EP3526245A1

EP3526245A1 - Purification process of fviii

Info

Publication number: EP3526245A1
Application number: EP17791442.1A
Authority: EP
Inventors: Tom Johnston; Ian Garner
Original assignee: Profactor Pharma Ltd
Current assignee: Profactor Pharma Ltd
Priority date: 2016-10-11
Filing date: 2017-10-11
Publication date: 2019-08-21
Also published as: EA201990926A1; ZA201902183B; CN109803982A; GB201617240D0; WO2018069700A1

Abstract

The present invention relates to the purification of recombinant blood coagulation factor VIII (FVIII) from in vitro culture employing chromatography techniques.

Description

PURIFICATION PROCESS OF FVIII

Field of the invention

The present invention relates to the purification of recombinant blood coagulation factor VIII (FVI II) from in vitro culture employing chromatography techniques.

Background to the invention

FVIII is a large, complex glycoprotein. It is synthesized as a 330-kDa protein with the domain structure A1 -A2-B-A3-C1 -C2 where both A and C domains have internal sequence homology and approximately 40% sequence identity to the A and C domains of factor V. The B-domain, which constitutes 38% of the total sequence, shares no sequence identity with other known proteins, including the B-domain of factor V. It is, however extensively glycosylated and contains 19 of the 26 asparagine (N)-linked glycosylation sites on the whole molecule.

WO2014/147386, for example, describes methods of expressing recombinant codon- optimised FVIII in vitro ior subsequent therapeutic use.

WO2009/057883 describes a process for purifying FVI II using a multimodal resin. The FVII I is described as being loaded on to the multimodal resin using high ionic strength (corresponding to a conductivity of 25 to 200 mS/cm at 25°C) and eluting using a buffer which includes at least one amino acid which is positively charged at pH 6 to 8, e.g. arginine, lysine or histidine.

Similarly to the above, WO2009/007451 also describes the use of a multimodal resin to purify FVIII. Elution from the multimodal resin is carried out using at least 1 .5M salt and at least 40% (w/v) ethylene glycol, propylene glycol or a mixture thereof, together with calcium ions. Initial loading of the resin does not require adjustment of pH or conductivity.

WO2006/103258 teaches a method of increasing the amount of a recombinant protein, such as FVIII, which may be obtained from a cell culture, which includes subjecting a suspension of cells, obtained from culture, to a non-physiologically increase concentration of at least one ionic substance. The increase in ionic strength may be affected by the addition of a salt and/or a charged amino acid. Various concentrations of the added substance are described, depending on the particular substance. Summary of the Invention

The present invention is based on the development of a purification process which may be, for example, simpler, cheaper, more environmentally friendly, less dangerous and/or faster than other purification processes known in the art.

In a first aspect there is provided a method for the purification or enrichment of FVIII or a B- domain deleted variant thereof (including modified versions thereof such as fusions with other protein moieties or pegylated molecules) from an in vitro cell culture, which comprises: providing a culture supernatant comprising said FVIII or the B-domain deleted variant thereof; contacting the supernatant containing FVIII or the B-domain deleted variant thereof with a multimodal resin in order to absorb FVIII or the B-domain deleted variant thereof to the multimodal resin; optionally washing the multimodal resin, having the absorbed FVIII or the B-domain deleted variant thereof, with an aqueous washing buffer; eluting FVIII or the B-domain deleted variant thereof containing fractions using an aqueous elution buffer comprising 4-methyl imidazole; and optionally collecting FVIII or the B-domain deleted variant thereof containing fractions in purified or enriched form.

The cell culture may be any suitable cell culture which is capable of expressing FVIII, such as a mammalian cell culture capable of expressing FVIII or B-domain deleted variant thereof, optionally co-expressing von Willebrand factor. The mammalian cell culture may be a cell culture which expresses a recombinant form of FVIII or B-domain deleted variant thereof. That is, the mammalian cells may have been modified so as to express an exogenously introduced FVIII gene or nucleic acid encoding a B-domain deleted variant thereof. In one embodiment the FVIII or B-domain deleted variant thereof is a codon optimised version of FVIII or B-domain deleted variant thereof as described, for example, in WO2014/147386, to which the skilled reader is directed and the entire contents of which are hereby incorporated by reference. Conveniently the cells which are used for culture and expression of the FVIII or B-domain deleted variant thereof are HEK 293, CHO, 3T3, BHK, MICK, Varo, Junket, Hela, as well as derivatives of such cells known in the art. In one embodiment the cells are CHO cells.

