Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/745—Blood coagulation or fibrinolysis factors
  • C07K14/75—Fibrinogen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/02—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using physical processes
  • A61L2/022—Filtration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/02—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using physical processes
  • A61L2/04—Heat
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/02—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using physical processes
  • A61L2/08—Radiation
  • A61L2/081—Gamma radiation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/02—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using physical processes
  • A61L2/08—Radiation
  • A61L2/10—Ultraviolet [UV] radiation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
  • B01D15/361—Ion-exchange
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3847—Multimodal interactions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
  • B01D61/027—Nanofiltration
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
  • B01D61/04—Feed pretreatment
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/58—Multistep processes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2103/00—Materials or objects being the target of disinfection or sterilisation
  • A61L2103/05—Living organisms or biological materials
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D2311/00—Details relating to membrane separation process operations and control
  • B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D2311/00—Details relating to membrane separation process operations and control
  • B01D2311/26—Further operations combined with membrane separation processes
  • B01D2311/2697—Chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D2315/00—Details relating to the membrane module operation
  • B01D2315/16—Diafiltration
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/14—Ultrafiltration; Microfiltration
  • B01D61/145—Ultrafiltration

Definitions

• the present invention relates to a fibrinogen filtration process, as well as the preparations obtained.
• Fibrinogen is an essential protein for blood coagulation, because its polymerization into insoluble fibrin formed at the end of the cascade of reactions which govern coagulation, results in the formation of a clot closing the vascular breach, responsible for bleeding. The placement of the clot is thus essential to ensure the cessation of bleeding.
• the fibrin formed at the level of the wound constitutes a fibrillar network which ensures tissue repair, therefore scarring.
• Congenital fibrinogen deficiencies can lead to serious pathologies. To treat these deficiencies, it is necessary to have fibrinogen concentrates which can be administered to patients in treatment. Other pathologies can also be treated by fibrinogen contributions, in particular in the event of massive blood loss, in the event of surgery or trauma for example, or following a decompensated consumption coagulopathy, for example the CIVD ( disseminated intravascular coagulation).
• CIVD disseminated intravascular coagulation
• compositions comprising fibrinogen in particular for therapeutic purposes, requires purification techniques leading to a product which is not only sufficiently purified of contaminants of diverse nature, such as the accompanying or co-purified proteins. , antibodies or proteases, but moreover virally secure and in terms of the ATNC covering the prion.
• certain conventional viral inactivation treatments consist of a heat treatment, for example pasteurization at 60 ° C. for 20 hours in the presence of protective stabilizers or dry heating of the lyophilized product, and / or a chemical treatment, such as as solvent-detergent, intended to make the fibrinogen compositions compatible with a therapeutic use.
• a heat treatment for example pasteurization at 60 ° C. for 20 hours in the presence of protective stabilizers or dry heating of the lyophilized product
• a chemical treatment such as as solvent-detergent
• the other biological security methods use viral elimination techniques, in particular using filtering.
• these filtration techniques require filters with a small pore size ( ⁇ 35 nanometers) which are hardly compatible with fibrinogen.
• Application EP1457497 describes a nanofiltration step requiring a preliminary freezing and thawing step followed by filtration which must be applied in order to remove aggregates, polymers or undesirable contaminants such as fibronectin, such a process also requires a prior dilution of the solution to less than 2 g / L to limit early clogging of the filters, which represents significant obstacles to the industrialization of such processes.
• low porosity filters such as the Planova 20N filter, which is conventionally used in the industry for biological safety, do not make it possible to achieve a charge on the filter sufficient to ensure an acceptable yield and industrial cost price.
• the methods using filters under such conditions therefore do not allow easy industrial implementation or use at high capacity, and represent a prohibitive cost in the implementation of a purification process on an industrial scale using batches of departure of several hundred or thousands of liters.
• such methods do not allow more than 0.2 kg of fibrinogen to be treated per m 2 of nanofilter membrane without including a preliminary stage of freezing / thawing and filtration of the product to be nanofiltrated.
• the development of a nanofiltration step of fibrinogen under conditions allowing its industrial implementation (sufficient flow rate, little clogging, acceptable cost price) is therefore known to be a difficulty.
• fibrinogen compositions must contain arginine to ensure their stability.
• the processes for obtaining fibrinogen compositions thus use arginine at different stages of the process, including during the elution of chromatography.
• application US2015 / 0366947 (Example 7) teaches that the nanofiltration of fibrinogen compositions obtained by elution from chromatography in a buffer comprising arginine would not be facilitated compared to the nanofiltration of fibrinogen compositions obtained by elution from chromatography in buffer not comprising arginine.
• the Applicant has therefore sought to develop a process for removing viruses and other undesirable contaminants (such as polymers, aggregates or prions) from a composition comprising fibrinogen, by filtration, which allows the obtaining a highly secure fibrinogen composition, said process being easy to implement on an industrial scale, and having good yield and an acceptable industrial cost price.
