EP2155784A1 - Chromatografisches verfahren zur gewinnung eines fviii/vwf-komplexes mit verschiedenen verhältnissen zwischen den beiden proteinen zur verwendung bei der behandlung von hämophilia a und willebrand-jürgens-krankheit - Google Patents

Chromatografisches verfahren zur gewinnung eines fviii/vwf-komplexes mit verschiedenen verhältnissen zwischen den beiden proteinen zur verwendung bei der behandlung von hämophilia a und willebrand-jürgens-krankheit

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Publication number
EP2155784A1
EP2155784A1 EP08750078A EP08750078A EP2155784A1 EP 2155784 A1 EP2155784 A1 EP 2155784A1 EP 08750078 A EP08750078 A EP 08750078A EP 08750078 A EP08750078 A EP 08750078A EP 2155784 A1 EP2155784 A1 EP 2155784A1
Authority
EP
European Patent Office
Prior art keywords
fviii
vwf
naci
process according
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.)
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Application number
EP08750078A
Other languages
English (en)
French (fr)
Inventor
Claudio Farina
Filippo Mori
Ilaria Nardini
Paola Rossi
Claudia Nardini
Rodolfo Franceschini
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.)
Kedrion SpA
Original Assignee
Kedrion SpA
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Filing date
Publication date
Application filed by Kedrion SpA filed Critical Kedrion SpA
Publication of EP2155784A1 publication Critical patent/EP2155784A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)

Definitions

  • FVIII Factor VIII
  • vWF a 256 KDa glycoprotein which circulates in plasma (0.1 ⁇ g/ml) bound to vWF.
  • This glycoprotein being very sensitive to proteolysis which causes its activation or degradation, plays a key role as co-factor for FIXa in the coagulation cascade.
  • the formation of the complex with vWF protects FVIII from its proteolytic inactivation.
  • a deficiency or an anomaly of FVIII leads to the pathology known as haemophilia A, characterized by repeated haemorrhagic episodes due to blood coagulation problems.
  • VWF is very important in the haemostatic process and in this respect, in addition to carrying out its function as a carrier protein for FVIII, it is essential for adhesion of thrombocytes to damaged endothelium/subendothelium.
  • vWF acts as a bridge linking specific receptors on thrombocyte surfaces (gplb, gpllb/llla) and endothelial components. Changes in vWF levels can also cause serious haemostatic anomalies.
  • Von Willebrand disease vWD is an autosomal hereditary disease of the blood, mainly caused by a deficiency or an abnormal multimeric composition of vWF.
  • vWF vWF
  • platelet adhesion to the damaged endothelium is less efficient and hence bleeding time is increased.
  • vWF plasma half- life is reduced, with similar or more serious symptomatic manifestations than those of haemophilia A.
  • the incidence of vWD is at least 0.8% in the world population, not accounting for differences between races or geographical distribution.
  • DDAVP desmopressin
  • FVIII/vWF concentrates Concentrates of FVIII/vWF are very important for the treatment of both haemophilia A and von Willebrand disease.
  • a vWF concentrate on its own is unable to restore FVIII content to normal levels in the first stage of bleeding, and in some cases can actually lead to thrombosis.
  • a FVIII/vWF concentrate must however possess a vWF with a multimeric pattern similar to that of plasma. (Haemostasis 1994; 24: 285-288).
  • stabilizers e.g. albumin
  • EP 0468181 describes a method for purifying FVIII from human plasma by ion exchange chromatography, eluting the FVIII at high ionic strength and at acidic pH, then collecting the eluate in the presence of a stabilizer such as heparin, albumin and PEG and lysine or histidine as antiproteases.
  • a stabilizer such as heparin, albumin and PEG and lysine or histidine as antiproteases.
  • albumin addition decreases the specific activity from 300-1200 U/mg protein to 18-24 U/mg protein.
  • US 4361509 describes the purification of FVIII by means of monoclonal antibodies which bind to the FVIII/vWF complex, then dissociating the FVIII from vWF by CaCI 2 .
  • the FVIII obtained, devoid of vWF, is however stable only after albumin addition.
  • a FVIII/vWF complex is purified by means of anion exchange chromatography, but in this case also the addition of heparin and albumin are needed to stabilize the product.
  • the purity of the FVIII/vWF complex is also very important, in which respect by reducing as far as possible the content of contaminating proteins, such as fibrinogen, fibronectin and IgG, allergic reactions or antibodies against the product itself occur less frequently.
  • Some preparations obtained by affinity chromatography (EP 90308104) have a low FVIII specific activity (66 U/mg).
  • the aim of the present invention is to provide a method for purifying a FVIII/vWF complex in which the vWF content can be easily modulated by suitably varying certain parameters of the chromatography process.
  • a chromatography method By this chromatography method various FVIII/vWF complexes can be obtained for use in different pathologies.
  • FVIIkvWF 1.25
  • the product can be used both for haemophilia A and for type 3 von Willebrand disease. Indeed, in this case, a product without FVIII may not be sufficient to reestablish initial levels of FVIII.
  • VWF concentrations of about 1.5 times greater than FVIII could instead be optimal for the various types of vWD.
  • Figure 1 shows the influence of wash buffer and column bed washings on the vWF recovery.
  • the present invention enables a FVIII concentrate to be obtained by means of an efficient, high yield method which can be easily scaled-up to industrial levels, and in which the vWF concentration can be modulated by using a single chromatography system and modifying only certain parameters of the buffers used for the chromatography.
  • Chromatographic separation is carried out on a synthetic hydrophilic support on which are attached long polymer chains (tentacle-like structure) at whose ends positively charged groups (TMAE - trimethylaminoethyl), suitable for strong anion exchange, are present; an example of a resin of this type is known as Fractogel EMD TMAE.
  • resins also enables high molecular weight proteins (>300 KDa), or adhesive proteins such as vWF, to easily bind to charged groups disposed at the ends of the long polymer chain, hence reducing interactions with the matrix which could result in a lower recovery of the protein and/or its inactivation.
  • high molecular weight proteins >300 KDa
  • adhesive proteins such as vWF
  • the biological fraction that contains the FVIII to be purified by chromatography can be obtained: from human plasma, from cryoprecipitate or from a plasma fraction obtained by a pre-purification treatment, such as absorption on aluminium hydroxide.
  • a chromatography column containing a resin of the aforesaid type is equilibrated with a buffer selected in the group consisting of citrate, phosphate and tris, having a pH comprised between 6.8 and 7.4, preferably 7.0, and containing NaCI at a concentration between 0.10 M and 0.15 M, preferably 0.12 M, and optionally comprising other substances such as glycine, calcium chloride.
  • the column equilibrated with the aforesaid buffer, is loaded with the biological fraction containing the FVIII to be purified.
  • the FVIII and the vWF bind preferentially to the resin while the contaminating proteins are for the most part eluted.
  • the first chromatography step consists of washing the resin with the equilibration buffer described above, so as to remove excess contaminating proteins which do not interact with the resin.
  • the preceding buffer is used though increasing its ionic strength (the equilibration buffer modified in this manner is known hereinafter as the “wash buffer”), so as to elute the weakly bound contaminating proteins and to partially elute the vWF.
  • the FVIII/vWF complex is eluted using the same buffer while further increasing the conductivity (this buffer is known hereinafter as the “elution buffer”) by means of NaCI or another salt at a concentration between 0.4 and 1.0 M, preferably 0.45 M.
  • the quantity of buffer used for washing the resin (normally comprised between 5 and 15 CV).
  • NaCI molarity during the aforesaid wash is between 0.2 and 0.25 in order to obtain, after subsequent elution, a final product with a FVIII/vWF ratio between about 0.68 and 2.47 (see table 1 ).
  • the presence of calcium ions at a suitable concentration in the wash buffer enables to obtain a FVIII/vWF concentrate with a very high R; the use of 8 mM CaCI 2 in the 0.25 M NaCI buffer enables to obtain a FVIIkvWF ratio of about 1 :0.5 without leading to a significant reduction in FVIII yield in the final product, which remains in the order of 85-90%. (Table 2).
  • the purification method of the present invention is highly efficient, reproducible, can be scaled-up to industrial levels and provides a complex between FVIII and vWF with high yields.
  • the product obtained has a high specific activity of FVIII, with low concentrations of the main contaminants such as fibrinogen, fibronectin, IgG, IgM, and the vWF exhibits a multimeric pattern with good percentages of high molecular weight multimers.
  • the present chromatography process leads to an enrichment of high molecular weight multimers in the finished product, while reducing the concentration of low molecular weight multimers in the step preceding elution of the FVIII/vWF complex.
  • FVIII/vWF complex purification according to the process of the present invention are given hereinafter.
  • the contaminating proteins were removed by washing the resin with the same buffer then treating the resin with about 10 CV of 10 mM citrate buffer at pH 7.0 with 0.25 M of NaCI, whereas the FVIII/vWF complex was eluted with the same buffer but with a 0.45 M NaCI concentration.
  • the supernatant (S1 ) obtained as in example 1 was loaded onto the Fractogel EMD TMAE resin equilibrated and washed with a 10 mM citrate buffer at pH 7.0 with NaCI at a 0.12 M concentration.
  • the contaminating proteins were removed by treating the resin with about 10 CV of the same buffer containing 0.26 M of NaCI, whereas the FVIII/vWF complex was eluted using NaCI at a 0.45 M concentration.
  • the activities of FVIIhC and vWF:Ag are summarized in Table C below.
  • the supernatant (S1 ) obtained as in example 1 was loaded onto the Fractogel EMD TMAE resin equilibrated and washed with a 10 mM citrate buffer at pH 7.0 with NaCI at a 0.12 M concentration.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
EP08750078A 2007-05-07 2008-05-06 Chromatografisches verfahren zur gewinnung eines fviii/vwf-komplexes mit verschiedenen verhältnissen zwischen den beiden proteinen zur verwendung bei der behandlung von hämophilia a und willebrand-jürgens-krankheit Withdrawn EP2155784A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT000007A ITLU20070007A1 (it) 2007-05-07 2007-05-07 Processo cromatografico per l'ottenimento di un complesso fviii/vwf con differenti rapporti tra le due proteine da utilizzare nella terapia della emofilia a e nella malattia di von willebrand.
PCT/EP2008/055526 WO2008135568A1 (en) 2007-05-07 2008-05-06 Chromatography process for obtaining a fviii/vwf complex with different ratios between the two proteins, for use in haemophilia a treatment and in von willebrand disease

