Process for preparing C1-esterase inhibitor

Abstract

Process for Preparing a C1-Esterase Inhibitor. The present invention relates to a process for preparing a C1-esterase inhibitor (C1-INH) comprising the following steps: a) obtaining a starting fraction selected from plasma fractions; b) purifying said starting fraction obtained by affinity chromatography, said affinity chromatography comprising an elution step with an elution buffer having a pH between 4.0 and 8.0; and c) recovering the eluate after chromatography b), and this eluate corresponds to the fraction comprising the C1-INH. Figure for abstract: none

Description

Title of the invention: Process for preparing Cl-esterase inhibitor

[0001] The invention relates to a process for preparing a Cl-esterase inhibitor, which includes an affinity chromatography step. The product (i.e., the concentrate) obtained is intended for therapeutic use.

[0002] Cl-esterase inhibitor or Cl-inhibitor (Cl-INH) is a protease inhibitor present in plasma that controls the activation of the first component of complement, Cl, and inactivates its subcomponents Clr and Cls. It also inhibits other serine proteases involved in coagulation, fibrinolytic, and contact systems, such as kallikrein, factor Xa and factor X11, as well as thrombin. It is also involved in the regulation of the kinin contact system.

[0003] Human plasma Cl-INH is a simple, glycosylated polypeptide chain with a molecular weight of 104 kDa.

[0004] Cl-INH deficiencies are associated with pathologies of varying severity, some of which can even be fatal. Patients with hereditary angioedema either lack Cl-INH (type I) or have Cl-INH with reduced activity (type II). Another form (type III) of hereditary angioedema is due to the abnormal binding of albumin to Cl-INH. Acquired Cl-INH deficiencies also exist, caused by abnormally high catabolism of the inhibitor or by the presence of autoantibodies. The disease manifests itself through edema of the face and extremities, the intestinal wall and the upper respiratory tract.

[0005] Various treatments are administered prophylactically to reduce the severity of attacks, using antifibrinolytic agents or hormones, but these are not effective against acute attacks and, moreover, they cause side effects incompatible with long-term treatment. For acute attacks, treatments with kallikrein inhibitors, such as aprotinin, are used, but these can cause allergies.

[0006] Replacement therapy with purified Cl-INH appears to be the treatment of choice for acute attacks as well as for long-term therapy or preoperative prophylaxis.

[0007] Furthermore, Cl-INH can be used as such (thanks to its protease inhibitor activity), but also as an excipient, to ensure the stability of therapeutic plasma products.

[0008] Currently some partially purified concentrates are available but all the preparation processes described are difficult to adapt to an industrial scale, either because of implementation difficulties or because of the cost of the materials to be used.

[0009] In all these processes, toxic stabilizers are added to the buffers to protect the activity of Cl-INH. Large-scale production processes have been developed over time, and involve in particular at least one ion-exchange chromatography step, such as DEAE-Sephadex® and / or CM-Sephadex®, followed by precipitation.

[0010] These various processes all include precipitation steps which must be followed by centrifugation; these are not very selective, difficult to implement on a large scale and have relatively low yields.

[0011] To circumvent these difficulties, the Applicant has succeeded in developing a process which does not include a precipitation step, which is easy to implement and which has a satisfactory yield.

[0012] Thus the process according to the present invention includes an affinity chromatography step.

[0013] The Cl-INH thus obtained can be used in therapy, or used as an excipient, in particular to ensure the stability of therapeutic plasma products.

[0014] The present invention therefore relates to a process for preparing a Cl-esterase inhibitor comprising the following steps:

[0015] a) obtaining a starting fraction chosen from the plasma fractions;

[0016] b) purification of said starting fraction obtained in a) by chromatography of affinity, said affinity chromatography comprising an elution step with an elution buffer having a pH between 4.0 and 8.0; and

[0017] c) recovery of the eluate after chromatography b), and this eluate corresponds to the fraction comprising the Cl-esterase inhibitor.

[0018] The process may also include a viral inactivation and / or elimination step.

[0019] Preferably, the process according to the invention comprises, after step c), a step d) of concentration and formulation of the eluate obtained in c).

[0020] Preferably, the process according to the invention comprises, after step d), a step e) of filtering the concentrated and formulated eluate obtained in d).

[0021] Preferably, the process according to the invention comprises, after step e), a step f) of storing the filtered eluate obtained in e).

[0022] The term "plasma fraction" means plasma, as well as any part or sub-part of plasma, that has undergone one or more purification steps. Plasma fractions thus include cryoprecipitated plasma supernatant (cryosupernatant), resuspended plasma cryoprecipitate, and fractions I to V obtained by ethanolic fractionation (according to the Cohn or Kistler & Nitschmann method or their derived methods), the supernatant and precipitate obtained after precipitation with caprylic acid and / or caprylate, chromatography eluates and unadsorbed fractions from chromatography columns, including multicolumn chromatography, and filtrates (i.e., or permeates, which are the fractions not retained after filtration, as opposed to retentates).

