[go: up one dir, main page]

WO2005077973A1 - Purification de proteines humaines de recombinaison - Google Patents

Purification de proteines humaines de recombinaison Download PDF

Info

Publication number
WO2005077973A1
WO2005077973A1 PCT/IN2005/000045 IN2005000045W WO2005077973A1 WO 2005077973 A1 WO2005077973 A1 WO 2005077973A1 IN 2005000045 W IN2005000045 W IN 2005000045W WO 2005077973 A1 WO2005077973 A1 WO 2005077973A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
recombinant
purification
proteins
monomer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IN2005/000045
Other languages
English (en)
Inventor
Sripad Gunwar
Murali Tummuru
Hemanth Nandigala
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
VIRCHOW BIOTECH PRIVATE Ltd
Original Assignee
VIRCHOW BIOTECH PRIVATE Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by VIRCHOW BIOTECH PRIVATE Ltd filed Critical VIRCHOW BIOTECH PRIVATE Ltd
Publication of WO2005077973A1 publication Critical patent/WO2005077973A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2

Definitions

  • the present invention provides a simple and economical method for the purification of recombinant proteins free from host proteins, wherein at least 90- 95% pure recombinant proteins can be obtained.
  • Biomolecules produced by rDNA technology can be endo or Intracellular proteins ('inside' the cell); Periplasmic (part of the cell wall) or extracellular proteins ('outside' the cell in the culture medium). They can be produced using a variety of organisms like E.coii; S.cerevisiae; P.pastoris etc.
  • the present invention relates specifically to endocellular rDNA derived biomolecules produced in E.coii as 'inclusion bodies'.
  • Endocellular proteins have to be purified from a cell extract containing a complex mixture of molecules (proteins, lipids, nucleic acids) that result from cell lysis. Then isolation, identification, and purification of the factor under investigation require a more or less complex technology that includes extraction, centrifugation and various separation techniques. This calls for different chromatographic techniques when the expected protein has to be highly purified. Chr ⁇ matography is of greatest interest because it produces very good separation and opens way to many prospects. It is based essentially upon the various interactions between the molecules to be separated and the stationary phase constituting the support. These different techniques can be used separately or in combination.
  • the present invention relates to improved RP-HPLC methods for purifying recombinant proteins using simple and novel refolding procedures circumventing the use of complex refolding procedures and purification protocols.
  • Inclusion bodies containing the protein of interest can be produced as a general flow chart as listed in Figure 1.
  • the present invention is limited to the use of Reversed Phase High performance Liquid Chromatography (RP-HPLC) as a purification and subsequently refolding step in the production of highly pure refolded active rDNA derived proteins.
  • RP-HPLC Reversed Phase High performance Liquid Chromatography
  • Ion exchange and gel filtration are widely used in commercial production of recombinant molecules. These are known techniques that practically grew up with protein chemistry.
  • Reversed phase was developed primarily for the analysis of small molecules and its potential was mostly unrealized until the introduction of wide pore RP adsorbents in the 1980's.
  • the present invention relates to improved RP-HPLC methods for purifying recombinant proteins using simple and novel refolding procedures circumventing the use of complex refolding procedures and purification protocols.
  • the RP-HPLC methods comprise using wide pore preparative polystyrene hydrophobic matrix and solvent system consisting of but not limited to acetonitrile; isopropanol; ethano! or methanol as an organic modifier and an organic acid like but not limited to trifluoroacetic; acetic acid; hepta fluorobutaric acid etc.
  • the bonded phase is wide pore polystyrene/divinylbenzene matrix with a pore size of 30um or more preferably 30um using trifluoroacetic as organic acid.
  • the organic acid can also be acetic acid or hepta fluorobutaric acid etc.
  • the concentration can be 0.1 % to 0.3% preferably in the pH range 3-7 more preferably pH 6.
  • the elution can be isocratic or gradient, preferably gradient either linear or nonlinear, wherein the concentration of solvent is 0-100% preferably 20-80%.
  • the solvent consists of but is not limited to acetonitrile; isopropanol; ethano! or methanol.
  • the volume of elution is preferably balanced with the amount of sample loaded on to the column.
  • the process has been demonstrated in at least three rDNA derived biomolecules, like but not limited to recombinant human platelet derived growth factor-B; recombinant human interferon alpha 2b; recombinant human interferon gamma 1 b.
