CN111175404A - High performance liquid chromatography detection method for charge heterogeneity impurities of recombinant protein drug - Google Patents
High performance liquid chromatography detection method for charge heterogeneity impurities of recombinant protein drug Download PDFInfo
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Abstract
The invention belongs to the technical field of drug detection, and particularly discloses a high performance liquid chromatography detection method for charge heterogeneity impurities of recombinant protein drugs. The invention adds a certain proportion of organic solvent into the mobile phase, namely, the organic phase gradient elution is added on the basis of the salt gradient elution, and the high performance liquid chromatography detection method is obtained by selecting chromatographic conditions, such as the selection of chromatographic columns, the components of the mobile phase A and the mobile phase B, the column temperature, the detection wavelength and the gradient elution conditions. Experiments prove that the method can effectively separate the target protein from the charge heterogeneity impurities, and has the advantages of good repeatability, simplicity, rapidness, high detection sensitivity, low cost and the like. In addition, the detection method has strong universality and can effectively separate and detect the charge heterogeneous impurities of the insulin recombinant protein drug sample.
Description
Technical Field
The invention relates to the technical field of drug detection, in particular to a high performance liquid chromatography detection method for charge heterogeneity impurities of recombinant protein drugs.
Background
Recombinant protein drugs expressed by eukaryotic systems all have certain posttranslational modifications, and the modified impurities have different Isoelectric points (pIs) and show different degrees of charge heterogeneity. The charge heterogeneity is a key quality attribute of the recombinant protein drugs, and has important influence on the stability, solubility, immunogenicity, in-vivo and in-vitro biological activity, pharmacokinetic function exertion and the like of the recombinant protein drugs. The situation of post-translational modification impurities in the drug can be known by analyzing the charge heterogeneity of the recombinant protein drug, and the analysis is one of essential items for quality control of the recombinant protein drug.
At present, three methods for detecting protein drug charge heterogeneity impurities are mainly used, namely plate gel isoelectric focusing electrophoresis, ion exchange chromatography and capillary isoelectric focusing electrophoresis (drug biotechnology, 2018,25 (5): 461-466). The isoelectric focusing electrophoresis method of the plate gel can quickly and intuitively display isoelectric point zones, has wide popularization range, but has the defects of complicated operation, low resolution and difficult accurate quantification. The resolution and accurate quantification of capillary isoelectric focusing electrophoresis are improved to a certain extent, but because all components subjected to focusing separation are required to be pushed to a detection window for detection through mechanical or electric field force after the focusing process, the spectral band is widened and deformed. The ion exchange chromatography can simultaneously separate charge isomers caused by different isoelectric points and inconsistent space charge distribution of proteins, but the ion exchange chromatography has uncertain mutual separation effect on different proteins due to the limitation of exchange chromatography media and the complexity of optimization of various experimental parameters.
In the industrial production process, the recombinant protein drug structure is changed in complexity and diversity, so that the selection of a recombinant protein drug charge heterogeneity impurity detection analysis method with high sensitivity and strong specificity is an effective means for controlling and stabilizing the production quality of the recombinant protein drug.
Disclosure of Invention
The invention mainly solves the technical problem of providing a high performance liquid chromatography detection method for charge heterogeneity impurities of recombinant protein drugs.
The recombinant protein medicament refers to a protein medicament obtained by recombinant DNA or recombinant RNA technology, such as an insulin-like recombinant protein medicament. The recombinant protein drug may produce post-translational modification impurities during the production process, thereby affecting the quality of the recombinant protein drug. Aiming at the detection of the charge heterogeneity impurities of the recombinant protein drugs, the aim of controlling the quality of the drugs can be achieved.
The sample to be detected by the method provided by the invention is a solution containing the recombinant protein drug. For example, the sample to be tested may be an injection solution containing the recombinant protein drug. For example, the sample to be tested may be a mixed solution obtained by mixing an injection solution containing the recombinant protein drug with water, an acid solution, an alkali solution, or a salt solution.
The invention provides a high performance liquid chromatography detection method for charge heterogeneity impurities of recombinant protein drugs, which comprises the following detection conditions: adopting weak cation column, and performing gradient elution with mobile phase A and mobile phase B; the mobile phase A is a salt solution of any one or more of acetate, citrate and phosphate, the mobile phase B is a salt solution of any one or more of acetate, citrate, phosphate, sodium salt and potassium salt, and an organic solvent is further added into the mobile phase B.
