WO1997026280A1 - Isolation du fibrinogene par chromatographie d'affinite - Google Patents

Isolation du fibrinogene par chromatographie d'affinite Download PDF

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Publication number
WO1997026280A1
WO1997026280A1 PCT/AU1997/000013 AU9700013W WO9726280A1 WO 1997026280 A1 WO1997026280 A1 WO 1997026280A1 AU 9700013 W AU9700013 W AU 9700013W WO 9726280 A1 WO9726280 A1 WO 9726280A1
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WIPO (PCT)
Prior art keywords
fibrinogen
solid support
resin
spacer
pro
Prior art date
Application number
PCT/AU1997/000013
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English (en)
Inventor
Jerry Kanellos
Hung Pham
Adrian Oates
Neil Goss
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Csl Limited
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 Csl Limited filed Critical Csl Limited
Priority to AU13601/97A priority Critical patent/AU1360197A/en
Publication of WO1997026280A1 publication Critical patent/WO1997026280A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
    • 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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen

Definitions

  • This invention relates to the production of fibrinogen, and in particular it relates to the isolation of fibrinogen from a fibrinogen-containing material such as plasma
  • fibrinogen-containing cell culture media arising from the production of fibrinogen by recombinant DNA techniques.
  • Fibrinogen interacts with a number of physiologically important proteins (Doolittle, 1984 9 ) such as plasminogen, thrombin, fibronectin, certain strains of staphylococcal bacteria and platelets.
  • a number of functional characteristics have been assigned to specific parts of the molecule including: the position of the fibrinopeptides released from the parent molecule by the catalytic action of thrombin, fibrin covalent stabilisation donor and acceptor sites, carbohydrate clusters, polymerisation sites, calcium binding sites and the attachment sites for fibronectin, plasminogen, bacteria and platelets.
  • Immobilised synthetic peptides comprising these sequences have been demonstrated to provide a useful affinity chromatography method for the quantitative isolation of fibrinogen out of anti-coagulated plasma (Kuyas et at., 1990 14 ).
  • the synthetic peptide tBOC-Gly-Pro-Arg-Pro-Lys-O-methylester was immobilised to carbodiimide-activated Fractogel, a solid hydrophilic polymer, and a quantitative method for the isolation of human fibrinogen from plasma was developed.
  • the peptide-Fractogel resin was pre-equilibrated in triethanolamine (TEA) buffer and anti- coagulated plasma passed through it.
  • TAA triethanolamine
  • the resin was then washed with TEA buffer containing 0.5% gelatin followed by at least 10 column volumes of TEA buffer containing 1M NaCl to reduce the non-specifically bound proteins.
  • the bound fibrinogen was finally eluted from the gel using three different solutions: 1. TEA buffer containing 6M urea; 2. 0.1M acetate buffer pH 4.25 containing 3M urea; and 3. 0.1 M acetate buffer pH 4.5 containing 2M urea.
  • the fibrinogen fractions were pooled and extensively dialysed against 0.05M Tris pH 7.4 containing 0.1M NaCl.
  • the present invention relates to the large scale separation by affinity chromatography of fibrinogen from other blood proteins in human blood plasma, fraction I precipitate, other plasma fractions containing fibrinogen or fibrinogen- containing culture media produced by recombinant DNA techniques. More specifically, it is an object of this invention to provide an affinity purification method for fibrinogen that involves a single chromatography step that does not rely on multiple wash buffers or multiple elution buffers containing urea to recover the bound fibrinogen.
  • Another specific object of this invention is to provide a relatively simple chromatographic method for obtaining fibrinogen preparations of high specific activity utilising a single washing procedure to remove non-specifically bound proteins and a single buffer for the recovery of bound fibrinogen.
  • fibrinogen may be isolated from a fibrinogen-containing material as described above in a single chromatographic step using a fibrinogen-binding peptide chemically coupled to a polysaccharide support to provide a fibrinogen preparation of high specific activity.
  • the method of this invention has been shown to be superior to other known affinity isolation procedures (including the procedure of Kuyas et al. 1990 14 , above) in that only mild elution buffers are required to recover the bound fibrinogen.