Typically, the supernatant is provided by centrifugation and/or filtration of the culture comprising FVIII or the B-domain deleted variant thereof expressing cells, in order to separate the culture supernatant from cells present within the culture. The separated cells may be returned to a culture, for further culturing, as required. Conveniently, the cell culture is not treated prior to the cell separation step, for example, by the addition of additional agents, such as ionic substances which are intended to increase the ionic strength of the culture and its supernatant and/or amino acids with charged side chains, such as arginine, histidine and/or lysine.

Prior to contacting the cell supernatant with the multimodal resin, the pH of the supernatant may be adjusted to between pH5.5 - 8.0, such as pH6 - 7, typically pH6.5, using a suitable reagent or buffer. Other reagents, such as amino acids (e.g. histidine), salts and/or a non- ionic surfactant, such as Tween 20, Tween 80 and/or Pluronic F68 may be added to the supernatant prior to contacting the multimodal resin.

In one embodiment, histidine, CaCI₂ and NaCI are added to the supernatant in a concentration ranging from: histidine in a concentration ranging from 0.05 to 0.2 M; NaCI in a concentration ranging from 0.25 to 0.75 M and CaCI2 in a concentration ranging from 0.05 to 0.2 M. A non-ionic surfactant may be added in an amount up to 1 % (v/v).

In one embodiment the supernatant is adjusted to pH6.5 with the addition of ^"l OOmM histidine (pH6.0), 100mM CaCI₂, 500mM NaCI, and 0.02% Tween 80.

The supernatant may also be further filtered (for example through a 0.1 - ^"Ι μιη filter or filters) in order to remove any small particles.

The term "multimodal resin" as used herein refers to a chromatographic material having a support and moieties bound to the support which moieties interact with chemical groups of the substances to be separated and are able to interact with FVIII, or the B-domain deleted variant thereof, in a mixture by ionic interactions and other types of interactions such as hydrogen bonding and/or hydrophobic interaction. In one embodiment the multi modal chromatography may be performed in a chromatographic column. Suitable "multimodal" chromatography resins include the following commercially available resins HEP Hypercel™; PPA Hypercel™ ; Capto Adhere™; Capto MMC™; MEP Hypercel™.

After binding of FVIII or B-domain deleted variant thereof to the multimodal resin, the multimodal resin may optionally be washed with a wash buffer comprising imidazole, typically in an amount of 0.05 - 0.2M, such as 0.1 M. The wash buffer may comprise reagents which are otherwise similar or the same as the reagents used to adapt the supernatant prior to contacting the multimodal resin. For example, the wash buffer may further comprise amino acids (e.g. histidine), salts and/or a surfactant(s). In one embodiment the wash buffer further comprises histidine, CaCI₂ and NaCI in a concentration ranging from: histidine in a concentration ranging from 0.005 to 0.02 M; NaCI in a concentration ranging from 0.2 to 0.4 M and CaCI₂ in a concentration ranging from 0.005 to 0.05 M. A surfactant such as Tween 80 may also be present at a concentration of 0.01 - 0.03% (v/v).

Elution of the FVIII or B-domain deleted variant thereof from the multimodal resin is carried out using an elution buffer comprising 4-methylimidazole, typically at a concentration of 0.5 - 1 M, such as 0.75M. The elution buffer may comprise reagents which are otherwise similar or salts and/or a surfactant(s). In one embodiment the elution buffer further comprises histidine, CaCI₂ and NaCI in a concentration ranging from: histidine in a concentration ranging from 0.01 to 0.03 M; NaCI in a concentration ranging from 0.4 to 0.8 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M. A surfactant such as Tween 80 at a concentration of 0.01 - 0.03% (v/v) and/or ethylene glycol (10 - 15% v/v) may also be present.