  • a skin wound is defined as a breach in the continuity of any body tissue caused by a minimal direct injury to the skin. There are many instances where a quick closure of the wounded skin will promote a beneficial response. Chronic, nonhealing wounds have plagued healthcare practitioners for decades.
  • PDGF-BB has been shown to be active in promoting wound healing in several " animal models.
  • Human platelet-derived growth factor has been shown to be the major mitogenic protein in serum for mesenchymal derived cells.
  • platelet extracts or purified PDGF-BB induces either cell multiplication or DNA synthesis in cultured smooth muscle ceils, fibroblasts and glial cells.
  • PDGF is a potent chemoattractant for cells that are responsive to it as a mitogen. This is also somewhat unusual in that mitogens generally do not also act as chemotactic agents.
  • PDGF-BB has therapeutic applications for the treatment of injuries, which require the proliferation of fibroblasts or smooth muscle cells to heal.
  • PDGF-BB has been shown to be active in promoting wound healing in several animal models.
  • Lynch et al disclose the use of insulin like growth factor (IGF-1) and purified PDGF- BB to promote healing of dermal wounds in pigs. These two growth factors showed a synergestic effect in promoting the healing.
  • Lynch et al also found that c combination PDGF-BB and IGF-1 promotes bone and cementum formation in a dog model of periodontitis.
  • Interferons are proteins naturally occurring in the body, which has antiviral, anti proliferative and immunoregulatory activity. In particular, it inhibits replication of a variety of RNA- and DNA-containing viruses, inhibits the growth of malignant cells, affects the expression of a variety of oncogenes and activates natural killer cells.
  • Recombinant human interferon alpha is used for the treatment of hairy cell leukemia, AIDS-related Kaposi's sarcoma and chronic hepatitis B and C.
  • interferon alpha leukocyte interferon
  • recombinant interferons are prepared from microbial source, e.g., E.
  • Recombinant DNA derived preparations should be highly pure and homogeneous with required biological activity. The purification of the recombinant material, therefore, plays a particularly important role.
  • the E.coii was used for the manufacture contains an expression vector incorporating the gene for expression of recombinant proteins.
  • E coli cells containing recombinant protein were harvested after fermentation, centrifuged, washed and lysed by sonicator/ bead beater.
  • the inclusion bodies were recovered by centrifugation and washed with buffer containing detergent. Finally, the inclusion bodies were purified by sucrose gradient centrifugation at 6000 rpm and inclusion body pellet was washed with distilled water and stored at -70 deg.C til! further use.
  • the human recombinant protein was extracted with 6M Guanidine Hydrochloride from inclusion body and loaded on to reverse phase polystyrene column (Resource 30 RPC) equilibrated with 30% Acetonitrile containing 0.1% TFA. The column was washed thoroughly with the starting buffer and the bound protein is eluted using 20- 80% Acetonitrile over 60 min. The monomeric form of recombinant protein is analyzed by SDS-PAGE. The pure monomer pooled fractions were concentrated and used for subsequent " purposes. The pooled fraction was diluted ten fold or higher in Tris CI buffer and left overnight for refolding. The refolded solution was concentrated by ultrafiltration and diafiltered extensively. The diafiltered sample was passed through 0.2u filter is almost 95% pure and has the activity comparing to the WHO standards. This protein is taken up for subsequent specific purifications depending on the nature of the biomolecule.
  • the method method herein described in the above examples is not limited to the examples given and is used for a variety of similar rDNA derived molecules like Recombinant Human lnterleukin-2.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention porte sur un procédé simple et économique de purification de certaines protéines humaines de recombinaison exemptes de protéines hôtes, au moins 90 à 95 % de protéines pures de recombinaison pouvant être ainsi obtenus. Le procédé met en oeuvre des méthodes RP-HPLC dans l'étape primaire utilisant une large matrice poreuse à base de polystyrène dans laquelle une forme monomère non pliée de protéine de recombinaison a été isolée et utilisée pour se replier d'une nouvelle façon. Ce procédé permet de convertir le monomère en forme active pliée. En fonction de la protéine, la forme pliée a été également purifiée soit par chromatographie sur gel, soit par chromatographie d'échange d'ions où l'on obtient une pureté supérieure à 98 %, ce qui correspond aux critères des protéines thérapeutiques.
PCT/IN2005/000045 2004-02-11 2005-02-11 Purification de proteines humaines de recombinaison Ceased WO2005077973A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN107/CHE/2004 2004-02-11
IN107CH2004 2004-02-11