• the invention therefore relates to a method of filtering a fibrinogen composition, comprising the following steps: a) purification by chromatography of the fibrinogen composition using an elution buffer comprising arginine;
• step b) optionally, at least one step of filtering the fibrinogen composition obtained by elution from chromatography in step a), on a filter having a pore size of between 0.08 ⁇ m and 0.22 ⁇ m,
• step a) filtration of the fibrinogen composition obtained by elution from chromatography in step a), or optionally obtained in b), on a symmetrical filter having a pore size of between 15 nm and 25 nm, preferably between 18 nm and 22 nm, and
• said filtration process being carried out without adding arginine after step a), at a capacity of at least 0.2 kg of fibrinogen / m 2 and said fibrinogen composition not being previously frozen and / or thawed.
• the method according to the invention does not require the addition of an adjustment step with arginine of the composition obtained after the chromatography step.
• Increasing concentrations of arginine in the elution buffer have been shown to increase the filtration capacity and do not clog the filter.
• a subsequent adjustment step with arginine is therefore not necessary, thus simplifying the industrial implementation of the process.
• the method according to the invention therefore advantageously allows filterability, on a symmetrical filter with a pore size of about 20 nm, of a composition comprising fibrinogen without prior freezing / thawing step, or prior dilution from a fibrinogen solution pre-purified by chromatography and eluted by buffer comprising arginine, and without addition of arginine after the purification step by chromatography.
• FIG. 1 shows the capacity in g of fibrinogen / m 2 of membrane as a function of the flow rate (L / H / m 2 ) of the process according to the invention (symmetrical filter) compared to a previous process (asymmetric filter)
• high capacity means a fibrinogen load per membrane surface, expressed in kg of fibrinogen per m 2 of membrane, greater than or equal to 0.2, preferably greater than or equal to 0 , 25, preferably greater than or equal to 0.3, preferably greater than or equal to 0.35, preferably greater than or equal to 0.4, preferably greater than or equal to 0.45, preferably greater than or equal to 0 , 5, preferably greater than or equal to 1, preferably greater than or equal to 2, preferably greater than or equal to 3, preferably greater than or equal to 5 kg / m 2 .
• the high capacity corresponds to a fibrinogen load per membrane surface of between 0.2 and 5 kg / m 2 , even more advantageously between 0.2 and 2.5 kg / m 2 .
• the Applicant has found that it was possible to obtain, on an industrial scale, fibrinogen compositions, highly secure, free of viruses, and in particular of small viruses, in particular non-enveloped, such than B19, and other undesirable contaminants (such as polymers, aggregates or prions), by the implementation of a flexible and simple filtration process which allows securing with a nanofiltration step of fibrinogen retaining its molecular integrity at an acceptable cost of production.
• a simple process, fast and at an acceptable industrial cost price is easily implemented on an industrial scale, which leads to increased optimization of the biological security of compositions comprising fibrinogen.
• a filtration process allows a high protein load with a high yield after filtration.
• the method according to the invention thus uses a fibrinogen composition, in particular from different sources.
• the fibrinogen composition can thus be obtained from blood plasma, preferably from plasma fractions, cell culture supernatant or milk from transgenic animals.
• the composition comprising fibrinogen (or fibrinogen composition) subjected to the process of the invention is blood plasma or a plasma fraction, preferably a plasma fraction obtained from pre-purified blood plasma .
• plasma fraction obtained from prepurified blood plasma means any part or sub-part of human blood plasma, having undergone one or more purification steps. Said plasma fractions thus include the supernatant of cryoprecipitate plasma, the cryoprecipitate of plasma (resuspended), fraction I obtained by ethanolic fractionation (according to the method of Cohn or of Kistler & Nitschmann), the eluates of chromatography and the non-adsorbed fractions of the chromatography columns, including multicolumn chromatographies, and the filtrates.
• the fibrinogen composition subjected to the process of the invention undergoes an additional chromatography step.
• the fibrinogen composition subjected to the process according to the invention is a chromatography eluate or a non-adsorbed fraction of a chromatography column, including multicolumn chromatography.
• the fibrinogen composition subjected to the method of the invention is a plasma fraction obtained from cryosupernatant or resuspended cryoprecipitate.
• the cryoprecipitate plasma supernatant corresponds to the liquid phase obtained after thawing of frozen plasma (cryoprecipitation).
• the cryosupernatant can be obtained by freezing blood plasma at a temperature between - 10 ° C and -40 ° C, then gentle thawing 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.
• Cryoprecipitate is a concentrate of fibrinogen, fibronectin, von Willebrand factor and factor VIII, while the cryosupernatant contains complement factors, dependent vitamin K factors such as protein C, protein S, protein Z, factor II, factor Vil, factor IX and factor X, fibrinogen, immunoglobulins and albumin.
• fibrinogen composition not being previously frozen and / or thawed
• fibrinogen composition which is subjected to step b) if it applies, or by default subjected directly to the 'step c), is not frozen and / or thawed before this step b) or c).
• the plasma fraction subjected to the method of the invention can be obtained according to the method described by the Applicant in application EP1739093.