  • a method for the recovery of fibrinogen from a fibrinogen-containing material which comprises contacting said fibrinogen-containing material with a fibrinogen-binding peptide coupled to a solid support, and subsequently eluting bound fibrinogen from said solid support, wherein said solid support is a polysaccharide support and said fibrinogen-binding peptide is coupled to the solid support through a spacer or linker moiety.
  • the fibrinogen-binding peptide is coupled to a polysaccharide support through a spacer or linker moiety.
  • the polysaccharide support is a polysaccharide gel or resin such as one of the cross-linked polysaccharide gels or resins sold under the trade names
  • the polysaccharide support is a Sepharose 4B resin, more particularly ether-diaminohexane (EAH)-Sepharose 4B resin.
  • the fibrinogen-binding peptide is preferably a peptide which consists of or contains one or more of the following sequences:
  • the invention extends to the use of fibrinogen-binding peptides which are modifications or variations of these particular peptide sequences.
  • one or more of the amino acid residues may be substituted with another of the 20 protein amino acids, with a non-protein amino acid, with a D amino acid or with a chemically modified amino acid.
  • the present invention encompasses the use of all such modified or variant peptides which retain effective fibrinogen-binding ability.
  • a suitable spacer or linker moiety is interposed between the fibrinogen-binding peptide and the polysaccharide support to facilitate effective binding of fibrinogen by reducing steric interference between the polysaccharide support and fibrinogen bound to the fibrinogen-binding peptide.
  • the spacer or linker is a chain greater than 7 atoms in length, more preferably about 10 atoms in length.
  • the free C-terminal carboxylic acid group of the pentapeptide GlyProArgProLys is coupled to the free primary amino groups of EAH-Sepharose 4B resin, using carbodiimide as a linker and to catalyse the coupling reaction.
  • the coupled pentapeptide retains a free N-terminal amino group.
  • the ability ofthe GlyProArgProLys-EAH-Sepharose 4B resin to bind to fibrinogen has been demonstrated and the bound fibrinogen has been successfully eluted from the affinity column using a single elution buffer.
  • EAH-Sepharose 4B has free amino groups at the end of a 10 atom long spacer arm for coupling ligands containing free carboxyl groups.
  • the spacer arm structure of EAH-Sepharose is shown in general formula I:
  • Ligands such as peptides of formula II below may be coupled to the spacer arm structure I of EAH-Sepharose in a simple one step procedure in the presence of a carbodiimide.
  • Suitable ligands are peptides of formula II:
  • R is selected from:
  • the synthetic peptide shown in formula II contains the known fibrinogen binding sequence Gly-Pro-Arg-Pro.
  • a lysine residue is preferably added to the sequence to:
  • the resulting Gly-Pro-Arg-Pro-Lys-EAH-Sepharose 4B has a significantly different affinity for fibrinogen allowing for a single wash buffer to be used to remove unbound proteins and a single elution buffer to collect the bound fibrinogen.
  • other known peptide binding sequences for fibrinogen may replace the Gly-Pro-Arg-Pro binding sequence shown in general formula III.
  • any fibrinogen-containing material may be used as a starting material in the method of the present invention, including by way of example, human blood plasma (preferably anti-coagulated), plasma cryoprecipitate, Cohn fraction I precipitate (preferably solubilised), other plasma fractions containing fibrinogen, and fibrinogen- containing cell culture media produced by recombinant DNA techniques.
  • a particularly preferred fibrinogen-containing starting material is solubilised Cohn fraction I precipitate.
  • a single elution buffer may be used to elute bound fibrinogen from the solid support.
  • the elution buffer contains a salt such as NaCl, particularly a gradient of 0 to 1M NaCl.
  • the coupling of the synthetic peptide to the carbodiimide-activated Fractogel support is controlled by use of a protected peptide and all the potential reactive functional groups present in the peptide molecule are blocked prior to the coupling step.
  • the synthetic peptide used by Kuyas et al. has a tBOC-blocked amino terminus and a O-methyl ester- blocked carboxy terminus, leaving the ⁇ amino group on the lysine residue free to react with the carbodiimide-activated Fractogel resin.
  • This procedure allows for one peptide molecule to react with one activated site on the resin, and the tBOC blocking group is then removed chemically using trifluoroacetic acid after the peptide has been coupled to the resin.
  • a peptide such as GlyProArgProLys which has a free N-terminal amino group on the glycine and a free C-terminal carboxylic acid group on the lysine which is used to drive the coupling reaction.
  • EAH Sepharose 4B has free primary amino groups for coupling ligands containing free carboxyl groups, and carbodiimide is used to promote condensation between the free amino groups on the EAH-Sepharose 4B and the free carboxyl group on the peptide to form a peptide link by acid catalysed removal of water.
  • a major difference between the two affinity supports appears to be the affinity the coupled peptide has for fibrinogen.
  • the method of Kuyas et a/. 14 produces a support with a strong affinity for fibrinogen.
  • the fibrinogen has a strong interaction with the peptide immobilised on the Fractogel resin requiring severe elution conditions to remove the bound protein.
  • the procedure of the present invention produces an immobilised peptide with a different affinity for fibrinogen, such that bound protein can be recovered with a single, high salt containing buffer.
  • EAH-Sepharose 4B has free primary amino groups at the end of 10 atom spacer arms which further distance the immobilised peptide from the solid support, and accordingly may contribute to a weaker affinity for the fibrinogen molecule enabling recovery under significantly less stringent conditions.
  • This Example describes the immobilisation of a peptide with the amino acid sequence, NH 2 -Gly-Pro-Arg-Pro-Lys-C0 2 H, and use of the immobilised peptide as an affinity chromatography approach for isolating fibrinogen.
  • EAH-Sepharose 4B gel 5 ml of drained EAH-Sepharose 4B gel (equivalent to 10 ml swollen gel) was first washed on a sintered glass filter with 200 ml of 0.5 M NaCl and then with MilliQ water. 184 ⁇ moles Gly-Pro-Arg-Pro-Lys was dissolved in 15 ml of MilliQ water and the final pH ofthe peptide solution was adjusted to 4.5. The washed gel was added to the peptide solution and 0.8g of water soluble carbodiimide, N-ethyl-N'-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) was then added.
  • EDC water soluble carbodiimide
  • the final volume of the reaction solution was made up to 25 ml and the pH adjusted to 4.5.
  • the coupling reaction was carried out at room temperature for 24 hours with end-to-end mixing. After the coupling period, the peptide-resin was collected by filtration and the filtrate was also collected and kept for amino acid analysis.
  • the amount of peptide 5 coupled to the EAH-Sepharose 4B was approximately 100 mg as determined by amino acid analysis.
  • solubilised fraction I was diluted (1/2 dilution) with triethanolamine (TEA) buffer containing 0.1 M NaCl and 0.5% gelatin.
  • TEA triethanolamine
  • the diluted fraction I solution was then mixed with the Gly-Pro-Arg-Pro-Lys-Sepharose 4B matrix, end-to-end for 1 hour at room temperature. After the incubation period, the Gly-Pro- Arg-Pro-Lys-EAH-Sepharose 4B matrix was collected on a sintered glass filter. The unbound proteins were removed by washing the peptide-matrix with 200 ml of TEA buffer.
  • the Gly-Pro-Arg-Pro-Lys-EAH-Sepharose 4B was then packed into a column (XK 16/20 Pharmacia) and was further washed with Tris(Hydroxymethyl)Methylamine (pH 7.4) at a flow rate of 0.5 ml/min for 3 hours. Elution of the bound proteins was carried out using a gradient from 0 to 1 M of NaCl over 4 hours at a flow rate of 0.5 ml/min and 10 ml fractions were collected. The gradient elution was monitored using a Waters 996 Photo Diode Array Detector and the chromatographic profile is shown in Figure 3. SDS-PAGE and immunoblots of the pooled fractions are illustrated in Figure 4.
  • Gly-Pro-Arg-Pro-Lys-EAH-Sepharose 4B matrix for fibrinogen was demonstrated by incubating commercial Fibrinogen (Kabi) and solubilised fraction I with EDC treated EAH-Sepharose 4B.
  • Fibrinogen standard was mixed with EDC treated EAH-Sepharose 4B end-to-end for 1 hour at room temperature.
  • the Sepharose 4B was then collected on a sintered glass funnel and extensively washed with Tris(Hydroxymethyl)Methylamine (pH 7.4).
  • the Gly-Pro-Arg-Pro-Lys-EAH- Sepharose 4B was then packed into a glass column (XK 16/20 Pharmacia) and was equilibrated with Tris(Hydroxymethyl)Methyiamine (pH 7.4) at a flow rate of 0.5 ml/min for 60 minutes. Elution of the bound proteins was carried out using a gradient from 0 to 1 M of NaCl over 1 h and 1 ml fractions were collected. The fractions were then analysed for clottable protein and the results obtained are presented in Table 1. TABLE 1

