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    • 专利摘要:
    The invention relates to a method for preparing a plasma protein concentrate from blood plasma using multicolumn chromatography.
    公开号:FR3018450A1
    申请号:FR1451997
    申请日:2014-03-11
    公开日:2015-09-18
    发明作者:Damien Bataille；Abdessatar Chtourou；Patrick Santambien
    申请人:LFB SA；
    IPC主号:
    专利说明:

    [0001] The invention relates to a process for the preparation of human plasma proteins for therapeutic use, from blood plasma or from a plasma fraction.
    [0002] BACKGROUND OF THE INVENTION Many pathologies are currently treated with fractions derived from blood plasma enriched in one or more plasma proteins. Thus, coagulation factors are generally used in replacement therapy, for the prevention or treatment of hemorrhages associated with deficits in coagulation factors. Fibrinogen is most often prescribed for the treatment of complications associated with constitutional or severe afibrinogenemia and haemorrhagic syndromes or risks of hemorrhages associated with hypofibrinogenemia. Albumin is intended for the restoration and maintenance of circulating blood volume (confirmed hypovolemia). Similarly, many pathologies are currently treated with immunoglobulin-enriched blood plasma fractions, and in particular immunoglobulin G (IgG), generally comprising more than 95% Ig. For example, immunoglobulin G-enriched blood plasma fractions, or IgG concentrates, are used to correct primary immunodeficiency deficits with defective antibody production, certain secondary immune deficits, such as leukemias, myelomas or recurrent infections, etc. The administration of Ig can also have a beneficial effect in the treatment of Idiopathic Thrombocytopenic Purpura (ITP) in children and adults, Guillain-Barré syndrome, demyelinating polyneuropathies, multifocal motor neuropathy, Chronic inflammatory demyelinating polyradiculoneuropathies (PIDC), Kawasaki disease, multiple sclerosis, corticosteroid dermatomyositis, acute myasthenia, Birdshot retinochoroiditis, neonatal jaundice due to fetal-maternal incompatibility the ABO system (hemolytic disease of the newborn by incompatibility ABO) etc.
    [0003] The multiple therapeutic indications, as well as the very high doses that can in some cases be prescribed (up to 2 g of immunoglobulins / kg / day), have led to situations of extreme stress on supplies, up to shortages in Europe and the United States of America. Also, many methods have been developed for the purification of plasma proteins from blood plasma. The industrial process for extracting plasma proteins for therapeutic use is called plasma fractionation. Fractionation techniques used today mostly start with a cryoprecipitation step, consisting of a low temperature plasma thawing to isolate a cryoprecipitate enriched in factor VIII, von Willebrand factor, fibrinogen and fibronectin. The supernatant fraction proteins (cryoprecipitate supernatant) can then be separated by sequential precipitation in the presence of ethanol (Cohn et al., 1946, J. Am Chem Soc 68, 459). Several variants of the original Cohn process also using ethanolic fractionation have been described. Thus, the publication by Tanaka K. et al., 2000 (Braz J Med Bio Res 2000, 33 (1): 27-30) discloses a method for purifying immunoglobulin G from "I + II + II" fractions. and "" obtained after ethanolic fractionation according to the method of Cohn, by separation by chromatography on three types of gels, two ion exchange gels (Q-Sepharose FF and CM-Sepharose FF) and a gel filtration step (Sephacryl S- 300 HR). An alternative route to ethanol precipitation has been described by Steinbuch et al. (Rev.
    [0004] Franc. And, Clin, and Biol. 1969, XIV, 1054) which involves precipitation with octanoic acid (or caprylic acid / caprylate). This precipitates most of the plasma proteins and keeps the immunoglobulins in the supernatant. The purification of these immunoglobulins is continued by adsorption ("batch") on an anion exchanger, DEAE-cellulose, which also leaves the immunoglobulins in the supernatant. This is then concentrated by ultrafiltration. However, these precipitations with ethanol or caprylic acid can cause some denaturation of proteins and the formation of protein aggregates (including immunoglobulin polymers), which can cause anaphylactic reactions. It is therefore necessary to carry out subsequent processing steps, for example with propiolactone or with reducing and alkylating agents, or at pH 4, or with PEG to precipitate the aggregates. These additional steps tend to decrease the yield of plasma proteins. Thus, most of the manufacturing processes claim an IgG yield of between 3.5 and 5.5 g of IgG per liter of fractionated plasma ("A modified caprylic acid method for manufacturing immunoglobulin G from human plasma with high yield and efficient virus clearance "- Vox Blood, 2006 Feb; 90 (2): 97-104. and" A new methodology for polyvalent intravenous immunoglobulin solution production with a Iwo-stage process of viral inactivation. "Brazilian Journal of Pharmaceutical Sciences - Vol. 4, out./dez., 2010).
    [0005] The implementation of ethanolic fractionation is also relatively expensive. Indeed, the fractionation of a single liter of plasma requires the use of 2L of ethanol. For the treatment of several thousand liters of plasma, it is therefore necessary to store very large quantities of ethanol directly at the production site, and moreover to refrigerate the facilities dedicated to ethanolic fractionation. In addition, the industrial installations must be adapted to the storage, treatment, disposal and possibly recycling of large volumes of solvent, which represents technical constraints and significant costs. The purity of the plasma proteins is an important stake to guarantee the best efficiency and safety of these during their use. Thus the ethanolic or caprylic fractionation must be completed by other purification steps to achieve the desired purity, but these operations are carried out to the detriment of the yield due to additional losses of product. For these additional steps, chromatography techniques are increasingly used because they are characterized by high purification rates coupled with high yields. It is therefore interesting to position these chromatography techniques as close as possible to the starting plasma in order to gain in yield and purity while maintaining the integrity of the proteins of interest. This is already applied for the capture of scarce plasma proteins such as factor XI, factor IX, factor VIII, von Willebrand factor, protein C, antithrombin III etc. But for abundant proteins such as polyvalent immunoglobulins, albumin, fibrinogen, the volumes of chromatography gel necessary for the capture of these molecules would be too great and would lead to unprofitable industrial tools (size of equipment and costs of gel ). While some describe the accumulation of cycles of chromatography for the treatment of large amounts of protein ("Description and Assessment of an Industrial Chromatography Unit for Preparing Human Plasma Albumin." - Biotechnology of Blood Proteins, 1993, Vol 227, pp. 176-181). these practices are not optimal and still not well developed. There is therefore still an important need to increase the yield of plasma protein production for therapeutic use, including immunoglobulins, while ensuring that the final product has a high purity and is free of potentially deleterious contaminants. SUMMARY OF THE INVENTION: In general, the subject of the present invention is a process for the preparation of a fraction of purified human plasma proteins with a high yield, and in particular greater than the yield of processes known to those skilled in the art. . For this, the Applicant has developed a process for preparing a purified human plasma protein concentrate from blood plasma, comprising at least one purification step by multicolumn chromatography, and in particular by multicolumn affinity chromatography or ion exchange. . More specifically, the process according to the invention proposes carrying out a chromatography step by fractionating the chromatography column usually used in several smaller columns, placed in series or managed independently in order to use the maximum of the functional groups present in the chromatography column. chromatography gel.
