Patent Application
20090047723
The present invention is directed to a method for purification of Factor VII protein using hydroxyapatite.
Factor VII (FVII), an important protein in the blood coagulation cascade, is a vitamin K-dependent plasma protein synthesized in the liver and secreted into the blood as a single-chain glycoprotein with a molecular weight of 53 kDa. The FVII zymogen is converted into an activated form (FVIIa) by proteolytic cleavage at a single site, R152-I153, resulting in two chains linked by a single disulfide bridge. Recombinant human FVIIa is commercially available from Novo Nordisk under the name NovoSeven® and is used for the treatment of bleeding episodes, e.g. in hemophilia or trauma. Recombinantly produced variants of human FVII have also been reported.
Purification of recombinant Factor VII (rFVII) or recombinant activated Factor VII (rFVIIa) is generally carried out using a combination of ion exchange and immuno-affinity chromatography based on a Ca2+-dependent recognition of the Gla region of FVII (amino acid residues 1 to 45 of human FVII). Although the immuno-affinity based purification step is highly selective and provides FVII protein of high purity, there are disadvantages to this step. For example, potential leaching of antibody into the drug product may affect the safety of the final drug, and the cost of producing the monoclonal antibody (mAb) immuno-affinity matrix is considerable as compared to more conventional, non-antibody based purification matrices.
While replacement of the immuno-affinity step with a different purification technique would be advantageous, this would require removal of non-FVII related contaminants as well as the ability to separate any unwanted isoforms of FVII that may be present in the culture supernatants.
We have explored the possibilities of using non-antibody based methods for purification of FVII protein and possible separation of isoforms of FVII captured from the culture supernatants, and it has surprisingly been found that excellent results are obtained using hydroxyapatite. The present invention thus relates to a method suitable for purifying rFVII or rFVIIa, comprising subjecting the rFVII or rFVIIa to liquid chromatography on a hydroxyapatite (HAP) column.
In the description and claims below, the follow definitions apply:
The term “FVII” or “FVII polypeptide” refers to a FVII molecule provided in single chain form.
The term “FVIIa” or “FVIIa polypeptide” refers to a FVIIa molecule provided in its activated two-chain form, wherein the peptide bond between R152 and I153 of the single-chain form has been cleaved.
The terms “rFVII” and “rFVIIa” refer to FVII and FVIIa molecules produced by recombinant techniques, respectively. These may have the wild-type human sequence or may be variants of the human sequence.
The terms “hFVII” and “hFVIIa” refer to wild-type human FVII and FVIIa, respectively.
Unless it is indicated otherwise or apparent from the context, the terms “FVII”, “FVII protein” and “Factor VII” as used herein are intended to include both the inactivated and activated forms of FVII, and to include the recombinant wild-type sequence of human FVII as well as variants thereof.
The FVII proteins that may be purified by the method of the invention include any FVII or FVIIa protein, in particular human recombinant FVII or FVIIa and variants thereof. The amino acid sequence of wild-type human FVII is well known and is disclosed, for example, in WO 01/58935. Variants of interest that may be purified by the method of the invention include, for example, those described in WO 01/58935, WO 03/093465, WO 2004/029091, WO 2004/111242, WO 99/20767, WO 00/66753, WO 88/10295, WO 92/15686, WO 02/29025, WO 01/70763, WO 01/83725, WO 02/02764, WO 02/22776, WO 02/38162, WO 02/077218, WO 03/027147, WO 03/037932, WO 2004/000366, WO 2004/029090, and WO 2004/108763.
The FVII or FVIIa variants may include one or more substitutions, insertions or deletions compared to wild-type human FVII, for example resulting in a variant that differs in 1-15 amino acid residues from the amino acid sequence of wild-type human FVII, typically in 1-10 or in 2-10 amino acid residues, e.g. in 1-8 or in 2-8 amino acid residues, such as in 3-7 or in 4-6 amino acid residues from the amino acid sequence, where the differences in amino acid sequence from the wild-type are typically substitutions. Such substitutions may be performed e.g. with the aim of introducing one or more in vivo glycosylation sites or PEGylation sites into the protein and/or for improving or otherwise modifying the clotting activity of the wild-type protein; such variants are described in WO 01/58935, WO 03/093465, WO 2004/029091 and WO 2004/111242. FVII or FVIIa variants may alternatively have a reduced clotting activity in order to function as anti-coagulants.
The FVII/FVIIa protein or variant thereof may be produced by any suitable organism, e.g. in mammalian, yeast or bacterial cells, although eukaryotic cells are preferred, more preferably mammalian cells such as CHO cells, HEK cells or BHK cells.
Hydroxyapatite (HAP) is a porous inorganic chromatography material based on calcium phosphate that is useful for purification of proteins, including enzymes and monoclonal antibodies, as well as nucleic acids. Since HAP consists of calcium phosphate, it contains both positive and negative charges. Liquid chromatography using HAP is typically performed within a pH range of about 5.5-9, e.g. about 6-9.
