Continuous pegylation reaction method for recombinant human erythropoietin (EPO)

Abstract

The invention relates to chemical modification of proteins, and discloses a continuous pegylation reaction method for recombinant human erythropoietin (EPO). The method comprises the following steps of: performing a primary pegylation reaction on EPO molecules in a first buffer solution; and further adding PEG into a reaction liquid obtained in the primary pegylation reaction in a second buffer solution which is different from the first buffer solution for performing a secondary pegylation reaction to obtain a final reaction liquid. A reaction method of multiple buffering systems is applied, so that continuous pegylation reaction of EPO is realized, and a pegylation reaction method with high mono-substitution rate and low multi-substitution rate is established.

Description

technical field [0001] The present invention relates to protein chemical modification, in particular to a continuous pegylation reaction method of recombinant human erythropoietin. Background technique [0002] Recombinant human erythropoietin (EPO for short), is a specific hematopoietic factor that protects and promotes the differentiation, proliferation and maturation of hematopoietic stem cells in the bone marrow into red blood cells. Renal anemia and non-renal anemia are known as genetic engineering drugs with the most mature technology, the most definite curative effect and the highest global sales. However, the drug has major problems such as frequent medication (one or three injections per week) and low biological activity, which brings great treatment pain and inconvenience to patients. Therefore, there is a need for further modification and improvement, that is, the development of long-acting EPO drugs. At present, one of the more mature long-acting EPO drugs in t...

AI Technical Summary

Problems solved by technology

At present, the relatively successful PEG-EPO drug in the world is "Mircera" listed by Roche. The single substitution reaction rate of its synthesis process is about 40%, and the content of multi-substituted products reaches 38%. The single-substituted product is PEG. -The target product of the EPO synthesis reaction has a relatively definite molecular structure and high drug activity, while the multi-substituted product is a by-product of the reaction, which will increase the difficulty of the subsequent purification process

In addition, compared with other existing PEGylated protein drugs, EPO has 9 reaction sites (such as figure 1 shown), including 8 lysine residues and an N-terminal amino group, it is easy to form multiple substitution products in the reaction, and it is more difficult to achieve specific site-directed PEGylation

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