WO2011027151A1 - Production stable de facteur viii - Google Patents

Production stable de facteur viii Download PDF

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Publication number
WO2011027151A1
WO2011027151A1 PCT/GB2010/051440 GB2010051440W WO2011027151A1 WO 2011027151 A1 WO2011027151 A1 WO 2011027151A1 GB 2010051440 W GB2010051440 W GB 2010051440W WO 2011027151 A1 WO2011027151 A1 WO 2011027151A1
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WO
WIPO (PCT)
Prior art keywords
factor viii
culture medium
cell culture
ions
calcium
Prior art date
Application number
PCT/GB2010/051440
Other languages
English (en)
Inventor
Jan Jezek
Barry Kingston Derham
Original Assignee
Arecor 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 Arecor Limited filed Critical Arecor Limited
Publication of WO2011027151A1 publication Critical patent/WO2011027151A1/fr

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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

  • This invention relates inter alia to the stabilisation of coagulation Factor VIII, particularly in aqueous liquid compositions for therapeutic applications and to improved methods of manufacture of recombinant Factor VIII.
  • Haemophilia A is a hereditary disorder in which the clotting ability of blood is impaired and excessive bleeding results.
  • Haemophilia A (often called classic haemophilia) is a deficiency in clotting factor VIII. Prolonged bleeding is the hallmark of haemophilia A. Small wounds and punctures are not usually a problem, but uncontrolled internal bleeding can result in pain, swelling and permanent damage especially to joints and muscles. Severity of symptoms can vary and severe forms become apparent early on. Mild cases may go unnoticed until later in life when there is excessive bleeding and clotting problems in response to surgery or trauma. Internal bleeding may happen anywhere, and bleeding into joints is common.
  • Factor VIII is a multi-domain glycoprotein which is essential to the blood clotting cascade and is used for the treatment of haemophilia A. Apart from treating bleeding episodes it is increasingly administered prophylactically to reduce long term damage to joints. Factor VIII is one of many proteins involved in the blood clotting cascade. Factor VIII is a co factor for Factor IXa which, in the presence of calcium ions and phospholipids, converts factor X to the activated form Xa. Factor VIII molecule consists of six key domains denoted Al, A2, A3, B, CI and C2. Most currently marketed Factor VIII products comprise all domains.
  • the native structure of Factor VIII comprises complexed calcium ions with an assumed 1 : 1 stoichiometry (i.e. one calcium ion per molecule of Factor VIII). Appropriate binding of calcium ions within the structure of Factor VIII is thus important for maintaining its structural integrity and coagulation activity.
  • RPMI media (Invitrogen), also extensively used to culture mammalian cells, contain 0.43 mM calcium nitrate.
  • US Patent No. 6,599,724 describes the additional beneficial effect of Cu 2+ ions and Zn 2+ ions at concentrations up to 5 ⁇ in the cell culture medium for production of Factor VIII.
  • the use of both Cu 2+ ions and particularly Zn 2+ ions is also relatively common as a component in commercially available cell culture media.
  • the D-MEM/F-12 medium (Invitrogen) contains 1.5 ⁇ zinc sulphate.
  • Factor VIII stability during manufacture there still exists a need to further improve Factor VIII stability during manufacture.
  • the invention is based on the discovery that it is particularly beneficial to provide the culture medium with an excess of Ca 2+ ions in the presence of a strong ligand, such as EDTA.
  • the strong ligand desirably binds undesired transition metal ions which may adversely impact on the Factor VIII potency in the culture medium.
  • an excess means that there are present free Ca 2+ ions which are not either complexed to Factor VIII or to the strong ligand (or any other ligand).
  • the concentration of the strong ligand does not exceed the concentration of calcium ion present in the composition.
  • the concentration of the strong ligand is less than half of the concentration of calcium ion, for example one tenth of the concentration of the calcium ion. The strong ligand is then practically absent in its free (i.e. not bound to metal ion) form.