[0023] According to the invention, the plasma fraction is obtained: - directly from the plasma; or - after a single purification step, the plasma fraction is then either the cryoprecipitated plasma supernatant (cryosupernatant) or the cryoprecipitate; or - after several purification steps carried out on the cryoprecipitated plasma supernatant (cryosupernatant) or on the cryoprecipitate.

[0024] Thus, according to the invention, the plasma fraction obtained after several purification steps can be, in particular: - One of the fractions I to V obtained by ethanolic fractionation (according to the Cohn or Kistler & Nitschmann method or their derived methods) of a cryoprecipitate plasma supernatant (cryosupernatant) or by ethanolic fractionation (according to the Cohn or Kistler & Nitschmann method or their derived methods) of a resuspended cryoprecipitate, - One of the eluates from chromatography and non-adsorbed fractions from chromatography columns, including multi-column chromatography, the chromatography being carried out on a cryoprecipitated plasma supernatant (cryosupernatant) or on a resuspended cryoprecipitate, - One of the filtration filtrates or retentates, the filtration being carried out on a cryoprecipitated plasma supernatant (cryosupernatant) or on a resuspended cryoprecipitate, - One of the eluates from chromatography and non-adsorbed fractions of chromatography columns, including multi-column chromatography, the chromatography being carried out on fractions I to V obtained by ethanolic fractionation (according to the Cohn or Kistler & Nitschmann method or their derived methods) of a cryoprecipitate supernatant (cryosupernatant) or by ethanolic fractionation (according to the Cohn or Kistler & Nitschmann method or their derived methods) of a resuspended cryoprecipitate, or - any other combination of purification steps.

[0025] Preferably, the plasma fraction is the cryosupernatant.

[0026] According to the invention, the "cryoprecipitated plasma supernatant," or "cryosupernatant," corresponds to the liquid phase obtained after thawing frozen plasma (cryoprecipitation). In particular, the cryosupernatant can be obtained by freezing blood plasma at a temperature between -10°C and -40°C, then gently thawing it at a temperature between 0°C and +6°C, preferably between 0°C and +1°C, followed by centrifuging the thawed plasma to separate the cryoprecipitate and the cryosupernatant. The cryoprecipitate is concentrated in fibrinogen, fibronectin, von Willebrand factor, and factor VIII, while the cryosupernatant contains complement factors, vitamin K-dependent factors such as protein C, protein S, protein Z, factor II, factor VII, factor IX, and factor X, fibrinogen, immunoglobulins, and albumin.

[0027] By "purification step" is meant any step in a process enabling the enrichment of a product of interest, and in particular a plasma protein, in a given fraction.

[0028] Preferably, the plasma according to the invention is human plasma. Step a)

[0029] Step a) includes obtaining a starting fraction selected from the plasma fractions.

[0030] Preferably, the starting fraction is a cryoprecipitated plasma supernatant (cryosupernatant).

[0031] Preferably, the starting fraction chosen from among the plasma fractions is a cryosupernatant. In this case, preferably, the cryosupernatant is subjected to a filtration step. The filter used preferably has a pore size ranging from 0.1 to 0.4 pm, preferably from 0.2 to 0.4 pm, preferably from 0.25 to 0.35 pm, and preferably from 0.3 pm.

[0032] In a particular embodiment of the invention, the starting fraction selected from the plasma fractions is a filtrate obtained by filtration-adsorption of the cryosupernatant. Typically, the filtrate obtained by filtration-adsorption of the cryosupernatant is devoid of Factor XI, which is retained on the depth filtration filter (retentate), and can be used as the starting fraction for a Factor XL purification process. Preferably, the filtration-adsorption is carried out on a depth filtration filter. The filter used is typically composed of cellulose, with diatomaceous earth as a filtration aid, in particular perlite. In a preferred embodiment, the filter used is a Sartoclear® DL20 filter (marketed by Sartorius Stedim Biotech GmbH). Other comparable commercially available filters can also be used.

[0033] In this particular embodiment, step a) includes a depth filtration step by passing the cryosurnageant over the filter.

[0034] This filtration step removes the majority of plasma proteins. Step b)

[0035] The starting fraction obtained in a) is subjected to an affinity chromatography step; this is step b).

[0036] Affinity chromatography consists of binding Cl-INH to an affinity chromatography resin comprising a ligand capable of binding specifically to a domain of Cl-INH, and then collecting this Cl-INH.

[0037] It is preferably carried out at room temperature (20-25°C).

[0038] This affinity chromatography of step b) can be carried out in one cycle or in several cycles. In the latter case, the Cl-INH obtained is pooled before being subjected to the subsequent steps of the process according to the invention.

[0039] The affinity chromatography of step b) according to the invention includes an elution step with an elution buffer having a pH between 4.0 and 8.0.

[0040] More specifically, preferably, step b) includes the following substeps:

[0041] bl) loading the column comprising an affinity chromatography resin with the starting fraction treated in b);

[0042] b2) chromatographic separation and removal of the filtrate; and

[0043] b3) elution.

[0044] Step c) then includes the recovery of the eluate, i.e. the fraction containing the Cl-INH.

[0045] Preferably, the affinity chromatography resin comprises a ligand including a camelid-derived variable domain (VHH) antibody fragment. In another particular embodiment, the ligand may comprise an aptamer.

[0046] Preferably, the affinity chromatography resin comprises a ligand including a camelid-derived variable-domain VHH antibody fragment. Preferably, the ligand is a VHH antibody fragment. Preferably, the resin comprises a matrix selected from a cross-linked poly(styrene-divinylbenzene) matrix and an agarose-based matrix. Preferably, the matrix is ​​in the form of beads.

[0047] Preferably, the resin comprises a cross-linked poly(styrene-divinylbenzene) matrix. Such a matrix is, for example, marketed by Thermo Fisher Scientific under the name POROS or POROS CaptureSelect™ Cl InhibitorXL Affinity Matrix. Preferably, the cross-linked poly(styrene-divinylbenzene) matrix is ​​in the form of medium-sized beads in a quantity of between 40 and 60 µm. Typically, the cross-linked poly(styrene-divinylbenzene) matrix beads are very These robust, incompressible tubes guarantee high flow rates and a linear pressure increase regardless of the flow rate. They are compatible with demanding feed streams such as plasma. Finally, they are chemically stable, allowing for rigorous cleaning.

[0048] An agarose-based matrix is ​​for example marketed by Thermo Fisher Scientific under the OEM reference Agarose or CaptureSelect™ Affinity Resin.

[0049] Preferably, the resin comprises a ligand capable of binding specifically to Cl-INH or to one of its fragments, preferably human, the ligand being conjugated to the resin.

[0050] Preferably, the resin comprises a ligand comprising a VHH antibody fragment capable of binding specifically to Cl-INH or to one of its fragments, preferably human, said VHH fragment being conjugated to a cross-linked poly(styrene-divinylbenzene) matrix or conjugated to an agarose-based matrix.

[0051] Typically, the ligand comprising a fragment of VHH antibody capable of binding specifically to Cl-INH or to one of its fragments has a molecular weight between 12 and 15 kDa (1 Da = 1 g / mol).

[0052] In another particular embodiment, the affinity chromatography resin comprises a ligand including an aptamer, and in particular a nucleic acid aptamer. The term "aptamer" as used here refers to a single-stranded nucleic acid molecule, DNA or RNA, and in particular a single-stranded nucleic acid molecule capable of binding specifically to the protein of interest, i.e., Cl-INH or one of its fragments, preferably human. Aptamers generally comprise between 5 and 120 nucleotides and can be selected in vitro by a process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment).

[0053] Aptamers have many advantages. Due to their oligonucleotide nature, aptamers have low immunogenicity and significant resistance to stringent physicochemical conditions (i.e., presence of DMSO, very acidic or very basic pH, use of organic solvents and / or high temperature), allowing for various strategies in the context of use as an affinity ligand.

[0054] French patent application FR 2 970 003, filed in the name of the Applicant, describes methods for manufacturing affinity supports with immobilized nucleic acid aptamers. In particular, this application describes a method for immobilizing nucleic acids comprising at least one reactive amine function, by grafting them onto a solid support having activated carboxylic acid groups on its surface.

[0055] Before loading the column with fraction a), the column is preferably balanced with a balancing buffer. Thus, preferably, at the beginning of step bl), The column containing an affinity chromatography resin is balanced with an equilibration buffer, then the balanced column is loaded with fraction a).

[0056] The balancing buffer is preferably an aqueous solution with a pH between 6 and 8, preferably between 6.5 and 7.7. The balancing buffer preferably comprises at least TRIS (tris(hydroxymethyl)aminomethane), preferably at a concentration between 40 and 60 mM, preferably between 45 and 55 mM. It has an osmolality between 80 and 400 mOsm / kg, preferably between 85 and 350 mOsm / kg.

[0057] Preferably, according to a first alternative, the balancing buffer is an aqueous solution with a pH between 6.5 and 7.7, preferably between 7.3 and 7.7, comprising at least TRIS in a concentration between 45 and 55 mM, and having an osmolality between 80 and 100 mOsm / kg, preferably between 85 and 95 mOsm / kg.

[0058] Preferably, according to a second alternative, the balancing buffer is an aqueous solution of pH between 6.5 and 7.7 comprising at least TRIS in a concentration between 45 and 55 mM and NaCl in a concentration between 100 and 150 mM, and having an osmolality between 200 and 400 mOsm / kg, preferably between 250 and 350 mOsm / kg.

[0059] Typically, the column is balanced with at least 4 volumes of balancing buffer, preferably 5 volumes.

[0060] At the end of step bl), the column is loaded with the starting fraction a).

[0061] Preferably, after step 1b) and before step b2), the affinity chromatography column loaded with the filtrate is washed. The wash buffer is the same as the equilibration buffer described above. Thus, preferably, the wash buffer is an aqueous solution with a pH between 6 and 8, preferably between 6.5 and 7.5; it preferably comprises at least TRIS, preferably in a concentration between 40 and 60 mM, preferably between 45 and 55 mM, and has an osmolality between 80 and 400 mOsm / kg, preferably between 85 and 350 mOsm / kg.

[0062] Then the fraction loaded onto the column is purified by chromatography; this is substep b2).

[0063] After chromatographic separation, the resin is washed to remove contaminants not bound to the resin. For example, the method includes washing the resin with a wash buffer before collecting the Cl-INH.

[0064] Affinity chromatography, by its function, allows it to act here also as a viral elimination step and can therefore also be used for biological safety.

[0065] Then the elements retained on the resin in substep b2) are eluted using a buffer to break the bond between the ligand and the Cl-INH; this is the elution (substep b3).

[0066] The elution buffer according to the invention has a pH between 4.0 and 8.0.

[0067] Different types of buffers can be used for elution. In particular, elution can be obtained at acidic, neutral or slightly basic pH, and buffers using different bases can therefore be used for elution.

[0068] Preferably, the elution buffer has a pH between 5.0 and 8.0, preferably between 5.8 and 8.0.

[0069] Advantageously, a TRIS buffer is used. Advantageously, the TRIS buffer is used at a molarity of 5 to 100 mM, advantageously from 10 to 90 mM, from 20 to 80 mM, from 30 to 70 mM, from 40 to 60 mM, from 45 to 55 mM, and particularly about 50 mM. Advantageously, the elution buffer comprises at least one electrolyte, preferably magnesium chloride (MgCl2), advantageously at a molarity of 1 to 5 M, preferably from 1.5 to 3 M. Optionally, the elution buffer also comprises a polyol, preferably a diol, preferably selected from C2 to C5 diols. The polyol is preferably selected from glycerin, diglycerin, ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, butylene glycol, pentylene glycol, hexylene glycol, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol and polyethylene glycols, in particular having 5 to 50 ethylene oxide groups and mixtures thereof.Preferably, the polyol is not propylene glycol, as this compound is less desirable for the purity and quality of the Cl-INH obtained.

[0070] Preferably, the elution buffer is a solution comprising 50 mM TRIS and MgCl22 M, optionally 20% by weight relative to the total weight of polyol buffer, and having a pH between 5.0 and 8.0. More preferably, the elution buffer is a solution comprising 50 mM TRIS and MgCl22 M, optionally 20% by weight relative to the total weight of polyol buffer, and having a pH between 7.0 and 8.0.

[0071] Preferably, alternatively, the elution buffer is a solution comprising 50 mM TRIS, MgCl22 M and 20% by weight relative to the total weight of polyol buffer, and having a pH between 5.8 and 8.0.

[0072] Preferably, alternatively, the elution buffer is a solution comprising 11.5 mM disodium phosphate (Na2HPO4), 7.5 mM monosodium phosphate (NaH2PO4) and an electrolyte, preferably 90 M NaCl, and having a pH between 4.0 and 8.0.

[0073] Preferably, between steps b2) and b3) (i.e. just before elution), at least one pre-elution step is added.

[0074] This pre-elution makes it possible in particular to improve the purity of the Cl-INH obtained. One or more pre-elutions may be added.

[0075] Different types of buffers can be used for pre-elution. Preferably, the pre-elution buffer has a pH between 5.0 and 8.0, preferably between 6.0 and 8.0. If several pre-elutions are carried out, the pre-elution buffers can be the same or different, preferably different.

[0076] Advantageously, the pre-elution buffer comprises at least one electrolyte, preferably selected from magnesium chloride (MgCl2), advantageously at a molarity of 0.1 to 2 M, preferably 0.2 to 1 M, sodium chloride (NaCl), advantageously at a molarity of 0.5 to 3 M, preferably 0.7 to 2 M, and mixtures thereof.

[0077] Advantageously, the pre-elution buffer comprises TRIS. Advantageously, the TRIS buffer is used at a molarity of 5 to 100 mM, advantageously from 10 to 90 mM, from 20 to 80 mM, from 30 to 70 mM, from 40 to 60 mM, from 45 to 55 mM and in particular from about 50 mM.

[0078] Preferably, the pre-elution buffer is a solution comprising 20.25 M MgCl and / or 1 M NaCl, and having a pH between 5.0 and 8.0. Preferably, the pre-elution buffer is a solution comprising 50 mM TRIS, 20.25 M MgCl and 1 M NaCl, and having a pH between 5.0 and 8.0. More preferably, the pre-elution buffer is a solution comprising 50 mM TRIS, 20.25 M MgCl and 1 M NaCl, and having a pH between 7.0 and 8.0.

[0079] Preferably, between steps b2) and b3) (i.e. just before elution), two pre-elution steps are added.

[0080] Advantageously, the buffer for the first pre-elution comprises at least one electrolyte, preferably magnesium chloride (MgCl2), advantageously at a molarity of 0.1 to 2 M, preferably 0.2 to 1 M.

[0081] Advantageously, the buffer for the second pre-elution comprises at least one electrolyte, preferably sodium chloride (NaCl), advantageously at a molarity of 0.5 to 3 M, preferably 0.7 to 2 M. Preferably, the buffer for the first pre-elution is a solution comprising 20.25 M MgCl and having a pH between 5.0 and 8.0. Preferably, the buffer for the first pre-elution is a solution comprising 50 mM TRIS, 20.25 M MgCl, and having a pH between 5.0 and 8.0 (preferably about 7.5).

[0082] Preferably, the buffer for the second pre-elution is a solution comprising 1 M NaCl.

[0083] At the end of the elution, the fraction containing Cl-INH is recovered; this is step c).

[0084] Thus, at the end of step c), at least one eluate comprising Cl-INH is obtained. Biological security

[0085] Preferably, according to a first embodiment, the process according to the invention comprises one or more biological safety steps. This biological safety step may take place between steps a) and b), and / or after step b); preferably, it takes place after step b), and / or preferably on the eluate recovered in step c).

[0086] According to another embodiment, the process according to the invention does not contain a viral inactivation or elimination step. In this case, typically, as indicated above, affinity chromatography, by its very function, also acts as a viral elimination step and can therefore also be used for biosafety.

[0087] The biological safety step aims to inactivate or eliminate infectious agents such as viruses and unconventional transmissible agents (UTAs) like prions, by inactivation or elimination. Viral inactivation often involves treatment with chemicals, for example with solvents and / or detergents, and / or with heat (pasteurization and / or dry heating) and / or with irradiation (Gamma and / or UVC) and / or with pH treatment (acidic pH treatment).

[0088] Preferably, the viral inactivation step includes at least one solvent and detergent treatment. The solvent and detergent treatment (generally referred to as Solvent / Detergent or S / D treatment) includes, in particular, treatment with tri-n-butylphosphate (TnBP) and / or a detergent selected from Triton X-100, Tween (preferably Tween 80), sodium cholate, and 2-[4-(2,4,4-trimethylpentane-2-yl)phenoxy]ethanol (Octoxinol). The viral inactivation step is preferably carried out for a duration of at least 8 hours. After a viral inactivation step by S / D treatment, it is typically important to add a step to remove the S / D by ion-exchange or affinity chromatography, preferably ion-exchange.

[0089] Viral elimination can be achieved by filtration (typically by filtration, depth filtration, nanofiltration...) or by partitioning during a purification step.

[0090] Nanofiltration can also be used to remove infectious agents, including viruses and T-cell antigens (TAAs). In the case of plasma proteins, nanofiltration generally refers to the filtration of the protein concentrate of interest through a filter with a pore size of less than 80 nm. Available filters include, for example, BioEx, Planova® 75 nm, Planova® 35 nm, Planova® 20 nm, or Planova® 15 nm (Asahi Corporation), Ultipor DV 50 or DV 20 (Pall Corporation), Virosart CPV (Sartorius), and Viresolve NFR or NFP (Millipore). Nanofiltration can advantageously be carried out on a single filter or on several filters in series of identical or decreasing grade (i.e. pore size).

[0091] The elimination of infectious agents can also be achieved by means of depth filtration. The available filters are, for example, filters composed of regenerated cellulose, to which filtration aids may have been added (such as cellite, perlite or kieselguhr earth) marketed by Cuno (Zeta+ VR series filters), Pall-Seitz (P-series Depth Filter) or Sartorius (Virosart CPV, Sartoclear P or Sartoclear DL depth filters).

[0092] According to the invention, the viral inactivation and / or elimination step is carried out directly on the eluate recovered in step c). Step d)

[0093] The process according to the invention may advantageously include one or more steps for concentrating and / or formulating the eluate obtained in c), optionally subjected to a viral inactivation or elimination step. Preferably, the concentration is carried out by ultrafiltration. For example, the ultrafiltration is membrane ultrafiltration.

[0094] It is also possible to provide for a step of adding one or more pharmaceutically acceptable stabilizers to the eluate obtained in c).

[0095] By pharmaceutically acceptable stabilizers, we mean formulations suitable for plasma protein concentrates, in particular excipients as described in applications FR0308403, and preferably formulations suitable for inhibitor Cl concentrates. Step e)

[0096] Preferably, the process according to the invention comprises, after step d), a step e) of filtering the concentrated and formulated eluate obtained in d).

[0097] Preferably, the filtration is carried out on a membrane with a pore size between 0.1 and 0.5 pm, preferably between 0.1 and 0.3 pm, preferably about 0.2 pm.

[0098] After filtration, the eluate is packaged, for example in containers or bags. Preferably, it is packaged at a concentration between 10 and 100 IU / ml, preferably between 20 and 50 IU / ml. Step f)

[0099] Step e) of filtration may optionally be followed by a step of freezing or lyophilizing the eluate obtained in e). This allows storage.

[0100] Thus, preferably, the process according to the invention comprises, after step e), a step f) of storing the filtered eluate obtained in e).

[0101] Preferably, storage is carried out by freezing at a temperature below -30°C, preferably below -35°C, typically below -60°C, or even below -70°C.

[0102] At the end of the process, a yield of at least 50% of Cl-INH is typically obtained.

[0103] The Cl-INH obtained by the process according to the invention can be used in therapy, or used as an excipient, in particular to ensure the stability of therapeutic blood products. In particular, it can be used as an excipient in the production of blood-derived medicinal products, preferably in the production of factor VIII (as marketed under the name Factane) and / or factor XI (as marketed under the name Hemoleven).

[0104] Thus, the eluate obtained in c), or the concentrated and formulated eluate obtained in d), or the eluate obtained in e), or the eluate stored after step f), can be mixed with a solution of FVIII or a solution of FXI, and the resulting mixture is then subjected to a filtration step, preferably nanofiltration. The Cl-INH thus undergoes nanofiltration within the mixture, but not alone.

[0105] Such nanofiltration generally refers to the filtration of the protein concentrate of interest through a filter with a pore size of less than 80 nm. Available filters include, for example, BioEx, Planova® 75 nm, Planova® 35 nm, Planova® 20 nm, or Planova® 15 nm (Asahi Corporation), Ultipor DV 50 or DV 20 (Pall Corporation), Virosart CPV (Sartorius), and Viresolve NFR or NFP (Millipore). Nanofiltration can advantageously be performed on a single filter or on several filters in series with identical or decreasing pore sizes.

[0106] Thus the invention also relates to a method for preparing a therapeutic plasma product solution, preferably a solution of FVIII or FXI, comprising the aforementioned method according to the invention, then mixing the eluate obtained in c), or the concentrated and formulated eluate obtained in d), or the eluate obtained in e), or the eluate stored at the end of step f), with a therapeutic plasma product solution, preferably FVIII or FXI, then the resulting mixture is subjected to a filtration step, preferably nanofiltration.

[0107] Preferably, the FVIII or FXI solution is stabilized by the addition of Cl-inhibitor, and optionally antithrombin and / or heparin, and undergoes a viral removal step, preferably by filtration through a 15 nm pore size filter. The FVIII or FXI solution is then packaged and lyophilized. The stabilized FVIII or FXI solution is, in particular, packaged as a pharmaceutical product.

[0108] In a preferred embodiment, the Factor VIII or XI solution is stabilized by adding 0.5 to 2 IU, preferably 1 to 2 IU, preferably 1.5 to 2 IU of Cl-INH per 100 IU of Factor VIII or XI; and optionally by adding 0.5 to 3 IU, preferably 1 to 3 IU, preferably 2 to 3 IU of antithrombin III, and / or by the addition of 0.5 to 4 IU, preferably 1 to 4 IU, preferably 2 to 4 IU of heparin.

[0109] The invention is now illustrated by the following examples. Examples

[0110] In the following examples, unless otherwise stated, the raw material (comprising the Cl-esterase inhibitor or Cl-INH or Cl-Inh or "Cl-inhibitor") is derived from the cryosupernatant (step a).

[0111] It is purified (step b) by affinity chromatography, said affinity chromatography comprising an elution step with an elution buffer having a pH between 4.0 and 8.0 (invention) or around 3.0 (comparative).

[0112] The eluate after chromatography is recovered (step c), and this eluate corresponds to the fraction comprising the Cl-INH.

[0113] Comparative example: Purification of the Ci-Inhibitor _ by a comparative process

[0114] Results of the trial on Gel POROS CaptureSelect™ Cl-InhXL Affinity Matrix

[0115] [Table 1] Raw Material Filtrate Sartoclear DL20 Starting Concentration: 0.16 g / L Cl Inh (obtained from cryosurnageant) Load 113 mL / 5 mL of gel 18.1 mg Cl Inh 3.62 g Cl Inh / L of gel Filtrate 150 mL at < 0.005 g / L Cl Inh < 0.75 mg Cl Inh Loss < 4% Gel Capacity > 3.5 g Cl Inh / L of gel Eluate Elution Buffer: Glycine 0.1 M adjusted to pH 3 with HCl pH 3.2 20 mL at 0.11 g / L Cl Inh 2.2 mg Cl Inh Yield = 12% Neutralization after elution with NaOH 0.1 M pH 8.2 20 mL at 0.14 g / L Cl Inh 2.8 mg Cl Inh Yield = 15.5% Conclusion :

[0116] Good fixation of Cl-INH to this gel.

[0117] Desorption of Cl-INH fixed on the gel is difficult; or the acid elution conditions (pH 3) are too drastic for the protein and its assay.

[0118] The yield is low.

[0119] Example 1: Purification of the Cl-Inhibitor by a process according to the invention

[0120] Results of the trial on Gel POROS N°2 CaptureSelect™ Cl-InhXL Affinity Matrix

[0121] [Tables2] Raw Material Filtrate Sartoclear DL20 Starting Concentration: 210 mg / L or 0.64 IU / mL Cl-INH Load 150 mL / 7 mL of gel 31.5 mg Cl-INH 4.5 g Cl-INH / L of gel 150 mL / 7 mL of gel 96 IU Cl-INH 13,700 IU Cl-INH / L of gel Filtrate 220 mL at 6.1 mg / L Cl-INH 1.34 mg Cl-INH Loss = 4% Gel Capacity = 4.3 g Cl-INH / L of gel ND Eluate Buffer Elution: TRIS 50 mM, MgCl2 2 M, Propylene Glycol 20% pH between 5.0 and 8.0 18 mL at 1.6 g / L Cl-INH 28.8 mg Cl-INH Yield = 91% 18 mL at 4.6 IU / mL Cl INH 82.8 IU Cl INH Yield = 86% Conclusion :

[0122] Good binding of Cl-INH to this gel. The capacity of the gel is calculated at 4.3 g Cl-INH / L of gel and 11,800 IU Cl-INH / L of gel.

[0123] The elution of Cl-INH is very efficient with an excellent yield: 91% in antigen and 86% in activity.

[0124] Example 2: Purification of the Ci-Inhibitor by a process according to the invention comprising a pre-elution step STEP 1: Raw material preparation (step a)

[0125] Raw material = Cryosurnatant obtained by filtration on SARTOPORE 2 (pore size 0.45 pm + 0.2 pm).

[0126] The DEPART FILTRE product has a Cl-INH activity of 0.84 IU / ml and a Cl-INH level by nephelometry of 200 mg / l.

[0127] STEP 2: Affinity chromatography according to the invention (POROS, XTO; step b)

[0128] POROS™ CaptureSelect™ Cl-InhXL Affinity Matrix Gel.

[0129] Equilibration Buffer: TRIS 50 mM, NaCl 115 mM; pH 7.43; 295 mOsm / kg.

[0130] Pre-Elution Buffer: TRIS 50 mM, MgCl2 0.25 M; 1 M NaCl; pH 7.49; 2640 mOsm / kg.

[0131] Elution buffer: 50 mM TRIS, 2 M MgCl2; pH 7.56; 5450 mOsm / kg. Cl Inhibitor Monitoring

[0132] [Tables3] STAGE VOLUME Activity Cl Inh Quantity of activity Cl Inh Rate Cl Inh Nephele Quantity Cl Inh Nephele STARTING FILTER 420 mL 0.84 IU / mL 353 IU 200 mg / L 84 mg PRE-ELUATE 50 mL 1.0 IU / mL 50 IU (14%) 220 mg / L 11 mg (13%) ELUATE 33 mL 6.7 IU / mL 221 IU (63%) 1.4 mg / mL 46 mg (55%) Fibrinogen Monitoring

[0133] [Tables4] STAGE VOLUME Nephel Fibrinogen Level Quantity Nephel Fibrinogen STARTING FIBER 420 mL 1.7 g / L 714 mg PRE-ELUATE 50 mL <140 mg / L 28 mg / L 1.4 mg (0.2%) ELUATE 33 mL <140 mg / L (56 mg / L) <4.6 mg (<0.7%) (1.8 mg (0.3%))

[0134] Then we recover the eluate (step c).

[0135] The affinity chromatography step (step b) according to the invention) allows more than 99% of the fibrinogen to be removed from the fraction before XTO.

[0136] There are 8.5 g of fibrinogen per gram of Cl Inhibitor before the XTO step, but only 40 mg of fibrinogen per gram of Cl Inhibitor in the total eluate.

[0137] Example 3: Purification of the Ci-Inhibitor by a process according to the invention comprising two pre-elution steps STEP 1: Raw material preparation (step a)

[0138] Raw material = Cryosurnatant obtained by filtration on SARTOPORE 2 (pore size 0.45 pm + 0.2 pm).

[0139] STEP 2: Affinity chromatography according to the invention (POROS, XTO; step b)

[0140] POROS™ CaptureSelect™ Cl-InhXL Affinity Matrix Gel

[0141] The cryosupernatant of step a) is loaded onto the column in an amount of about 2.8g / L of gel.

[0142] Equilibration Buffer: TRIS 50 mM, NaCl 115 mM; pH 7.5.

[0143] Pre-Elution Buffer 1: 50 mM TRIS, 0.25 M MgCl2; pH 7.5.

[0144] Pre-Elution Buffer 2: NaCl 1 M.

[0145] Elution buffer: 50 mM TRIS, 2 M MgCl2; pH 7.5.

[0146] 3 elution cycles are carried out, then the Cl-INH are pooled (recovered, step c), and this eluate corresponds to the fraction containing Cl-INH. The yields are as follows:

[0147] [Tables5] Stages Cl-INH Yield Activity Nephele Cl-INH Yield Fibrinogen (mg Fg / g Cl-INH) POROS cycle 1 100% 87% 35 POROS cycle 2 67% 62% 75 POROS cycle 3 98% 86% 23 Conclusion :

[0148] The elution of Cl-INH is very efficient with an excellent yield.

[0149] Example 4: Purification of the Cl-Inhibitor by a process according to the invention comprising two pre-elution steps

[0150] The same process as in Example 3 is carried out on 3 batches of POROS gel.

[0151] The results are as follows:

[0152] [Tableauxô] Lot of gel 1 2 3 Static capacity supplier 4.6 g / L 3.83 g / L 3.51 g / L Load 3.4 g / L gel 13 kIU / L gel 3.4 g / L gel 12.5 kIU / L gel 3.0 g / L gel 13 kIU / L gel Cl-INH yield Activity 92% 95% 100% Cl-INH yield Nephele 88% 89% 93% Conclusion :

[0153] The elution of Cl-INH is very efficient with an excellent yield.

Claims

Demands

1. A process for preparing Cl-esterase inhibitor (Cl-INH) comprising the following steps: a) obtaining a starting fraction selected from plasma fractions; b) purifying said starting fraction obtained in a) by affinity chromatography, said affinity chromatography comprising an elution step with an elution buffer having a pH between 4.0 and 8.0; and c) recovering the eluate after chromatography b), and this eluate corresponds to the fraction comprising Cl-INH.

2. A process according to claim 1, comprising, after step c), a step d) of concentration and formulation of the eluate obtained in c), preferably the concentration is done by ultrafiltration.

3. A process according to claim 2, which comprises, after step d), a step e) of filtering the concentrated and formulated eluate obtained in d).

4. A method according to claim 3, comprising, after step e), a step f) of storing the filtered eluate obtained in e), preferably after freezing or freeze-drying the eluate obtained in e).

5. A process according to any one of the preceding claims, wherein the plasma fractions of step a) are obtained (i) directly from the plasma; or (ii) after a single purification step, the plasma fraction then being either the cryoprecipitated plasma supernatant or the cryoprecipitate; or (iii) after several purification steps carried out on the cryoprecipitated plasma supernatant or on the cryoprecipitate.

6. A method according to any one of the preceding claims, wherein the starting fraction of step a) is a cryoprecipitated plasma supernatant.

7. A process according to any one of the preceding claims, comprising one or more biological safety steps; preferably said biological safety step comprising at least one treatment with chemicals and / or heat and / or irradiation and / or pH treatment.

8. A method according to any one of the preceding claims, wherein step b) comprises the following substeps: b1) loading of the column comprising an affinity chromatography resin with the starting fraction treated in b); b2) chromatographic separation and removal of the filtrate; and b3) elution.

9. A method according to claim 8, wherein the affinity chromatography resin comprises a ligand comprising a camelid-derived variable domain VHH antibody fragment, or a ligand comprising an aptamer.

10. A method according to any one of claims 8 or 9, wherein the resin comprises a matrix selected from a crosslinked poly(styrene-divinylbenzene) matrix and an agarose-based matrix.

11. A process according to any one of claims 8 to 10, wherein the elution in step b3) is carried out with an elution buffer having a pH between 5.0 and 8.0, and comprising a TRIS buffer and at least one electrolyte; optionally, the elution buffer also comprises a polyol, preferably selected from C2 to C5 diols.

12. A process according to claim 11, wherein the elution buffer is a solution comprising 50 mM TRIS and MgCl22 M and having a pH between 5.0 and 8.0, preferably between 5.8 and 8.0; or the elution buffer is a solution comprising 50 mM TRIS, MgCl22 M and 20% by weight relative to the total weight of polyol buffer, and having a pH between 5.8 and 8.

0.

13. A process according to any one of claims 8 to 12, wherein, between steps b2) and b3), at least one pre-elution step is added, preferably the pre-elution buffer has a pH between 5.0 and 8.0, and comprises at least one electrolyte selected from magnesium chloride (MgCl2), sodium chloride (NaCl), and mixtures thereof; preferably the pre-elution buffer comprises TRIS; advantageously two pre-elution steps are added between steps b2) and b3), and advantageously the buffer for the first pre-elution comprises at least MgCl2, and the buffer for the second pre-elution comprises at least NaCl.

14. A method for preparing a therapeutic plasma product solution, preferably a solution of FVIII or FXI, comprising the method according to any one of claims 1 or 5 to 13, and then mixing the eluate obtained in c), or the concentrated eluate and formulated obtained in d) according to claim 2, or of the eluate obtained in e) according to claim 3, or of the eluate stored at the end of step f) according to claim 4, with a therapeutic plasma product solution, preferably FVIII or FXI, and then the resulting mixture is subjected to a filtration step, preferably nanofiltration.