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A protamine and process technology, applied in the field of protamine purification process, can solve the problem of low protamine purity, and achieve the effects of enhancing competitiveness, providing production efficiency and improving quality
Active Publication Date: 2014-01-15
XUZHOU WANBANG JINQIAO PHARMA +1
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[0008] The purpose of the present invention is to provide a protamine purification process suitable for large-scale production, mainly to solve the problem of low protamine purity in the prior art
Method used
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Embodiment 1
[0037] A kind of purification process of protamine comprises the following steps:
[0038] a. Dissolving protamine in a buffer solution with pH 4.0 to 5.0, filtering, and taking the clear liquid to obtain a protamine sample;
[0039] b. Select the reverse column;
[0040] c. Load the protamine and equilibrate the reverse phase chromatography with a buffer solution of pH 4.0~5.0;
[0041] d. Phase elution: phase A, buffer solution with pH 4.0~5.0,
[0042] B phase, the organic buffer solution of pH4.0~5.0, the organic solvent concentration in described B phase is 10~20% (volume percentage), and described organic solvent is selected from acetonitrile, methanol, ethanol,
[0043] In the first stage, the eluent B is 15%~20%, isocratic elution, and the eluent I is collected.
[0044] In the second stage, the eluent B is 25%~35%, isocratic elution, and the eluent II is collected.
[0045] In the third stage, the eluent B is 100%, isocratic elution, and the eluent III is coll...
Embodiment 2
[0049] On the basis of Example 1, the buffer solution system with a pH of 4.0-5.0 is an ammonium acetate-acetic acid buffer solution system.
Embodiment 3
[0051] On the basis of the above examples, in the step e, the amount of ethanol added is 2-5 times (volume ratio) of the eluent II.
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Abstract
The invention relates to the pharmaceutical field, particularly relates to the field of protein purification, more particularly relates to a purification technology of protamine, and aims to solve the problem of low purity of the protamine in the prior art. According to the purification technology of the protamine, by a reverse silica gel filler chromatography separation mode and ratio control of water phase and an organic phase in an eluant, non-functional impurity proteins in the protamine can be removed by one-step chromatography, the product quality is improved, reaches an international level, and meets relevant provisions of United States pharmacopeia (USP) and British pharmacopoeia (BP), and the product safety is improved.
Description
[0001] technical field [0002] The invention relates to the field of pharmacy, in particular to the field of protein purification, more specifically to the purification process of protamine. [0003] Background technique [0004] Protamine is a polycationic antibacterial short peptide found in the sperm nucleus of fish. It is a small and simple spherical basic protein with a small molecular weight, usually below 10,000, and generally consists of about 30 amino acids. Composition, the molecule is rich in proline and arginine, of which more than 2 / 3 is arginine, the isoelectric point is 10-12, soluble in water and dilute acid, and will not solidify when heated. According to its amino acid composition, these protamines are roughly divided into the following three categories: (1) Monoprotamine (Monoprotamine): the only basic amino acid is arginine. Protamine, protamine of rainbow trout, salmon protamine of salmon, etc.; (2) Diprotamine: a basic amino acid containing arginine,...
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