  • the simultaneous presence of calcium ion and the strong ligand has the benefit of removing traces of other ions especially transition metal ions (such as cupric or ferric ions, also zinc and manganese ions) which may otherwise be present in the cell culture medium composition as contaminants and contribute to detrimental oxidation or aggregation processes.
  • transition metal ions such as cupric or ferric ions, also zinc and manganese ions
  • the absence of trace metals other than calcium is very likely to inhibit the activity of proteases produced by the transfected cells. It was confirmed in our experiments that the presence of Ca 2+ ions especially at concentrations between 1 to 10 mM e.g. 1 to 5 mM is beneficial in the cell culture medium for Factor VIII production.
  • an aspect of the invention provides a cell culture medium for recombinant Factor VIII production, characterized in that the culture medium contains calcium ions and a strong ligand such as EDTA.
  • the calcium ions may be provided as an additional source.
  • the total concentration of calcium ions in the culture medium is, for example, between 1 to 10 mM e.g between 2 to 5 mM and the total concentration of strong ligand (such as EDTA) in the culture medium is, for example, between 0.1 to 5 mM e.g. between 0.1 to 0.5 mM.
  • a cell culture medium that comprises (i) calcium ions at a concentration between 1 to 10 mM, (ii) a strong ligand such as EDTA at a concentration between 0.1 to 5 mM, wherein the concentration of the strong ligand does not exceed the concentration of calcium ion and (iii) conventional culture components, including amino acids, sugars, vitamins or growth factors, necessary to ensure growth of the cells.
  • the cell culture medium according to this aspect of the present invention can be used for recombinant protein production using a wide range of cells such as mammalian, bacterial, yeast or insect cells.
  • a process for the production of recombinant Factor VIII comprising culturing cells capable of expressing recombinant Factor VIII in a cell culture medium as described herein.
  • a process for the production of recombinant Factor VIII comprising culturing cells engineered to express recombinant Factor VIII in a cell culture medium containing calcium ions and a strong ligand wherein the stability or potency of recombinant Factor VIII in the cell culture medium is enhanced relative to the absence of the strong ligand or calcium ions and the strong ligand.
  • the culture composition comprises water, cells engineered to produce Factor VIII, TRIS calcium ions, and EDTA, and other conventional cell culture components.
  • the composition consists essentially of these components.
  • a composition consisting essentially of the stated components is intended to exclude compositions that contain excipients or additives that result in the reduction of Factor VIII potency during fermentation.
  • a composition consisting essentially of the stated components is intended to exclude a composition which contains an excipient having a pKa within 1 pH unit of the pH of the formulation for example at pH of between 6.5 and 7.5 e.g. pH 7.0.
  • Use of a strong ligand in amounts which exceed the concentration of free metal ions (e.g. calcium ions) present in the formulation is also suitably avoided.
  • the invention is applicable to recombinant Factor VIII.
  • coagulation Factor VIII and “Factor VIII” are used herein to encompass a protein molecule, either produced by recombinant technology with biological activity (i.e. blood clotting activity) identical or similar to that of the native human Factor VIII.
  • biological activity i.e. blood clotting activity
  • coagulation factor VIII and “Factor VIII” encompass both molecules containing all native domains of Factor VIII (Al , A2, A3, B, CI and C2) and molecules in which one or more domains have been deleted without significantly affecting the blood clotting activity.
  • coagulation Factor VIII and “Factor VIII” encompass both molecules comprising domains with amino acid sequence identical to the native human Factor VIII as well as analogues in which mutations of the amino acid sequence have been implemented without significantly affecting the coagulation activity.