The HAP column may be equilibrated with a low ionic strength buffer, e.g. about 5-10 mM, with a suitable buffer being e.g. a phosphate buffer such as sodium phosphate or alternatively a non-phosphate buffer such as imidazole, TRIS, histidine, MES (2-(N-morpholino)ethane sulfonic acid) or borate. The buffer may optionally include a salt such as NaCl.
The column itself may have any suitable size and volume, depending on e.g. the amount of protein and supernatant to be purified. Persons skilled in the art will be readily able to select a suitable column based on the actual purification needs.
It has been found that HAP is particularly well-suited for purification of rFVII as well as separation of unwanted FVII isoforms. The FVII protein binds tightly to HAP at low buffer concentrations, e.g. a phosphate concentration of from about 1 mM, such as from about 5 mm, e.g. from about 10 mM, and up to about 20 mM, at a pH of from about 5.5 to about 7.5, e.g. about 6.0-7.5, or in the absence of phosphate at a pH of from about 5.5 to about 9.0 or 9.5, typically about 6.0-7.6. The presence of a low concentration of CaCl2, e.g. a concentration of up to about 1 mM, or a higher concentration, e.g. up to about 5, 10 or 20 mM, or even higher such as up to about 50 mM, does not prevent binding of the FVII protein to the HAP matrix.
Elution of non-FVII impurities and unwanted FVII isoforms, for example isoforms with an insufficient level of gamma-carboxylation in the Gla-region of FVII, can be achieved by use of mild elution conditions at a pH of from about 5.5 to about 7.5, e.g. washing with a phosphate concentration of from about 10 mM to about 50 mM or higher, such as up to about 100 or 150 mM, at a pH of about 6.0-7.0. A further alternative for elution of non-FVII impurities and unwanted FVII isoforms is use of a high concentration of NaCl, e.g. up to 1 M or even higher, such as up to about 1.5 M, in the presence of phosphate.
Elution of the FVII protein can be accomplished by increasing the phosphate concentration, the FVII protein being eluted from the column using an appropriate combination of pH and phosphate, for example a gradient starting at about 10-20 mM phosphate and increasing to a maximum phosphate concentration of e.g. about 400-500 mM at a pH of about 5.5 or higher, typically about 6.0 or higher, e.g. up to about 9.0 or 9.5, such as up to about 8.6. The phosphate concentration may be increased in either a linear or stepwise manner as is generally known in the art. Alternatively, elution may be performed in a stepwise manner or with a gradient using an increasing concentration of a non-phosphate displacer in a similar manner to elution with a phosphate buffer. In this case, the displacer could e.g. be calcium in the form of calcium chloride or calcium sulfate, with a pH of e.g. about pH 5.5-9.5, for example 6.0-9.0. Elution with phosphate will often be preferred.
Regeneration of the HAP column may be performed with a phosphate buffer, e.g. in a concentration of about 0.5 M.
Under certain elution conditions the FVII protein elutes in discrete peaks, possibly due to separation of different isoforms. In this case, different approaches are possible for separating the different fractions. One approach is to increase the phosphate or other displacer concentration as described above while maintaining a steady pH. After reaching the maximum phosphate/displacer concentration the pH can then be increased stepwise, typically by increasing the pH in a single step by a value of 1-3, e.g. about 1.5-2.5, such as about 2, over the pH used during the gradient elution. For example, the FVII protein may be eluted at pH of about 6-7 and a maximum concentration of phosphate or other displacer of 400-500 μM, after which the pH is increased from about 6-7 to about 8-9, e.g. from about 6 to about 8 or from about 7 to about 9, while maintaining the high phosphate/displacer level.
Alternatively, elution may be performed using a combined concentration and pH gradient in which both the phosphate/displacer concentration and the pH are increased simultaneously. This may take place starting with a low phosphate/displacer concentration of, e.g. about 10-20 mM, or after the phosphate/displacer concentration is initially raised to an intermediate level of e.g. about 100 or 150 mM. For example, after an initial increase of the phosphate concentration to an intermediate level of 100-150 mM, a phosphate concentration gradient starting at this level and increasing to e.g. about 400-500 mM can be used together with a pH gradient, typically starting with an initial pH of about 5.5-7.0 and increasing to a final pH of about 7.5-9.0, e.g. starting with a pH of about 6 and increasing to a pH of about 8. A variant of this approach is to use a stepwise elution in which the eluent concentration and/or the pH are altered so as to go directly from the intermediate level to final elution conditions. An example of such a stepwise elution is to go from a phosphate/displacer concentration of e.g. about 100 or 150 mM and a pH of e.g. about 6 in a single step to a phosphate/displacer concentration of e.g. about 400 or 500 mM and a pH of e.g. about 5 or 9. If desired, the combined phosphate/displacer and pH gradient can further be combined with a salt gradient, e.g. starting with a NaCl concentration of 0.5-1.5 M, such as about 1.0 M, and decreasing typically down to 0 M.
The FVII protein eluted by the method of the invention has been found to have a purity of greater than about 80%, and in some cases about 90% or more, as determined by LDS PAGE (lithium dodecyl sulfate-polyacrylamide gel electrophoresis).
The invention will be further described with reference to the following non-limiting examples.
The recombinant human FVII protein applied onto the HAP column was produced in CHO-K1 cells. Culture supernatants were sterile-filtered, ultra-filtered and dia-filtered against 10 nM Tris pH 8.6. The sample was subsequently captured on a Q-Sepharose™ FF column previously equilibrated with 10 mM Tris, pH 8.6. After washing in 10 mM Tris, 100 mM NaCl, pH 8.6 the bound FVII protein was eluted in 10 mM Tris, 35 mM CaCl2, 25 mM NaCl, pH 8.6. This sample was desalted to lower the conductivity in 20 mM Tris-HCl, 0.5 mM CaCl2, pH 7.5 prior to application onto the hydroxyapatite column.
Ceramic hydroxyapatite type 1 was from Biorad (cat #157-0400). The columns were packed at volumes of 3-10 ml with column diameters of 5 or 10 mm (Amersham Biosciences) in 0.2 M Na-Phosphate, pH 9-10 and subsequently equilibrated with 10 mM Tris, pH 8.6. The columns were run at room temperature and typically at up to 30 CV/h.
FVII eluted from an anion exchange capture as described above was bound in 20 mM Tris-HCl, 0.5 mM CaCl2, pH 7.5 to the hydroxyapatite column. The bound protein was washed first in 20 mM Tris-HCl, pH 7.5, then using 20 nM Na-Phosphate, pH 6.0. Bound protein was then eluted with a gradient of 20-500 mM Na-Phosphate at pH 6.0 followed by a step of 500 mM Na-Phosphate pH 8.0.
The FVII protein eluted in two well-separated pools, one late in the gradient and one with the pH 8.0 step. The protein was >90% pure in both pools as estimated on SDS-PAGE (
The recombinant human FVII protein applied onto the HAP column was produced in CHO-K1 cells. Culture supernatants were sterile-filtered, ultra-filtered and dia-filtered against 25 mM imidazole, 25 mM NaCl, pH 7.0. 5 mM EDTA was subsequently added to the dia-filtered sample prior to capture on a Q-Sepharose™ FF column previously equilibrated with 25 mM imidazole, 25 mM NaCl, pH 7.0. After washing in 25 mM imidazole, 25 mM NaCl, 5 mM CaCl2, pH 7.0, the bound FVII protein was eluted in 25 mM imidazole, 75 mM CaCl2, 5 mM NaCl, pH 7.0. This sample was diluted in 25 mM imidazole, pH 6.5 to lower the conductivity prior to application onto the hydroxyapatite column.
Ceramic hydroxyapatite was as described above in Example 1. The columns were packed and run as described above, equilibrating with 25 mM imidazole, pH 6.5, with a column volume of 30 ml and a column diameter of 16 mm. The presence of FVII isomers is estimated by the ratio between two ELISAs, directed towards the Gla domain (“Sigma ELISA”) and the PD domain (amino acid residues 153-406 of human FVII) (“PD ELISA”), respectively. The Sigma ELISA recognizes FVII protein with correctly folded and highly gamma-carboxylated Gla domains, and is thus a quantitative estimate of the population of FVII with functional Gla domains. The PD ELISA recognizes all FVII molecules in the sample irrespective of the condition of the Gla domain. The ratio of the two ELISAs is an indicator of the purity of the FVII population with a functional Gla domain.
FVII eluted from an anion exchange capture as described above was bound in 25 mM imidazole, approx. 6 mM NaCl, approx. 18 nM CaCl2, pH 6.5 to the hydroxyapatite column. The bound protein was washed first in 25 mM imidazole, pH 6.5, then using 100 nm Na-Phosphate, pH 6.3, followed by 150 nM Na-Phosphate, 1 M NaCl to elute contaminants and unwanted FVII isoforms. FVII protein containing the desired highly gamma-carboxylated isoforms was then eluted using Na-Phosphate, 1 M NaCl, with a Na-Phosphate concentration gradient of from 150 mM to 500 mM together with a pH gradient of from 6.3 to 8.0.
The FVII protein eluted under two different sets of conditions: one in moderate Na-Phosphate concentrations of up to 150 mM, with an NaCl concentration up to 1 M, at a pH between 6.0 to 6.5, and one where FVII is eluted in the combined phosphate/pH/salt gradient ending at 500 mM Na-Phosphate, and pH 8.0. The protein was >80% pure in the final product pool (
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/DK06/00024 | 1/13/2006 | WO | 00 | 4/3/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60644126 | Jan 2005 | US |
