Method for preparing c1-esterase inhibitor

Abstract

The present invention relates to a method for preparing C1-esterase inhibitor (C1-INH) comprising the following steps: a) obtaining a starting fraction selected from plasma fractions; b) purifying said obtained starting fraction by affinity chromatography, said affinity chromatography comprising an elution step with an elution buffer having a pH of between 4.0 and 8.0; and c) recovering the eluate after chromatography step b), said eluate corresponding to the fraction comprising C1-INH.

Description

[0001] Process for preparing C1-esterase inhibitor

[0002] The invention relates to a process for preparing a C1-esterase inhibitor, which includes an affinity chromatography step. The product (i.e., the concentrate) obtained is intended for therapeutic use.

[0003] C1-esterase inhibitor, or C1 inhibitor (C1-INH), is a protease inhibitor present in plasma that controls the activation of the first complement component, C1, and inactivates its subcomponents C1r and C1s. It also inhibits other serine proteases involved in coagulation, fibrinolytic, and contact systems, such as kallikrein, factor Xa, and factor XIa, as well as thrombin. It is also involved in the regulation of the kinin contact system.

[0004] Human plasma C1-INH is a simple, glycosylated polypeptide chain with a molecular weight of 104 kDa.

[0005] C1-INH deficiencies are associated with pathologies of varying severity, some of which can be fatal. Patients with hereditary angioedema either lack C1-INH (type I) or have C1-INH with reduced activity (type II). Another form (type III) of hereditary angioedema is due to the abnormal binding of albumin to C1-INH. Acquired C1-INH deficiencies also exist, caused by abnormally high catabolism of the inhibitor or by the presence of autoantibodies. The disease manifests as edema of the face and extremities, the intestinal wall, and the upper respiratory tract.

[0006] Various treatments are administered prophylactically to reduce the severity of attacks, including antifibrinolytic agents or hormones, but these are ineffective against acute attacks and, moreover, cause side effects incompatible with long-term treatment. For acute attacks, treatments with kallikrein inhibitors, such as aprotinin, are used, but these can cause allergic reactions.

[0007] Replacement therapy with purified C1-INH appears to be the treatment of choice for acute attacks, as well as for long-term therapy and preoperative prophylaxis. Furthermore, C1-INH can be used on its own (due to its protease inhibitor activity) and 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 C1-INH. Large-scale production processes have been developed over time, and involve 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 that does not include a precipitation step, is easy to implement and offers a satisfactory yield.

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

[0013] The C1-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 C1-esterase inhibitor comprising the following steps: a) obtaining a starting fraction selected from the 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 the C1-esterase inhibitor.

[0015] The process may also include a viral inactivation and / or elimination step. Preferably, the process according to the invention includes, after step c), a step d) of concentration and formulation of the eluate obtained in c).

[0016] Preferably, the process according to the invention comprises, after step d), a step e) of filtering the concentrated and formulated eluate obtained in d).

[0017] Preferably, the process according to the invention includes, after step e), a step f) of storing the filtered eluate obtained in e).

[0018] The term "plasma fraction" refers to plasma and 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, fractions I to V obtained by ethanolic fractionation (according to the Cohn or Kistler & Nitschmann method or their derivatives), 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).

[0019] According to the invention, the plasma fraction is obtained: directly from the plasma; or after a single purification step, the plasma fraction then being 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.

[0020] Thus, according to the invention, the plasma fraction obtained after several purification steps can be, in particular:

[0021] 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,

[0022] 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.

[0023] One of the eluates of 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 method of Cohn or Kistler & Nitschmann or their derived methods) of a cryoprecipitated plasma supernatant (cryosupernatant) or by ethanolic fractionation (according to the method of Cohn or Kistler & Nitschmann or their derived methods) of a resuspended cryoprecipitate, or any other combination of purification steps.

[0024] Preferably, the plasma fraction is the cryosupernatant.