Publications (1)

Publication Number Publication Date
EP2155784A1 true EP2155784A1 (de) 2010-02-24

Family

ID=39736960

Family Applications (1)

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EP08750078A Withdrawn EP2155784A1 (de) 2007-05-07 2008-05-06 Chromatografisches verfahren zur gewinnung eines fviii/vwf-komplexes mit verschiedenen verhältnissen zwischen den beiden proteinen zur verwendung bei der behandlung von hämophilia a und willebrand-jürgens-krankheit

Country Status (3)

Country Link
EP (1) EP2155784A1 (de)
IT (1) ITLU20070007A1 (de)
WO (1) WO2008135568A1 (de)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1396528B1 (it) 2009-01-19 2012-12-14 Kedrion Spa Nuovo processo di purificazione altamente selettivo di due proteine plasmatiche: von willebrand factor (vwf) e fibronectina (fn).
AU2012290004B2 (en) * 2011-08-02 2014-11-27 Takeda Pharmaceutical Company Limited Systems and methods to increase protein yield from recombinant manufacturing processes
GB201506113D0 (en) 2015-04-10 2015-05-27 Ge Healthcare Bio Sciences Ab Method for chromatography
GB201506117D0 (en) 2015-04-10 2015-05-27 Ge Healthcare Bio Sciences Ab Method for chromatography

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2673632A1 (fr) * 1991-03-08 1992-09-11 Lille Transfusion Sanguine Procede de preparation de concentre de facteur von willebrand humain de tres haute purete, approprie a un usage therapeutique.
AT403764B (de) * 1996-03-15 1998-05-25 Immuno Ag Stabiler faktor viii/vwf-komplex
US6531577B1 (en) * 1997-12-15 2003-03-11 Hemasure Denmark A/S von Willebrand factor (vWF)-containing preparation, process for preparing vWF-containing preparations, and use of such preparations
ES2228052T3 (es) * 1998-06-01 2005-04-01 Genentech, Inc. Separacion demonomeros de anticuerpos de sus multimeros utilizando cromatografia de intercambio de iones.
EP1148063A1 (de) * 2000-04-18 2001-10-24 Octapharma AG Haemostatisch aktives vWF enthaltendes Präparat und Verfahren zu seiner Herstellung

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2008135568A1 *

Also Published As

Publication number Publication date
ITLU20070007A1 (it) 2008-11-08
WO2008135568A1 (en) 2008-11-13

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