Publications (1)

Publication Number Publication Date
WO2005077973A1 true WO2005077973A1 (fr) 2005-08-25

Family

ID=34856858

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2005/000045 Ceased WO2005077973A1 (fr) 2004-02-11 2005-02-11 Purification de proteines humaines de recombinaison

Country Status (1)

Country Link
WO (1) WO2005077973A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10071182B2 (en) 2014-10-14 2018-09-11 Samuel E. Lynch Methods for treating wounds
WO2024264071A1 (fr) 2023-06-23 2024-12-26 Lynch Samuel E Procédés et compositions pour la réparation, le rajeunissement et le confort de la peau

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1439191A1 (fr) * 2004-01-19 2004-07-21 Ares Trading S.A. Procédé de purification des protéines produites par la voie bactérienne

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1439191A1 (fr) * 2004-01-19 2004-07-21 Ares Trading S.A. Procédé de purification des protéines produites par la voie bactérienne

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CARR D.: "The Handbook of Analysis and Purification of Peptides and Proteins by Reversed-Phase HPLC.", 2002, GRACEVYDAG. *
MUSACCHIO A. ET AL: "Recombinant Opoc meningococcal protein folded in vitro, elicits bactericidal antibodies after immunization.", VACCINE., vol. 15, no. 6-7, 1997, pages 751 - 758 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10071182B2 (en) 2014-10-14 2018-09-11 Samuel E. Lynch Methods for treating wounds
WO2024264071A1 (fr) 2023-06-23 2024-12-26 Lynch Samuel E Procédés et compositions pour la réparation, le rajeunissement et le confort de la peau

Similar Documents

Publication Publication Date Title
US4667016A (en) Erythropoietin purification
EP3398964B1 (fr) Méthode chromatographique d'isolement et de purification de sérumalbumine humaine recombinée de pureté élevée
US4432895A (en) Monomeric interferons
KR101831300B1 (ko) 재조합 대장균으로부터 인간 과립구 콜로니 자극인자를 정제하는 방법
JP3894570B2 (ja) Ｉｇｆ−ｉの精製方法
EP0401941A2 (fr) Antigène de surface du virus de l'hépatite B, et sa production
EP0228452B1 (fr) Purification de proteines
JP6742300B2 (ja) ｒＨｕ−ＧＣＳＦの精製のための新規プロセス
CN103288953A (zh) 一种血浆中功能性蛋白质的分离纯化方法
MXPA02006303A (es) Proceso para la purificacion de proteinas farmacologicamente activas a traves de cromografia del intercambio cationico.
EP2051989A1 (fr) Purification de protÉines en une seule Étape par IMAC (MCAC)
JP2002128795A (ja) ヒト血清アルブミン多量体の除去方法
JP6232130B2 (ja) ダルベポエチンアルファの精製方法
CN102702341A (zh) 基于cho细胞表达系统的重组人神经生长因子纯化方法
Khan et al. Large-scale production of recombinant proteins: human leukocyte interferon
WO2005077973A1 (fr) Purification de proteines humaines de recombinaison
JP2009501195A (ja) Ｇ−ｃｓｆを精製する方法
JPS6261040B2 (fr)
Dasari et al. Optimization of the downstream process for high recovery of rhG-CSF from inclusion bodies expressed in Escherichia coli
WO2020234742A1 (fr) Purification du facteur de stimulation de colonies de granulocytes
KR100531670B1 (ko) 인체 인터페론 알파의 제조방법
Wang et al. Renaturation with simultaneous purification of rhG‐CSF from Escherichia coli by ion exchange chromatography
Misterova et al. Optimization of a Purification Method for the Recombinant Platelet-Derived Growth Factor rhPDGF-BB Expressed in the Methylotrophic Yeast Pichia Pastoris
Wang et al. Chromatographic refolding of proteins: molecular action and column control
CN111434676A (zh) 一种乙型肝炎病毒HBV来源HBx蛋白的制备方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase