WO1989005652A1 - Produit du facteur ix pur - Google Patents

Produit du facteur ix pur Download PDF

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Publication number
WO1989005652A1
WO1989005652A1 PCT/US1988/003686 US8803686W WO8905652A1 WO 1989005652 A1 WO1989005652 A1 WO 1989005652A1 US 8803686 W US8803686 W US 8803686W WO 8905652 A1 WO8905652 A1 WO 8905652A1
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WO
WIPO (PCT)
Prior art keywords
factor
product
protein
fix
buffer
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/US1988/003686
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English (en)
Inventor
John Tharakan
Dudley Strickland
David Clark
William Drohan
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.)
American National Red Cross
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American National Red Cross
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Filing date
Publication date
Application filed by American National Red Cross filed Critical American National Red Cross
Publication of WO1989005652A1 publication Critical patent/WO1989005652A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is related to the preparation of pure Factor IX product from human plasma or plasma concentrates. More particularly, the present invention is related to the pure preparation of Factor IX product free from all detectable thrombogenic factors and any other contaminating human plasma protein.
  • the Factor VII content depends upon the fractionation procedure, for example concentrates derived from calcium phosphate adsorption contain substantial amounts and those prepared by ion-exchange chro atography contain less (White et al, Blood 49:159-170, 1977).
  • the potency of Factors II, IX and X is unaffected by Factor VII content (Menache et al, Patent No. 4,447,416, 1984). It has been reported that although clinically efficacious, the use of Factor IX Complex concentrates, particularly when given in large amounts over a protracted period, produces adverse reactions of thrombosis and/or disseminated intravascular coagulation (DIC) (Kingdon - et al, Thro bos. Diathes.
  • DIC disseminated intravascular coagulation
  • mice 9 59:401-407, 1982.
  • Thrombosis and Haemostasis 56:271-276, 1986 and the FIX in particular (ieb an et al, PNAS 82:3879-3883, 1985; Bessos et al, Throm. Res. 40:863-867, 1985; Smith et al, Throm. Res. 33:211-224, 1984; and Thompson Blood 62:1027-1034, 1983).
  • polyclonal antibodies have also been used (Bessos et al. Thrombosis and Haemostasis 56:86-89, 1986).
  • Figure 1 shows chromatographic results obtained from the purification procedure in accordance with the present invention.
  • the full circles represent protein concentration as measured by absorbance at 280 nm.
  • the full triangles represent FIX activity as measured by a single stage clotting assay as described in Example 2 herein;
  • Figure 2 shows the characterization of monoclonal antibody-purified ' FIX.
  • Figures are as described; Figure 3 shows the characterization of monoclonal antibody purified FIX. Western blots against inter-alpha- rypsin inhibitor, factor IX and murine IgG. Commassie Blue stained SDS polyacrylamide gel is also shown, including standards. The antibody to murine IgG was shown previously to be reactive against mouse IgG; and Figure 4 shows the results of HPLC of monoclonal antibody purified FIX product. B was increased to 80% as explained in Example 4. The full scale corresponds to an absorbance at 211nm of 1.0.
  • mice 22 immunized mice were fused with the mouse myeloma cell
  • Antibody Screening Assay Cell supernants were assayed by using a solid phase enzyme-linked immunoabsorbent assay (ELISA). Purified human FIX was coated to the wells of microtiter plates (Immunolon 2, Dynatech) at a concentration of 1 mg/mL in 50 mM aHC ⁇ 3, pH 9.5, overnight at 4°C. Following washing, the plates were coated with bovine serum albumin (10 mg/mL) in the same buffer for 1 hour at 37°C.
  • ELISA solid phase enzyme-linked immunoabsorbent assay
  • the plates were washed and then incubated with cell supernatants in a 50 mM sodium phosphate buffer, pH 7.4 containing 0.15 M NaCl/0.02% Tween 20/10 mM CaCl2 « Binding of antibodies to the antigen-coated well was detected by adding an antimouse IgG-alkaline phosphate conjugate.
  • the purified antibody contained a single band of approximately 150 kDa upon SDS-PAGE under nonreducing conditions. Analysis of the purified antibody by SDS-PAGE under reducing condition revealed that the molecule is comprised of two subunits, with approximate molecular weights of 50 kDa and 25 kDa, which is characteristic of the heavy and light chains of IgG.
  • the purified antibody was subtyped by the Ouchterlony technique and identified as belonging to an IgGl subclass with a kappa light chain.
  • Purification Process The coupled gel is poured into a column. The column is equilibrated in buffer containing 10 mM Mg 2+ .
  • the sample to be applied can be any source of FIX including plasma,.
  • the sample is equilibrated so that an excess of Mg2 + or Ca 2+ is present and is then applied to the column.
  • the column is then washed until the absorbance of the column effluent is less than 0.1. Any other means of detecting protein may also be utilized.
  • Absorbance at 280 nm is chosen as an example of a relatively straight forward and direct method of on-line protein estimation. After the absorbance reaches a value ⁇ 0.1, the washing buffer is changed to a buffer to chelate the metal ion. This may be an EDTA based buffer, a citrate based buffer or any other chelating buffer well known to one of ordinary skill in the art.
  • the eluation results in a single protein peak as monitored by tracking the absorbance at 280 nm.
  • the eluted protein is then assayed for FIX by activity assay, tested for purity using SDS-PAGE, N-terminal sequencing and western blotting. Other assay methods could also be used.
  • Sepharose CNBr4B was used as the immobilization substrate.
  • 2.5 grams of freeze dried resin was suspended in ImM HC1 and washed for 15 minutes in ImM HC1.
  • 31 is of protease inactivated (56°C for 30 minutes) monoclonal antibody solution (0.86 mg/ml protein, >90% IgG SDS-PAGE) was then mixed with the gel and allowed to mix end-over-end for 2 hours at room temperature.
  • the gel was then washed with 20 mM PO4, 100 mM NaCl, pH 8 and any remaining active groups were blocked with 200 mM glycine, pH 8 for 2 hours.
  • the gel was then washed in turn with acetate buffer (0.1M, 0.5M NaCl pH 4) and phosphate buffer (20 mM, 100 mM NaCl, pH 8) in sequence several times. - This resulted in coupling of 2.9 mg protein/ml of gel.
  • the product was then stored in the cold in phosphate buffer containing 0.2% azide for further use.
  • the coupled gel was poured into an Amicon G10 x 150 Column, forming a bed of volume 7.5 cm 3 .
  • the column was equilibrated with equilibration buffer (10 mM MgCl2, 100 mM NaCl, 20 mM TRIS, pH 7.5 with 0.2% NaN 3 ) using a flow rate of about 0.4 ml/min.
  • the sample to be applied was an eluate from a DEAE Sephadex adsorption of cryo-poor plasma (Run # 283E016 from American Red Cross at Hyland Therapeutics). This sample was equilibrated to 40 mM MgCl2 in order to balance to 20 mM concentration of citrate ion that the sample was in. The sample was then applied at the same flow rate of 0.4 ml/min giving a mean residence time in the column of about 18 minutes. Fractions were collected and assayed for protein content and factor IX clotting activity.
  • the column was subsequently washed with washing buffer (10 mM MgCl2, 1 M NaCl, 20 mM TRIS, pH 7.5 with 0.2% NaN 3 ) until the absorbance of the wash was below 0.02 at 280 nm. At this point the buffer was changed to elution buffer (20 mM Sodium Citrate, 110 mM NaCl, pH 6.8 with 0.2% NaN 3 ). Elution fractions were also collected and assayed for protein and factor IX clotting activity. The profile of the adsorption, washing and elution are shown in Figure 1. Both the protein content and the clotting activity are included. Elution yielded a single protein peak as shown in Figure 1. The analysis in terms of FIX specific activity and percent recovery is shown in Table 1.
  • the starting material was lot number 283E016 from the American Red Cross utilizing the pilot facility of Baxter Travenol at Glendale, California, which was eluate from a preliminary adsorption of cryosupernatant plasma on DEAE-Sephadex.
  • the activity of Factor IX adsorbed and eluted was determined by one stage coagulation assays as described in Example 2. 1050 TJ FIX was loaded onto the column, and 141 U was present in the drop through. Total protein was deter- mined by absorbance at 280 nm.
  • Factor IX activity was measured by a standard one-stage coagulation assay as described by Biggs (Human Blood Coagulation Haemostasis and Thrombosis ed. 1, Oxford: Balckwell Scientific, p614, 1972). Samples to be assayed are diluted 1:10, 1:20, 1:40, and 1:80 in a dilution buffer.
  • the dilution buffer contains 0.05M Imidazole, 0.1 NaCl, pH 7.4 containing 0.1% w/v Bovine serum albumin (Sigma, RIA grade) and 0.01% v/v Tween 20 (Sigma).
  • FIX activity is below about 1 U/ml, corresponding to that of pooled plasma.
  • the standard used was fresh frozen pooled plasma, consisting of a pool of not less than 10 donor units.