Preferably, the mobile phase A is a salt solution of any one or more of sodium acetate, potassium acetate, sodium citrate, disodium hydrogen phosphate and sodium dihydrogen phosphate, and is preferably a sodium acetate solution; the mobile phase B is a salt solution of any one or more of sodium acetate, potassium acetate, sodium citrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium sulfate, potassium chloride and potassium nitrate; the organic solvent is any one of ethanol, methanol and acetonitrile.
Preferably, the pH value of the mobile phase A salt solution is 5.0-6.0, preferably 5.0-5.6. Preferably, the mobile phase A adopted by the invention is sodium acetate solution, and the concentration of the sodium acetate solution is preferably 10-30 mM, and more preferably 20 mM.
Preferably, the volume percentage concentration of the organic solvent in the mobile phase B is 40-60%, preferably 40-55%, and more preferably 50%. The organic solvent is preferably ethanol.
Further preferably, the mobile phase B is a mixed solution of sodium acetate, sodium chloride and ethanol, or a mixed solution of sodium acetate, potassium chloride and ethanol, or a mixed solution of sodium acetate, sodium chloride and methanol, or a mixed solution of sodium acetate, sodium chloride and acetonitrile, or a mixed solution of sodium citrate, sodium chloride and ethanol, or a mixed solution of potassium acetate, potassium chloride and acetonitrile; preferably, the mixed solution of sodium acetate, sodium chloride and ethanol, more preferably, the concentration of the sodium acetate in the mixed solution is 5-20 mM, the concentration of the sodium chloride is 0.4-0.6M, and the volume percentage concentration of the ethanol is 40-60%. As a preferred embodiment, the composition of the mobile phase B is: 10mM sodium acetate, 0.5M NaCl, 50% (volume percent) ethanol.
As a preferred embodiment, the gradient elution is in particular: eluting with mobile phase A, and eluting with mobile phase B.
Further preferably, the gradient elution conditions are: the flow rate of the mobile phase is 0.5-1.0 ml/min, the 100% mobile phase A is adopted for elution for 4-10 minutes, and then the 100% mobile phase B is adopted for elution until the total elution time is 15-20 minutes.
As a preferred embodiment, the mobile phase B consists of: 10mM sodium acetate, 0.5M NaCl, 50% by volume ethanol; and/or the mobile phase A is 20mM sodium acetate solution.
Preferably, the weak cation column is any one of a ProPac WCX-10 analytical column, a Bio WCX analytical column and a ProteomixWCX analytical column. The gauge is preferably 4mm × 250 mm.
Preferably, the detection conditions further comprise: a diode array detector or an ultraviolet detector is adopted;
and/or the temperature of the chromatographic column is 25-45 ℃ during detection, and preferably 30 ℃;
and/or the detection wavelength is 210-220 nm;
and/or the sample introduction amount is 5-10 mu l.
As a preferred embodiment of the invention, the high performance liquid chromatography detection method for the charge heterogeneity impurities of the recombinant protein drugs comprises the following detection conditions:
an instrument is adopted: the Agilent 1260 liquid chromatograph is provided with a diode array detector or an ultraviolet detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250 mm;
mobile phase A: 20mM sodium acetate;
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 50% ethanol;
performing gradient elution with mobile phase A and mobile phase B, wherein the elution conditions shown in the following table 1 or table 2 can be adopted in the elution procedure;
flow rate: 0.5 ml/min;
sample introduction amount: 5-10 mul;
column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
TABLE 1
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 4 | 0 | 100 |
| 16 | 0 | 100 |
TABLE 2
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 6 | 0 | 100 |
| 16 | 0 | 100 |
The high performance liquid chromatography detection method can be applied to detecting the charge heterogeneity impurities of the recombinant protein drugs; preferably, the recombinant protein drug charge-heterogeneity impurity is a charge-heterogeneity impurity of an insulin-based recombinant protein drug.