    [0006] The present invention also relates to a method for preparing a human immunoglobulin (Ig) concentrate with a higher yield compared to methods known to those skilled in the art. For this, the Applicant has developed a process for the preparation of purified Ig, in which the initial fractionation step with ethanol and / or caprylic acid is replaced by a multicolumn chromatography capture step, and in particular by multicolumn affinity chromatography or ion exchange. No step of removing protein contaminants by precipitation is carried out before chromatography. Blood plasma or cryosurning blood plasma is directly subjected to said multicolumn chromatography. In the case where the multicolumn chromatography is anion-exchange chromatography, the process according to the invention may advantageously comprise a step of fractionation with caprylic acid and / or with ethanol following this chromatography. The invention therefore relates to a process for preparing a purified plasma protein concentrate for therapeutic use from blood plasma comprising a purification step according to which blood plasma or a plasma fraction is subjected to multicolumn chromatography. In particular, the subject of the invention is a process for the preparation of human immunoglobulin concentrates for therapeutic use from blood plasma comprising a step of purification of immunoglobulins according to which cryoprecipitated plasma or plasma supernatant is subjected to multicolumn chromatography. 'affinity. In another exemplary embodiment, the immunoglobulin purification step is carried out by subjecting plasma or cryoprecipitated plasma supernatant to anion exchange multicolumn chromatography. It is then possible to provide a subsequent purification step by caprylic acid precipitation after which the supernatant containing the immunoglobulins is recovered. The invention also relates to a method for preparing human albumin concentrates and / or fibrinogen for therapeutic use from blood plasma comprising a step of purifying albumin and / or fibrinogen according to which plasma is subjected to or plasma supernatant cryoprecipitated to multicolumn chromatography, and especially by multicolumn affinity chromatography or ion exchange. According to the invention, such a method may also comprise a subsequent additional step according to which the albumin-containing and / or fibrinogen-containing fraction is subjected to a precipitation purification stage, at the end of which the contaminants remain in solution.
    [0007] In general, according to the invention, it is possible to carry out the step of purifying a plasma protein for therapeutic use by multicolumn chromatography directly from blood plasma, or cryosurning agent, or an ethanolic fraction. or caprylic, possibly virally secured, or any product resulting from an intermediate purification step and in particular a filtration, another chromatography, or even a multicolumn chromatography, etc. In an example of a particular embodiment, the process according to the invention consists of a process for preparing a concentrate of albumin and / or fibrinogen for therapeutic use from blood plasma comprising a plasma supernatant purification step. cryoprecipitated by multicolumn affinity chromatography or anion exchange, optionally followed by a step of removing contaminants present in the fraction comprising albumin and / or fibrinogen by precipitating said fraction with ethanol. In one particular embodiment, the process according to the invention consists of a process for preparing a therapeutic-use immunoglobulin concentrate from blood plasma comprising successively: (a) a first step of inactivation or elimination viral solvent / plasma detergent; (b) a step of purifying cryoprecipitated plasma supernatant by multicolumn affinity chromatography of the inactivated plasma from step (a); (c) optionally a step of removing anti-A and anti-B antibodies, especially by multicolumn affinity chromatography, the immunoglobulin concentrate from step (b); (d) optionally a second step of inactivation or viral elimination by nanofiltration of the immunoglobulin concentrate from step (b) or (c); and (e) a step of adding one or more pharmaceutically acceptable stabilizers to the immunoglobulin concentrate from step (b), (c) or (d). In another particular embodiment, the method according to the invention consists of a process for the preparation of an immunoglobulin concentrate for therapeutic use from blood plasma comprising successively: (a ') a step of purification of cryoprecipitated plasma supernatant by multicolumn affinity chromatography; (b ') a first step of inactivation or viral solvent / detergent removal of the immunoglobulin concentrate obtained in step (a'); (c ') optionally a step of removing anti-A and anti-B antibodies, especially by multicolumn affinity chromatography, the immunoglobulin concentrate from step (b'); (d ') optionally a second step of inactivation or viral elimination by nanofiltration of the immunoglobulin concentrate from step (b') or (c '); and (e ') a step of adding one or more pharmaceutically acceptable stabilizers to the immunoglobulin concentrate from step (b'), (c ') or (d').
    [0008] In another example of a particular embodiment, the method according to the invention consists of a process for preparing an immunoglobulin concentrate for therapeutic use from blood plasma comprising successively: (A) an ethanolic fractionation step and / or caprylic cryoprecipitate plasma supernatant; (B) optionally a first step of inactivation or viral solvent / detergent removal of the solution obtained in step (A); (C) a purification step by anion exchange multicolumn chromatography of the solution obtained in step (A) or (B); (D) optionally a step of removing anti-A and anti-B antibodies, in particular by multicolumn affinity chromatography, from the immunoglobulin concentrate obtained at the end of step (C); (E) optionally a second step of inactivation or viral elimination by nanofiltration of the immunoglobulin concentrate obtained at the end of step (C) or (D); and (F) a step of adding one or more pharmaceutically acceptable stabilizers to the immunoglobulin concentrate from step (C), (D) or (E).
    [0009] The invention also relates to a blood plasma fractionation method comprising the steps of directly subjecting blood plasma or cryoprecipitated plasma supernatant to: multicolumn affinity chromatography wherein at least one affinity ligand is a ligand specifically binding immunoglobulins, after which the fraction containing the immunoglobulins is recovered; and / or multicolumn anion-exchange chromatography and / or mixed-mode chromatography mode, optionally highly salt-tolerant, after which the fraction containing fibrinogen and / or albumin, optionally followed by a purification step of said fraction, not retained from the preceding multicolumn chromatography, by precipitation and / or by chromatography.
    [0010] The invention also relates to a method for fractionating blood plasma comprising the steps of directly subjecting blood plasma or cryoprecipitated plasma supernatant to: multicolumn affinity chromatography wherein at least one affinity ligand is a ligand specifically binding immunoglobulins, after which the fraction containing the immunoglobulins is recovered; and / or multicolumn affinity chromatography in which at least one affinity ligand is a ligand specifically binding fibrinogen, after which the fibrinogen-containing fraction is recovered; and / or multicolumn affinity chromatography in which at least one affinity ligand is a ligand specifically binding albumin, after which the albumin-containing fraction is recovered. Advantageously, the multicolumn affinity chromatography targeting the immunoglobulins is carried out first, or the multicolumn affinity chromatography targeting albumin and / or fibrinogen being made from the fraction resulting from this first fractionation stage and free from Ig.