  • 100 yl of FIX-deficient plasma (George King, Biomedical) and 100 ⁇ l of diluted sample were placed in a Coagamate X-2 tray (General Diagnostics) and the tray placed in a Coagamate X-2 coagulation machine.
  • APTT Reagent General Diagnostics
  • CaCl2 CaCl2
  • Factor II and X assays were performed similar to the assay for Factor IX except for two changes.
  • the deficient plasma utilized was factor II and VII deficient (Sigma) for the Factor II assay and Factor II and VII deficient (Sigma) for the Factor X assay.
  • Russels Viper Venom (RW, Sigma) and CaCl2 were used instead of APTT and CaCl2 « These assays are quite standard in the art. Protein C was assayed using a chromogenic substrate and the procedure outlined by Odegaard et al, (Haemostasis, 17:109-113, 1987) as well as a coagulation assay (Francis et al. Am. J. Clin. Path.
  • the results of the assay for Factor II, VII, IX, X and Protein C are shown in Table II.
  • the data shows duplicate clot times measured as described and indicate quite clearly that clotting factors II, VII, X and Protein C are not detectable by any of the coagulation assays.
  • Factor IX was determined independently since the concentration was too high and a 100-fold dilution was necessary to obtain meaningful results.
  • a buffer clotting time is not provided because it is not useful for comparisons.
  • the clot time for Factors II, VII and X for the buffer are all less than the clot time for the monoclonal antibody purified product indicating the absence of any of these vitamin-K dependent coagulation factors in the product.
  • the buffer clot time for Protein C is greater than the clot time for the monoclonal purified product, indicating the absence of any protein C in the product.
  • SDS- polyacrylamide gel electrophoresis was performed by the method of Laemmli (Nature 227:680, 1970) utilizing the Pharmacia Phastgel system and the Hoeffer Mighty Small Vertical Slab (MSVS) Gel Model SE200. In the Heoffer MSVS, a 4% acrylamide stacking gel preceded a 9% acrylamide separating gel.
  • the Pharmacia Phastgel system utilized an 8-25% gradient gel. Proteins were diluted to the appropriate concentration in TRIS-buffered saline 0.1% SDS with or without 2-mercaptoethanol.
  • the membranes were cut into strips to be probed with polyclonal rabbit antisera to human FIX (Accurate, lot 014A), prothrombin (Behring, lot 010508), FX (Diagnostic Stago, lot 114) and human protein C (Diagnostica Stago, lot 6006E15).
  • the membranes were incubated with these antibodies for two hours, washed extensively and then incubated with goat-anti-rabbit conjugated to horse radish peroxidase (Biorad. lot 31816). After extensive washing, the strips were subsequently developed with color reagent containing H2O2.
  • Figure 2 is an SDS-PAGE gel along with western blots against Factors II, IX, X and Protein C, both in the starting material loaded on the immunoaffinity column as. well as the final product.
  • the starting material in this case was eluate from DEAE Sephadex adsorption of cryo-supernatant poor plasma. As the results clearly indicate, the starting material contains all the three contaminants that were tested.
  • FIX product which, by all the criteria employed herein, is found to be completely free of detectable amounts of any human plasma protein, unlike any other FIX preparation heretofore known in the art.
  • pure FIX of the present invention is obtained in high yields and the product, being devoid of any and all known thrombogenic contaminants, is of course, non-thrombogenic and is safely useful for replacement therapy in treating patients afflicted with haemophilia B; the purity of the product having been demonstrated by four different methods: N-terminal sequence analysis.;. Laemmli gel electrophoresis. Western Blot analysis and coagulation assays.
  • a pharmaceutical composition for replacement therapy in accordance with the present invention comprises therapeutic amount of the pure FIX of the present invention and pharmaceutically acceptable carrier such as physiological saline, non-toxic sterile buffers and the like.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un produit du facteur IX pur (FIX) isolé du plasma ou de concentrés de plasma. Cette protéine est exempte d'autres protéines humaines. Aucun autre facteur de coagulation détectable n'est présent. De plus, un procédé d'immunoaffinité montre qu'il n'y a pas de fuite d'un anticorps monoclonal murin.
PCT/US1988/003686 1987-12-21 1988-10-20 Produit du facteur ix pur Ceased WO1989005652A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13536287A 1987-12-21 1987-12-21
US135,362 1987-12-21