Aiming at the defect of the existing detection method for detecting the charge heterogeneity impurities of the recombinant protein drugs, the invention researches and develops a high performance liquid chromatography detection method by adding a certain proportion of organic phase into a salt gradient elution mobile phase. The detection method is characterized in that a certain proportion of organic solvent is added into the mobile phase, namely, the organic phase gradient elution is added on the basis of the salt gradient elution, and chromatographic conditions, such as chromatographic columns, mobile phase A and mobile phase B, are selected, so that the high performance liquid chromatography detection method is obtained. Experiments prove that the method can effectively separate the target protein from the charge heterogeneity impurities, and has the advantages of good repeatability, simplicity, rapidness, high detection sensitivity, low cost and the like. In addition, the detection method has strong universality and can effectively separate and detect the charge heterogeneous impurities of the insulin recombinant protein drug sample.
Drawings
FIG. 1 is a chromatogram obtained in example 1 of the present invention;
FIG. 2 is a chromatogram obtained in example 3 of the present invention;
FIG. 3 is a chromatogram obtained in example 4 of the present invention;
FIG. 4 is a chromatogram obtained in example 5 of the present invention;
FIG. 5 is a chromatogram obtained in example 6 of the present invention;
FIG. 6 is a chromatogram obtained in example 7 of the present invention;
FIG. 7 is a chromatogram obtained in example 8 of the present invention;
FIG. 8 is a chromatogram obtained in example 9 of the present invention;
FIG. 9 is a chromatogram obtained in example 10 of the present invention;
FIG. 10 is a chromatogram obtained in example 11 of the present invention.
Detailed Description
The technical solution of the present invention will be described in detail by specific examples.
Example 1
The present example is a test for detecting the charge heterogeneity impurities of insulin glargine injection, and the experimental process is as follows:
(1) treatment of the test article: taking 400 mu l of insulin glargine injection, adding 600 mu l of 0.01M HCl, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate (pH 5.6);
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 50% (volume percent) ethanol;
flow rate: 0.5 ml/min;
sample introduction amount: 5 mu l of the solution;
column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
Gradient elution was performed with mobile phase a and mobile phase B, and the elution procedure was as in table 1.
(3) Repeatability measurements and results: the repeatability solution was measured 3 times, the peak area percentage mean of the impurity peak was 3.831%, the relative standard deviation was 2.5%, specifically shown in the chromatogram of fig. 1, and the analysis results are specifically shown in table 3.
TABLE 3 repeatability test results
| Repetitive solution | Main Peak (%) | Impurity peak (%) | Degree of separation of main peak from |
| 1 | 96.201 | 3.799 | 1.061 |
| 2 | 96.246 | 3.754 | 1.021 |
| 3 | 96.061 | 3.939 | 1.053 |
| Mean value | 96.169 | 3.831 | N/A |
| RSD(％) | 0.1 | 2.5 | N/A |
Example 2
The present example is a test for detecting charge heterogeneity impurities in degummed insulin injection, and the test process is as follows:
(1) treatment of the test article: taking 300 mu l of degummed insulin injection, adding 400 mu l of ultrapure water, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate (pH 5.0);
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 50% (volume percent) ethanol;
flow rate: 0.5 ml/min;
sample introduction amount: 10 mu l of the mixture;
column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
Gradient elution was performed with mobile phase a and mobile phase B, and the elution procedure was as in table 2.
(3) Repeatability measurements and results: the repeated solution was measured 3 times, and the peak area percentage mean of the impurity peak was 9.809%, and the relative standard deviation was 1.1%.
Example 3
In this embodiment, the influence of no organic solvent added in the mobile phase B on the detection effect is examined, and the specific experimental process is as follows:
(1) system applicability solution: weighing 15.2mg of insulin glargine in a 10ml volumetric flask, adding 2ml of 1M HCl, reacting for 4 hours at room temperature, adding 2ml of 1M NaOH for neutralization, diluting with 0.01M HCl to a constant volume, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate (pH 5.6);
mobile phase B: 10mM sodium acetate, 0.5M NaCl;
flow rate: 0.5 ml/min;
sample introduction amount: 5 mu l of the solution;
column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
Gradient elution was performed with mobile phase a and mobile phase B, and the elution procedure was as in table 1.
(3) And (3) measuring results: the mobile phase B contained 0% ethanol (i.e. no organic solvent ethanol was added, and a simple salt gradient was used for elution), and the protein was not eluted during this elution time, and the chromatogram obtained is shown in fig. 2.
Example 4
In this example, the influence of changing the organic solvent in the mobile phase B from ethanol to acetonitrile on the detection effect is examined, and the specific experimental process is as follows:
(1) system applicability solution: weighing 15.2mg of insulin glargine in a 10ml volumetric flask, adding 2ml of 1M HCl, reacting for 4 hours at room temperature, adding 2ml of 1M NaOH for neutralization, diluting with 0.01M HCl to a constant volume, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate (pH 5.6);
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 50% (volume percent) acetonitrile;
flow rate: 0.5 ml/min;
sample introduction amount: 5 mu l of the solution;
column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
Gradient elution was performed with mobile phase a and mobile phase B, and the elution procedure was as in table 1.
(3) And (3) measuring results: when mobile phase B contains 50% acetonitrile, the protein can be eluted, but impurity peaks are not separated, and the obtained chromatogram is shown in FIG. 3.
Example 5
In this example, the influence of changing the organic solvent in the mobile phase B from ethanol to methanol on the detection effect is examined, and the specific experimental process is as follows:
(1) system applicability solution: weighing 15.2mg of insulin glargine in a 10ml volumetric flask, adding 2ml of 1M HCl, reacting for 4 hours at room temperature, adding 2ml of 1M NaOH for neutralization, diluting with 0.01M HCl to a constant volume, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate (pH 5.6);
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 50% (volume percent) methanol;
flow rate: 0.5 ml/min;
sample introduction amount: 5 mu l of the solution;
column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
Gradient elution was performed with mobile phase a and mobile phase B, and the elution procedure was as in table 1.
(3) And (3) measuring results: when the mobile phase B contains 50% of methanol, the protein can be eluted, the separation degree of the main peak and the impurity peak 1 is 1.478, but the total impurity is less than that of the mobile phase containing 50% of ethanol, and the obtained chromatogram is shown in figure 4.
Example 6
In this embodiment, the influence of increasing the volume percentage concentration of ethanol in the mobile phase B to 60% on the detection effect is examined, and the specific experimental process is as follows:
(1) system applicability solution: weighing 15.2mg of insulin glargine in a 10ml volumetric flask, adding 2ml of 1M HCl, reacting for 4 hours at room temperature, adding 2ml of 1M NaOH for neutralization, diluting with 0.01M HCl to a constant volume, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate (pH 5.6);
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 60% (volume percent) ethanol;
flow rate: 0.5 ml/min;
sample introduction amount: 5 mu l of the solution;
column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
Gradient elution was performed with mobile phase a and mobile phase B, and the elution procedure was as in table 1.
(3) And (3) measuring results: when the mobile phase B contains 60% ethanol, the separation degree of the main peak and the impurity peak 1 is 1.373, but when the ethanol concentration reaches 60%, the blank is interfered at the impurity, and the obtained chromatogram is shown in FIG. 5.
Example 7
In this embodiment, the influence of increasing the volume percentage concentration of ethanol in the mobile phase B to 55% on the detection effect is examined, and the specific experimental process is as follows:
(1) system applicability solution: weighing 15.2mg of insulin glargine in a 10ml volumetric flask, adding 2ml of 1M HCl, reacting for 4 hours at room temperature, adding 2ml of 1M NaOH for neutralization, diluting with 0.01M HCl to a constant volume, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate (pH 5.6);
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 55% (volume percent) ethanol;
flow rate: 0.5 ml/min;
sample introduction amount: 5 mu l of the solution;
column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
Gradient elution was performed with mobile phase a and mobile phase B, and the elution procedure was as in table 1.
(3) And (3) measuring results: when the mobile phase B contains 55% of ethanol, the separation degree of the main peak and the impurity peak 1 is 1.380, but when the ethanol concentration reaches 55%, the blank is interfered at the impurity position before the main peak, and the obtained chromatogram is shown in FIG. 6.
Example 8
In this embodiment, the effect of reducing the volume percentage concentration of ethanol in the mobile phase B to 40% on the detection effect is examined, and the specific experimental process is as follows:
(1) system applicability solution: weighing 15.2mg of insulin glargine in a 10ml volumetric flask, adding 2ml of 1M HCl, reacting for 4 hours at room temperature, adding 2ml of 1M NaOH for neutralization, diluting with 0.01M HCl to a constant volume, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate (pH 5.6);
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 40% (volume percent) ethanol;
flow rate: 0.5 ml/min;
sample introduction amount: 5 mu l of the solution;
column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
Gradient elution was performed with mobile phase a and mobile phase B, and the elution procedure was as in table 1.
(3) And (3) measuring results: when the mobile phase B contains 40% ethanol, the separation degree of the main peak from the impurity peak 1 is 1.043, but when the ethanol concentration is 40%, the blank interferes after the main peak but has no influence on the impurity detection, and the obtained chromatogram is shown in fig. 7.
Example 9
In this embodiment, the influence of reducing the volume percentage concentration of ethanol in the mobile phase B to 45% on the detection effect is examined, and the specific experimental process is as follows:
(1) system applicability solution: weighing 15.2mg of insulin glargine in a 10ml volumetric flask, adding 2ml of 1M HCl, reacting for 4 hours at room temperature, adding 2ml of 1M NaOH for neutralization, diluting with 0.01M HCl to a constant volume, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate (pH 5.6);
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 45% (volume percent) ethanol;
flow rate: 0.5 ml/min;
sample introduction amount: 5 mu l of the solution;
column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
Gradient elution was performed with mobile phase a and mobile phase B, and the elution procedure was as in table 1.
(3) And (3) measuring results: when the mobile phase B contains 45% ethanol, the separation degree of the main peak from the impurity peak 1 is 1.299, but when the ethanol concentration is 45%, the blank has interference after the main peak but has no influence on the impurity detection, and the obtained chromatogram is shown in fig. 8.
Example 10
In this embodiment, the influence of gradient elution time and mobile phase flow rate on the detection effect is examined, and the specific experimental process is as follows:
(1) system applicability solution: weighing 15.2mg of insulin glargine in a 10ml volumetric flask, adding 2ml of 1M HCl, reacting for 4 hours at room temperature, adding 2ml of 1M NaOH for neutralization, diluting with 0.01M HCl to a constant volume, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate (pH 5.6);
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 50% (volume percent) ethanol;
sample introduction amount: 5 mu l of the solution;
column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
The flow rates were set to 0.5ml/min and 0.8ml/min, respectively.
Elution gradient: the elution gradient at a flow rate of 0.5ml/min is as in Table 1; the elution gradient at a flow rate of 0.8ml/min is given in Table 4 below.
TABLE 4
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 8 | 0 | 100 |
| 16 | 0 | 100 |
(3) And (3) measuring results: the degrees of separation of the total impurity and main peaks from impurity peak 1 at 0.5ml/min were: 7.473% and 1.325; the degrees of separation of the total impurity and main peaks from impurity peak 1 at 0.8ml/min were: 6.208% and 1.770, the resulting chromatograms are shown in FIG. 9.
Example 11
The influence of the column temperature on the detection effect is investigated in the embodiment, and the specific experimental process is as follows:
(1) system applicability solution: weighing 15.2mg of insulin glargine in a 10ml volumetric flask, adding 2ml of 1M HCl, reacting for 4 hours at room temperature, adding 2ml of 1M NaOH for neutralization, diluting with 0.01M HCl to a constant volume, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate (pH 5.6);
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 50% (volume percent) ethanol;
flow rate: 0.5 ml/min;
sample introduction amount: 5 mu l of the solution;
setting the column temperature: 30 ℃, 35 ℃ and 40 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
The elution gradient is as in Table 1.
(3) And (3) measuring results: the column temperature of 30 ℃, 35 ℃ and 40 ℃ has no obvious influence on elution and separation of main peaks and impurities, and the obtained chromatogram is shown in figure 10.
Example 12
In this embodiment, the influence of the pH value of the mobile phase a on the detection effect is examined, and the specific experimental process is as follows:
(1) the test solution is insulin degu injection; taking 300 mu l of degummed insulin injection, adding 400 mu l of ultrapure water, and uniformly mixing to obtain a sample to be detected;
(2) determining chromatographic conditions:
the instrument comprises the following steps: the Agilent 1260 liquid chromatograph is provided with a diode array detector;
a chromatographic column: ProPac WCX-10, BioLC, 4X 250mm (Thermo);
mobile phase A: 20mM sodium acetate, pH5.0, 5.1, 5.2, 5.3, 5.4, 5.6;
mobile phase B: 10mM sodium acetate, 0.5M NaCl, 50% (volume percent) ethanol;
flow rate: 0.5 ml/min;
sample introduction amount: 5 mu l of the solution;
setting the column temperature: 30 ℃;
detection wavelength: 214 nm;
and (3) post-operation: and (5) 12 min.