    [0011] The subject of the invention is also an immunoglobulin concentrate having an immunoglobulin distribution profile that is similar or identical to the distribution profile of the immunoglobulins in the plasma. Preferably, the immunoglobulin concentrate according to the invention is a concentrate of immunoglobulins G exhibiting between 50 and 70% of IgG1, 25 to 35% of IgG2, 2 to 8% of IgG3 and 1 to 8% of IgG4. . The subject of the invention is also an immunoglobulin concentrate having an antigenic repertoire similar or identical to the antigenic repertoire of the plasma. Preferably, the immunoglobulin concentrate according to the invention has an antigenic repertoire similar to and / or greater than the antigenic repertoire of the concentrates of the prior art. Detailed Description By "plasma protein" is meant according to the invention any protein, and more particularly any protein of industrial or therapeutic interest, contained in the blood plasma. Blood plasma proteins include albumin, alpha / macroglobulin, antichymotrypsin, antithrombin, antitrypsin, Apo A, Apo B, Apo C, Apo D, Apo E, Apo F, Apo G, beta XIIa, C 1-inhibitor, protein C-reactive, C7, Clr, Cls, C2C3, C4, C4bP, C5, C6, Clq, C8, C9, carboxypeptidase N, ceruloplasmin, factor B, factor D, factor H, factor I, factor IX, factor V , factor VII, factor VIIa, factor VIII, factor X, factor XI, factor XII, factor XIII, fibrinogen, fibronectin, haptoglobin, hemopexin, heparin cofactor II, histidine rich GP, IgA, IgD, IgE, IgG, ITI, IgM, kininase II, kininogen HPM, lysozyme, PAI 2, PAI I, PCI, plasmin, plasmin inhibitor, plasminogen, prealbumin, prekallikrein, properdin, protease nexin INH, protein C, protein S, protein Z, prothrombin, serum amyloid protein ( SAP), TFPI, thiol-proteinase, thrombomodulin, tissue factor (TF), TPA, transcolabamine II, transcortin, transferrin, vitronectin, and the von Willebrand factor. In particular, plasma proteins include coagulation proteins, that is, plasma proteins involved in the chain of cascade reactions resulting in the formation of a blood clot. Coagulation proteins include Factor I (fibrinogen), Factor II (prothrombin), Factor V (proaccelerin), Factor VII (proconvertin), Factor VIII (antihemophilic factor A), Factor IX (factor antihemophilic B), factor X (Stuart factor), factor XI (Rosenthal factor or PTA), factor XII (Hageman factor), factor XIII (fibrin stabilizing factor or FSF), PK (prekallikrein) KHPM ( high molecular weight kininogen), factor III (thromboplastin or tissue factor), heparin cofactor II (HCII), protein C (PC), thrombomodulin (TM), protein S (PS), von Willebrand (Wf) and tissue factor pathway inhibitor (TFPI), or tissue factors. In some embodiments, the plasma protein is an enzymatically active coagulation protein. Enzymatic coagulation proteins include activated forms of factor II (prothrombin), factor VII (proconvertin), factor IX (antihemophilic factor B), factor X (Stuart factor), factor XI (Rosenthal factor or PTA) , factor XII (Hageman factor), factor XIII (fibrin stabilizing factor or FSF) and PK (prekallikrein).
    [0012] In the context of the invention, the term "human immunoglobulins" or "human Ig" means polyvalent immunoglobulins which may be immunoglobulins A (IgA), immunoglobulins E (IgE), immunoglobulins M (IgM) or immunoglobulins G (IgG). The human immunoglobulins according to the invention are advantageously IgG, regardless of their subclass (IgG1, IgG2, IgG3 and IgG4). It may be whole immunoglobulins, or any intermediate fraction obtained during the process of manufacturing polyvalent immunoglobulins. By "plasma fraction" is meant any part or subpart of the plasma, having undergone one or more purification steps. The plasma fractions thus include cryoprecipitated plasma supernatant, plasma cryoprecipitate (resuspended), fractions I to V obtained by ethanolic fractionation (according to the method of Cohn or Kistler & Nitschmann), the supernatant and the precipitate obtained after caprylic acid and / or caprylate precipitation, chromatographic eluates and non-adsorbed fractions of the chromatography columns, and the filtrates. According to the invention, the "cryoprecipitated plasma supernatant", or "cryosurnant", corresponds to the liquid phase obtained after thawing frozen plasma (cryoprecipitation). In particular, the cryosurnant can be obtained by freezing blood plasma at a temperature between -10 ° C and -40 ° C, and then soft 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 cryosurnant. Cryoprecipitate is concentrated in fibrinogen, fibronectin, von Willebrand factor and factor VIII, while cryosurnant 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 and immunoglobulins and albumin. By "purification step" is meant any step of a process for the enrichment of a product of interest, and in particular a plasma protein, in a given fraction.
    [0013] The preparation process according to the invention is based mainly on the use of a multicolumn chromatography step for the purification of a therapeutic plasma protein from blood plasma or any plasma fraction. The implementation of such a multicolumn chromatography step is particularly suitable for the preparation of an immunoglobulin concentrate. Indeed, it is possible, according to the invention, to directly subject human blood plasma or cryosurnant of human blood plasma to multicolumn chromatography. Of course, it is also possible to provide a filtration and / or ethanolic / caprylic fractionation step upstream of the purification by multicolumn chromatography.
    [0014] The use of such multicolumn chromatography generally allows a reduction on an industrial scale of the cost price of plasma proteins for therapeutic use. In particular, multicolumn chromatography requires a lesser amount of gel than conventional chromatography. Similarly, the elution, washing and sanitization phases being performed on smaller columns, buffer requirements are halved compared to conventional chromatography. If, the principle of fractionation of plasma molecules by cascading chromatographies has already been described (John Curling et al., "A Comparative Study of Cohn and Chromatography Fractionation Using a" Novation-Offscape -Cascade Process "" PPB Congress May 2005), he The use of multicolumn chromatography according to the invention makes the principle of cascade chromatography much more attractive, by allowing a very limited industrial interest because of the low capacities and associated productivities. a better capacity and a better productivity According to the invention, several multicolumn chromatography techniques can be used, in particular the "Simulated Mobile Bed" or "SMB" (Simulated Moving Bed) technology from which the technology derives. SMCC (Sequential Multicolumn Chromatography), which are particularly suitable for implementation of the method according to the invention. Examples of implementation of multicolumn chromatography are described in patent applications WO2007 / 144476 and WO2009 / 122281.
    [0015] According to the invention, the multicolumn chromatography may be affinity chromatography, ion exchange (anions or cations), hydrophobic, mixed mode or size exclusion. Preferably, the multicolumn chromatography is a multicolumn affinity chromatography, mixed mode or anion exchange.
    [0016] In an example of a particular implementation of the invention, the multicolumn chromatography is a radial chromatography, that is to say using radial columns. According to the invention, it is possible to use radial columns having a ratio of at least two between the outer diameter surface (inlet side) larger and the inner diameter surface (outlet side) smaller. These columns make it possible to increase the loading rates, making the process particularly advantageous when there are large plasma volumes to be treated and / or that the plasma protein of interest, such as immunoglobulins, must be purified rapidly in order to maintain its molecular integrity. Likewise, this makes it possible to reduce the duration of the purification steps and thus to multiply the batches per unit of time.
    [0017] Depending on the needs, and more particularly the volumes of plasma to be treated, it is possible to use chromatography columns of a few milliliters (for an implementation at the laboratory scale for example), for example from 5 to 20 ml, to several hundred or thousands of liters (on an industrial scale), for example from 200 to 2000 L. For each range of columns, it is also possible to adapt the height of the gel bed forming the stationary phase, for example 6 cm, 12 cm or 18 cm. The person skilled in the art knows, depending on the volumes to be treated and / or the desired flow rate, to adapt the number of columns, their dimensions and the gel heights.
    [0018] In a particular example, and especially for the preparation of an immunoglobulin concentrate directly from cryosurnant, multicolumn affinity chromatography is advantageously used. It is known that most of the affinity gels (forming the stationary phase of affinity chromatography), especially those used industrially, have a maximum load capacity of Ig four to five times lower than an exchange gel. ions (20-30 gL-1 against 80-100 gL-1). It is therefore generally accepted that in order to treat large plasma volumes the amount of affinity gel to be used is such that its use is economically unsuitable.
