EP3277703A1

EP3277703A1 - Method for the purification of a therapeutic recombinant protein from transgenic milk

Info

Publication number: EP3277703A1
Application number: EP16731191.9A
Authority: EP
Inventors: Sandrine DE MARCO
Original assignee: LFB SA
Current assignee: LFB SA
Priority date: 2015-04-02
Filing date: 2016-04-01
Publication date: 2018-02-07
Also published as: FR3034419A1; WO2016156752A1; FR3034419B1; US20180079779A1; AR104167A1

Abstract

The invention relates to a method for the purification of a therapeutic protein from transgenic milk, comprising a clarification step using a salt of poly(diallyldimethylammonium).

Description

PROCESS FOR PURIFYING RECOMBINANT THERAPEUTIC PROTEIN FROM TRANSGENIC MILK

Field of the invention

The invention relates to the purification of therapeutic proteins produced recombinantly in a biological fluid derived from a transgenic animal.

Technological background

Biomedicines, especially therapeutic proteins, are a major source of innovation and today account for nearly half of the new molecules registered worldwide. In May 2013, nearly 170 biomedicines were marketed in France, among which recombinant monoclonal antibodies are an important therapeutic class, particularly for the treatment of cancer and certain autoimmune diseases or for the prevention of transplant rejection (Leem , Biomedicines in France and state of the art, 2013).

The vast majority of therapeutic proteins are produced recombinantly. There are many expression systems such as unicellular organisms (bacteria or yeasts), insect cells (baculovirus / insect cell system) or transgenic plants. Nevertheless, these expression systems have many limitations, particularly in connection with imperfect protein folding, the impossibility of producing complex proteins such as immunoglobulins or incomplete glycosylation or different from that found in humans.

The most used expression systems in the pharmaceutical industry are, at present, mammalian cells. For example, the active ingredient of MabThera®, a chimeric anti-CD20 antibody for the treatment of non-Hodgkin's lymphoma, is recombinantly produced in the CHO (Chinese Ovary Hamster) cell line.

Mammalian cells allow the production of recombinant proteins with glycosylation patterns very close to those of human endogenous proteins but generally offer low production yields.

The industrial need to produce therapeutic proteins with high efficiency has led the pharmaceutical industry to consider the use of mammalian transgenic animals, such as cows, rabbits or goats, as expression systems. In this approach, the expression of the recombinant protein is directed at the mammary epithelial cells. The recombinant protein is thus secreted in milk and can be recovered from this fluid by extraction and purification processes. For example, Atryn®, a recombinant human antithrombin approved by the FDA and ΕΜΕΑ for the preventive treatment of venous thromboembolism in patients with congenital antithrombin deficiency, is produced in the milk of transgenic goats (Houdebine , Comparative Immunology Microbiology & Infectious Disease, 2009, 32, 107-121).

Several applications and patents describe the preparation of therapeutic proteins in transgenic milk. In particular, EP 0 741 515 and WO2004 / 050847 describe the production of recombinant antibodies. As a further example, the work of Wei et al. concerning the expression of the chHabl8 chimeric antibody in the milk of transgenic mice (Transgenic Res 2011, 20, 321-330). Although transgenic animals provide satisfactory levels of expression, the extraction and purification of the recombinant protein from milk remains one of the limiting steps of these expression systems.

Indeed, milk is a complex biological fluid containing about 87% by weight of water and 13% by weight of dry matter. It comprises lipids, essentially triglycerides, present in emulsion form, proteins, including casein, sugars, in particular lactose, and secondary constituents such as vitamins and trace elements. There are generally three phases in milk:

■ whey comprising carbohydrates, oligoelements, seroproteins and water-soluble vitamins, in which are dispersed:

^■ a lipid phase or cream essentially composed of lipids as an emulsion of fat globules of about 2-6 μιη in diameter, and

^■ a colloidal micellar phase resulting from the combination of casein proteins and calcium phosphate salts.

The recombinant protein may, depending on its nature, be present in the whey, be associated with the fat globules and / or be trapped in the casein micelles, which further complicates its extraction.

Methods for extracting recombinant antibodies from transgenic milk have been proposed. For example, the application WO 97/42835 describes a process for extracting monoclonal antibodies from goat's milk comprising a step of tangential ultrafiltration of the raw milk, in the presence of EDTA, followed by a step of chromato graphy. permeate on a protein G affinity support. The application WO 2004/050847 mentions, in turn, that it would be possible to extract the recombinant antibodies present in a transgenic milk by implementing steps skimming, centrifugation, sedimentation and / or fractionation. Nevertheless, the application WO 2004/050847 does not provide any specific example of implementation of a purification process. Similarly, the application WO 2007/106078 describes a method for extracting a recombinant protein present in a transgenic milk comprising a deep filtration step, but does not propose any concrete application to the purification of recombinant antibodies.

There is therefore, at present, a need for novel methods for purifying recombinant therapeutic proteins from a transgenic milk or protein solution derived from a transgenic milk.

Summary of the invention

The invention relates to a method for purifying a recombinant protein from a transgenic milk or a protein solution obtained from a transgenic milk, comprising a step of clarification using a salt poly (diallyldimethylammonium), preferably poly (diallyldimethylammonium chloride) (pDADMAC). In some embodiments, the recombinant protein is selected from hormones, cytokines, proteins involved in the immune response, antibodies, and coagulation factors and inhibitors. Preferably, the recombinant protein is an antibody.

In some embodiments of the method according to the invention, the clarification step comprises:

adding the poly (diallyldimethylammonium) salt to said transgenic milk or said solution so as to form a liquid phase comprising the recombinant protein, and a flocculate, and

separating the flocculate and the liquid phase so as to recover the liquid phase comprising the recombinant protein.

The final content of the poly (diallyldimethylammonium) salt in the milk or solution may range from 0.01 g / L to 20.0 g / L. Said milk or said solution may be incubated at a temperature of 20 ° C to 60 ° C, after the addition of the poly (diallyldimethylammonium) salt. The separation of the liquid phase and the flocculate can be carried out by a method chosen from mechanical pressing and dewatering. In some embodiments, the clarification step is performed on a crude transgenic milk that has optionally undergone one or more treatments selected from freezing, thawing, and pasteurizing.

The method according to the invention may further comprise at least one additional purification step preferably selected from the group consisting of ultrafiltration, tangential ultrafiltration, microfiltration, diafiltration, reverse phase chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, cation exchange resin chromatography, anion exchange chromatography, affinity chromatography, multimodal chromatography, size exclusion chromatography and combinations thereof.

In one embodiment, the purification process comprises at least one multimodal chromatography step. By way of example, the process may comprise two multimodal chromatography steps

In another embodiment, the purification method according to the invention comprises a step of elimination and / or inactivation of residual pathogens.

The method according to the invention can comprise one, several or all of the following characteristics:

the milk solution is a crude transgenic milk, and / or

the recombinant protein is a human, humanized or chimeric antibody, and / or

the pDADMA salt is a pDADMAC having a mean molecular weight of between 100,000 and 500,000 g.mol ^-1 , and / or

in the precipitation step, the final concentration of pDADMA salt is 2.0 g / L at 5.0 g / L, and / or

the process comprises at least one multimodal chromatography step carried out after the clarification step, and / or

the method comprises at least one purification step selected from microfiltration, ultrafiltration, tangential ultrafiltration, microfiltration, diafiltration, reverse phase chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, cations, anion exchange chromatography, and affinity chromatography, size exclusion chromatography, and / or

the method comprises a pasteurization step carried out before the clarification step, and / or the step of clarification by adding the salt of pDADMA is the only step of clarification of the process, and / or

the method does not include a skimming step performed upstream of the clarification step by adding the pDADMA salt.

The purification method according to the invention may, for example, comprise the combination of the following steps:

at. pasteurizing the transgenic milk or said solution,

b. a step of clarification of the pasteurized transgenic milk or of said pasteurized solution by adding a salt of pDADMA, preferably pDADMAC,

vs. implementing a multimodal chromato graphie step on the clarified solution obtained in step c, possibly after adjusting the pH or the conductivity of said solution,

d. optionally, carrying out one or more additional purification steps on the recombinant protein solution resulting from step d., preferably chosen from the group consisting of ultrafiltration, tangential ultrafiltration, microfiltration, diafiltration , reverse phase chromato graphy, hydrophobic interaction chromatography, hydroxyapatite chromatography, cation exchange resin chromatography, anion exchange chromatography, affinity chromatography, size exclusion chromatography, and combinations thereof, and

e. at least one step of inactivation and / or elimination of residual pathogens, preferably by sterilizing nanofiltration. The invention also relates to a method for preparing a pharmaceutical composition comprising a recombinant protein, said method comprising:

providing a recombinant protein by carrying out a purification method according to the invention, as defined above, and

mixing the recombinant protein with one or more pharmaceutically acceptable excipients.

A further object according to the invention is the use of a poly (diallyldimethylammonium) salt, preferably pDADMAC, as a clarifying agent for the purification of a therapeutic protein from a transgenic milk, or of a protein solution obtained from a transgenic milk. figures

Figures 1A, 1B and 1C show the results of the transgenic milk clarification experiments described in Example 1. The term "Flocculent" refers to pDADMAC.

Figure 1A shows the clarity of the supernatant for each clarifying agent tested. The clarity corresponds to 1 / turbidity, the turbidity of each supernatant having been determined by absorbance at 400 nm. The higher the value of clarity, the better the clarifying agent. Figure 1B shows the recombinant antibody content (mg) in the supernatant obtained for each clarifying agent. The antibody content was determined by nephelemetry.

Figure 1C shows the protein purity of antibodies (relative to total proteins) in each supernatant; the protein purity of antibodies was determined from SDS PAGE gels according to the Laemmli technique.

FIG. 2 shows the electrophoresis gel obtained in SDS PAGE (staining with Coomasie blue) for the supernatants resulting from the various clarification conditions tested and summarized in the table of Example 2. For each compound, the left gel corresponds to at the "low" concentration tested and the gel on the right corresponds to the high concentration tested.

Figure 3 shows a diagram describing the steps of a purification process according to the invention. The implementation of this method is exemplified in Example 4.

detailed description

To the Inventors' knowledge, poly (diallyldimethylammonium chloride) (pDADMAC) is a flocculation agent mainly used in the chemical industry, particularly in the field of water treatment and the paper industry.

The inventors have surprisingly shown that poly (diallyldimethylammonium chloride) (pDADMAC) can be used as a clarifying agent for the purification of a recombinant therapeutic protein from a transgenic milk. Indeed, pDADMAC makes it possible to precipitate a large proportion of casein moles and fat globules present in the milk while maintaining the recombinant protein in solution. In particular, the inventors have shown that the addition of pDADMAC in a transgenic milk comprising a recombinant antibody induces the formation of a multiphase system comprising:

a supernatant in the form of a solution of low turbidity, almost clear, rich in recombinant antibodies, and a phase with a high turbidity comprising most of the casein micelles and fat globules of the starting milk, in the form of a flocculate (which may also be called floc or dairy deposit).

The supernatant rich in recombinant antibodies can be recovered by a conventional liquid / solid separation step, for example by a mechanical pressing, dewatering or filtration step.

Thus, by a single step of clarification, the inventors obtained a pre-purified recombinant antibody solution, cleared of almost all the fat globules and caseins present in the starting milk and having a turbidity of less than 0 , 1, with a yield of about 80% recombinant antibody.

^■ General Embodiment of the purification process according to the invention

Thus, a first object according to the invention is the use of a poly (diallyldimethylammonium) salt as clarifying agent for the purification of a recombinant protein from a transgenic milk, or a solution protein obtained from a transgenic milk.