  • the composition preferably contains sufficient calcium ions to optimize Factor VIII potency and preferably is present in the composition in excess to that required for complexation with protein and more particularly with protein and any strong ligands in the composition. Accordingly, calcium is preferably added in an amount and in a suitable form to provide calcium ions (free and complexed) at a concentration between 0.5 to 30 mM, preferably between 0.5 to 20 mM, most preferably between 1 mM to 10 mM e.g. between 1 mM to 5 mM e.g. between 2 mM to 5 mM.
  • the calcium can be added to the composition, for example as a salt.
  • a preferred example of a calcium salt includes calcium chloride.
  • calcium carbonate and calcium hydrogen carbonate is preferably excluded.
  • magnesium ion such as magnesium chloride
  • magnesium can be added.
  • magnesium can be excluded.
  • the composition contains a strong ligand in an amount sufficiently low to allow the presence of free calcium ions in the composition.
  • ligand is used herein to encompass any compound capable of binding metal ions resulting in formation of complex ions.
  • the ligands are divided to "weak ligands", “medium- strength ligands” and "strong ligands”.
  • a weak ligand has a stability constant of a complex with calcium ion log K ⁇ 0.5; a medium-strength ligand has stability constant of a complex with calcium ion log K between 0.5 to 2; a strong ligand has stability constant of a complex with calcium ion log K > 2. All stability constants are those measured at 25°C.
  • a strong ligand is preferably added to the composition to control or minimize undesirable protein-metal ion complexation.
  • the preferred amount of ligand to be added is that which binds undesirable metal ions (e.g., ions of residual or trace transition metals, such as copper, zinc or iron, or manganese).
  • the strong ligand desirably has a binding affinity for transition metal ions such as Cu , Zn , Fe ) or Mn which exceeds its binding affinity for Ca ions. Binding affinity is suitably measured in terms of the stability constant of a complex of the strong ligand with said ion measured at 25°C.
  • the stability constants of ETDA complexes with Ca 2+ , Cu 2+ , Zn 2+ , Fe 3+ ions are respectively 10.7, 16.5, 18.8 and 25.7 at 25 °C.
  • the cell culture medium is preferably substantially free of uncomplexed transition metal ions (eg ions of copper, zinc, iron or manganese in which by "uncomplexed” is meant ions which are not either complexed to protein or to the strong ligand.
  • uncomplexed transition metal ions
  • substantially free is means that suitably the level of these ions in uncomplexed form is below 1 ⁇ , e.g. below 0.1 ⁇ or below 0.05 ⁇ .
  • the preferred amount of ligand is preferably not so great as to compete and prevent desirable calcium ion complexation to the Factor VIII protein nor to bind all calcium ions in the composition.
  • This preferred range of ligand is defined herein as an "effective amount.”
  • the stability constants of metal- ligand complexes can be obtained from a comprehensive database published by the US National Institute of Standards and Technology (NIST Standard Reference Database 46, R. M. Smith and A. E. Martell: Critically Selected Stability Constants of Metal Complexes Database). The art of using the stability constants in the context of the present invention is described in detail in WO2009/133200 which is incorporated herein by reference.
  • Suitable strong ligands include: EDTA (10.81), citrate (3.48), methionine (2.04), cysteine (2.5), malate (2.06), and sulphite (2.62).
  • EDTA 10.81
  • citrate 3.48
  • methionine 2.04
  • cysteine 2.5
  • malate 2.06
  • sulphite 2.62
  • the selection of ligands is described generally in WO2009/133200, which is incorporated herein by reference.
  • the strong ligand such as EDTA is present at a concentration allowing the presence of free calcium ions in the composition.
  • a preferred composition comprises EDTA at a concentration between 0.001 mM to 2 mM.
  • the pH of the culture composition is preferably about 6.5 - 7.5. In another embodiment the culture composition is at a pH of about 6.25.
  • WO2008/084237 describes methods of controlling the pH of an aqueous composition utilizing displaced buffers. This publication is incorporated herein by reference in its entirety. Displaced buffers are buffers having ionisable groups having a pKa within 1-5 e.g. 1-4 e.g. 1-3 pH units of the pH of the composition and having no pKa values within 1 pH unit of the pH of the composition.