    [0019] However, the Applicant has discovered that this disadvantage can be countered by fractionating the chromatography column into several smaller columns and in particular into 3 to 8 columns, preferably 3 to 5 columns. The use of a plurality of chromatography columns makes it possible to place in series of a main column (s) guard columns which will capture the plasma proteins of interest which have leaked from the main column (s) when loading the protein of interest. The affinity ligands present in this main column (es) are then saturated so as to reach the maximum capacity of the chromatography gel. This column (es) is (are) then output (s) of the circuit to be eluted (s) separately and allow recovery of the proteins of interest. The guard columns in turn becoming the main column (s). Successive cycles of 3 to 50, preferably 5 to 30 and most preferably 7 to 15 maximize the use of the affinity ligands and thereby reduce the total gel volume required for capture and control. elution of the molecule of interest. Advantageously, the affinity ligand is chosen from antibodies, antibody fragments, antibody derivatives or chemical ligands such as peptides, mimetic peptides, peptoids, nanofitins or else oligonucleotide ligands such as the aptamers. For example, it is possible to use a gel comprising a crosslinked agarose matrix, making it possible to work at high flow rates, in combination with an affinity ligand able to bind the protein of interest. In a particular embodiment, the affinity ligand makes it possible to bind the Fc fragment of human Ig or any sequence conserved within the immunoglobulins. In another particular embodiment, the affinity ligand used is a recombinant protein recognizing the Fc fragment of Ig and coupled to a matrix, for example the IgSelect gel from GE Healthcare. Advantageously, the ligand may be chosen so as to specifically recognize a class of immunoglobulins (for example immunoglobulins G) and / or to specifically recognize one or more immunoglobulin subclasses (for example IgG1 and / or IgG2 and / or IgG3 and / or IgG4). In another particular embodiment, the affinity ligand used is protein G, which has an affinity for IgG and advantageously has no affinity for IgA. In yet another particular embodiment, the affinity ligand used is protein A which specifically binds IgG1, IgG2 and IgG4 but does not capture IgG3.
    [0020] In a particular embodiment, the affinity ligand of the multicolumn affinity chromatography has an affinity for immunoglobulins G, and is advantageously chosen from ligands having an affinity for IgG1, IgG2, IgG3 and IgG4. More particularly, the IgG concentrate obtained according to the process of the invention advantageously has a distribution profile of the IgG subclasses similar to that of the plasma. In particular, the IgG concentrate according to the invention advantageously comprises between 50 and 70% of IgG1, 25 to 35% of IgG2, 2 to 8% of IgG3 and 1 to 8% of IgG4 or even more preferred between 60 and 70% IgG1, 30 to 35% IgG2, 3 to 6% IgG3 and 2 to 5% IgG4. In a particular embodiment, the concentrate obtained according to the process of the invention may have a slight decrease of the IgG3 and / or IgG4 subclasses relative to the plasma, the product nevertheless remaining comparable in therapeutic efficacy to a product having a repertory of IgG subclasses similar to that of plasma. In another particular embodiment, the affinity ligand of the multicolumn affinity chromatography has an affinity for fibrinogen or albumin. More particularly, the invention also relates to a process for the preparation of albumin and / or human fibrinogen concentrates directly from blood plasma, according to which said plasma or cryoprecipitated plasma supernatant is subjected to multicolumn chromatography. Affinity in which the affinity ligand (s) has affinity for fibrinogen and / or albumin. The ligand used can be in particular any commercially available ligand, for example the CaptureSelect HSA affinity ligand (Life Technologies) or the CaptureSelect Fibrinogen affinity matrix ligand (Life Technologies). Advantageously, the affinity ligand as chosen for the process of the invention is resistant to sanitary conditions and / or intensive reuse compatible with industrial use. In a particular embodiment, the affinity ligand is thus advantageously chosen from peptides and / or peptoids (derived from combinatorial biology, such as phage display), nanofitins (extracted from extremophilic bacteria), and / or aptamers. .
    [0021] In a particular example, the affinity ligand is an aptamer, and in particular a nucleic aptamer. The term "aptamer" as used herein refers to a molecule of single-stranded nucleic acid, DNA or RNA, and in particular a single-stranded nucleic acid molecule capable of specifically binding to the protein of interest, for example to a immunoglobulin by binding to the Fc fragment of human Ig or conserved immunoglobulin sequences. Advantageously, the aptamer may be chosen so as to specifically recognize an immunoglobulin class (for example immunoglobulins G) and / or to specifically recognize one or more immunoglobulin subclasses (for example IgG1 and / or IgG2 and / or IgG3 and / or IgG4). Aptamers generally comprise between 5 and 120 nucleotides and can be selected in vitro by a process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). According to the invention, the term "nucleic aptamer" is intended to mean a single-stranded nucleic acid, and in particular a single-stranded nucleic acid specifically binding to the Fc fragment of human IgGs. Aptamers have many advantages. Because of their oligonucleotide nature, aptamers possess low immunogenicity and high resistance to stringent physicochemical conditions (presence of DMSO, very acidic or very basic pH, use of organic solvents or high temperature) allowing various sanitization strategies in the context of use as an affinity ligand. It is thus possible to increase the life of the affinity gels by carrying out sanitization steps to clean the chromatography columns and limit their clogging and reduce the risk of viral or prion contamination, despite the multiple cycles. and the charges increased compared with conventional chromatography. Aptamers, thanks to their nucleic acid structure, are particularly adapted to basic pH sanitizations, allowing reuse from 100 to 200 cycles. The patent application FR 2 970 003, in the name of the Applicant, describes methods of manufacturing affinity supports with immobilized nucleic aptamers. In particular, this application describes a method of immobilizing nucleic acids comprising at least one reactive amine function, by grafting on a solid support having, on its surface, activated carboxylic acid groups. In a particular embodiment, the multicolumn chromatography can be advantageously applied to one or more successive chromatography columns. Thus, the non-retained fraction of the first multicolumn chromatography can be used to purify the other plasma proteins of interest in the most appropriate order.
    [0022] According to the invention, the process for preparing human plasma protein concentrates for therapeutic use may also include at least one of the following steps: (i) a viral inactivation or viral elimination step; (ii) an ion exchange chromatography step; (iii) a step of removing anti-A and anti-B antibodies, in particular by affinity chromatography; (iv) a precipitation step with caprylic acid and / or caprylate; (v) a concentration step by ultrafiltration; (vi) a formulation step. Advantageously, the preparation method according to the invention makes it possible to obtain a human protein concentrate for therapeutic use and may comprise at least one subsequent stage among steps (i) to (vi).
    [0023] In a particular embodiment, the preparation method makes it possible to obtain a human immunoglobulin concentrate for therapeutic use, optionally comprising one or more subsequent steps from steps (i) to (vi). For example, the human plasma protein concentrate and / or the immunoglobulin concentrate obtained by directly subjecting blood plasma or cryosurnant to multicolumn chromatography, affinity or anion exchange in particular can advantageously undergo at least one step of elimination or inactivation of at least one infectious agent. Among the infectious agents referred to in step (i), mention may be made of viruses and NCTAs (unconventional transmissible agents) such as prions. Viral inactivation often includes treatment with chemicals, for example solvent, and / or detergent and / or heat (pasteurization and / or dry heating) and / or irradiation (Gamma and / or UVC) and / or by pH treatment (treatment with acidic pH). Preferably, step (i) according to the invention comprises at least one treatment with solvent and detergent. The treatment with solvent and detergent (generally called Solvent / Detergent or S / D treatment) comprises in particular the treatment with tri-n-butyl phosphate (TnBP) and / or a detergent which is chosen from Triton X-100, Tween ( preferably Tween 80), sodium cholate and 2- [4- (2,4,4-trimethylpentan-2-yl) phenoxy] ethanol (Octoxinol). Nanofiltration can also be used to remove infectious agents, including viruses and NCTAs. In the case of plasma proteins, nanofiltration generally refers to the filtration of the protein concentrate of interest through a filter with a pore size of less than 80 nm. The filters available are, for example, BioEx filters, Planova® 75 nm, Planova® 35 nm, Planova® 20 nm or Planova® 15 nm (Asahi corporation), Ultipor DV 50 or DV 20 (Pall Corporation), Virosart CPV (Sartorius) , Viresolve NFR or NFP (Millipore). The nanofiltration can be advantageously carried out on a single filter or on several filters in series of identical or decreasing porosity. The removal of infectious agents can also be accomplished by means of depth filtration. The available filters are, for example, regenerated cellulose composite filters, in which filter aids may have been added (such as cellite, perlite or Kieselguhr earths) marketed by Cuno (Zeta + VR series filters), Pall-Seitz (P-series Depth Filter) or Sartorius (Virosart CPV, Sartoclear P depth filters). In a particular embodiment, a viral inactivation step may advantageously be carried out directly on the plasma or the cryoprecipitated plasma supernatant or the resuspended plasma cryoprecipitate, so that all the proteins to be purified benefit from the upstream treatment. multicolumn chromatography. In another particular embodiment, the crude plasma and / or the cryoprecipitated plasma supernatant and / or the resuspended plasma cryoprecipitate may be subjected to a depth filtration or to a filtration sequence, for example on a polypropylene filter. or media equivalent of 61.1m, 1 lm, then 0.45 - 0.2 iam, upstream of multicolumn chromatography. Such a preliminary step advantageously makes it possible to prevent and / or reduce premature clogging of the columns used for multicolumn chromatography. In another particular embodiment, the depth filter (s) used are delipidant depth filters (for example Cuno or Pall-Seitz) allowing a reduction of the lipids in the treated plasma fraction. Advantageously, step (i) of inactivation or viral elimination is followed by a step (ii) of ion exchange chromatography.
    [0024] In a particular example, the ion exchange chromatography step (ii) is anion exchange chromatography or cation chromatography. Such steps of anion exchange chromatography or cation are described in patent applications EP 0 703 922 and WO 99/64462 in the name of the Applicant. In a particular example, step (ii) of anion exchange chromatography can be carried out on a crosslinked polysaccharide gel or a vinyl or acrylic polymer, grafted with DEAE or TMAE or QAE groups, as described in the applications WO2002 / 092632 and WO2013 / 007740 in the name of the Applicant. In another particular example, multicolumn anion-exchange and / or mixed-mode chromatography is performed on a high salt tolerance matrix for capturing the plasma protein in a medium having a high salinity such as plasma or fraction. non-retention of a chromatography. Advantageously, the multicolumn chromatography with anion exchange and / or mixed mode with high salt tolerance is carried out on the STAR AX gel from PALL BIOSEPRA, Capto MMC from GE Healthcare or the Merck ESHMUNO HCX gel or any known equivalent gel from the USSR. skilled person.
    [0025] In particular, for the preparation of an immunoglobulin concentrate for therapeutic use, step (ii) of anion exchange chromatography may comprise: the adjustment to a pH of 8 to 10 of the solution having undergone the treatment solvent-detergent (step (i)); - Its charge on the chromatography column previously equilibrated in buffer at pH 8 to 10 which allows the adsorption of immunoglobulins and the passage of non-adsorbed proteins in the effluent; washing with the same buffer until all the unadsorbed proteins and the detergent solvent mixture have been removed; and elution of the immunoglobulins by an appropriate buffer. This elution can be carried out with phosphate buffer at pH between 4 and 7, and preferably at pH 6.2 to elute the immunoglobulins.
    [0026] The method according to the invention may also comprise a step (iii) of removing anti-A and / or anti-B antibodies. In a particular example, the step (iii) of removing anti-A and / or anti-B antibodies is carried out on the solution obtained at the end of step (ii). This step may for example be carried out according to the method described in the patent application WO 2007/077365 in the name of the Applicant. Thus, in the case of the preparation of an immunoglobulin concentrate for therapeutic use, the solution obtained in step (ii) may be subjected to a step of removing the anti-A and anti-B antibodies by chromatography. immunoaffinity by percolation of said polyvalent immunoglobulin concentrate on a support whose matrix is grafted with antigenically similar oligosaccharide groups to the blood groups A and / or B, or on a mixture of supports whose matrices are grafted with oligosaccharide groups antigenically similar to the groups In a particular embodiment, the step (iii) of removing anti-A and / or anti-B antibodies is carried out on a multicolumn affinity chromatography.
    [0027] In certain cases, the process according to the invention may comprise a step (iv) of precipitation with caprylic acid and / or caprylate. In general, the addition of caprylic acid and / or caprylate in a slightly acidic medium makes it possible to precipitate the plasma proteins, with the exception of the Ig which remain in the supernatant. According to the invention, it is possible to proceed to this step (iv) of caprylic precipitation before or after the purification step 30, by multicolumn chromatography.
    [0028] In an example of a particular embodiment, the step of purifying the plasma protein of interest by multicolumn anion exchange chromatography, is followed by a subsequent step of precipitation with caprylic acid and / or caprylate. Such a combination of multicolumn ion exchange chromatography / caprylic acid precipitation may be particularly advantageous for the preparation of an Ig (and especially IgG) concentrate and / or an albumin and / or fibrinogen concentrate. . In particular, for the preparation of an immunoglobulin concentrate for therapeutic use, step (iv) of caprylic acid precipitation may comprise the steps of - optionally adjusting the pH of the plasma fraction to a value between 3.0 and 6.0, preferably at pH 4; - Add caprylic acid to the plasma fraction; - Centrifuge or filter to recover the supernatant enriched with IgG. The process according to the invention may advantageously comprise one or more ultrafiltration concentration steps (v). For example, during the preparation of an Ig concentrate, it is possible to subject the fraction enriched in Ig, resulting from the multicolumn chromatography step, possibly previously subjected to one and / or the other of the steps (i) to (iv), membrane ultrafiltration. It is also possible to provide a step (vi) of adding one or more pharmaceutically acceptable stabilizers to the human plasma protein concentrate. According to the invention, it is possible to provide a step (i) of additional viral security, by nanofiltration, before the step (vi) of formulation. By pharmaceutically acceptable stabilizers is meant formulations adapted to plasma protein concentrates, in particular the excipients as described in applications FR 03 08403, and preferentially the formulations adapted to immunoglobulin concentrates, and in particular the excipients as described in the applications FR 03 04388, FR 08 59117. FR 10 54721, FR 10 55825 in the name of the Applicant. The formulation step (vi) may optionally be followed by a step (vii) of freezing or lyophilizing said pharmaceutical preparation obtained in step (vi). Advantageously, the process according to the invention allows the preparation of immunoglobulin concentrates, and / or fibrinogen and / or albumin, in particular by avoiding cold storage steps. Advantageously, the process according to the invention makes it possible to obtain an immunoglobulin concentrate with a yield greater than 5 g / l of plasma, preferably with a yield greater than 7 g / l of plasma, and of even more preferred way with a yield greater than 9g.L-1 of plasma. Very advantageously, the method according to the invention is optimized in order to obtain a yield of immunoglobulins close to 10 -12 g.L-1 of initial plasma. Advantageously, the immunoglobulin concentrate obtained by the process according to the invention has an antigenic repertoire similar or identical to the antigenic repertoire of the plasma. In a particular embodiment, the immunoglobulin concentrate obtained by the process according to the invention has an antigenic repertoire similar to and / or greater than the antigenic repertoire of the concentrates of the prior art. In fact, the limited losses of immunoglobulins during the process according to the invention advantageously make it possible to keep a distribution of immunoglobulins similar to that of the plasma and to maintain a broad antigenic panel. The method according to the invention is thus particularly advantageous for the production of immunoglobulin fractions directed against a specific antigenic target.
    [0029] The following example illustrates the invention without limiting its scope. Example: preparation of immunoglobulin concentrates by multicolumn affinity chromatography Material & Cryo-supernatant method Two lots of approximately 5L of human plasma cryosurnant (lots 13500-13L-06002 and 1300-13L-06007) comprising between 8 and 10 g / L-1 IgG are aliquoted into 700 mL fractions and then frozen at -80 ° C. The characteristics of the batches used are reproduced in Table 1 below. Table 1: Distribution of IgG Subclasses in Cryosurants Material IgG IgG1 IgG2 IgG3 IgG4 IgG3 Total IgG1 First Total (%) (%) (%) (%) Cryo 13L06002 100.0 Supernatant 57.9 32.0 3.5 6.6 Cryo 13L06007 100.0 Supernatant 57.2 32.3 4.0 6.6 In a test, the required volume of cryosurnant is thawed for 20-30 min in a water bath at room temperature. 37 ° C without the product exceeding the internal temperature of 25 ° C. Buffer Solutions The composition of the buffer solutions used in the various steps of the affinity chromatography process is summarized in Table 2 below.
    [0030] Table 2: Buffer solutions for affinity chromatography Phases Composition Target values Equilibration and washing Na2HPO4, 12 H2O: 16.7mM NaH2PO4, 2 H2O: 3.34 mM 150 mM NaCl pH = 7.4 Elution Glycine 0.1M pH = 3 Adjustment Eluate Glycine 0.1M pH = 11 The composition of the buffer solutions used during the various steps of the ion exchange chromatography process is summarized in Table 3 below.
    [0031] Table 3: Buffer solutions for ion exchange chromatography. Phases Composition Target values Pre-equilibration Glycine, NaCl pH: 9.0 +/- 0.1 conductivity: 8.5 +/- 0.5 mS / cm Equilibration and Glycine, NaCl pH: 9.0 +/- 0, 1 Washing conductivity: 1000 +/- 100, uS / cm Elution Na2HPO4, 12H20: NaH2PO4, 2H20 pH: 6.2 +/- 0.1 conductivity: 1600 +/- 100, uS / cm Gels of chromatography For chromatography affinity, a GE Healthcare Ig Select® gel (lots # 10035817 and # 10017479) is used. The affinity ligand used is a ligand of the company BioAffinityCompany, which specifically binds the Fc fragment of human IgG. This ligand is a recombinant protein of 14kD, coupled to the base of the matrix by a long carbon spacer arm (spacer) that facilitates the adsorption of Ig. This is coupled to the spacer via multipoint amide bonds. For ion exchange chromatography, a strong Fractogel® EMD TMAE anion exchange gel (batch No. 09L03583) is used. This gel consists of a crosslinked polymethacrylate resin on which trimethylaminoethyl (TMAE) groups are grafted. Columns For multicolumn affinity chromatography, from 3 to 5 radial columns of the company Proxcys, referenced RFC 5.0-6.0 (lot 1303B014 - gel volume: 10 ml, gel height: 12 cm, ratio of inlet diameter / output diameter: 2/1) were used in combination with a BioSc Lab (Novasep). For TMAE chromatography, a column XK 16 (GE Healthcare) gel volume 41 mL height 20.4 cm, and surface 2.0 cm 2 was used with an AKTA Purifier 10 (GE Healthcare). Ultrafiltration A Millipore Biomax 30 (PES) cassette is used with a 30 kDa cut-off and a surface area of 50 cm 2 (reference C1PA45544).
    [0032] Sterilizing Filtration Sartorius Minisart filters with a surface area of 5 cm 2 and a porosity of 0.45 μm and 0.2 μm are used (references 16537 and 16532).
    [0033] Multicolumn affinity chromatography assays The tests were carried out on 4 columns, connected in series, with a loading of 26 g of IgG.L-1 gel, a contact time of 5 minutes, with a fixing pH of between 7 hours. , 3 and 7.8, the elution being carried out with a solution of 0.1M glycine pH3. The gel was regenerated after each chromatography. The regeneration consisted in passing 2 VC of 2M sodium chloride solution. Monitoring of the chromatography was performed by recording the OD at 280 nm and calculating the yield of IgG (nephelometry assay).
    [0034] The working rate was 2.3 mL.min-1 which corresponds to a contact time of 2 min during adsorption. The progress of the multicolumn affinity chromatography steps is summarized in Table 4 below. Table 4: Stages of multicolumn affinity chromatography Step Solution Volume Remarks Balancing Buffer Balancing At least 2VC pH check (7.4 ± 0.2) Adsorption Cryosurning 3 mL to 22 mL according to test Collection of the non-adsorbed fraction by OD tracking 280 nm Wash Buffer Balancing 2 to 4 VC Until return Baseline Elution Glycine 0.1M pH 3.0 2 VC Gather until return to base line. Regeneration NaC12 M 2 VC / Rebalancing of the column Buffer Balancing At least 2VC pH check (7.4 ± 0.2) Storage Ethanol 20% Minimum 2VC Not performed if immediate reuse of the VC column: column volume In order to limit the risk of IgG aggregation at acidic pH, the eluates of the affinity columns obtained at optimal pH for IgGs (pH = 9) were adjusted using an adjustment buffer (0.1M glycine pH 11 ). S / D Treatment and TMAE Chromatography S / D treatment is performed for approximately 30 min to inactivate enveloped viruses.
    [0035] The product is then adjusted in pH and conductivity before injection on the TMAE gel. The unadsorbed fraction of the TMAE gel is then recovered (fraction removed), after return to the baseline and washing of the column, the gel eluate is recovered for the next phase.
    [0036] Ultrafiltration The ultrafiltration step makes it possible to dialyze and concentrate the eluate of the TMAE column at an intermediate concentration of 80-120.L-1. Formulation The product was formulated by adding the formulation buffer (mannitol, glycine, Polysorbate80). The concentration of the final product is adjusted to 50 g / L.
    [0037] The product obtained is sterile filtered through 0.45 and 0.22 μm filters. It is then sampled and stored at 4 ° C. Results The analyzes were carried out on the final product in order to be compared with a IgG immunoglobulin at 50 G / L, (reference product) obtained according to a process having in the first stages of purification a fractionation with ethanol - IgG assay total and subclasses by Nephelemetry (Table 6). IgA, IgM and IgE assay by ELISA (Enzyme-Linked Immunosorbent Assay) (Table 7). Table 5: Distribution of IgG Subclasses Total IgG Assay (%) IgG1 (%) IgG2 (%) IgG3 (%) IgG4 (%) Final Product (IgSelect Charge 26 gL-1) 100.0 53.4 33 , 4 1.2 1.7 Product of 100.0 59.7 36.8 2.4 1.9 Reference Table 6: Analysis of contaminant protein content IgG (gL-1) IgA assay (mg.L- 1) Ig M (mg.L-1) Ig E (UI.mL-1) Final Product (Charge 57.6 <13 <8 1.5 IgSelect 26 gL-1) Product of 46.1 8.5 <8 <0.8 Reference It is noted that these conditions of IgG purification directly from cryosurnant give a product of equivalent quality to the reference product. In addition, as shown in Table 6 above, all IgG subclasses are conserved, with a distribution approximating that of the plasma. More interestingly, multicolumn affinity chromatography using IgSelect gel has a step yield greater than 90%, and 7.4g recovered IgG per liter of cryosurnant. Table 7: Ig affinity yield Select Multicolumn IgG Charge Assay (gL-1) Volume (L) Ig G (mg) Yield Step (%) IgSelect 26 g / L / g IgG per liter Cryosurning plasma 8.0 880 6500 100 In conclusion, the purification of IgGs directly from plasma cryosurning by multicolumn chromatography is quite feasible on an industrial scale, in particular to treat volumes of several thousand liters of plasma a day. For example, for the treatment of an industrial batch of 4500L of human plasma, it is possible to use 4 columns of 50L and 12cm of gel height, with a charge of cryosurning corresponding to 27g of IgG per liter of gel, a flow rate of linear adsorption of between 100 and 300 cm.h-1, and a linear flow rate for gel washing and elution steps greater, between 200 and 600 cm / hour configurations composed of 3 columns of 70 liters of gel, or 5 columns of 40 liters of gel are possible, the number of cycles to achieve to treat the entire batch being determined according to the volume of raw material used. Such an implementation, even on an industrial scale, makes it possible to obtain an immunoglobulin concentrate that meets the regulatory requirements and has all the qualities required for pharmaceutical use (purity, biological safety, distribution of IgG subclasses). ...).
    权利要求:
    Claims (27)
    [0001]
    REVENDICATIONS1. A process for preparing a purified plasma protein concentrate for therapeutic use from blood plasma comprising a purification step in which blood plasma or a plasma fraction is subjected to multicolumn chromatography.
    [0002]
    The method according to claim 1, wherein the plasma fraction is selected from cryoprecipitated plasma supernatant, resuspended plasma cryoprecipitate, ethanolic fraction I to V fractions, supernatant and precipitate obtained after precipitation at room temperature. caprylic acid and / or caprylate, eluates or non-retained fractions of chromatographies, filtrates.
    [0003]
    3. A process according to claim 1 or 2, wherein the purified plasma protein concentrate for therapeutic use contains a plasma protein selected from immunoglobulins, albumin, coagulation factors such as fibrinogen (factor 1), the factor VII, factor VIII, factor IX, factor XI, factor XIII, von Willebrand factor, prothrombin complex or PPSB (factors II, VII, IX, X). activated prothrombin complex, biological glues, and protease inhibitors such as alpha-1 antitrypsin, C1-esterase inhibitor or antithrombin, alone or in admixture. alpha / macroglobulin, antichymotrypsin, antitrypsin, Apo A, Apo B, Apo C. Apo D. Apo E. Apo F. Apo G, beta XIIa, C-reactive protein, C7, Cl r, Cl s, C2 C3, C4, C4bP, C5, Cb, Cl, C8, C9, Carboxypeptidase N, Ceruloplasmin, Factor B, Factor D, Factor H. Fibronectin, Haptoglobin, Hemopexin, Heparin Cofactor II, Histidine Rich GP. Kininase II. Kininogen HPM, Lysozyme, PAI 2, PAI, PCI, Plasmin, Plasmin Inhibitor, Plasminogen, Prealbumin, Prekallikrein, Properdine, Nexin Inh Protein. Protein C, Protein S, Protein Z, serum amyloid protein (SAP), TFPI, Thiol proteinase. Thrombomodulin, Tissue Factor (TF), TPA, Transcolabamine II, Transcortin, Transferrin, Vitronectin. 30
    [0004]
    The method according to any one of the preceding claims, wherein the purified plasma protein concentrate for therapeutic use is an immunoglobulin concentrate.
    [0005]
    5. Method according to one of claims 1 to 3, wherein the purified plasma protein concentrate for therapeutic use is a concentrate of fibrinogen and / or albumin.
    [0006]
    The method of any one of the preceding claims, wherein the purified plasma protein concentrate for therapeutic use is a concentrate of immunoglobulins and albumin and / or fibrinogen.
    [0007]
    The method of any of the preceding claims, wherein the multicolumn chromatography is a multicolumn affinity chromatography.
    [0008]
    The method of any one of the preceding claims wherein cryoprecipitated plasma or plasma supernatant is directly subjected to a human immunoglobulin purification step consisting of multicolumn affinity chromatography.
    [0009]
    The method of claim 8, wherein the affinity ligand of the multicolumn affinity chromatography is selected from ligands having affinity for IgG1, EgG2, IgG3 and IG4.
    [0010]
    10. Method according to one of claims 7 to 9, wherein the multicolumn affinity chromatography uses an affinity ligand selected from antibodies, antibody fragments, antibody derivatives, chemical ligands such as: as peptides, mimetic peptides, peptoids, nanofitins, and oligonucleotide ligands such as aptamers.
    [0011]
    11. Method according to one of claims 7 to 10, wherein the multicolumn affinity chromatography uses an affinity ligand selected from the affinity ligands resistant to sanitization conditions and / or intensive reuse compatible with a industrial use, especially among peptides, peptoids, nanofitines and aptamers.
    [0012]
    The process according to any one of the preceding claims, wherein the multicolumn chromatography is a multicolumn ion exchange chromatography, such as anion exchange or cation exchange multicolumn chromatography, or hydrophobic multicolumn chromatography, or chromatography. mixed-mode multicolumns or multicolumn size exclusion chromatography.
    [0013]
    13. The process as claimed in claim 12, in which the anion-exchange multicolumn chromatography is carried out on a crosslinked polysaccharide gel or a vinyl or acrylic polymer grafted with DEAE or TMAE or QAE groups.
    [0014]
    The method of claim 13, wherein the multicolumn anion exchange and / or mixed mode chromatography is performed on a high salt tolerance matrix to capture the plasma protein in a medium having a high salinity such as plasma. or the non-retained fraction of a chromatography.
    [0015]
    15. Method according to one of claims 1 to 14, wherein plasma or cryoprecipitated plasma supernatant is directly subjected to a step of purification of human immunoglobulins consisting of anion exchange multicolumn chromatography, and optionally in which one subjecting the fraction containing said human immunoglobulins to a subsequent purification step by caprylic acid precipitation and recovering the supernatant containing the immunoglobulins.
    [0016]
    The method according to one of claims 1 to 15, wherein plasma or cryoprecipitated plasma supernatant is directly subjected to a purification step of albumin and / or fibrinogen consisting of multicolumn affinity chromatography or anion exchange multicolumn chromatography, and optionally in which the fraction containing albumin and / or fibrinogen is subjected to a subsequent purification step by caprylic acid precipitation and the albumin-containing fraction is recovered and / or fibrinogen.
    [0017]
    17. Method according to one of the preceding claims. in which the multicolumn chromatography comprises from 3 to 8 columns in series, and preferably from 3 to 5 columns, and / or in which the columns of the multicolumn chromatography are radial columns.
    [0018]
    18. Method according to one of the preceding claims. comprising at least one of the following additional steps - a delipidation and / or filtration step; a step of precipitation with caprylic acid; a viral inactivation or viral elimination step optionally comprises a solvent-detergent and / or nanofiltration treatment; an ion exchange chromatography step, and in particular an anion exchange or cation chromatography step, the anion exchange chromatography step preferably being carried out on a crosslinked polysaccharide or polymer gel; vinyl, grafted with DEAE or TMAE or QAE groups; a step of removing anti-A and anti-B antibodies, in particular by affinity chromatography such as multicolumn affinity chromatography; a concentration step by ultrafiltration; - a formulation step.
