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Method for isolating recombinant human nerve growth factor from mammalian cell culture

A cell culture and mammalian technology, applied in the field of separation of recombinant human nerve growth factor, to achieve the effect of improving recovery rate, shortening production cycle, and less operation steps

Inactive Publication Date: 2017-03-08
SINOBIOWAY BIOMEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Although murine nerve growth factor (mNGF) is highly homologous to human nerve growth factor (hNGF), the animal-derived protein is potentially immunogenic and production requires large numbers of qualified male mouse submandibular glands

Method used

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  • Method for isolating recombinant human nerve growth factor from mammalian cell culture
  • Method for isolating recombinant human nerve growth factor from mammalian cell culture
  • Method for isolating recombinant human nerve growth factor from mammalian cell culture

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Embodiment 1

[0050] 1. Cation Exchange Chromatography

[0051] ÄKTA Purifer100 chromatography system was used for affinity chromatography, and the chromatographic column was HiTrap SP Sepharos FastFlow (SP FF) 1 mL. The instrument is operated according to the operation guide, the A1 pump is Buffer A (50 mmol / L phosphate buffer, pH6.0), the B1 pump is Buffer B (50 mmol / L phosphate buffer, 0.8mol / L NaCl, pH6.0 ).

[0052] Cleaning: Rinse the SP FF resin column for 5 column volumes (CV) with 0.1mol / L NaOH and a flow rate of 1 ml / min, and then wash 5 cv with ultrapure water at a flow rate of 1 ml / min to remove impurities on the SP FF resin .

[0053] Equilibration: Flush Buffer A for 10CV at a flow rate of 1 ml / min to maintain the SP FF resin in an environment suitable for hNGF binding.

[0054] Sample loading: Clarify the filtered fermentation broth, dilute the fermentation broth with deionized water until the conductivity is lower than 10 mS / cm, and adjust the pH to 6.0. The hNG was boun...

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Abstract

The invention discloses a method for isolating recombinant human nerve growth factor from mammalian cell culture, comprising the steps of carrying out strong cation exchange chromatography, to be specific, sampling supernate of clarified mammalian cell culture to strong cation exchange chromatography resin, eluting with elution buffer to obtain first recombinant nerve growth factor; carrying out hydrophobic chromatography, to be specific, treating the first recombinant nerve growth factor, sampling to hydrophobic chromatography resin, and eluting with elution buffer to obtain second recombinant nerve growth factor. The method has few operation steps, the production cycle is shortened, recovery rate is increased, the purity is high, the uniformity is good, and in-vitro bioactivity is very high, generally higher than 1.5*105 U / mg, which is far higher than that (0.5*105 U / mg) of mouse nerve growth factors for injection in the market.

Description

technical field [0001] The invention relates to a biological purification method, in particular to a method for isolating recombinant human nerve growth factor from mammalian cell culture. Background technique [0002] Nerve growth factor (NGF) is the earliest neurotrophic factor discovered. It has a wide range of functions and plays an important role in the growth, development, differentiation and regeneration of central and peripheral nerve cells. The β subunit is the active region of NGF. The so-called β-hNGF is a mature peptide of 118 amino acids. In the human body, the expression of β-NGF has the following processes: 1. The coding sequence of β-NGF (723 bp) is translated into prepro-hNGF (241aa), including signal peptide (1-18aa), guide peptide (19-121aa), Mature peptide (122-241aa); 2. On the endoplasmic reticulum, the signal peptide is hydrolyzed to form pro-hNGF, including the guide peptide and mature peptide. There are 4 amino acid residues (118-121aa) Arg-Ser-Lys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/48C07K1/20C07K1/18
CPCC07K14/48
Inventor 张文宇黄奋飞章永垒白羊陈星陈胜亮阮卡葛平辉邹有土马燕玲王明灶
Owner SINOBIOWAY BIOMEDICINE
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