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High performance liquid chromatography detection method of recombinant protein drug charge heterogeneous impurities

A technology of high performance liquid chromatography and detection method, which is applied in the field of high performance liquid chromatography detection of impurities with heterogeneity in charge of recombinant protein drugs, can solve the problems of deformation, low resolution, complicated operation and the like

Active Publication Date: 2020-05-19
DONGGUAN HEC BIOPHARMACEUTICAL R&D CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Slab gel isoelectric focusing electrophoresis method can quickly and intuitively display the isoelectric point zone, and is widely used, but its operation is cumbersome, the resolution is low, and accurate quantification is difficult
Capillary isoelectric focusing electrophoresis has been improved in terms of resolution and accurate quantification. However, after the focusing process, the separated components need to be pushed to the detection window by mechanical or electric field force after the focusing process, which will lead to band broadening and out of shape
Ion-exchange chromatography can simultaneously separate charge isomers due to different isoelectric points of proteins and inconsistent space charge distribution. The effect of mutual separation is also uncertain

Method used

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  • High performance liquid chromatography detection method of recombinant protein drug charge heterogeneous impurities
  • High performance liquid chromatography detection method of recombinant protein drug charge heterogeneous impurities
  • High performance liquid chromatography detection method of recombinant protein drug charge heterogeneous impurities

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] This embodiment is a detection experiment of charge heterogeneity impurities in insulin glargine injection, and the experimental process is as follows:

[0051] (1) Treatment of the test product: Take 400 μl of insulin glargine injection, add it to 600 μl of 0.01M HCl, mix well, and obtain the sample to be tested;

[0052] (2) Determine the chromatographic conditions:

[0053]Instrument: Agilent 1260 liquid chromatograph equipped with diode array detector;

[0054] Chromatographic column: ProPac WCX-10, BioLC, 4×250mm (Thermo);

[0055] Mobile phase A: 20mM sodium acetate (pH5.6);

[0056] Mobile phase B: 10mM sodium acetate, 0.5M NaCl, 50% (volume percentage) ethanol;

[0057] Flow rate: 0.5ml / min;

[0058] Injection volume: 5μl;

[0059] Column temperature: 30°C;

[0060] Detection wavelength: 214nm;

[0061] Last run: 12min.

[0062] Gradient elution was carried out with mobile phase A and mobile phase B, and the elution procedure was the same as in Table 1. ...

Embodiment 2

[0067] This example is an experiment for detecting charge heterogeneity impurities in insulin degludec injection, and the experiment process is as follows:

[0068] (1) Treatment of the test product: Take 300 μl of insulin degludec injection, add 400 μl of ultrapure water, mix well, and obtain the sample to be tested;

[0069] (2) Determine the chromatographic conditions:

[0070] Instrument: Agilent 1260 liquid chromatograph equipped with diode array detector;

[0071] Chromatographic column: ProPac WCX-10, BioLC, 4×250mm (Thermo);

[0072] Mobile phase A: 20mM sodium acetate (pH5.0);

[0073] Mobile phase B: 10mM sodium acetate, 0.5M NaCl, 50% (volume percentage) ethanol;

[0074] Flow rate: 0.5ml / min;

[0075] Injection volume: 10μl;

[0076] Column temperature: 30°C;

[0077] Detection wavelength: 214nm;

[0078] Last run: 12min.

[0079] Gradient elution was carried out with mobile phase A and mobile phase B, and the elution procedure was the same as in Table 2. ...

Embodiment 3

[0082] In this embodiment, the influence of not adding an organic solvent on the detection effect in the mobile phase B is investigated. The specific experimental process is as follows:

[0083] (1) System suitability solution: Weigh 15.2mg of insulin glargine into a 10ml volumetric flask, add 2ml of 1M HCl, react at room temperature for 4h, add 2ml of 1M NaOH to neutralize, dilute with 0.01M HCl to volume, and mix well to obtain Sample to be tested;

[0084] (2) Determine the chromatographic conditions:

[0085] Instrument: Agilent 1260 liquid chromatograph equipped with diode array detector;

[0086] Chromatographic column: ProPac WCX-10, BioLC, 4×250mm (Thermo);

[0087] Mobile phase A: 20mM sodium acetate (pH5.6);

[0088] Mobile phase B: 10mM sodium acetate, 0.5M NaCl;

[0089] Flow rate: 0.5ml / min;

[0090] Injection volume: 5μl;

[0091] Column temperature: 30°C;

[0092] Detection wavelength: 214nm;

[0093] Last run: 12min.

[0094] Gradient elution was carri...

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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 of recombinant protein drug charge heterogeneous impurities. The method comprises steps of adding a certain proportion of organic solvent into a mobile phase, adding organic phase gradient elution on the basis of salt gradient elution, and selecting chromatographic conditions, such as chromatographic column, mobile phase A and mobile phase B components, column temperature, detection wavelength and gradient elution conditions, thereby obtaining thehigh performance liquid chromatography detection method. Tests prove that the method can effectively separate the target protein from the charge heterogeneous impurities, and the method has advantagesof good repeatability, simplicity, rapidness, high detection sensitivity, low cost and the like, in addition, the detection method is high in universality, and charge heterogeneity impurities of insulin recombinant protein drug samples can be effectively separated and detected.

Description

technical field [0001] The invention relates to the technical field of drug detection, in particular to a high-performance liquid chromatography detection method for recombinant protein drug charge heterogeneity impurities. Background technique [0002] Recombinant protein drugs expressed by eukaryotic systems all have a certain degree of post-translational modification, and these modified impurities have different isoelectric points (Isoelectric point, pI), showing different degrees of charge heterogeneity. Charge heterogeneity is a key quality attribute of recombinant protein drugs, which has an important impact on the stability, solubility, immunogenicity, biological activity in vivo and in vitro, and pharmacokinetic function of recombinant protein drugs. Analyzing the charge heterogeneity of recombinant protein drugs can understand the situation of post-translational modification impurities in drugs, which is one of the essential items for the quality control of recombin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/34
CPCG01N30/02G01N30/34
Inventor 詹丽婷郭林峰李晓平封启媛冯艳陈俊英徐军陈小锋李文佳
Owner DONGGUAN HEC BIOPHARMACEUTICAL R&D CO LTD
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