Optionally, a virus inactivation step may be provided and this may be carried out on the eluate or fractions from the multimodal resin, which comprise FVIII or the B-domain deleted variant thereof. Typically the virus inactivation step may comprise a solvent/detergent process well known to the skilled addressee. One such suitable solvent/detergent process employs the use of tri-n-butyl phosphate and Triton-X-100. tri-n-butyl phosphate may be provided in a concentration ranging from 0.15 - 0.6% (e.g.0.3%) and Triton-X-100 may be provided in a concentration ranging from 0.5 - 1 .5% (e.g.1%). Typically the solvent/detergent reagent is contacted with the eluate/fractions for 15min - 60min, such as 30min at a temperature of 4 - 30°C, such as 22°C+/- 2°C A suitable process is described in Roberts, Biologicals, 2008, 36/5, p330-335, to which the skilled reader is directed and the entire contents of which are hereby incorporated by way of reference. Following elution and optional collection of FVIII or the B-domain deleted variant thereof containing fractions in purified or enriched form, the FVIII or the B-domain variant thereof may be further purified or enriched by use of a FVI II affinity chromatography step wherein the affinity is provided by a protein ligand such as an antibody fragment specifically reactive with an epitope which is present on the FVIII or the B-domain deleted variant thereof.

Thus, a method purifying or enriching FVI II or a B-domain deleted variant thereof from an in vitro cell culture may further comprise: contacting a solution comprising FVIII or the B-domain deleted variant thereof with an immunoaffinity matrix which binds to the FVIII or the B-domain deleted variant polypeptide; optionally washing the immunoaffinity matrix, having FVIII or the B-domain deleted variant thereof adsorbed thereto, with an aqueous washing buffer; eluting FVIII or the B-domain deleted variant thereof containing fractions by an aqueous elution buffer comprising imidazole; and optionally collecting FVIII or the B-domain deleted variant thereof containing fractions in purified or enriched form.

Advantageously the solution comprising FVI II or the B-domain deleted variant thereof may be a diluted solution of the FVI II or the B-domain deleted variant thereof containing elution fractions/solution obtained from the multimodal resin elution. In this way, buffer exchange is not required, which simplifies the process and may also lead to increased purification efficiency.

Dilution may be carried out with a dilution buffer (pH6 - 7, e.g. pH6.5) which comprises an amino acid(s) (e.g. histidine) and/or salts In one embodiment the dilution buffer comprises histidine, CaCI₂ and NaCI in a concentration ranging from : histidine in a concentration ranging from 0.01 to 0.03 M; NaCI in a concentration ranging from 0.2 to 0.4 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M. A surfactant such as Tween 80 may also be present at a concentration of 0.01 - 0.03% (v/v).

After binding of FVIII or B-domain deleted variant thereof to the immunoaffinity matrix, the immunoaffinity matrix may optionally be washed with a wash buffer. The wash buffer may comprise reagents which are otherwise similar or the same as the reagents used for the dilution buffer. For example, the wash buffer may comprise an amino acid(s) (e.g. histidine), salt(s) and/or a surfactant(s). In one embodiment the wash buffer comprises histidine, CaCI₂ and NaCI in a concentration ranging from: histidine in a concentration ranging from 0.01 to 0.03 M; NaCI in a concentration ranging from 0.5 to 1 .5 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M. A surfactant such as Tween 80 may also be present at a concentration of 0.01 - 0.03% (v/v)

Elution of the FVIII or B-domain deleted variant thereof from the immunoaffinity matrix is carried out using an elution buffer comprising imidazole, typically at a concentration of 0.5 - 1 M, such as 0.75M. The elution buffer may further comprise reagents which are otherwise similar or the same as the reagents used in the dilution buffer and further comprising ethylene glycol in an amount of 35-65%, such as 50% (v/v). For example, the elution buffer may further comprise an amino acid(s) (e.g. histidine), salt(s) and/or a surfactant(s). In one embodiment the elution buffer further comprises histidine, CaCI₂ and NaCI in a concentration ranging from: histidine in a concentration ranging from 0.01 to 0.03 M; NaCI in a concentration ranging from 0.2 to 0.4 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M. A surfactant such as Tween 80 also be present at a concentration of 0.01 - 0.03% (v/v).

Following elution and optional collection of FVIII or the B-domain deleted variant thereof containing fractions in purified or enriched form, the FVIII or the B-domain variant thereof may be further purified or enriched by use of an anion exchange chromatography step, such as by using Q-Sepharose, Amberlite or Dowex resins.

Advantageously the solution comprising FVIII or the B-domain deleted variant thereof may be a diluted solution of the FVIII or the B-domain deleted variant thereof containing elution fractions/solution obtained from the immunoaffinity purification process. In this way, buffer exchange is not required, which simplifies the process and may also lead to increased purification efficiency.

