Process for purifying recombinant human Fc fusion pegylated interferon

A technology of interferon and process, applied in the field of purification process, can solve the problems of complex purification process, difficulty in amplification, complex purification process of fusion interferon, etc.

Inactive Publication Date: 2011-06-15
泰州新生源生物医药有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fusion interferon purification process in this patent is complex (purification steps include: centrifugation, cationic chromatography, hydrophobic chromatography, anionic chromatography, molecular sieve chromatography, etc.), and the recovery rate is low (33%)
Complicated purification process, low recovery rate, and difficult amplification are the main problems in the purification of fusion proteins

Method used

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  • Process for purifying recombinant human Fc fusion pegylated interferon
  • Process for purifying recombinant human Fc fusion pegylated interferon
  • Process for purifying recombinant human Fc fusion pegylated interferon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Pilot purification study of Fc-IFN (experimental batch number: 090712)

[0056] 1. Experimental purpose: Based on the conditions of the small test in the purification laboratory, the integration and optimization of the pilot test will be carried out.

[0057] 2. Chromatographic conditions: see Table 5.

[0058] 3. Experimental materials:

[0059] Protein A Sepharose FF (2.6cm×15cm);

[0060] Q Sepharose FF (2.6cm×10cm);

[0061] 10K ultrafiltration module;

[0062] Solution A: 20mM sodium dihydrogen phosphate / disodium hydrogen phosphate (pH7.5)+0.1M NaCl;

[0063] Solution B: 20mM citric acid-trisodium citrate (pH3.5);

[0064] Solution C: 20mM sodium dihydrogen phosphate / disodium hydrogen phosphate (pH7.5);

[0065] Solution D: 20mM sodium dihydrogen phosphate / disodium hydrogen phosphate (pH7.5)+1M NaCl;

[0066] Solution E: 10mM CH3COONH4+0.15M NaCl.

[0067] 4. Results and analysis:

[0068] Chromatography and electrophoresis results are shown in ...

Embodiment 2

[0073] Example 2. Fc-IFN pilot purification research (experimental batch number: 090728)

[0074] 1. Experimental purpose: Based on the conditions of the small test in the purification laboratory, the integration and optimization of the pilot test will be carried out.

[0075] 2. Chromatographic conditions: see Table 5.

[0076] 3. Experimental materials: see Example 1.

[0077] Chromatography and electrophoresis results are shown in Figure 7 , 8 , 9, 10 and Table 7.

[0078] Figure 10 .Purified sample electrophoresis pattern (experimental batch number: 090728)

[0079] 1. Marker, 2. Cell culture medium, 3. G-25 chromatographic elution

[0080] Table 7: Analysis of Fc-IFN pilot purification study results (experimental batch number: 090728)

[0081]

Embodiment 3

[0082] Example 3. Fc-IFN pilot purification research (experimental batch number: 090814)

[0083] 1. Experimental purpose: Based on the conditions of the small test in the purification laboratory, the integration and optimization of the pilot test will be carried out.

[0084] 2. Chromatographic conditions: see Table 5.

[0085] 3. Experimental materials: see Example 1.

[0086] Chromatography and electrophoresis results are shown in Figure 11 , 12 , 13, 14 and Table 8.

[0087] Figure 14 .030814 electrophoresis pattern (experimental batch number: 090814)

[0088] 1.Marker, 2.G-25 chromatographic elution

[0089] Table 8: Analysis of Fc-IFN pilot purification research results (experimental batch number: 090814)

[0090]

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Abstract

The invention relates to a process for purifying Fc fusion protein. The Fc fusion protein involved in the invention comprises but is not limited to recombinant human Fc fusion pegylated interferon (Fc-IFN). The process for purifying the Fc fusion protein combines the factors of the characteristics of Fc fusion protein, the requirements of purity and yield of purified samples, and the requirements of the amplification of the subsequent purification process and the preparation and formula and the like. A purification process which is suitable for industrial production is determined through laboratory research and scaled-up pilot test research. The process comprises the steps of centrifuging and pre-treatment, primary purification of ProteinA SepharoseFF chromatography and refined purification of Q-Sepharose FF and G25 Sephadex chromatography. The purity of the target protein obtained by purification reaches over 95 percent, the protein purification yield reaches over 40 percent, and the purity and yield of the target protein are improved. Research results prove that: the purification process is simple and convenient and suitable for industrial scale-up production, and the purity and the yield of the target protein are high.

Description

technical field [0001] The invention relates to the field of purification technology. Specifically, the present invention relates to a purification process suitable for recombinant human Fc fusion long-acting interferon (Fc-IFN), which optimizes the purification process according to the characteristics of the target fusion protein and improves the purity and yield of the target protein. Background technique [0002] Interferon (IFN) is a complex group of proteins naturally formed by eukaryotic cells in response to various stimuli. This protein has multiple biological activities, including anti-proliferative, immunomodulatory, antiviral and differentiation-inducing effects. [0003] The half-life (half-life, t 1 / 2 ) is short, only about 2h. In order to maintain its effective blood concentration and achieve the purpose of treatment, long-term multiple administrations are required. Therefore, the interferon treatment plan is generally a large dose and frequent injections. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N5/10C07K19/00C07K1/22C07K1/18C07K1/16
Inventor 任军黄阳滨邵珂朱学义李艳管章委杨静
Owner 泰州新生源生物医药有限公司
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