[0025] According to the invention, the "cryoprecipitated plasma supernatant," or "cryosupernatant," corresponds to the liquid phase obtained after thawing frozen plasma (cryoprecipitation). Specifically, 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 centrifugation of 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.

[0026] By "purification step" we mean any step in a process that allows the enrichment of a product of interest, and in particular a plasma protein, in a given fraction.

[0027] Preferably, the plasma according to the invention is human plasma.

[0028] Step a) involves obtaining a starting fraction chosen from the plasma fractions. Preferably, the starting fraction is a cryoprecipitated plasma supernatant (cryosupernatant).

[0029] 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.

[0030] 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 free of Factor XI, which is retained on the depth filtration filter (retentate), and can be used as the starting fraction for a Factor XI 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, in particular perlite, as a filtration aid. 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.

[0031] In this particular embodiment, step a) includes a depth filtration step by passing the cryosurnageant over the filter.

[0032] This filtration step removes the majority of plasma proteins.

[0033] Step b)

[0034] The starting fraction obtained in a) is subjected to an affinity chromatography step; this is step b).

[0035] Affinity chromatography involves binding C1-INH to an affinity chromatography resin comprising a ligand capable of binding specifically to a domain of C1-INH, and then collecting this C1-INH.

[0036] It is preferably carried out at room temperature (20-25°C).

[0037] This affinity chromatography in step b) can be performed in one cycle or in several cycles. In the latter case, the C1-INH obtained is pooled before being subjected to the subsequent steps of the process according to the invention. The affinity chromatography in step b) according to the invention includes an elution step with an elution buffer having a pH between 4.0 and 8.0.

[0038] More specifically, preferably, step b) includes the following substeps: b1) loading 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.

[0039] Step c) then includes the recovery of the eluate, i.e. the fraction containing the C1-INH.

[0040] 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.

[0041] 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.

[0042] 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™ C1 InhibitorXL Affinity Matrix. Preferably, the cross-linked poly(styrene-divinylbenzene) matrix is ​​in the form of medium-sized beads with a count between 40 and 60 µm. Typically, cross-linked poly(styrene-divinylbenzene) matrix beads are very robust, incompressible, and ensure 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 thorough cleaning.

[0043] For example, an agarose-based matrix is ​​marketed by Thermo Fisher Scientific under the OEM reference Agarose or CaptureSelect™ Affinity Resin.

[0044] Preferably, the resin comprises a ligand capable of binding specifically to C1-INH or one of its fragments, preferably human, the ligand being conjugated to the resin.

[0045] Preferably, the resin comprises a ligand including a VHH antibody fragment capable of binding specifically to C1-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.

[0046] Typically, the ligand comprising a VHH antibody fragment capable of binding specifically to C1-INH or one of its fragments has a molecular weight between 12 and 15 kDa (1 Da = 1 g / mol).

[0047] 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., C1-INH or one of its fragments, preferably human. Aptamers generally comprise between 5 and 120 nucleotides and can be selected in vitro by a method known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment).

[0048] Aptamers offer numerous advantages. Due to their oligonucleotide nature, aptamers possess 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 diverse strategies in the context of use as an affinity ligand.

[0049] 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.

[0050] Before loading the column with fraction a), the column is preferably equilibrated with an equilibrating buffer. Thus, preferably, at the beginning of step b1), the column containing an affinity chromatography resin is equilibrated with an equilibrating buffer, and then the equilibrated column is loaded with fraction a).

[0051] 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.

[0052] 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.

[0053] Preferably, according to a second alternative, the balancing buffer is an aqueous solution with a 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.

[0054] Typically, the column is balanced with at least 4 volumes of balancing buffer, preferably 5 volumes.

[0055] At the end of step b1), the column is loaded with the starting fraction a).

[0056] Preferably, after step b1) 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.

[0057] Then the fraction loaded onto the column is purified by chromatography; this is sub-step b2).

[0058] After chromatographic separation, the resin is washed to remove non-resin-bound contaminants. For example, the method involves washing the resin with a wash buffer before collecting the C1-INH.

