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A preparation process for increasing the natural conformation content of recombinant human interferon α2b

A recombinant human interferon, a natural technology, applied in the preparation method of interferon, peptide, cytokine/lymphokine/interferon, etc., can solve the problems of increased content of miscellaneous peaks and small retention time

Active Publication Date: 2017-05-17
ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high oxidative environment and alkaline conditions, the thioether group of methionine may be oxidized to sulfoxide or sulfone, as shown in the literature (Liu Yundong, "Refolding, separation, purification and stabilization of complex interferon inclusion bodies Sexual research") thinks that the oxidation of methionine will cause "impurity peaks with shorter retention times than the natural structure on the reversed phase", such as Figure 4 As shown, in the example of the refolding solution containing GSH / GSSG, the content of the miscellaneous peak increased significantly

Method used

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  • A preparation process for increasing the natural conformation content of recombinant human interferon α2b
  • A preparation process for increasing the natural conformation content of recombinant human interferon α2b
  • A preparation process for increasing the natural conformation content of recombinant human interferon α2b

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Embodiment 1

[0035]Add 6M guanidine hydrochloride / 0.1M boric acid to recombinant human interferon α2b cells at a ratio of 1:3 (weight / volume), stir and lyse at 4°C for 4 hours;

[0036] The above lysate was dialyzed with 50 mM Tris-hydrochloric acid, pH 8.3, and 1 mM EDTA. The ratio of lysate to dialysate was 1:40, during which the dialysate was changed 4 times, the dialysis time was 48 hours, and the temperature was 2-8°C. The obtained protein solution was centrifuged at 8000rpm / 15min to obtain the supernatant.

[0037] Affinity chromatography adsorption and elution: adjust the pH of the above supernatant to 7.4, centrifuge and apply to a monoclonal antibody affinity chromatography column, wash with PBS, elute with 1% acetic acid, and collect the eluate. Calculate the protein amount, and use RP-HPLC, SDS-PAGE to detect the N configuration content, the results are as follows figure 2 And shown in Table 1.

Embodiment 2

[0039] Add 6M guanidine hydrochloride / 0.1M boric acid to recombinant human interferon α2b cells at a ratio of 1:3 (weight / volume), stir and lyse at 4°C for 4 hours.

[0040] The above lysate is dialyzed sequentially with the first and second dialysates at a temperature of 2-8°C. The ratio of lysate to dialysate was 1:40, during which the first and second dialysates were replaced twice each, and the total time of dialysis was 48 hours. Wherein, the first dialysate is 50mM Tris-hydrochloric acid, pH8.3, 1mM EDTA, 3mMGSSG / 1mMGSH, and the second dialyzate is 20mM Tris-hydrochloric acid, pH8.3, 1mM EDTA. The obtained protein solution was centrifuged at 8000rpm / 15min to obtain the supernatant.

[0041] Affinity chromatography adsorption and elution: adjust the pH of the above supernatant to 7.4, centrifuge and apply to a monoclonal antibody affinity chromatography column, wash with PBS, elute with 1% acetic acid, and collect the eluate. Calculate the amount of protein, and use RP-...

Embodiment 3

[0043] Add 6M guanidine hydrochloride / 0.1M boric acid to recombinant human interferon α2b cells at a ratio of 1:3 (weight / volume), stir and lyse at 4°C for 4 hours. The above-mentioned lysate is dialyzed with the first and second dialysates in sequence. The temperature during dialysis with the first dialysate was 4° C., and the temperature during dialysis with the second dialysate was 28° C. The ratio of lysate to dialysate was 1:40, and the first and second dialysates were replaced twice during the period. The dialysis time for the first dialysate was 24 hours, and the time for the second dialysate was 24 hours. Wherein the first dialysate is 0.1MPBS, pH7.4, and the second dialysate is 40mM sodium acetate / acetic acid, pH3.6. The obtained protein solution was centrifuged at 8000rpm / 15min to obtain the supernatant.

[0044] Affinity chromatography adsorption and elution: adjust the pH of the above supernatant to 7.4, centrifuge and apply to a monoclonal antibody affinity chro...

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Abstract

The invention discloses a method for improving native conformation content of recombinant human interferon alpha2b. The method comprises the following operations: dialyzing bacterium lysate of split recombinant human interferon alpha2b expression bacteria by using first dialysate of which the pH is not lower than 7.0 and second dialysate of which the pH is lower than 4 sequentially; then separating and purifying so as to obtain the recombinant human interferon alpha2b with high native conformation content; carrying out renaturation on the interferon alpha2b by adopting a two-step dialysis method, wherein a low-pH dialysis scheme is adopted in the second step with an initial target of further reducing the guanidine hydrochloride concentration, replacing a buffer system and removing impure protein with an unstable acid; but by an accident, an experiment finds that the yield of native conformation interferon can be greatly improved by the two-step renaturation method when the renaturation rate is not reduced. Transformation of an SMM and an aggregate or an intermediate state into the native conformation is promoted, and the SMM and aggregate content is reduced.

Description

technical field [0001] The invention relates to the field of protein drug production, in particular to a method for increasing the natural conformation content of recombinant human interferon α2b. Background technique [0002] Interferon (Interferon, IFN), discovered by British scientists Isaacs and LinderMann in 1957, is a class of proteins with broad-spectrum antiviral activity. According to their amino acid structure, antigenicity and cell origin, they can be divided into three categories: alpha interferon, beta interferon and gamma interferon. Among them, alpha interferon is a type of interferon that is widely used. It is mainly used clinically to treat viral diseases such as hepatitis B and hepatitis C; it is also used in combination with some tumor chemotherapy drugs. [0003] There are at least 20 different subtypes of interferon-α, and its family members have similar structural characteristics: the number of amino acids is 165 or 166, the amino acid composition is s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/56C07K1/22C07K1/14
CPCC07K14/56
Inventor 宋礼华王荣海许培董世建周乐春李增礼沈毅
Owner ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD