogen, and atomization temperature is 25 DEG C；Flow velocity 1.5ml/min, column temperature：25 DEG C, the chromatographic peak of acetic acid copaxone
Separating degree with the chromatographic peak of mannitol is 4.6；Acetic acid copaxone tailing factor 2.2.(Fig. 4 c)
Embodiment 5. measures the acetic acid copaxone in the loose sample of Kappa with the chromatographic process of the present invention
The card of 6 lot numbers produced using chromatographic of the present invention to TEVA companies in different regions different time
Handkerchief pine (Copaxone) analysis of pharmaceutical dosage forms result such as table 4 below,
Loose preparation (Copaxone) the detection data summarization of 4. 5 crowdes of TEVA of table Kappa
From the point of view of the smart condition of the analyses of 5 wholesale bright business's preparations, its acetic acid lattice takes over the content of thunder or suitable consistent, and 6
The RSD%=2.46 for the relative detector response criticized；6 crowdes of RSD%=0.42 of its mannitol.Illustrate method of the present invention,
It can be good at the detection and demarcation suitable for acetic acid copaxone sample.
It is last, it is necessary to explanation, above-described embodiment is only supplied skilled artisan understands that technical scheme
Particular content, is not used as limiting the scope of the present invention.
Claims (8)
1. a kind of special chromatographic detection method of concentration for detecting acetic acid copaxone in the loose formulation samples of Kappa, it is characterised in that
Using the Corona CAD EFI fog detectors of Thermo scientific Dionex liquid chromatographs, chromatographic column is：
Kromasil 60-5CN, 4.6 × 250mm, 5um are detected to the acetic acid copaxone concentration in sample；
The compound method of mobile phase during detection sample is that buffer salt solution mixes with organic phase according to buffer salt solution with organic
The ratio of agent is 4：1~16v/v is mixed；The pH value for adjusting mobile phase is 2.0-5.0；Described organic phase is acetonitrile or methanol；
Described buffer salt is ammonium formate, ammonium acetate；The concentration of the buffer salt solution is 0.1-0.5mol/l.
2. according to the method described in claim 1, it is characterised in that described mobile phase pH is 3.0；Organic phase is acetonitrile, is delayed
Salt is rushed for ammonium formate, buffer concentration is 0.2mol/l.
3. according to any described method of claim 1 or 2, it is characterised in that 20-60 DEG C of column temperature when chromatogram is detected.
4. method according to claim 3, it is characterised in that column temperature when chromatogram is detected is 25 DEG C.
5. according to any described method in claim 1,2 or 4, it is characterised in that the sample introduction concentration of detection sample is 0.01-
0.5mg/ml。
6. method according to claim 3, it is characterised in that the sample introduction concentration of detection sample is 0.01-0.5mg/ml.
7. method according to claim 5, it is characterised in that the sample introduction concentration of detection sample is 0.1mg/ml.
8. method according to claim 6, it is characterised in that the sample introduction concentration of detection sample is 0.1mg/ml.
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Citations (3)
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130210054A1 (en) * | 2012-02-09 | 2013-08-15 | Momenta Pharmaceuticals, Inc. | Amino Acid Copolymer Assay |
-
2014
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Patent Citations (3)
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| Direct Measurement of Mass Balance in the Digestion of a Protein;Christopher Crafts等;《LC GC NORTH AMERICA》;20120601;第1-3段 * |
| les.《Analytica Chimica Acta》.2013,第795卷第88-98页. * |
| 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 profi * |
| 反相液相色谱在蛋白质及多肽分离分析中的应用;朱晓囡等;《分析化学 》;20040228;第32卷(第2期);第248-254页 * |
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