• the plasma fraction used is preferably obtained as follows:
• human plasma cryosupernatant is used.
• This plasma cryosupernatant is subjected to ethanolic precipitation by Cohn's method, according to conditions known to those skilled in the art, in particular such that the concentration of ethanol in the plasma considered is 8 to 10% (v / v).
• the supernatant and the precipitate thus obtained are then centrifuged.
• the precipitate constitutes fraction I of Cohn consisting mainly of fibrinogen (purity about 70%).
• This prepurified Cohn fraction I is resuspended and washed by dispersion.
• the purified precipitate paste (fraction I of purified Cohn) is recovered and then dissolved.
• the solution thus obtained is then subjected to an elimination of the procoagulant factors by treatment with alumina gel at a pH of 6.9-7.1.
• this prepurified solution is subjected to a first viral inactivation treatment by solvent-detergent in the presence of Tween®-TnBP.
• the prepurified solution thus obtained is injected onto a chromatographic column filled with an DEAE Macroprep anion exchange gel (Bio-Rad, France), previously balanced in a buffer consisting of sodium chloride and trisodium citrate, adjusted to a pH of 8 , 0.
• DEAE Macroprep anion exchange gel Bio-Rad, France
• the fibrinogen is eluted by an appropriate elution buffer, for example containing 1 M sodium chloride and a mixture consisting of trisodium citrate, lysine, glycine, arginine and isoleucine, adjusted to a pH 7.5.
• the eluate thus recovered constitutes the plasma fraction used for nanofiltration.
• the chromatography step is carried out by affinity, mixed-mode or ion exchange chromatography.
• the chromatographic purification is an ion exchange chromatography.
• it is carried out on an ion exchange matrix based on natural or synthetic polymer, resin or gel, onto which anion exchange groups of weak base type, preferably DEAE, are grafted.
• the chromatographic purification comprises a first step of loading a composition of fibrinogen, in particular of the solubilized plasma fraction, on an anion exchanger of weak base type, said exchanger being beforehand balanced by a buffer of predetermined ionic strength of basic pH. Said buffer is called a balancing buffer.
• the elution buffer comprises arginine in arginine is preferably at least 200 mM, at least 300 mM, at least 400 mM, at least 500 mM, at least 600 mM , at least 700 mM, at least 800 mM, at least 900 mM, at least 1 M.
• the arginine concentration of the elution buffer is preferably between 200 and 800 mM, between 200 and 700 mM, between 200 and 600 mM, between 200 and 500 mM, between 200 and 400 mM, between 200 and 300 mM.
• the arginine concentration of the elution buffer is preferably between 300 and 800 mM, between 400 and 800 mM, between 500 and 800 mM, between 600 and 800 mM, between 700 and 800 mM.
• the arginine concentration of the elution buffer is preferably between 300 and 800 mM, between 400 and 700 mM, between 400 and 600 mM.
• the elution buffer can also contain other suitable excipients, such as salts and / or amino acids, for example trisodium citrate, Tris, lysine, glycine, and / or isoleucine.
• suitable excipients such as salts and / or amino acids, for example trisodium citrate, Tris, lysine, glycine, and / or isoleucine.
• the protein concentration in the eluate is of the order of 2 to 5 g / l.
• the chromatographic purification is an affinity chromatography.
• the chromatographic purification comprises a first step of loading a fibrinogen composition, obtained from the cryosupernatant or from the resuspended cryoprecipitate, onto an affinity resin, said resin being beforehand balanced by a buffer of predetermined ionic strength of pH adapted. Said buffer is called a balancing buffer.
• the solubilized plasma fraction is loaded onto any affinity matrix, resin or gel, onto which are grafted chemical or synthetic ligands such as antibodies, antibody fragments, antibody derivatives or chemical ligands such as peptides, mimetic peptides, peptoids, nanofitins or even oligonucleotide ligands such as aptamers.
• the chromatographic support is available under the name CaptureSelect Fibrinogen (Life Technologies).
• the chromatographic support is obtained according to the method described in application WO2018007530.
• the plasma fraction subjected to the method of the invention can thus be obtained according to the method described by the Applicant in application WO2015 / 136217 or application WO2018007530.
• the affinity chromatography can be carried out by continuous chromatography of the SMB (Simulated Moving Bed) type, for example with the NOVASEP SMCC technology (Sequential MultiColumns Chromatography).
• SMB Simulated Moving Bed
• NOVASEP SMCC Sequential MultiColumns Chromatography
• the size of the columns and of the chromatography equipment is reduced significantly (on the order of 10 times).
• the need for resin per batch of fibrinogen can be reduced by 10 to 50% in general.
• the eluates generated in continuous chromatography can either be used continuously for the following stages with or without in-line concentration using in-line concentration equipment of the Cadence Pall type or equivalent from other suppliers.
• a variant consists in pooling the eluates before the continuation of the process, with a possible reconcentration of the eluates before their use.
• the affinity chromatography is carried out on the fibrinogen solution having undergone the viral inactivation treatment, thus the viral inactivation solution is found in the non-adsorbed fraction of chromatography and is removed at the same time as the fibrinogen is purified.
• the elution buffer comprises arginine; the arginine concentration is preferably at least 200 mM at least 300 mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, at least 1 Mr.
• the arginine concentration of the elution buffer is preferably between 200 and 800 mM, between 200 and 700 mM, between 200 and 600 mM, between 200 and 500 mM, between 200 and 400 mM , between 200 and 300 mM.
• the arginine concentration of the elution buffer is preferably between 300 and 800 mM, between 400 and 800 mM, between 500 and 800 mM, between 600 and 800 mM, between 700 and 800 mM.
• the arginine concentration of the elution buffer is preferably between 300 and 800 mM, between 400 and 700 mM, between 400 and 600 mM.
• the elution buffer can also contain other suitable excipients, such as salts and / or amino acids, for example trisodium citrate, Tris, lysine, glycine, and / or isoleucine.
• suitable excipients such as salts and / or amino acids, for example trisodium citrate, Tris, lysine, glycine, and / or isoleucine.
• the elution buffer can consist either of a modification of the pH and / or of the ionic strength.
• the composition comprising fibrinogen comes from milk of transgenic animals, for example obtained according to the method described in WO00 / 17234 or in WO00 / 17239.
• the fibrinogen composition subjected to the process according to the invention has a purity greater than or equal to 70%, preferably greater than or equal to 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%.
• the fibrinogen composition subjected to the method according to the invention advantageously does not comprise other co-purified proteins, advantageously no FXIII and / or fibronectin and / or prothrombin (Fil) and / or thrombin, and / or plasminogen and / or plasmin.
• the fibrinogen composition subjected to the process according to the invention is advantageously devoid of FXIII.
• the fibrinogen composition subjected to the process according to the invention can also comprise one or more accompanying proteins, optionally co-purified.
• the fibrinogen composition subjected to the process according to the invention advantageously comprises FXIII.
• the fibrinogen composition subjected to the process according to the invention does not include multimeric forms of fibrinogen, advantageously no fibrinogen polymers or fibrinogen aggregates.
• the fibrinogen composition subjected to the process according to the invention is concentrated to more than 1 g of fibrinogen / L of solution, preferably to more than 2 g of fibrinogen / L of solution, even more so preferred to more than 3g of fibrinogen / L of solution, to more than 3.5g of fibrinogen / L of solution, to more than 4g of fibrinogen / L of solution, to more than 4.5g of fibrinogen / L of solution.
• the fibrinogen composition subjected to the process according to the invention is concentrated between 2g and 5g of fibrinogen / L of solution.
• it is used without prior dilution. Indeed, preferably, the method according to the invention does not require a prior dilution step of the fibrinogen composition.
• the method according to the invention optionally comprises a step b), according to which at least one fibrinogen filtration step is carried out on a filter having a pore size between 0.08pm and 0.22pm.
• step b) comprises two stages of filtration of fibrinogen on a filter having a pore size of between 0.08pm and 0.22pm.
• the first filtration is carried out on a filter having a pore size of between 0.15 ⁇ m and 0.22 ⁇ m, preferably around 0.2 ⁇ m.
• the second filtration is carried out on a filter having a pore size of between 0.08 ⁇ m and 0.15 ⁇ m, preferably around 0.1 ⁇ m.
• PES polyethersulfone
• the sequence of filters is, prior to step b), balanced with the buffer of the preliminary purification steps, in particular with the elution buffer for the chromatography optionally supplemented with amino acids.
• a fibrinogen composition is recovered.
• the method according to the invention comprises filtering the fibrinogen composition optionally obtained in b), on a symmetrical filter having a pore size between 15 nm and 25 nm: this is step c).
• step b) If step b) is carried out, then the fibrinogen solution obtained in b) is passed through a symmetrical filter having pores with a diameter between 15 nm and 25 nm, preferably 20 nm, and the resulting solution of fibrinogen is recovered. If step b) is not carried out, then the fibrinogen composition obtained by elution from chromatography in step a) is directly passed through a symmetrical filter having pores of diameter between 15 nm and 25 nm, preferably 20 nm, and the resulting fibrinogen solution is recovered.
• step c) is typically carried out at a pressure between 200 and 4000 mbar.
• the nanofiltration of step b) is typically carried out at a pressure between 200 and 1000 mbar, or between 2000 and 4000 mbar.
• the filters used can be defined by their average pore size in nm, by the viruses retained by the filter, by a molecular weight threshold or by the type of symmetry of their membrane.
• the filters used can therefore be filters or any other equivalent filter sold:
• filters from the Planova® range made up of a hollow fiber membrane formed from cellulose regenerated with cuprammonium and marketed by Asahi-Kasei (Planova® 15N, Planova® 20N), and those of the Ultipor® range, composed of a polyvinylidene fluoride membrane modified on the surface and are marketed by Pall (Ultipor DV20, Pegasus SV4), or any other equivalent filter marketed;
• Such filters defined by the viruses retained by the filter, include the Planova BioEX PVDF filters (retention of parvoviruses, modified hydrophilic polyvinylidene fluoride membrane) sold by the company Asahi Kasei Bioprocess, the Pegasus SV4 filters, or Ultipor VF (retention parvoviruses, modified hydrophilic polyvinylidene fluoride membrane) marketed by Pall, Viresolve® NFP filters (retention of parvoriruses, surface-modified polyvinylidene fluoride membrane), Viresolve Pro (retention of parvoriruses, double-layer polyethersulfone membrane) and Viresolve® NFR (retention of retroviruses, polyethersulfone membrane) marketed by Millipore, and Virosart® CPV (retention of canine
• asymmetric filters such as filters from the Planova® range, marketed by Asahi-Kasei (Planova® 15N, Planova® 20N, Planova BioEx), Viresolve NFP and Viresolve Pro (marketed by Merck Millipore), Virosart HF (marketed by Sartorius Stedim) .
• symmetrical filters such as the Pegasus SV4 or Ultipor DV20 filter (marketed by Pall), the Virosart CPV filter (marketed by Sartorius Stedim).
• symmetrical filter means a filter having an equivalent porosity between the internal surface (in contact with the solution to be filtered) and external of the filter. This is in contrast to asymmetric filters for which the internal surface of the filter is often more porous than the external surface.
• the pore size in nm can be measured by a person skilled in the art according to known techniques.
• the nanofiltration carried out in step c) is carried out using a symmetrical type filter.
• the nanofiltration of step c) is carried out using a symmetrical filter, such as the Pegasus SV4 or Ultipor DV20 filter (marketed by Pall), the Virosart CPV filter (marketed by Sartorius Stedim).
• a symmetrical filter such as the Pegasus SV4 or Ultipor DV20 filter (marketed by Pall), the Virosart CPV filter (marketed by Sartorius Stedim).
• the symmetrical filter is in pleated mounting.
• the symmetrical filter is advantageously characterized by a hydrophilic polyvinylidenedifluoride membrane (PVDF).
• PVDF hydrophilic polyvinylidenedifluoride membrane
• the symmetrical filter in pleated assembly characterized by a hydrophilic polyvinylidenedifluoride membrane (PVDF) is a Pegasus SV4 filter (sold by Pall).
• the Applicant has advantageously demonstrated that the symmetrical type filters, such as filters similar to the Pegasus SV4 or Ultipor DV20 filter (marketed by Pall) or Virosart CPV (marketed by Sartorius Stedim) made it possible to carry out nanofiltration of fibrinogen with a load of at least 0.2 kg of fibrinogen / m 2 of membrane and said fibrinogen composition not being previously frozen and / or thawed and obtaining better results than with an asymmetric type filter such as filters from the Planova® range, marketed by Asahi-Kasei (Planova® 15N, Planova® 20N).
• an asymmetric type filter such as filters from the Planova® range, marketed by Asahi-Kasei (Planova® 15N, Planova® 20N).
• this step c) makes it possible to filter a substantial volume of fibrinogen solution, with a very good yield, ie at least 90%.
• This substantial volume corresponds to a capacity of at least 0.2 kg of fibrinogen per m 2 and can go up to at least 5 kg per m 2 .
• the filtration capacity of a fibrinogen composition is advantageously increased by adding increasing concentrations of arginine in the elution buffer for the chromatography carried out prior to the filtration sequence .
• the chromatography elution buffer carried out in step a) comprises an arginine concentration of at least 200 mM and said filtration method has a capacity of at least 0.25 kg / m 2 .
• the chromatography elution buffer carried out in step a) comprises an arginine concentration of at least 200 mM and said filtration method has a capacity of at least 0.30 kg / m 2
• the elution buffer for the chromatography carried out in step a) comprises an arginine concentration of at least 200 mM and said filtration process has a capacity of at least 0.35 kg / m 2 .
• the chromatography elution buffer carried out in step a) comprises an arginine concentration of at least 400 mM and said filtration process has a capacity of at least 0.25 kg / m 2
• the chromatography elution buffer carried out in step a) comprises an arginine concentration of at least 400 mM and said filtration process has a capacity of at least 0.30 kg / m 2 .
• the chromatography elution buffer carried out in step a) comprises an arginine concentration of at least 400 mM and said filtration process has a capacity of at least 0.35 kg / m 2 .
• the filtration capacity is measured by any method known to those skilled in the art. Typically, this is determined as follows: The fibrinogen solution to be nanofiltered is prepurified by chromatography according to the method described in EP1739093. The concentration of the starting fibrinogen solution is 3 g / L.
• the filtration capacity of the filter is determined by analyzing the clogging profile; the maximum filtration capacity corresponds to the quantity of fibrinogen associated with a filtration rate less than 25% of the initial rate.
