WO2011027151A1

WO2011027151A1 - Stable manufacture of factor viii

Info

Publication number: WO2011027151A1
Application number: PCT/GB2010/051440
Authority: WO
Inventors: Jan Jezek; Barry Kingston Derham
Original assignee: Arecor Limited
Priority date: 2009-09-04
Filing date: 2010-09-02
Publication date: 2011-03-10
Also published as: GB0915481D0

Abstract

There is provided inter alia 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.

Description

STABLE MANUFACTURE OF FACTOR VIII

FIELD OF THE INVENTION

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.

BACKGROUND TO THE INVENTION

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. However, the B-domain was shown dispensable for the coagulation activity, and one of the currently marketed Factor VIII products (ReFacto) is produced with the B- domain deleted. 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.

The beneficial effect of the presence of Ca²⁺ ions at a concentration of at least 0.1 mM in a cell culture medium for the production of recombinant Factor VIII is generally well recognized (see for example, US Patent No. 6,599,724). In fact, a number of commonly used, commercially available cell culture media contain a source of calcium ions, for example in the form of calcium chloride, as one of the essential components. For example, the D-MEM/F-12 medium (Invitrogen), a very commonly used medium for culturing mammalian cells, including those transfected to produce Factor VIII (see Doering et al. (2002) J. Biol. Chem. 277(41), 38345-38349) contains 1.05 mM calcium chloride. Similarly, RPMI media (Invitrogen), also extensively used to culture mammalian cells, contain 0.43 mM calcium nitrate. In addition, US Patent No. 6,599,724 describes the additional beneficial effect of Cu²⁺ ions and Zn²⁺ ions at concentrations up to 5 μΜ in the cell culture medium for production of Factor VIII. In fact, the use of both Cu²⁺ ions and particularly Zn²⁺ ions is also relatively common as a component in commercially available cell culture media. For example, the D-MEM/F-12 medium (Invitrogen) contains 1.5 μΜ zinc sulphate. However there still exists a need to further improve Factor VIII stability during manufacture.

SUMMARY OF THE INVENTION

The invention is based on the discovery that it is particularly beneficial to provide the culture medium with an excess of Ca²⁺ 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. In this context, "an excess" means that there are present free Ca²⁺ ions which are not either complexed to Factor VIII or to the strong ligand (or any other ligand). For example it is suitable to use Ca²⁺ ions at a concentration between 1 to 10 mM e.g. 1 to 5 mM e.g. 2 to 5 mM in combination with a strong ligand, such as EDTA at a concentration e.g. between 0.1 to 5 mM e.g. between 0.1 to 2 mM e.g. between 0.1 to 0.5 mM in the culture medium. It is critical, however, that the concentration of the strong ligand does not exceed the concentration of calcium ion present in the composition. Preferably 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. As just noted, it is believed that 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. In addition, 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 Ca²⁺ 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. Most commonly used, commercially available cell culture media contain a source of calcium ions, but supplementing them with additional source of calcium ions in order to increase the overall concentration of calcium ions to, say, 1 to 10 mM e.g. 1 to 5 mM can increase the yield of Factor VIII. Without being limited by theory, it is believed that this effect is due to improving the stability of Factor VIII following its expression and secretion by the transfected cells into the cell culture medium.

Thus 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.

In another aspect of the present invention there is provided 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.

In another aspect of the invention there is provided 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.

In another aspect of the invention there is provided 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.

In another aspect of the invention there is provided use of an additional source of calcium ions and a strong ligand such as EDTA to increase the stability of Factor VIII or potency of Factor VIII activity in a culture medium for Factor VIII production. DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the culture composition comprises water, cells engineered to produce Factor VIII, TRIS calcium ions, and EDTA, and other conventional cell culture components. In preferred embodiments, 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. For example, 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. The terms "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. The terms "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. The terms "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. As will be explained below, calcium carbonate and calcium hydrogen carbonate is preferably excluded. Optionally, magnesium ion, such as magnesium chloride, can be added. In another embodiment, 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. The term "ligand" is used herein to encompass any compound capable of binding metal ions resulting in formation of complex ions. For the purpose of this invention the ligands are divided to "weak ligands", "medium- strength ligands" and "strong ligands". The terms of "weak ligand", "medium- strength ligand" and "strong ligand" are defined based on the stability constants of their complexes with calcium ion, as follows: 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. Thus, 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. For example, the stability constants of ETDA complexes with Ca²⁺, Cu²⁺, Zn²⁺, Fe³⁺ 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. By "substantially free" is means that suitably the level of these ions in uncomplexed form is below 1 μΜ, e.g. below 0.1 μΜ or below 0.05 μΜ.