  • Suitable displaced buffer combinations include one displaced buffer having an ionisable group with a pKa above the pH of the composition and one displaced buffer with an ionisable group with a pKa below the pH of the composition.
  • a displaced buffer may contain two ionisable groups, one with a pKa above the pH of the composition and one with an ionisable group with a pKa below the pH of the composition.
  • Preferred buffers can be selected in accordance with the teachings of that reference.
  • An important aspect of the present invention lies in controlling the metal ions, e.g., adding calcium ions and avoiding excess or free forms of medium-strength and strong ligands, thus ensuring the presence of free calcium ions in the solution.
  • Buffers e.g., displaced buffers, are preferably selected among weak ligands in relation to calcium ion binding.
  • the potency of Factor VIII can be estimated in vitro by measuring the coagulation time in the activated partial thromboplastin time (APTT) test or by a specific Factor VIII chromogenic assay as described herein.
  • Factor VIII potency may be measured using a CA-50 semi-automated coagulometer (Sysmex Corporation) and the APTT procedure provided by Dade Behring Inc. (OTXW G13 E0535 (623) H 1, April 2001 edition) for determination of coagulation Factor VIII.
  • APTT activated partial thromboplastin time
  • mammalian cell cultures are used for the recombinant protein production and more preferably either Baby Hamster Kidney (BHK) cells or Chinese Hamster Ovary (CHO) cells are used.
  • BHK Baby Hamster Kidney
  • CHO Chinese Hamster Ovary
  • the concentration of Factor VIII in the culture medium may suitably be in the range between 1-50 ⁇ g/ml, for example 1-10 ⁇ g/ml.
  • TRIS base or TRIS hydrochloride can be used as a source of TRIS.
  • Calcium chloride is the preferred source of calcium ions, but other soluble salts of calcium can also be used (however preferably not the carbonate or hydrogen carbonate salts).
  • free form of a ligand is used herein to describe molecules of a ligand which is not bound to a metal cation in a particular composition comprising ligand molecules and metal ion molecules.
  • One of ordinary skill in the art will be able to calculate the proportion of free ligand from stability constants of the ligand-metal-ion complex providing overall concentrations of all ligands and all metal ions in the composition are known.
  • a pH "around” or “about” 6.25 means a pH range within which the rates of major degradation processes are not considerably different from those measurable at pH 6.25, preferably a pH range between 5.9 to 6.6, most preferably 6.1 to 6.4.
  • Example 1 Effect of the presence of calcium ions (with and without EDTA) on the activity of Factor VIII in cell culture medium
  • the effect of calcium ions and EDTA was investigated on the Factor VIII activity measurable in expression media containing calls expressing Factor VIII.
  • Baby hamster kidney (BHK) cells (adherent) transfected with Factor VIII gene were maintained in D- MEM/F-12 (Invitrogen) comprising 100 ⁇ g/mL geneticin, penicillin G and streptomycin. Cells were seeded in this medium and allowed to reach 70-80% confluence. For the Factor VIII production, the cells were transferred into a serum-free AIM-V Medium (Invitrogen).
  • the AIM-V contained streptomycin sulfate at 50 ⁇ g/mL and gentamicin at 10 ⁇ g/mL.
  • This basic composition was used as a control medium and compared with two other media of the same basic composition spiked with (i) 3 mM calcium chloride and (ii) 3 mM calcium chloride and 0.5 mM EDTA.
  • Cells were allowed to express Factor VIII for 4 days. After this time the cells were removed and the activity of Factor VIII was followed in the medium for up to 20 days at ambient temperature. It was shown that the presence of the additional source of calcium or calcium/EDTA did not affect significantly the production of Factor VIII in the AIM-V Medium. However, it was shown that the presence of the additional source of calcium ions resulted in considerably higher preservation of Factor VIII activity in the medium compared with the control medium in the absence of the additional calcium ion (3 mM).
  • the invention embraces all combinations of preferred and more preferred groups and embodiments of groups recited above.

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  • 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)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention porte, entre autre, sur un milieu de culture cellulaire pour une production de facteur VIII recombinant caractérisé en ce que le milieu de culture contient des ions calcium et un ligand fort tel que l'EDTA.
PCT/GB2010/051440 2009-09-04 2010-09-02 Production stable de facteur viii WO2011027151A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US23994209P 2009-09-04 2009-09-04
GB0915481A GB0915481D0 (en) 2009-09-04 2009-09-04 Stable manufacture of factor V111
GB0915481.6 2009-09-04
US61/239,942 2009-09-04

Publications (1)

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WO2011027151A1 true WO2011027151A1 (fr) 2011-03-10

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016508376A (ja) * 2013-02-26 2016-03-22 バイエル・ヘルスケア・エルエルシーＢａｙｅｒ ＨｅａｌｔｈＣａｒｅＬＬＣ 組換えタンパク質産生の増加のための製剤及び方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009122A1 (fr) 1989-12-15 1991-06-27 Kabivitrum Ab Derive du facteur humain viii de recombinaison
WO1996030041A1 (fr) * 1995-03-31 1996-10-03 Pharmacia & Upjohn Ab Formulation de protein comprenant le facteur de coagulation viii ou ix dans une solution aqueuse
US6338964B1 (en) * 1999-05-07 2002-01-15 Bayer Corporation Process and medium for mammalian cell culture under low dissolved carbon dioxide concentration
US6599724B1 (en) 1999-07-13 2003-07-29 Biovitrum Ab Stable factor VIII compositions
US6936441B2 (en) 1997-06-20 2005-08-30 Baxter Aktiengesellschaft Recombinant cell clones having increased stability and methods of making and using the same
WO2008084237A2 (fr) 2007-01-11 2008-07-17 Arecor Limited Stabilisation de protéines
EP2113564A1 (fr) * 2008-05-01 2009-11-04 Arecor Limited Formule pour protéines

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009122A1 (fr) 1989-12-15 1991-06-27 Kabivitrum Ab Derive du facteur humain viii de recombinaison
WO1996030041A1 (fr) * 1995-03-31 1996-10-03 Pharmacia & Upjohn Ab Formulation de protein comprenant le facteur de coagulation viii ou ix dans une solution aqueuse
US6936441B2 (en) 1997-06-20 2005-08-30 Baxter Aktiengesellschaft Recombinant cell clones having increased stability and methods of making and using the same
US6338964B1 (en) * 1999-05-07 2002-01-15 Bayer Corporation Process and medium for mammalian cell culture under low dissolved carbon dioxide concentration
US6599724B1 (en) 1999-07-13 2003-07-29 Biovitrum Ab Stable factor VIII compositions
WO2008084237A2 (fr) 2007-01-11 2008-07-17 Arecor Limited Stabilisation de protéines
EP2113564A1 (fr) * 2008-05-01 2009-11-04 Arecor Limited Formule pour protéines
WO2009133200A1 (fr) 2008-05-01 2009-11-05 Arecor Limited Formulation de protéine

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DOERING C B ET AL: "High level expression of recombinant porcine coagulation factor VIII", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, INC, US, vol. 277, no. 41, 23 July 2002 (2002-07-23), pages 38345 - 38349, XP002379313, ISSN: 0021-9258, DOI: DOI:10.1074/JBC.M206959200 *
DOERING ET AL., J. BIOL. CHEM., vol. 277, no. 41, 2002, pages 38345 - 38349
FATOUROS ANGELICA ET AL: "Recombinant factor VIII SQ: Influence of oxygen, metal ions, pH and ionic strength on its stability in aqueous solution", INTERNATIONAL JOURNAL OF PHARMACEUTICS, ELSEVIER BV, NL, vol. 155, no. 1, 1 January 1997 (1997-01-01), pages 121 - 131, XP002475005, ISSN: 0378-5173, DOI: DOI:10.1016/S0378-5173(97)00155-5 *
GIBSON D.H., COORDINATION CHEMISTRY REVIEWS, vol. 185-186, 1999, pages 335 - 355
SUDHAKAR KATAKAM ET AL: "Effects of copper on the structure and function of factor VIII subunits: Evidence for an auxiliary role for copper ions in cofactor activity", BIOCHEMISTRY, vol. 37, no. 19, 12 May 1998 (1998-05-12), pages 6874 - 6882, XP002612106, ISSN: 0006-2960 *
TOOLE, NATURE, vol. 312, 1984, pages 342 - 347
WOOD, NATURE, vol. 312, 1984, pages 330 - 337

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016508376A (ja) * 2013-02-26 2016-03-22 バイエル・ヘルスケア・エルエルシーＢａｙｅｒ ＨｅａｌｔｈＣａｒｅＬＬＣ 組換えタンパク質産生の増加のための製剤及び方法

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