Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1986-10-10
1989-05-09
Hazel, Blondel
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
435 70, 435811, 530351, 424 855, C12P 2100, C07K 1526, A61K 4502
Patent
active
048289906
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a method for purifying a physiologically active polypeptide produced by a recombinant DNA technology without denaturation or decomposition by proteases, more particularly, said polypeptide being produced by a microorganism transformed by a plasmid vector bearing a gene coding for a polypeptide having physiological activities. This Invention in particular provides an effective method for purifying a desired polypeptide from a culture mixture of a microorganism capable of producing a polypeptide having interferon activities, especially human immune (or gamma) interferon activities.
BACKGROUND ART
Interferon proteins have been classified into three types, alpha, beta and gamma (abbreviated to IFN-.alpha., IFN-.beta. and IFN-.gamma. respectively) based on antigenic and structural differences. Gamma interferon has a number of characteristics that differentiate it from alpha and beta interferons. Among these differences are antigenic distinctiveness and greater activity with regard to immunoregulation and anti-tumor effects. Human gamma interferon (referred to herein as "h-IFN-.gamma.") may be produced by T lymphocytes stimulated by mutagens or by antigens to which they are sensitized. It may also be obtained through cloning and expression techniques now well known to the art.
Recently, it has become possible by the progress in genetic engineering to produce many physiologically active polypeptides from microorganisms or animal cells, although these substances have been produced by separation and purification from an organism. However, it cannot yet be said that a method has been established for extracting and purifying the intended substance with a purity sufficient to be used for drugs and without causing denaturation or decomposition.
Gamma interferon-containing cells, however obtained, are collected and are disrupted by various means such as osmotic shock, ultrasonic vibration, grinding or high shear disruption and the disrupted cell-gamma interferon mixture is then processed to isolate the gamma interferon. The insoluble debris is separated by centrifugation and the gamma interferon-containing supernatant is collected for purification.
Although disclosure has been made of certain technology for such production methods, e.g. a method extracting and purifying the polypeptide produced by recombinant microorganism by using guanidine hydrochloride and urea (Japanese Patent Public Disclosure No. 161321/1984 and U.S. Pat. No. 4,476,049) and a purification method using a monoclonal antibody (Japanese Patent Public Disclosure No. 186995/1984), the intended substance is not always adequately purified without being subjected to denaturation and without its activity being lost.
European Patent Application 0,087,686 discloses a three-step process for purifying human immune interferions from the cell-free supernatant or extract from the crude interferon source. In the first step (for naturally occurring interferon), an affinity column, such as Concanavalin-A Sepharose is used, followed by chromatography on a carboxymethyl silica column using an increasing salt gradient and finally, on a silica gel permeation column. If sufficient purity is not obtained, concentration and chromatography on either the TSK or CM column is used.
European Patent Application 0,063,482 disclosed a purification process employing chromatographic methods using (1) Controlled Pore Glass beads; (2) Concanavalin-A Sepharose; (3) Heparin-Sepharose or Procion Red-agarose; and (4) gel filtration.
European Patent Applications 0,107,498 and 0,077,670 disclose a purification scheme employing (1) polyethyleneimine precipitation; (2) pH precipitation of bacterial proteins; (3) concentration and dialysis; (4) chromatography on (a) carboxymethyl cellulose; (b) a calcium phosphate gel; (c) a carboxymethyl cellulose; and (d) gel filtration resins.
These purification processes require a multitude of steps, cause degradation of the interferon by degradation or aggregation of the interferon molecule, or otherwise result
REFERENCES:
patent: 4266024 (1981-05-01), Swetly et al.
patent: 4476049 (1984-10-01), Kung
patent: 4751078 (1988-06-01), Nogabhushar et al.
Chemical Abstracts, vol. 93, Abstract No. 90967t, 1980.
Biological Abstracts, vol. 73, Abstract No. 85856, 1982.
Chemical Abstracts; vol. 81, Abstract No. 87854a, 1974.
Higashi Naoki
Nagabhushan Tattanahalli L.
Okada Tsutomu
Shirasawa Hounai
Sugimoto Shunjiro
Hazel Blondel
Magatti Anita W.
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