The elution gradient is as in Table 1.
(3) And (3) measuring results: the pH of the mobile phase is from 5.0 to 5.6, and the protein can be well eluted; however, when the pH of the mobile phase is 5.2-5.6, the sample needs to be subjected to simple acidification pretreatment (depolymerizing the degummed insulin double hexamer into monomers), i.e. 1ml of degummed insulin injection is added with 3 μ l of 6M HCl and mixed uniformly. Therefore, the pH range of the mobile phase A can be selected from 5.0-5.6.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A high performance liquid chromatography detection method for charge heterogeneity impurities of recombinant protein drugs is characterized in that the detection conditions comprise: adopting weak cation column, and performing gradient elution with mobile phase A and mobile phase B; the mobile phase A is a salt solution of any one or more of acetate, citrate and phosphate, the mobile phase B is a salt solution of any one or more of acetate, citrate, phosphate, sodium salt and potassium salt, and an organic solvent is further added into the mobile phase B.
2. The detection method according to claim 1, wherein the mobile phase A is a salt solution of any one or more of sodium acetate, potassium acetate, sodium citrate, disodium hydrogen phosphate and sodium dihydrogen phosphate, preferably a sodium acetate solution; the mobile phase B is a salt solution of any one or more of sodium acetate, potassium acetate, sodium citrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium sulfate, potassium chloride and potassium nitrate; the organic solvent is any one of ethanol, methanol and acetonitrile.
3. The detection method according to claim 1 or 2, wherein the pH value of the mobile phase A salt solution is 5.0 to 6.0, preferably 5.0 to 5.6.
4. The detection method according to any one of claims 1 to 3, wherein the volume percentage concentration of the organic solvent in the mobile phase B is 40 to 60%, preferably 40 to 55%.
5. The detection method according to claim 4, wherein the mobile phase B is a mixed solution of sodium acetate, sodium chloride and ethanol, or a mixed solution of sodium acetate, potassium chloride and ethanol, or a mixed solution of sodium acetate, sodium chloride and methanol, or a mixed solution of sodium acetate, sodium chloride and acetonitrile, or a mixed solution of sodium citrate, sodium chloride and ethanol, or a mixed solution of potassium acetate, potassium chloride and acetonitrile; preferably, the mixed solution of sodium acetate, sodium chloride and ethanol, more preferably, the concentration of the sodium acetate in the mixed solution is 5-20 mM, the concentration of the sodium chloride is 0.4-0.6M, and the volume percentage concentration of the ethanol is 40-60%.
6. The detection method according to claim 5, wherein the gradient elution is in particular: eluting with a mobile phase A, and then eluting with a mobile phase B; preferably, the gradient elution conditions are: the flow rate of the mobile phase is 0.5-1.0 ml/min, the 100% mobile phase A is adopted for elution for 4-10 minutes, and then the 100% mobile phase B is adopted for elution until the total elution time is 15-20 minutes.
7. The detection method according to claim 6, wherein the mobile phase B consists of: 10mM sodium acetate, 0.5M NaCl, 50% by volume ethanol; and/or the mobile phase A is 20mM sodium acetate solution.
8. The detection method according to claim 7, wherein the weak cation column is any one of a ProPac WCX-10 analytical column, a Bio WCX analytical column, and a Proteomix WCX analytical column.
9. The detection method according to claim 8, wherein the detection condition further comprises: a diode array detector or an ultraviolet detector is adopted;
and/or the temperature of the chromatographic column is 25-45 ℃ during detection;
and/or the detection wavelength is 210-220 nm;
and/or the sample introduction amount is 5-10 mu l.
10. The use of the high performance liquid chromatography detection method of any one of claims 1-9 for detecting charge-heterogeneity impurities in recombinant protein drugs;
preferably, the recombinant protein drug charge-heterogeneity impurity is a charge-heterogeneity impurity of an insulin-based recombinant protein drug.
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