However, 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. Examples of suitable strong ligands (calcium ion Log K's are in parentheses) include: EDTA (10.81), citrate (3.48), methionine (2.04), cysteine (2.5), malate (2.06), and 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. For example, 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. Alternatively 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.

It is important to realize in the context of the present invention that molecules of dissolved gases are also ligands capable of forming co-ordinate bonds with metal ions. This is particularly the case of carbon dioxide. Carbon dioxide-metal complexes are reviewed in Gibson D.H.: Coordination Chemistry Reviews, 185-186 (1999) 335-355. Apart from direct binding to a metal ion carbon dioxide can also contribute to metal binding indirectly by giving rise to various carbonate species that are capable of metal-binding. This is due to the fact that in aqueous solutions carbon dioxide always exists in equilibrium with carbonic acid and various carbonate anions:

CO2 + H2O H2CO3 H⁺ + HCO3- ^T^~¾ H⁺ + CO3² Experiments showed that the presence of carbon dioxide in the headspace, even at a low partial pressure, is detrimental to the stability of aqueous Factor VIII stored under the headspace. This is the reason why use of calcium as calcium carbonate or calcium hydrogen carbonate is suitably avoided.

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. For example, 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. Such methods are accepted by the US and the European Pharmacopoeia.

Preferably, 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.

Production of recombinant Factor VIII is, for example, described in Wood (1984)

Nature 312, 330-337 and Toole (1984) Nature 312, 342-347. Techniques for preparing cells engineered to express recombinant Factor VIII are described, for example, in Doering et al. (2002) J. Biol. Chem. 277(41), 38345-38349 or in US Patent No. 6,936,441. Production of a Factor VIII derivative being devoid of much of the B domain is described in WO91/09122. The contents of these references/patent documents are herein incorporated by reference in their entirety.

The concentration of Factor VIII in the culture medium may suitably be in the range between 1-50 μg/ml, for example 1-10 μg/ml.

Either 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).

The term "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.

One skilled in the art will appreciate that in the context of the present invention 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.

The invention is illustrated in the following examples: 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 μg/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 μg/mL and gentamicin at 10 μg/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). Importantly, the preservation of Factor VIII was even more considerable in the medium spiked both with 3 mM calcium chloride and 0.5 mM EDTA (Table 1). Such stabilisation of Factor VIII in the cell culture medium could potentially improve the yield of recombinant factor VIII production considerably. Table 1. Effect of CaCl₂ on the preservation of Factor VIII activity expressed by transfected BHK cells in AIM-V media.

As can be seen from Table 1, the addition of calcium ions (with or without strong ligand EDTA) did not have a significant effect on the Factor VIII activity during the expression phase. However, the activity declined less rapidly in the subsequent phase, following removal of the cells, in the presence of added calcium ions suggesting that the addition of calcium ions improves the stability of Factor VIII in the culture medium. The effect of adding the strong ligand EDTA is to increase the stability yet further.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The invention embraces all combinations of preferred and more preferred groups and embodiments of groups recited above.

Claims

What is claimed:

1 A cell culture medium for recombinant Factor VIII production characterized in that the culture medium contains calcium ions and a strong ligand.

2. A cell culture medium according to claim 1, wherein the total concentration of calcium ions in the culture medium is between 1 to 10 mM and the concentration of strong ligand in the culture medium is between 0.1 to 5 mM.

3. A cell culture medium according to claim 2, wherein the total concentration of calcium ions is between 2 to 5 mM.

4. A cell culture medium according to any one of claims 1 to 3 wherein the strong ligand is EDTA.

5. A cell culture medium according to claim 4 wherein the concentration of EDTA is between 0.1 to 0.5 mM.

6. A cell culture medium according to any one of claims 1 to 5 which is substantially free of uncomplexed transition metal ions.

7. A cell culture medium according to any one of claims 1 to 6, comprising mammalian cells.

8. A process for the production of recombinant Factor VIII, comprising culturing cells capable of expressing recombinant Factor VIII in a cell culture medium according to any one of claims 1 to 7.

9. 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 additional source of calcium ions and the strong ligand.

10. Use of an additional source of calcium ions and a strong ligand such as EDTA to increase the stability of Factor VIII or potency of Factor VIII activity in a culture medium for Factor VIII production.

11. Use according to claim 10 wherein the strong ligand is EDTA.