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  • Chemical & Material Sciences (AREA)
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  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
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Abstract

L'invention concerne un procédé pour extraire du fibrinogène à partir d'un matériau contenant du fibrinogène. Ce procédé consiste à placer un matériau contenant du fibrinogène en contact avec un peptide se liant au fibrinogène et couplé à un support solide, et à éluer ensuite le fibrinogène lié du support solide. Ce support est un support polysaccharide et le peptide se liant au fibrinogène est couplé au support solide par l'intermédiaire d'une fraction d'un espaceur ou d'un bras.
PCT/AU1997/000013 1996-01-16 1997-01-14 Isolation du fibrinogene par chromatographie d'affinite WO1997026280A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU13601/97A AU1360197A (en) 1996-01-16 1997-01-14 Isolation of fibrinogen by affinity chromatography

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPN7564 1996-01-16
AUPN7564A AUPN756496A0 (en) 1996-01-16 1996-01-16 Isolation of fibrinogen by affinity chromatography

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WO1997026280A1 true WO1997026280A1 (fr) 1997-07-24

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012104638A1 (fr) * 2011-02-01 2012-08-09 Haemostatix Limited Agents thérapeutiques ayant une liaison au fibrinogène améliorée
US8741846B2 (en) 2008-06-23 2014-06-03 Bio-Products & Bio-Engineering Ag Storage-stable, functionally intact fibrinogen
WO2016084097A1 (fr) 2014-11-13 2016-06-02 Hemarus Therapeutics Ltd. Procédé de production de fibrinogène et de thrombine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5043288A (en) * 1988-06-20 1991-08-27 Motsenbocker Marvin A Immobilize molecular binding partners to contact activating supports

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5043288A (en) * 1988-06-20 1991-08-27 Motsenbocker Marvin A Immobilize molecular binding partners to contact activating supports

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
THROMBOSIS & HAEMOSTASIS, Vol. 63, No. 3, (1990), KUYAS C. et al., "Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Lys-Fractogel", pages 439-444. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8741846B2 (en) 2008-06-23 2014-06-03 Bio-Products & Bio-Engineering Ag Storage-stable, functionally intact fibrinogen
WO2012104638A1 (fr) * 2011-02-01 2012-08-09 Haemostatix Limited Agents thérapeutiques ayant une liaison au fibrinogène améliorée
US9724379B2 (en) 2011-02-01 2017-08-08 Haemostatix Limited Therapeutic agents with improved fibrinogen binding
GB2488023B (en) * 2011-02-01 2019-06-12 Haemostatix Ltd Therapeutic agents with improved fibrinogen binding
WO2016084097A1 (fr) 2014-11-13 2016-06-02 Hemarus Therapeutics Ltd. Procédé de production de fibrinogène et de thrombine
US9932388B2 (en) 2014-11-13 2018-04-03 Hemarus Therapeutics Limited Chromatographic process for producing high purity fibrinogen and thrombin

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