    [0019]
    The method according to any of the preceding claims, further comprising a formulation step comprising adding one or more pharmaceutically acceptable stabilizers to the purified plasma protein concentrate for therapeutic use, and optionally a freeze or lyophilization step. of the pharmaceutical preparation obtained at the end of the formulation step.
    [0020]
    The method of any one of the preceding claims comprising successively (a) a first solvent / detergent inactivation step of the plasma; (b) a step of purifying immunoglobulins from the inactivated plasma obtained in step (a) by multicolumn affinity chromatography; then (c) optionally a step of removing anti-A and anti-B antibodies, in particular by affinity chromatography, from the immunoglobulin concentrate from step (b); then (d) optionally a second step of viral elimination by nanofiltration of the immunoglobulin concentrate from step (b) or (c); and (e) a step of adding one or more pharmaceutically acceptable stabilizers to the immunoglobulin concentrate from step (b), (c) or (d).
    [0021]
    21. A method according to any one of the preceding claims comprising successively (a ') a step of purifying immunoglobulins from cryoprecipitated plasma supernatant by multicolumn affinity chromatography; then (b ') a first solvent / detergent inactivation step of the immunoglobulin concentrate obtained in step (a'); then I O (c ') optionally a step of removing anti-A and anti-B antibodies, in particular by affinity chromatography, the immunoglobulin concentrate from step (b'); then (of) optionally a second step of viral elimination by nanofiltration of the immunoglobulin concentrate from step (h ') or (C); and (e ') a step of adding one or more pharmaceutically acceptable stabilizers to the immunoglobulin concentrate from step (b'), (c ') or (d').
    [0022]
    22. Method according to one of the preceding claims comprising successively (A) a step of ethanolic and / or caprylic fractionation of cryoprecipitated plasma supernatant; then (B) optionally a first step of inactivation or viral solvent / detergent removal of the solution obtained in step (A); then (C) a step of purifying immunoglobulins by anion exchange multicolumn chromatography of the solution obtained in step (A) or (B); then (D) optionally a step of removing anti-A and anti-B antibodies, in particular by affinity chromatography, from the immunoglobulin concentrate obtained at the end of step (C); then (E) optionally a second step of viral elimination by nanofiltration of the immunoglobulin concentrate obtained at the end of step (C) or (D); and (F) a step of adding one or more pharmaceutically acceptable stabilizers to the immunoglobulin concentrate from step (C). (D) or (E) .5
    [0023]
    23. The method according to one of the preceding claims, comprising the steps of subjecting blood plasma or cryoprecipitated plasma supernatant to: multicolumn affinity chromatography in which at least one affinity ligand specifically binding immunoglobulins, from which the fraction containing the immunoglobulins is recovered; and / or multicolumn anion exchange chromatography, after which the fraction containing fibrinogen and / or albumin is recovered, and optionally a subsequent step of purification by caprylic acid precipitation of the fraction containing albumin and / or fibrinogen, and / or optionally at least one of the following subsequent steps: a viral inactivation or viral elimination step, an ion exchange chromatography step, a step of elimination of anti-A and anti-B antibodies, in particular by affinity chromatography, an ultrafiltration concentration step, a formulation step.
    [0024]
    24. A method according to any one of the preceding claims, wherein the cryoprecipitated plasma supernatant has been previously obtained by precipitation of blood plasma at a temperature between -10 ° C and -40 ° C, followed by gentle thawing at a temperature between 0 ° C and + 1 ° C, followed by centrifugation of the thawed plasma to separate the cryoprecipitate and the supernatant.
    [0025]
    25. A blood plasma fractionation method comprising the steps of directly subjecting blood plasma or cryoprecipitated plasma supernatant or a plasma fraction to: multicolumn affinity chromatography wherein at least one ligand of affinity is a ligand specifically binding the immunoglobulins, after which the fraction containing the immunoglobulins is recovered; and multicolumn anion exchange chromatography and / or mixed mode, optionally highly salt-tolerant, at the end of which the fraction containing albumin and / or fibrinogen is recovered; and optionally a purification step of said non-retained fraction of the above multicolumn chromatography containing fibrinogen and / or albumin by precipitation and / or by chromatography.
    [0026]
    26. A method for fractionating blood plasma comprising the steps of directly subjecting blood plasma or cryoprecipitated plasma supernatant or plasma fraction to: multicolumn affinity chromatography wherein at least one affinity ligand is a binding ligand immunoglobulins, at the end of which the immunoglobulin-containing fraction is recovered: and / or multicolumn affinity chromatography in which at least one affinity ligand is a ligand binding specifically fibrinogen, after which one recovers the fraction containing the fibrinogen; and / or multicolumn affinity chromatography in which at least one affinity ligand is a ligand specifically binding albumin, after which the albumin-containing fraction is recovered.
    [0027]
    27. Fractionation method according to one of claims 23 to 26, wherein the multicolumn affinity chromatography binding immunoglobulins is performed upstream of the multicolumn chromatography (s) for recovering albumin and / or fibrinogen.
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    同族专利:
    公开号 | 公开日
    EP3116527A1|2017-01-18|
    CN106103473A|2016-11-09|
    ES2759259T3|2020-05-08|
    AU2015228649A1|2016-09-15|
    MX2016011641A|2016-10-31|
    FR3018450B1|2016-04-15|
    US20170073396A1|2017-03-16|
    CA2942203A1|2015-09-17|
    EP3116527B1|2019-09-04|
    WO2015136217A1|2015-09-17|
    KR20160130495A|2016-11-11|
    TW201620925A|2016-06-16|
    IL247508D0|2016-11-30|
    JP2017508759A|2017-03-30|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1451997A|FR3018450B1|2014-03-11|2014-03-11|PROCESS FOR THE PREPARATION OF HUMAN PLASMA PROTEINS|FR1451997A| FR3018450B1|2014-03-11|2014-03-11|PROCESS FOR THE PREPARATION OF HUMAN PLASMA PROTEINS|
    AU2015228649A| AU2015228649A1|2014-03-11|2015-03-10|Method for preparing human plasma proteins|
    CN201580012945.8A| CN106103473A|2014-03-11|2015-03-10|The method preparing human plasma protein fraction|
    PCT/FR2015/050598| WO2015136217A1|2014-03-11|2015-03-10|Method for preparing human plasma proteins|
    EP15717016.8A| EP3116527B1|2014-03-11|2015-03-10|Method for preparing human plasma proteins|
    CA2942203A| CA2942203A1|2014-03-11|2015-03-10|Method for preparing human plasma proteins|
    ES15717016T| ES2759259T3|2014-03-11|2015-03-10|Procedure for preparing human plasma proteins|
    US15/125,483| US20170073396A1|2014-03-11|2015-03-10|Method for preparing human plasma proteins|
    JP2016556862A| JP2017508759A|2014-03-11|2015-03-10|Methods for preparing human plasma proteins|
    KR1020167028103A| KR20160130495A|2014-03-11|2015-03-10|Method for preparing human plasma proteins|
    MX2016011641A| MX2016011641A|2014-03-11|2015-03-10|Method for preparing human plasma proteins.|
    TW104107803A| TW201620925A|2014-03-11|2015-03-11|Method for preparing human plasma proteins|
    IL247508A| IL247508D0|2014-03-11|2016-08-28|Method for preparing human plasma proteins|
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