[0059] 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.

[0060] Then, the elements retained on the resin in substep b2) are eluted using a buffer that breaks the bond between the ligand and C1-INH; this is the elution (substep b3). The elution buffer according to the invention has a pH between 4.0 and 8.0. Different types of buffers can be used for the elution. In particular, the elution can be carried out at acidic, neutral, or slightly basic pH, and buffers using different bases can therefore be used for the elution.

[0061] Preferably, the elution buffer has a pH between 5.0 and 8.0, preferably between 5.8 and 8.0.

[0062] Advantageously, a TRIS buffer is used. Advantageously, the TRIS buffer is used at a molarity of 5 to 100 mM, advantageously 10 to 90 mM, 20 to 80 mM, 30 to 70 mM, 40 to 60 mM, 45 to 55 mM, and especially 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 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 C1-INH obtained.

[0063] Preferably, the elution buffer is a solution comprising 50 mM TRIS and 2 M MgCh, 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 2 M MgCh2, optionally 20% by weight relative to the total weight of polyol buffer, and having a pH between 7.0 and 8.0.

[0064] Preferably, alternatively, the elution buffer is a solution comprising 50 mM TRIS, 2 M MgCh and 20% by weight relative to the total weight of polyol buffer, and having a pH between 5.8 and 8.0.

[0065] 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.

[0066] Preferably, between steps b2) and b3) (i.e. just before elution), at least one pre-elution step is added.

[0067] This pre-elution helps to improve the purity of the C1-INH obtained. One or more pre-elutions may be added.

[0068] 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 performed, the pre-elution buffers can be the same or different, preferably different.

[0069] 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.

[0070] 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 especially from about 50 mM.

[0071] Preferably, the pre-elution buffer is a solution comprising 0.25 M MgCh 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, 0.25 M MgCh 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, 0.25 M MgCh and 1 M NaCl, and having a pH between 7.0 and 8.0.

[0072] Preferably, between steps b2) and b3) (i.e. just before elution), two pre-elution steps are added.

[0073] 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.

[0074] 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 0.25 M MgCh 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, 0.25 M MgCh, and having a pH between 5.0 and 8.0 (preferably about 7.5).

[0075] Preferably, the buffer for the second pre-elution is a solution containing 1 M NaCl.

[0076] At the end of the elution, the fraction containing C1-INH is recovered; this is step c).

[0077] Thus, at the end of step c), at least one eluate containing C1-INH is obtained. Biological safety

[0078] 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).

[0079] According to another embodiment, the process according to the invention does not include 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.

[0080] The biological safety step aims to inactivate or eliminate infectious agents such as viruses and unconventional transmissible agents (UTAs) like prions, through inactivation or elimination. Viral inactivation often involves treatment with chemicals, for example, solvents and / or detergents, and / or heat (pasteurization and / or dry heating), and / or irradiation (gamma and / or UVC), and / or pH treatment (acidic pH treatment).

[0081] Preferably, the viral inactivation step includes at least one solvent and detergent treatment. 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.

[0082] Viral removal can be achieved by filtration (typically by filtration, depth filtration, nanofiltration...) or by partitioning during a purification step.

[0083] 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 filtering the protein concentrate of interest through a filter with a pore size of less than 80 nm. Examples of available filters include BioEx, Planova® 75 nm, Planova® 35 nm, Planova® 20 nm, or Planova® 15 nm (Asahi Corporation), Ultipor DV 50 or DV 20 (Pali Corporation), Virosart CPV (Sartorius), and Viresolve NFR or NFP (Millipore). Nanofiltration can advantageously be performed on a single filter or on multiple filters in series of the same or decreasing pore size.

[0084] The elimination of infectious agents can also be achieved by means of depth filtration. Available filters include, for example, filters made 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).

[0085] According to the invention, the viral inactivation and / or elimination step is carried out directly on the eluate recovered in step c).

[0086] Step d)

[0087] 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.

[0088] It is also possible to include a step of adding one or more pharmaceutically acceptable stabilizers to the eluate obtained in c).

[0089] Pharmaceutically acceptable stabilizers are defined as formulations suitable for plasma protein concentrates, including excipients as described in applications FR0308403, and preferably formulations suitable for C1 inhibitor concentrates.

[0090] Step e)

[0091] Preferably, the process according to the invention comprises, after step d), a step e) of filtering the concentrated and formulated eluate obtained in d).

[0092] Preferably, 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.

[0093] 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.

[0094] Step f) Step e) of filtration may optionally be followed by a freezing or freeze-drying step of the eluate obtained in e). This allows for storage.

[0095] Thus, preferably, the process according to the invention includes, after step e), a step f) of storing the filtered eluate obtained in e).

[0096] 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.

[0097] At the end of the process, a yield of at least 50% of C1-INH is typically obtained.

[0098] The C1-INH obtained by the process according to the invention can be used in therapy or as an excipient, particularly to ensure the stability of therapeutic blood products. Specifically, 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).

[0099] 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 C1-INH thus undergoes nanofiltration within the mixture, but not alone.

[0100] Nanofiltration generally refers to the filtration of the protein concentrate of interest through a filter with a pore size smaller 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 (Pali 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.

[0101] 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 after step f), with a therapeutic plasma product solution, preferably FVIII or FXI, and then subjecting the resulting mixture to a filtration step, preferably nanofiltration. Preferably, the FVIII or FXI solution is stabilized by the addition of a Ci-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.

[0102] In a preferred embodiment, the Factor VIII or XI solution is stabilized by the addition of 0.5 to 2 IU, preferably 1 to 2 IU, preferably 1.5 to 2 IU of C1-INH per 100 IU of Factor VIII or XI; and optionally by the addition of 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.

[0103] The invention is now illustrated by the following examples.

[0104] Examples

[0105] In the following examples, unless otherwise stated, the raw material (including the C1-esterase inhibitor or C1-INH or C1-lnh or "Ci-inhibitor") is derived from the cryosupernatant (step a).

[0106] 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).

[0107] The eluate after chromatography is recovered (step c), and this eluate corresponds to the fraction including C1-INH.

[0108] Comparative example: Purification of C1 inhibitor by a comparative process

[0109] Results of the trial on Gel PORCS CaptureSelect™ C1-lnhXL Affinity Matrix

[0110] [Table 1]

[0111] Conclusion :

[0112] C1-INH adheres well to this gel.

[0113] Desorption of C1-INH fixed on the gel is difficult; or the acid elution conditions (pH 3) are too drastic for the protein and its assay.

[0114] The yield is low.

[0115] Example 1: Purification of the C1 Inhibitor by a process according to the invention. Results of the test on POROS Gel No. 2 CaptureSelect™ C1-lnhXL Affinity Matrix

[0116] [Table 2]

[0117] Conclusion :

[0118] Good binding of C1-INH to this gel. The gel capacity is calculated at 4.3 g C1-INH / L of gel and 11,800 IU C1-INH / L of gel.

[0119] The elution of C1-INH is highly efficient with an excellent yield: 91% antigen and 86% activity. Example 2: Purification of C1-Inhibitor by a process according to the invention comprising a pre-elution step

[0120] STEP 1: Raw material preparation (step a)

[0121] Raw material = Cryosurnatant obtained by filtration on SARTOPORE 2 (pore size 0.45 pm + 0.2 pm).

[0122] The DEPART FILTRE product has a C1-INH activity of 0.84 IU / ml and a C1-INH level by nephelometry of 200 mg / l.

[0123] STEP 2: Affinity chromatography according to the invention (POROS, XTO; step b)

[0124] POROS™ CaptureSelect™ C1-lnhXL Affinity Matrix Gel.

[0125] Equilibration Buffer: 50 mM TRIS, 115 mM NaCl; pH 7.43; 295 mOsm / kg.

[0126] Pre-Elution Buffer: 50 mM TRIS, 0.25 M MgCh; 1 M NaCl; pH 7.49; 2640 mOsm / kg.

[0127] Elution buffer: 50 mM TRIS, 2 M MgCh; pH 7.56; 5450 mOsm / kg.

[0128] C1 Inhibitor Monitoring

[0129] [Table 3]

[0130] Fibrinogen Monitoring

[0131] [Table 4]

[0132] Then we recover the eluate (step c).

[0133] 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.

[0134] There are 8.5 g of fibrinogen per gram of C1 Inhibitor before the XTO step, but only 40 mg of fibrinogen per gram of C1 Inhibitor in the total eluate.

[0135] Example 3: Purification of the C1 inhibitor by a process according to the invention comprising two pre-elution steps

[0136] STEP 1: Raw material preparation (step a)

[0137] Raw material = Cryosurnatant obtained by filtration on SARTOPORE 2 (pore size 0.45 pm + 0.2 pm).

[0138] STEP 2: Affinity chromatography according to the invention (POROS, XTO; step b)

[0139] POROS™ CaptureSelect™ C1-lnhXL Affinity Matrix Gel

[0140] The cryosupernatant of step a) is loaded onto the column in an amount of approximately 2.8g / L of gel.

[0141] Equilibration Buffer: 50 mM TRIS, 115 mM NaCl; pH 7.5.

[0142] Pre-Elution Buffer 1: 50 mM TRIS, 0.25 M MgCh; pH 7.5.

[0143] Pre-Elution Buffer 2: NaCl 1 M.

[0144] Elution buffer: 50 mM TRIS, 2 M MgCh; pH 7.5.

[0145] Three elution cycles are performed, then the C1-INH is pooled (recovered, step c), and this eluate corresponds to the fraction containing the C1-INH. The yields are as follows:

[0146] [Table 5]

[0147] Conclusion :

[0148] The elution of C1-INH is very efficient with an excellent yield. Example 4: Purification of C1-Inhibitor by a process according to the invention comprising two pre-elution steps

[0149] The same procedure as in example 3 is performed on 3 batches of POROS gel. The results are as follows:

[0150] [Table 6]

[0151] Conclusion :

[0152] The elution of C1-INH is very efficient with an excellent yield.

Claims

DEMANDS 1. 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 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 the C1-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 carried out by ultrafiltration.

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

4. A process according to claim 3, comprising, after step e), a step f) of storing the filtered eluate obtained in e), preferably after freezing or lyophilizing 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 takes 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); preferably, said biological safety step comprises at least one treatment with chemicals, for example, with a solvent and / or detergent, and / or with heat (pasteurization). and / or dry heating) and / or by irradiation (Gamma and / or UVC) and / or by pH treatment (acidic pH treatment).

8. A method according to any one of the preceding claims, wherein step b) comprises the following substeps: b1) loading 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, preferably between 5.8 and 8.0, and comprising a TRIS buffer and at least one electrolyte, preferably magnesium chloride (MgCl2); optionally, the elution buffer also comprises a polyol, preferably a diol, 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 2 M MgCh 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, 2 M MgCh 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 having a pH between 5.0 and 8.0, preferably between 6.0 and 8.0, preferably the pre-elution buffer comprising 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 their mixtures; preferably the pre-elution buffer comprises TRIS, 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; advantageously two pre-elution steps are added between steps b2) and b3), and advantageously the buffer of the first pre-elution comprises at least one electrolyte, preferably MgCl2, advantageously at a molarity of 0.1 to 2 M, preferably from 0.2 to 1 M, and the buffer of the second pre-elution comprises at least one electrolyte, preferably NaCl, advantageously at a molarity of 0.5 to 3 M, preferably from 0.7 to 2 M; Preferably the buffer for the first pre-elution is a solution comprising 50 mM TRIS, 0.25 M MgCh, and having a pH between 5.0 and 8.0 and the buffer for the second pre-elution is a solution comprising 1 M NaCl.

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

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