Dilution may be carried out with a dilution buffer (pH7 - 8, e.g. pH7.5) which comprises imidazole, salt(s) and/or surfactant(s). In one embodiment the dilution buffer comprises imidazole, CaCI₂ and a surfactant in a concentration ranging from: imidazole in a concentration ranging from 0.005 to 0.02 M; CaCI₂ in a concentration ranging from 0.002 to 0.01 M and a surfactant such as Tween 80 in a concertation range from 0.01 - 0.03%.

Optionally, prior to contacting the diluted solution with the anion exchange resin, the solution may be subjected to nanofiltration in order to ensure any viruses or inactivated virus material is removed from the solution. Suitable nanofiltration filters are manufactured by AsahiKASEI under the Planova trademark. Alternatively, this step may be conducted after the anion- exchange step.

The diluted solution is contacted with the anion exchange resin in order to bind FVIII or B- domain deleted variant thereof. After binding of FVIII or B-domain deleted variant thereof to the anion exchange matrix, the matrix may optionally be washed with a wash buffer. The wash buffer may comprise reagents which are otherwise similar or the same as the reagents used for the dilution buffer. For example, the wash buffer may comprise imidazole, salt(s) and/or a surfactant(s). In one embodiment the wash buffer comprises imidazole, CaCI₂ and NaCI: imidazole in a concentration ranging from 0.01 to 0.03 M; NaCI in a concentration ranging from 0.1 to 0.5 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M. A surfactant such as Tween 80 may also be present at a concentration of 0.01 - 0.03% (v/v).

Elution of the FVIII or B-domain deleted variant thereof from the anion exchange matrix is carried out using an elution buffer comprising histidine, typically at a concentration of 0.01 - 0.05M, such as 0.02M. The elution buffer may further comprise reagents which are otherwise similar or the same as the reagents used in the wash buffer. For example, the elution buffer may further comprise salt(s) and/or a surfactant(s). In one embodiment the elution buffer further comprises CaCI₂ and NaCI in a concentration ranging from : NaCI in a concentration ranging from 0.3 to 0.6 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M. A surfactant such as Tween 80 may also be present at a concentration of 0.01 - 0.03% (v/v).

Following elution and optional collection of FVIII or the B-domain deleted variant thereof containing fractions in purified or enriched form, the FVIII or the B-domain variant thereof may be further purified or enriched by use of a gel filtration chromatography step, such as by using Superdex, Sephacryl, Sephadex, Sepharose or Superose gel filtration media.

Advantageously the solution comprising FVI II or the B-domain deleted variant thereof may be a concentrated solution of the FVI II or the B-domain deleted variant thereof containing elution fractions/solution obtained from the anion exchange purification process. In this way, buffer exchange is not required, which simplifies the process and may also lead to increased purification efficiency. The solution may be concentrated by evaporation or more typically by centrifugation through a concentrator membrane, such as a Spin X UF concentrator. Conveniently, the solution may be concentrated by reducing the original solution volume by at least 25%, more preferably 40% or even 50%. The concentrated solution is contacted with the gel filtration media in order to retain FVIII or B-domain deleted variant thereof.

Elution of the FVIII or B-domain deleted variant thereof from the gel filtration media is carried out using a buffer which serves to remove FVIII or B-domain deleted variant thereof from the gel filtration media and provide the FVIII or B-domain deleted variant thereof directly in a formulation buffer. The elution/formulation buffer comprises histidine, sucrose, CaCI₂, NaCI and a surfactant in a concentration ranging from: histidine in a concentration 0.005 - 0.02 M histidine at a pH6.5 - 7.5; sucrose in a concentration 0.005 - 0.02 M; NaCI in a concentration ranging from 0.3 to 0.6 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M. A surfactant such as Tween 80 may also be present at a concentration of 0.01 - 0.03% (v/v).

In one embodiment of the invention, a process of the present invention may comprise the following purification steps:

i. Cell separation;

ii. Supernatant pH and salt adjustment;

iii. Multimodal chromatography (such as by using Capto MMC);

iv. Optional solvent/detergent treatment (such as by employing tri-n-butyl phosphate and Triton X-100 );

v. Immunoaffinity chromatography (such as by employing the GE Healthcare VINSelect ligand);

vi. Optionally one or more nanofiltration steps to remove viruses (such as using a Planova 35N VRF filter)

vii. Anion exchange chromatography (such as by using Q Sepharose);

viii. Optional nanofiltration to remove viruses (such as using a Planova 35N VRF filter); and

ix. Gel filtration chromatography (such as by using Superdex 200).

