Spectral detection method and application of recombinant human erythropoietin PEGylation reaction solution

A PEGylation and spectral detection technology, which is applied in the measurement of color/spectral properties, etc., can solve the problems of increasing the risk of changing the quality of the PEG-EPO reaction solution, and taking a long time for detection and analysis, so as to improve the UV absorbance and save labor costs. and time cost, the effect of improving accuracy

Active Publication Date: 2016-06-29
SHENZHEN SCIPROGEN BIO PHARMA
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Problems solved by technology

Usually, the HPLC method is used to detect the PEGylation reaction rate of each EPO PEGylation reaction solution, and each detection time is close to 1h. In view of the large number of EPO PEGylation reaction solutions that need to be detected, a detection and analysis time-consuming Longer problem and increased risk of quality change of PEG-EPO reaction solution during storage

Method used

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  • Spectral detection method and application of recombinant human erythropoietin PEGylation reaction solution
  • Spectral detection method and application of recombinant human erythropoietin PEGylation reaction solution
  • Spectral detection method and application of recombinant human erythropoietin PEGylation reaction solution

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preparation example Construction

[0118] The preparation method of the PEGylation reaction solution of EPO is basically the same as that of Experiment 1, except that 0.00, 0.05, 0.10, 0.15 and 0.20 mL of 40 mM ammonium sulfate solution were added to the reaction 1 mg / mL EPO solution, and mixed. After the reaction was completed, samples were taken for HPLC testing (by the method of Experiment 2), and the results are listed in Table 8. From the results in Table 8, it can be found that with the increase of the amount of ammonium sulfate solution added, the PEGylation reaction rate of EPO decreased significantly, and the free EPO content in the resulting EPO PEGylation reaction solution finally increased by more than two times. At the same time, the ratio of the single-substitution ratio to the multi-substitution ratio of the reaction also increased exponentially, and finally increased to 7.3:1.0, and the multi-substitution ratio at this time was far below 10%. This fully proves that the result of the PEGylation r...

Embodiment

[0125] Apply the multi-stage dilution UV-Vis detection method in the above experiments 15-16 to the production process of PEG-EPO, and quickly identify each reaction solution before combining multiple reaction solutions.

[0126] According to the PEGylation synthesis method of EPO in Experiment 1, 6 PEGylation reactions of EPO were carried out continuously, and the reaction amounts of EPO were respectively: 5mg (1 reaction), 200mg (1 reaction) and 400mg (4 reactions), and the obtained The serial numbers of the PEGylation reaction solution of EPO are respectively set as: 15, 16, 17, 18, 19, 20. Except for the pre-reaction with a reaction volume of 5 mg, the other 5 EPO PEGylation reaction solutions were tested and passed the test and then combined to form a batch of 1800 mg EPO PEGylation reaction solutions. The detection method before the reaction solution is combined is as follows:

[0127] (1) Perform HPLC detection on the pre-reaction sample with a reaction volume of 5 mg ...

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Abstract

The invention relates to the production technical field of PEGylated recombinant human erythropoietin (PEG-EPO), in particular to a spectral detection method of PEG-EPO reaction solution and a rapid identification method before combining multiple reaction solutions. The detection system is phosphate buffer solution, nano-gold sol chromogen, and diluted PEG-EPO reaction solution; after measuring the absorbance at 260nm, perform linear fitting between the absorbance and the dilution factor and calculate the absorbance decrease coefficient (k 260 ). By comparing the k of each reaction solution 260 Value can quickly judge whether the PEGylation reaction rate of each reaction solution is the same or close. Combined with the detection results of high performance liquid chromatography, it can be quickly obtained 260 The PEGylation reaction rate of the reaction solution of the value. Therefore, the present invention greatly shortens the detection and analysis time before the PEG-EPO reaction solution is combined, improves the efficiency of the PEG-EPO drug production process, and saves labor costs and time costs. Moreover, the detection method of the present invention has good sensitivity, reliability and reproducibility, and can be applied in the actual production process of PEG-EPO medicine.

Description

technical field [0001] The invention relates to the technical field of production of PEGylated recombinant human erythropoietin, in particular to a spectral detection method of a recombinant human erythropoietin PEGylation reaction solution and its application. Background technique [0002] Erythropoiesis-stimulating Agents (ESAs) are very effective drugs for the treatment of renal anemia. ESAs can fundamentally correct the symptoms of anemia, significantly restore the patient's physical fitness, and improve the quality of life of the patient. In addition, eliminating the symptoms of anemia can significantly reduce morbidity and mortality from other related diseases. However, due to the relatively short in vivo half-life of ESAs, patients typically require one to three doses per week. In order to overcome this shortcoming, in recent years, researchers have carried out chemical modification studies on the protein structure of recombinant human erythropoietin (EPO) to improv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/33
Inventor 张涤平陈贤光张向荣吴园园王康黄俊龙黄健盛光阳
Owner SHENZHEN SCIPROGEN BIO PHARMA
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