Publications (1)

Publication Number Publication Date
WO1989005652A1 true WO1989005652A1 (fr) 1989-06-29

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WO (1) WO1989005652A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5714583A (en) * 1995-06-07 1998-02-03 Genetics Institute, Inc. Factor IX purification methods

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4405603A (en) * 1980-11-29 1983-09-20 Behringwerke Aktiengesellschaft Method for rendering factors IX and X hepatitis-safe with calcium ions
US4786726A (en) * 1986-01-06 1988-11-22 Blood Systems, Inc. Factor IX therapeutic blood product, means and methods of preparing same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4405603A (en) * 1980-11-29 1983-09-20 Behringwerke Aktiengesellschaft Method for rendering factors IX and X hepatitis-safe with calcium ions
US4786726A (en) * 1986-01-06 1988-11-22 Blood Systems, Inc. Factor IX therapeutic blood product, means and methods of preparing same

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Vol. 26, No. 17, issued 25 August 1987 (USA), LIN et al., "Expression of Human Factor IX and its Subfragments in Escherichia coli and Generation of Antibodies to the Subfragments", see pages 5267 to 5274. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE, Vol. 82, issued June 1985 (USA), LIEBMAN et al., "Immunoaffinity purification of factor IX (Christmas factor) by using conformation-specific antibodies directed against the factor IX-metal complex", see pages 3879 to 3883. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 253, No. 17, issued 10 September 1978 (USA), OSTERUD et al., "Human Blood Coagulation Factor IX", see pages 5946 to 5951. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 261, No. 21, issued 25 July 1986 (USA), KAUFMAN et al., "Expression, Purification, and Characterization of Recombinant gamma-Carboxylated Factor IX Synthesized in Chinese Hamster Ovary Cells", see pages 9622 to 9628. *
THROMBOSIS RESEARCH, Vol. 33, issued 1 January 1984 (USA), SMITH et al., "Monoclonal antibodies to Factor IX: characterization and use in immunoassays for Factor IX", see pages 211 to 224. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5714583A (en) * 1995-06-07 1998-02-03 Genetics Institute, Inc. Factor IX purification methods
US6627737B1 (en) 1995-06-07 2003-09-30 Genetics Institute, Llc Factor IX purification methods

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