Advantageously no buffer exchange is required and only addition of reagents, dilution and/or concentration steps are required before each chromatography step.

Detailed Description of the Invention

The present invention will now be further described with reference to the following figure and non-limiting examples: Figure 1 shows a schematic flow diagram of a typical purification process in accordance with the present invention; and

Figure 2 shows an SDS PAGE gel (4-12% BT) photograph of samples taken across a purification process of the present invention.

Gel Key:

1 : Molecular mass markers

2: Thawed cell culture supernatant (untreated)

3: Supernatant post centrifugation

4: Supernatant post pH adjustment to pH 6.5

5: Supernatant post depth and 0.45 micron filtration

6: Capto MMC breakthrough material

7: Capto MMC eluted material/VIII Select immunoaffinity load

8: VIII Select immunoaffinity eluted material/Q Sepharose load

9: Q Sepharose wash material

10: Q Sepharose eluted material

1 1 : Concentrated pre Superdex 200 material

12: Early (High Mw) Superdex 200 fraction

13: FVIII active Superdex 200 fraction

14: Late (Low Mw) Superdex 200 fraction

15: ReFacto standard

16: Molecular mass markers.

A preferred purification process will be described hereinafter. A flow diagram of the process is shown in Figure 1 .

Example 1

Fermentation culture, volume (750 imLs) stored frozen at -80°C containing rHFVIII and previously filtered during harvest using a 0.2 micron filter was adjusted to achieve a pH of 6.0 +/- 0.5 by the addition of 100mM Histidine, pH 6.0, 100mM CaCI₂, 500mM NaCI, 0.2% Tween 80. This was followed by filtration through a 0.1 -0.85 micron depth filter and subsequent 0.45 micron end filter. Filtrate was utilised directly in subsequent purification steps using a GE Healthcare Akta AVANT 25 chromatography system.

Initially, sample was applied with a 5ml/min flow rate to a GE Healthcare CaptoMMC HiScale 16 column (1 .6cm diameter x 10.8cm bed height = 21 .7ml packed resin volume) previously equilibrated with 10mM Histidine, pH 6.5, 10mM CaCI₂, 100mM NaCI, 0.02% Tween 80. Elution of bound sample from this is achieved by applying a buffer of 20mM Histidine, pH 7.5, 10mM CaCI₂, 650mM NaCI, 750mM 4-Methylimidazole, 12.5% ethylene glycol, 0.02% Tween 80.

Concentrated sample was then applied to 1 .17ml GE Healthcare Vlll-Select resin packed into a 1 cm OmniFit column. Following column equilibration with 10mM Histidine, pH 6.5, 10mM CaCI₂, 100mM NaCI, 0.02% Tween 80. Sample was diluted 2 parts to one part with 20mM Histidine, pH 6.5, 10mM CaCI₂, 100mM NaCI, 0.02% Tween 80. Following application, the column was washed with 20mM Histidine, pH 6.5, 10mM CaCI₂, 1 M NaCI, 0.02% Tween 80. Sample was eluted with 20mM Histidine, pH 7.0, 10mM CaCI₂, 300mM NaCI, 750mM imidazole, 50% ethylene glycol, 0.02% Tween 80.

Eluted sample was then applied to a GE 1 ml Hi-Trap Q-Sepharose Fast Flow column anion exchange chromatography column following dilution of 1 part sample with 2 parts buffer containing 10mM Imidazole, pH 7.5, 5mM CaCI₂, 0.02% Tween 80. Bound sample was eluted with 20mM Histidine, pH 6.0, 10mM CaCI₂, 400mM NaCI, 0.02% Tween 80.

Samples containing FVIII were further purified using Superdex 200 gel filtration chromatography utilising a GE Superdex 200 10/300 GL column. The column was equilibrated with 2 column volumes of buffer containing 9.6mM Histidine, pH 7.0, 8.8mM Sucrose, 1 .7mM CaCI₂, 308mM NaCI, 0.01 % Tween 80 (as per FVIII formulation buffer of Osterberg, 2001 ). Collected fractions were sampled by FVIII ELISA, activity assay and SDS-

PAGE analysis before being stored at -80°C.

An SDS page of samples taken at the various stages of a sample purified in accordance with the above purification process is shown in Figure 2. As can be seen the active samples as run on lane 13 are very similar to the Refracto FVIII product as run on lane 15, demonstrating that the purification process is efficient in obtaining a purified FVIII or B domain deleted variant thereof.

Claims

1 . A method for the purification or enrichment of FVIII or a B-domain deleted variant thereof (including modified versions thereof such as fusions with other protein moieties or pegylated molecules) from an in vitro cell culture, which comprises:

providing a culture supernatant comprising said FVIII or the B-domain deleted variant thereof;

contacting the supernatant containing FVIII or the B-domain deleted variant thereof with a multimodal resin in order to absorb FVIII or the B-domain deleted variant thereof to the multimodal resin;

optionally washing the multimodal resin, having the absorbed FVIII or the B-domain deleted variant thereof, with an aqueous washing buffer;

eluting FVIII or the B-domain deleted variant thereof containing fractions using an aqueous elution buffer comprising 4-methyl imidazole; and

optionally collecting FVIII or the B-domain deleted variant thereof containing fractions in purified or enriched form.

2. The method according to claim 1 wherein the cell culture is any suitable cell culture which is capable of expressing FVIII, such as a mammalian cell culture capable of expressing FVIII or B-domain deleted variant thereof, optionally co-expressing von Willebrand factor.

3. The method according to claim 2 wherein the cell culture expressed a recombinant form of FVIII or B-domain deleted variant thereof.

4. The method according to claim 3 wherein the recombinant FVIII or B-domain deleted variant thereof is a codon optimised version of FVIII or B-domain deleted variant thereof.

5. The method according to any of claims 2 - 4 wherein the cell culture is a culture of CHO, HEK 293, 3T3, BHK, MICK, Vara, Junket, or Hela cells, as well as derivatives of such cells known in the art.

6. The method according to any preceding claim wherein the supernatant is provided by centrifugation and/or filtration of the culture comprising FVIII or the B-domain deleted variant thereof expressing cells, in order to separate the culture supernatant from cells present within the culture.

7. The method according to any preceding claim wherein the cell culture is not treated prior to the cell separation step, for example, by the addition of additional agents, such as ionic substances which are intended to increase the ionic strength of the culture and its supernatant and/or amino acids with charged side chains, such a arginine, histidine and/or lysine.

8. The method according to any preceding claim wherein prior to contacting the cell supernatant with the multimodal resin, the pH of the supernatant is adjusted to between pH5.5 - 8.0, such as pH6 - 7, typically pH6.5, using a suitable reagent or buffer.

9. The method according to any preceding claim, wherein histidine, CaCI₂ and NaCI are added to the supernatant in a concentration ranging from: histidine in a concentration ranging from 0.05 to 0.2 M; NaCI in a concentration ranging from 0.25 to 0.75 M and CaCI₂ in a concentration ranging from 0.05 to 0.2 M, and optionally a non-ionic surfactant in an amount up to 1 % (v/v).

10. The method according to claim 9 wherein the supernatant is adjusted to pH6.5 with the addition of 100mM histidine (ph6.0), 100mM CaCI₂, 500mM NaCI, and 0.02% Tween 80.

1 1 . The method according to any preceding claim wherein the multimodal resin is washed with a wash buffer comprising imidazole, typically in an amount of 0.05 - 0.2M, such as 0.1 M.

12. The method according to claim 1 1 wherein the wash buffer further comprises histidine, CaCI₂ and NaCI in a concentration ranging from: histidine in a concentration ranging from 0.005 to 0.02 M; NaCI in a concentration ranging from 0.2 to 0.4 M and CaCI₂ in a concentration ranging from 0.005 to 0.05 M and optionally a surfactant such as Tween 80 at a concentration of 0.01 - 0.03% (v/v).

13. The method according to any preceding claim wherein elution of the FVIII or B- domain deleted variant thereof from the multimodal resin is carried out using an elution buffer comprising 4-methylimidazole, typically at a concentration of 0.5 - 1 M, such as 0.75M.

14. The method according to claim 13 wherein the elution buffer further comprises histidine, CaCI₂ and NaCI in a concentration ranging from: histidine in a concentration ranging from 0.01 to 0.03 M; NaCI in a concentration ranging from 0.4 to 0.8 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M and optionally a surfactant such as Tween 80 at a concentration of 0.01 - 0.03% (v/v) and/or ethylene glycol (10 - 15% v/v).

15. The method according to claims 13 or 14 further comprising a virus inactivation step carried out on the eluate or fractions from the multimodal resin, which comprise FVIII or the B-domain deleted variant thereof.

16. The method according to claims 13 - 15 wherein following elution and optional collection of FVIII or the B-domain deleted variant thereof containing fractions in purified or enriched form, the FVIII or the B-domain variant thereof is further purified or enriched by use of a FVIII affinity chromatography step wherein the affinity is provided by a protein ligand such as an antibody fragment specifically reactive with an epitope which is present on the FVIII or the B-domain deleted variant thereof.

17. The method according to claim 16 wherein the affinity chromatography step comprises: contacting a solution comprising FVIII or the B-domain deleted variant thereof with an immunoaffinity matrix which binds to the FVIII or the B-domain deleted variant polypeptide;

optionally washing the immunoaffinity matrix, having FVIII or the B-domain deleted variant thereof adsorbed thereto, with an aqueous washing buffer;

eluting FVIII or the B-domain deleted variant thereof containing fractions by an aqueous elution buffer comprising imidazole; and

18. The method according to claim 17 wherein the solution comprising FVIII or the B- domain deleted variant thereof is a diluted solution of the FVIII or the B-domain deleted variant thereof containing elution fractions/solution obtained from the multimodal resin elution.

19. The method according to claim 18 wherein the solution is diluted in a dilution buffer (pH 6 - 7, e.g. pH6.5) which comprises an amino acid(s) (e.g. histidine) and/or salts.

20. The method according to claim 19 wherein the dilution buffer comprises histidine, CaCI₂ and NaCI in a concentration ranging from: histidine in a concentration ranging from 0.01 to 0.03 M; NaCI in a concentration ranging from 0.2 to 0.4 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M, optionally comprising a surfactant such as Tween 80 may also be present at a concentration of 0.01 - 0.03% (v/v).

21 . The method of claim 20 wherein after binding of FVIII or B-domain deleted variant thereof to the immunoaffinity matrix, the immunoaffinity matrix is washed with a wash buffer.

22. The method according to claim 21 wherein the wash buffer comprises an amino acid(s) (e.g. histidine), salt(s) and/or a surfactant(s).

23. The method according to claim 22 wherein the wash buffer comprises histidine, CaCI₂ and NaCI in a concentration ranging from: histidine in a concentration ranging from 0.01 to 0.03 M; NaCI in a concentration ranging from 0.5 to 1 .5 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M and optional a surfactant such as Tween 80 may also be present at a concentration of 0.01 - 0.03% (v/v).

24. The method according to claims 17 - 23 wherein elution of the FVIII or B-domain deleted variant thereof from the immunoaffinity matrix is carried out using an elution buffer comprising imidazole, typically at a concentration of 0.5 - 1 M, such as 0.75M.

25. The method according to claim 24 wherein the elution buffer further comprises reagents which are otherwise similar or the same as the reagents used in the dilution buffer and further comprising ethylene glycol in an amount of 35-65%, such as 50% (v/v).

26. The method according to claim 25 wherein the elution buffer further comprises an amino acid(s) (e.g. histidine), salt(s) and/or a surfactant(s). In one embodiment the elution buffer further comprises histidine, CaCI₂ and NaCI in a concentration ranging from: histidine in a concentration ranging from 0.01 to 0.03 M; NaCI in a concentration ranging from 0.2 to 0.4 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M and optionally a surfactant such as Tween 80 also be present at a concentration of 0.01 - 0.03% (v/v).

27. The method according to any of claims 17 - 26 wherein following elution and optional collection of FVIII or the B-domain deleted variant thereof containing fractions in purified or enriched form, the FVIII or the B-domain variant thereof is further purified or enriched by use of an anion exchange chromatography step.

28. The method according to claim 27 wherein the solution comprising FVIII or the B- domain deleted variant thereof is a diluted solution of the FVIII or the B-domain deleted variant thereof containing elution fractions/solution obtained from the immunoaffinity purification process.

29. The method according to claim 28 wherein dilution is carried out with a dilution buffer (pH 7 - 8, e.g. pH7.5) which comprises imidazole, salt(s) and/or surfactant(s).

30. The method according to claim 29 whereinln the dilution buffer comprises imidazole, CaCI₂ and a surfactant in a concentration ranging from: imidazole in a concentration ranging from 0.005 to 0.02 M; CaCI₂ in a concentration ranging from 0.002 to 0.01 M and a surfactant such as Tween 80 in a concertation range from 0.01 - 0.03%.

31 . The method according to claim 30 wherein, prior to contacting the diluted solution with the anion exchange resin, the solution is subjected to nanofiltration.

32. The method according to claims 28 - 31 wherein the diluted solution is contacted with the anion exchange resin in order to bind FVIII or B-domain deleted variant thereof.

33. The method according to claim 32 wherein after binding of FVIII or B-domain deleted variant thereof to the anion exchange matrix, the matrix is washed with a wash buffer.

34. The method according to claim 33 wherein the wash buffer comprises reagents which are otherwise similar or the same as the reagents used for the dilution buffer.

35. The method according to claim 34 wherein the wash buffer comprises imidazole, salt(s) and/or a surfactant(s).

36. The method according to claim 35 wherein the wash buffer comprises imidazole, CaCI₂ and NaCI: imidazole in a concentration ranging from 0.01 to 0.03 M; NaCI in a concentration ranging from 0.1 to 0.5 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M and optionally a surfactant such as Tween 80 may also be present at a concentration of 0.01 - 0.03% (v/v).

37. The method according to claims 27 - 36 wherein elution of the FVIII or B-domain deleted variant thereof from the anion exchange matrix is carried out using an elution buffer comprising histidine, typically at a concentration of 0.01 - 0.05M, such as 0.02M.

38. The method according to claim 37 wherein the elution buffer further comprises reagents which are otherwise similar or the same as the reagents used in the wash buffer.

39. The method according to claim 38 wherein, the elution buffer further comprises salt(s) and/or a surfactant(s).

40. The method according to claim 39 wherein the elution buffer further comprises CaCI₂ and NaCI in a concentration ranging from: NaCI in a concentration ranging from 0.3 to 0.6 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M and a surfactant such as Tween 80 may also be present at a concentration of 0.01 - 0.03% (v/v).

41 . The method according to claims 27 - 40 wherein following elution and optional collection of FVIII or the B-domain deleted variant thereof containing fractions in purified or enriched form, the FVIII or the B-domain variant thereof is further purified or enriched by use of a gel filtration chromatography step.

42. The method according to claim 41 wherein the solution comprising FVIII or the B- domain deleted variant thereof which is subjected to gel filtration is a concentrated solution of the FVIII or the B-domain deleted variant thereof containing elution fractions/solution obtained from the anion exchange purification process.

43. The method according to claim 42 wherein the solution is concentrated by evaporation or more typically by centrifugation through a concentrator membrane.

44. The method according to claims 42 -43 wherein the concentrated solution is contacted with the gel filtration media in order to retain FVIII or B-domain deleted variant thereof.

45. The method of claim 44 wherein elution of the FVIII or B-domain deleted variant thereof from the gel filtration media is carried out using a buffer which serves to remove FVIII or B-domain deleted variant thereof from the gel filtration media and provide the FVIII or B-domain deleted variant thereof directly in a formulation buffer.

46. The method according to claim 45 wherein the elution/formulation buffer comprises histidine, sucrose, CaCI₂, NaCI and a surfactant in a concentration ranging from: histidine in a concentration 0.005 - 0.02 histidine at a pH 6.5 - 7.5; sucrose in a concentration 0.005 - 0.02; NaCI in a concentration ranging from 0.3 to 0.6 M and CaCI₂ in a concentration ranging from 0.005 to 0.02 M and optionally a surfactant such as Tween 80 may also be present at a concentration of 0.01 - 0.03% (v/v).

47. A method for the purification or enrichment of FVIII or a B-domain deleted variant thereof (including modified versions thereof such as fusions with other protein moieties or pegylated molecules) from an in vitro cell culture, which comprises: x. Cell separation;

xi. Supernatant pH and salt adjustment;

xii. Multimodal chromatography (such as by using Capto MMC);

xiii Optional solvent/detergent treatment (such as by employing tri-n- butyl phosphate and Triton X-IOO );

xiv Immunoaffinity chromatography (such as by employing the GE Healthcare VINSelect ligand);

xv. Optionally one or more nanofiltration steps to remove viruses (such as using a Planova 35N VRF filter)

xvi. Anion exchange chromatography (such as by using Q Sepharose);

xvii. Optional nanofiltration to remove viruses (such as using a Planova 35N VRF filter); and

xviii. Gel filtration chromatography (such as by using Superdex 200).