• the process for filtering a fibrinogen composition according to the invention comprises the following steps:
• A) obtaining the fibrinogen composition said fibrinogen composition being chosen from a cell culture supernatant, milk from transgenic animals, the cryoprecipitate plasma supernatant, the resuspended plasma cryoprecipitate, the fraction I obtained by ethanolic fractionation according to the method of Cohn or of Kistler & Nitschmann, the supernatant and the precipitate obtained after precipitation of a plasma fraction with aluminum hydroxide and / or precipitation at low temperature, and the chromatography eluates and the non-adsorbed fractions chromatography columns obtained from a plasma fraction, a cell culture supernatant or a milk from transgenic animals,
• said filtration process being carried out, without adding arginine after step a), at high capacity and said fibrinogen composition not being previously frozen and / or thawed.
• the process for filtering a fibrinogen composition according to the invention comprises the following steps:
• said filtration process being carried out without adding arginine after step a), at high capacity and said fibrinogen composition not being previously frozen and / or thawed.
• the fibrinogen solution optionally obtained in b) is passed through a filter having pores of diameter between 15 nm and 50 nm prior to step c): this is step b ').
• the filters used can be defined by their average pore size in nm, by the viruses retained by the filter, by a molecular weight threshold or by the type of symmetry of their membrane.
• the filters used can therefore be filters or any other equivalent filter sold:
• filters from the Planova® range made up of a hollow fiber membrane formed from cellulose regenerated with cuprammonium and marketed by Asahi-Kasei (Planova® 15N, Planova® 20N), and those of the Ultipor® range, composed of a membrane of polyvinylidene fluoride modified on the surface and are sold by Pall (Ultipor DV20, Pegasus SV4), or any other equivalent filter sold;
• Such filters defined by the viruses retained by the filter, include the Planova BioEX PVDF filters (retention of parvoviruses, modified hydrophilic polyvinylidene fluoride membrane) sold by the company Asahi Kasei Bioprocess, the Pegasus SV4 filters, or Ultipor VF (retention parvoviruses, modified hydrophilic polyvinylidene fluoride membrane) marketed by Pall, Viresolve® NFP filters (retention of parvoriruses, surface-modified polyvinylidene fluoride membrane), Viresolve Pro (retention of parvoriruses, double-layer polyethersulfone membrane) and Viresolve® NFR (retention of retroviruses, polyethersulfone membrane) marketed by Millipore, and Virosart® CPV (retention of canine
• asymmetric filters such as filters from the Planova® range, marketed by Asahi-Kasei (Planova® 15N, Planova® 20N, Planova BioEx), Viresolve NFP and Viresolve Pro (marketed by Merck Millipore), Virosart HF (marketed by Sartorius Stedim) .
• symmetrical filters such as the Pegasus SV4 or Ultipor DV20 filter (marketed by Pall), the Virosart CPV filter (marketed by Sartorius Stedim).
• symmetrical filter is meant a filter having an equivalent porosity between the internal surface (in contact with the solution to be filtered) and the external surface of the filter.
• the nanofiltration of step b ') is carried out using filters having pores with a diameter between 25 nm and 50 nm, preferably 35 nm.
• the nanofiltration of step b ') is then carried out using the Planova 35 N filter sold by Asahi Kasei Bioprocess or STyLUX by Meissner (40 nm).
• the nanofiltration of step b ’) is carried out using symmetrical filters having pores with a diameter between 15 nm and 25 nm, preferably 20 nm.
• the nanofiltration of step b ’) is then carried out using a symmetrical membrane filter, such as the Pegasus SV4 or Ultipor DV20 filter (marketed by Pall) or Virosart CPV (marketed by Sartorius Stedim).
• the nanofiltration of step b ') and of step c) is carried out on filters of decreasing porosity, advantageously on a filter of porosity 35 nm followed by a symmetrical filter with a porosity of 20 nm.
• the nanofiltration of step b ') is then carried out using the Planova 35 N filter marketed by Asahi Kasei Bioprocess then a symmetrical membrane filter, such as the Pegasus SV4 or Ultipor DV20 filter (marketed by Pall) or Virosart CPV (marketed by Sartorius Stedim).
• the nanofiltration of step b ’) and of step c) is carried out on filters of the same pore size, advantageously on 2 identical filters.
• the nanofiltration of step b ’) is then carried out using a symmetrical membrane filter, such as the Pegasus SV4 or Ultipor DV20 filter (marketed by Pall) or Virosart CPV (marketed by Sartorius Stedim).
• the nanofiltration of step b ’) is typically carried out at a pressure between 200 and 4000 mbar.
• the nanofiltration of step b ’) is typically carried out at a pressure between 200 and 1000 mbar, or between 2000 and 4000 mbar.
• the invention relates to a process for filtering a fibrinogen composition, comprising the following steps:
• said filtration process being carried out without adding arginine after step a), at high capacity and said fibrinogen composition not being previously frozen and / or thawed.
• the solution obtained comprises fibrinogen, and is highly secure.
• step c) of the method according to the invention allows the elimination of at least 2log, advantageously at least 3log, even more advantageously at least 4log, preferably at at least 5 log or at least 6log of small viruses such as parvovirus B19.
• the process for filtering a fibrinogen composition according to the invention comprises the following steps:
• A) obtaining the fibrinogen composition said fibrinogen composition being chosen from a cell culture supernatant, milk from transgenic animals, the cryoprecipitate plasma supernatant, the resuspended plasma cryoprecipitate, the fraction I obtained by ethanolic fractionation according to the method of Cohn or Kistler & Nitschmann, the supernatant and the precipitate obtained after precipitation of a plasma fraction with aluminum hydroxide and / or precipitation at low temperature, and the chromatography eluates and the non-adsorbed fractions chromatography columns obtained from a plasma fraction, a cell culture supernatant or a milk from transgenic animals,
• said filtration process being carried out without adding arginine after step a), at high capacity and said fibrinogen composition not being previously frozen and / or thawed.
• the process for filtering a fibrinogen composition according to the invention comprises the following steps:
• said filtration process being carried out without adding arginine after step a), at high capacity and said fibrinogen composition not being previously frozen and / or thawed.
• the solution obtained in step d) can then be concentrated, for example by ultrafiltration, to contents typically between 10 and 40, preferably between 15 and 25 g of total protein / l, determined by measurements. classics known to those skilled in the art.
• the fibrinogen solution obtained, optionally concentrated, can be subjected to a diafiltration step.
• This step is intended to remove any excess inorganic salt used to obtain solutions having a ionic strength of at most 0.2 M.
• This step may also prove necessary in order to formulate the fibrinogen under optimal conditions.
• the buffer is advantageously suitable either for keeping the fibrinogen composition in liquid form (ready-to-use liquid formulation) or for keeping in lyophilized form (formulation suitable for preservation during the lyophilization step and optionally for 'dry heating step). This allows in this case on the one hand, dry heating of the fibrinogen without risk of denaturation, and, on the other hand, rapid solubilization when the fibrinogen is lyophilized thereafter, typically in 3 to 8 minutes.
• the respective solutions can optionally be lyophilized according to conventional methods and usual conditions.
• the lyophilisates can then be reconstituted in an aqueous medium compatible with clinical use, preferably in purified water for injection (PPI), and directly injected intravenously.
• PPI purified water for injection
• Viral inactivation often includes treatment with chemicals, for example by solvent and / or detergent and / or by heat (pasteurization and / or dry heating) and / or by irradiation (gamma rays and / or UVC )
• This step can be carried out by a conventional chemical viral inactivation treatment, preferably consisting of a solvent-detergent treatment (generally called S / D treatment).
• the viral inactivation chemical agents are preferably mixtures of polysorbate and Tri (n-butyl) phosphate (TnBP), or mixtures of Triton (octoxynol) and TnBP, whose typical concentrations are between 0.1 and 2 %.
• This viral inactivation can be integrated at any stage of the process, but it is judiciously carried out before stage a) of chromatographic purification. In this way, it will contribute to the effective elimination of inactivating agents.
• an additional step of dry viral inactivation heat treatment can be used, carried out on the fibrinogen lyophilisates obtained after the lyophilization step.
• the operating conditions are typically around 80 ° C for 72 hours.
• the elimination of infectious agents can also be carried out by means of deep filtration.
• the filters available are for example filters composed of regenerated cellulose, in which filter aids may have been added, such as cellite, perlite or Kieselguhr earth.
• filters are sold in particular by Cuno (Zeta + VR serial filters), Pall-Seitz (P-series Depth Filter) or Sartorius Sartoclear P depth filters).
• the implementation of the method leads to highly secure fibrinogen solutions, free of viral particles and / or ATNC type contaminants.
• the invention therefore relates to a fibrinogen solution capable of being obtained by the method described above.
• the fibrinogen solution capable of being obtained by the process described above advantageously has a purity greater than or equal to 95% and is advantageously stable without the addition of stabilizing protein such as albumin.
• the fibrinogen solution capable of being obtained by the process described above advantageously has an integral fibrinogen activity with in particular a coagulable fibrinogen / antigenic fibrinogen ratio> 0.9, or even equal to 1.0.
• Example 1 Evaluation of the fibrinogen filtration on a 20 nm symmetrical filter according to the invention
• composition comprising prepurified fibrinogen is obtained according to the method described in application EP1739093.
• Pegasus SV4 filter from Pall Life Sciences, surface 0.00096 m 2 (Pegasus VF SV4, 10MCFSV4, surface 9.6 cm 2 ).
• the solution comprising fibrinogen is concentrated at 3g / L and more than 90% pure
• the filtration pressure was kept constant at 2.1 Bar on the 20 nm filter during all of the filtration.
• the filtration sequence is balanced in the elution buffer of the ion exchange chromatography described in patent EP1739093 comprising arginine.
• the clogging profile of fibrinogen on the 20 nm pore size filter is linear with a decrease proportional to the filtered volume up to 181 L / m 2 .
• the filtration sequence applied made it possible to filter an amount equivalent to 0.5 kg of fibrinogen per m 2 of 20 nm pore size membrane in 6 hours. Higher filterability could have been achieved by extending the filtration time.
• the yield of this nanofiltration is greater than 90%.
