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Method of purifying and combining human interleukins 12

A technology for interleukin and cell culture, applied in the field of purification of recombinant human interleukin 12, can solve the problems of difficult industrial application, complicated process, large pore size, etc., achieve high recovery rate and purity, low cost of separation and purification, and easy operation Effect

Active Publication Date: 2007-09-12
广州市茵良强生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, U.S. Patent No. 5,853,714 uses four column chromatography and one ultrafiltration process for purification, but the medium S-200 Sephacryl High Resolution used in molecular sieve chromatography has too large a pore size, and the separation effect is not ideal
Another example is that the purification method mentioned in U.S. Patent US6830751 includes three column chromatography, which is further improved on the basis of patent US5853714; Chinese patent CN200410080518.7 includes three column chromatography and an ultrafiltration process, the patent It can be seen from the ion exchange chromatogram that the peak of the impurity protein is relatively high, which affects the service life of the ion exchanger, and there are many kinds of eluents and equilibrium liquids used, the whole process is very cumbersome and difficult for industrial application

Method used

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  • Method of purifying and combining human interleukins 12
  • Method of purifying and combining human interleukins 12
  • Method of purifying and combining human interleukins 12

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Purification of embodiment 1 rhIL-12

[0026] 1. Sample filtration

[0027] The cell culture supernatant was filtered with a 0.45 μm microporous membrane to remove impurities, and the filtrate was diluted 4-fold with 10 mM PB, pH=6.0 for later use.

[0028] 2. Purification of rhIL-12 by cationic column chromatography

[0029] Select SP Sepharose High Performance cation exchange column, and equilibrate with 2 to 3 column volumes of equilibration buffer (20mM PB, pH6.0). After the column is fully equilibrated, load the dilution obtained in step 1. The equilibration buffer washes the column until A 280 Reach the baseline or stabilize to the vicinity of the baseline; wash the chromatographic column with eluent I (20mM PB, 0.30M NaCl, pH6.0), collect the elution peak I, and fully wash the chromatographic column until the baseline is stable; continue to use The eluent II (20mM PB, 0.60M NaCl, pH6.0) was used to wash the column, and the elution peak II was collected, and th...

Embodiment 2

[0038] Example 2 Purification of rhIL-12

[0039] 1. Sample filtration

[0040] The cell culture supernatant was filtered with a 0.45 μm microporous membrane to remove impurities, and the filtrate was diluted 4 times with 10 mM Tris-Cl, pH 8.0 for use.

[0041] 2. Purification of rhIL-12 by anion column chromatography

[0042] Choose DEAE Sepharose Fast Flow anion exchange column, equilibrate 2 to 3 column volumes with equilibration buffer (20mMTris-Cl, pH8.0), and load the rhIL-12-containing dilution obtained in step 1 after the chromatographic column is fully equilibrated. After loading the sample, wash the column with equilibration buffer until A 280 Reach the baseline or stabilize to the vicinity of the baseline; wash the chromatographic column with eluent I (20mM Tris-Cl, 0.15M NaCl, pH8.0), collect the elution peak I, and fully wash the chromatographic column until the baseline is stable; Continue to wash the chromatographic column with eluent II (20mM Tris-Cl, 0.28M ...

Embodiment 3

[0051] Example 3 Purification of rhIL-12

[0052] 1. Sample filtration

[0053] The cell culture supernatant was filtered with a 0.45 μm microporous membrane to remove impurities, and the filtrate was diluted 4 times with 10 mM PB, pH=6.5 for use.

[0054] 2. Purification of rhIL-12 by cationic column chromatography

[0055] Select Capto S cation exchange column, and equilibrate 2 to 3 column volumes with equilibration buffer (20mM PB, pH6.5). The liquid washes the column until A 280 Reach the baseline or stabilize to the vicinity of the baseline; wash the chromatographic column with eluent I (20mM PB, 0.40M NaCl, pH6.5), collect the elution peak I, and fully wash the chromatographic column until the baseline is stable; continue to use Eluent II (20mM PB, 0.70MNaCl, pH6.5) washes the chromatographic column, collects the elution peak II, and fully washes the chromatographic column until the baseline is stable; , pH6.5) to wash the chromatographic column, collect the elution...

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Abstract

This invention discloses a method to purify rhIL-12, belonging to the protein purification technology, which technical points are that the cell culture supernatant of rhIL-12 is conducted filtering, cation or anion exchange chromatography, precipitation of ammonium sulfate, anion or cation exchange chromatography and molecular sieve chromatography, and during the precipitation of ammonium sulfate, pH is far away from the isoelectric point of rhIL-12. This method uses ammonium sulfate precipitation to remove most of hybridproteins, making the future separation simple and effective.

Description

technical field [0001] The invention relates to a protein purification method, in particular to a method for purifying recombinant human interleukin-12. Background technique [0002] Interleukin-12 (IL-12), also known as cytotoxic lymphoid maturation factor (cytotoxic lymphocyte maturation factor, CLMF) or natural killer cell stimulation factor (natural killer cell stimulation factor, NKSF), is a human important cytokines. It has a wide range of immune effects; such as promoting the proliferation and killing of natural killer cells (naturekiller, NK) and T cells, promoting the secretion of cytokines, and inducing the differentiation of antigen-specific helper T cells (helper Tcell TH) into Th1. IL-12 is a cytokine with various immunomodulatory functions. As a functional bridge, it connects the early non-specific innate immunity and the later antigen-specific adaptive immunity, and plays an important role in infection, tumor and autoimmune diseases. At present, it is widel...

Claims

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Application Information

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IPC IPC(8): C07K14/54
Inventor 蒲勤李毅赵峰杨愈丰吴思纹叶倩君夏书奇曾振飞王翠玲郭凯旋粟宽源曾水娣于源邱向南张宜俊
Owner 广州市茵良强生物科技有限公司
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