• the method according to the invention therefore allows filterability, on a symmetrical filter with a pore size of approximately 20 nm, of a composition comprising fibrinogen without prior freezing / thawing step, or prior dilution from a fibrinogen solution pre-purified by chromatography and eluted by buffer comprising arginine.
• Example 2 Evaluation of the fibrinogen filtration on a 20 nm symmetrical filter Pali DV 20 according to the invention
• composition comprising prepurified fibrinogen is obtained according to the method described in application EP1739093.
• the solution comprising fibrinogen is concentrated to 3.1 g / L and more than 90% pure
• the filtration pressure was kept constant at 2.0 Bar on the 20 nm filter throughout the filtration.
• the filtration sequence is balanced in the elution buffer for the ion exchange chromatography described in patent EP1739093 comprising arginine.
• the clogging profile of fibrinogen on the 20 nm pore size filter is linear with a decrease proportional to the filtered volume up to 136 L / m 2 .
• the filtration sequence applied made it possible to filter an amount equivalent to 0.4 kg of fibrinogen per m 2 of 20 nm pore size membrane in 15 hours. Higher filterability could have been achieved by extending the filtration time.
• Example 3 Evaluation of the fibrinogen filtration on a 20 nm symmetrical filter according to the invention with 20-50 nm prefiltration
• composition comprising prepurified fibrinogen is obtained according to the method described in application EP1739093.
• Pegasus SV4 filter from Pall Life Sciences, surface 0.00096 m 2 (Pegasus VF SV4, 10MCFSV4, surface 9.6 cm 2 ).
• the solution comprising fibrinogen is concentrated to 3g / L and more than 90% pure
• the filtration pressure was kept constant at 2.1 Bar on the 20 nm filter during all of the filtration.
• the filtration sequence is balanced in the elution buffer for the ion exchange chromatography described in patent EP1739093.
• the clogging profile of fibrinogen on the 20 nm pore size filter is linear with a decrease proportional to the filtered volume up to 276 L / m 2 .
• the filtration sequence applied made it possible to filter an amount equivalent to 0.8 kg of fibrinogen per m 2 of 20 nm pore size membrane in 18 hours.
• the yield of this nanofiltration is greater than 90%.
• the method according to the invention therefore allows filterability, on a symmetrical filter with a pore size of approximately 20 nm, of a composition comprising fibrinogen without prior freezing / thawing step, or prior dilution from a fibrinogen solution pre-purified by chromatography and eluted by buffer comprising arginine.
• Example 4 Comparison of the method according to the invention with a method of the prior art on an asymmetric filter of about 20 nm
• composition comprising pre-purified fibrinogen is obtained according to the method described in application EP1739093.
• the chromatography eluate obtained according to EP1739093 is eluted in a buffer comprising 200 mM of arginine.
• the fibrinogen composition is prefiltered using a PES (Polyether sulfone) model Sartopore 2 filter with a porosity of 0.2 - 0.1 pm (100 nm).
• PES Polyether sulfone
• the prefiltered composition is then filtered:
• Planova 20N filter sold by the company Asahi at 395 ⁇ 23 mbar.
• the asymmetric nanofilters tested on a solution of fibrinogen prepurified by chromatography having a concentration of at least 2 g / L show filterabilities of less than 0.1 Kg of fibrinogen per m 2 while the method according to the invention on a symmetrical filter allows achieve filterability greater than 0.2 kg of fibrinogen per m 2 .
• composition comprising pre-purified fibrinogen is obtained according to the method described in application EP1739093. [0166] III - Preparation of the filter balancing buffer:
• the fibrinogen composition is prefiltered using a Minisart High flow model PES (Polyether sulfone) filter with a porosity of 0.2 - 0.1 pm (100 nm).
• PES Polyether sulfone
• Nanofiltration is carried out on the Pegasus SV4 filter from Pall Life Sciences at a pressure of 2.1 +/- 0.1 bar.
• the viral load is measured and produced using the PPV Ultrapure Gold 1% (v / v) product.
• a sample is taken from the nanofiltered fraction after filtration of 79.2 L / m 2 of solution (loaded volume).
• Example 7 Evaluation of the fibrinogen filtration capacity on a 20 nm symmetrical filter as a function of increasing concentrations of arginine in the elution buffer for the chromatography step
• the fibrinogen compositions tested are prepurified by affinity chromatography according to the method described in application EP1739093, using an elution buffer comprising 50 mM of sodium citrate and increasing concentrations of arginine HCL (150 mM, 200 mM , 400 mM)
• a filtration sequence as described in Example 1 is then carried out on the eluate in order to study the clogging profile of fibrinogen on the nanofilter with a porosity of 20 nm.
• composition eluted by buffer chromatography comprising increasing concentrations of arginine, without additional addition of arginine before the nanofiltration step, allows an increase in the nanofiltration capacity 20 nm of fibrinogen.
• increasing concentrations of arginine in the elution buffer according to the invention do not cause clogging of the filter and allow the nanofiltration capacity of the process to be increased, without adding any arginine after the purification step by chromatography. .

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