More specifically, the subject of the invention is a process for purifying a recombinant protein from a transgenic milk or a protein solution obtained from a transgenic milk, comprising a step of clarification with the aid of a poly (diallyldimethylammonium) salt.

For the purposes of the invention, the term "clarification" means reducing the turbidity of a solution. In other words, a clarification step makes it possible to reduce the quantity of particles suspended in a solution. In the case of a milk solution, the clarification step makes it possible to eliminate, at least partially, the casein colloids and / or the suspended fat globules.

For the purposes of the invention, the term "recombinant protein" means a protein whose expression in the transgenic animal results from the introduction of a heterologous gene into its genome. Methods for producing transgenic animals expressing a recombinant protein in their milk are well known to those skilled in the art. By way of example, a person skilled in the art can refer to patent EP 0 741 515, to the application WO 2004/050847 or to the review article by Houdebine, 2009, supra, whose teachings are incorporated here by reference. The heterologous gene coding for the recombinant protein is preferably under the control of a promoter directing its expression in mammary epithelial cells. For example, it may be a promoter of the casein, a lactalbumin promoter, a WAP (whey acidic protein) promoter, or a beta-lactoglobulin promoter, this list not being exhaustive. The transgenic animal may be selected from a goat, a cow, a rabbit, a sow, a mouse, a sheep or a camel. Preferably, the transgenic animal is a goat, a sow or a rabbit.

The recombinant protein can be of any type. Preferably, it is a recombinant protein intended for use in human or veterinary medicine. It can be a human protein or a variant of a human protein.

By way of example of "recombinant proteins" of interest which it is desired to express in a transgenic animal, mention may be made of hormones, cytokines, proteins involved in the immune response, for example factor H, therapeutic antibodies. antibody fragments such as the Fc fragment or Fab or F (ab ') 2 fragments or monodomain fragments (dAb, nanobodies), as well as coagulation factors and inhibitors. Cytokines include, inter alia, interferons, interleukins, chemokines also called chemokines, tumor necrosis factor, growth factors. Hormones include, among others, insulin, erythropoietin, steroid hormones, growth hormone. Coagulation factors include factor VII, (FVII), factor VIII (FVIII), factor X (FX), factor IX (Factor IX), factor XI (FXI), factor XII (FXII), factor XIII (FXIII), factor II (prothrombin), antithrombin III (AT III), heparin cofactor II (Hal), protein C (PC), thrombomodulin (TM), protein S (PS), factor V (FV), von Willebrand factor (vWF), tissue factor pathway inhibitor (TFPI). According to one particular embodiment, the recombinant protein may be a variant of a wild-type protein, that is to say a protein exhibiting one or more mutations with respect to the wild-type protein. In another embodiment, the recombinant protein may be a chimeric or fusion protein, for example comprising protein domains from different wild proteins. In a preferred embodiment, the recombinant protein that is desired to be purified is a recombinant antibody.

The term "antibody" shall be understood to include any polypeptide comprising a binding domain derived from a variable portion of an immunoglobulin, said binding domain being capable of specifically recognizing an epitope and comprising one or more CDR domains (Complementary Determining Regions). ). Preferably, the recombinant antibody according to the invention comprises at least one variable part coupled to an Fc region. By "Fc" or "Fc region" is meant a polypeptide comprising the constant region of a immunoglobulin (with the exception of the first constant domain CH1). For IgG, the Fc part comprises the CH2 and CH3 domains and possibly the lower part of the hinge region ("hinge region"). The recombinant antibody according to the invention may be a chimeric antibody, a humanized antibody, a human antibody or a fusion protein comprising at least one variable region of an immunoglobulin fused to a polypeptide of interest, for example a polypeptide having a therapeutic activity, a polypeptide allowing cell or tissue targeting, or a polypeptide facilitating the subsequent purification of the antibody. Preferably, the recombinant antibody is a human, chimeric or humanized isotype G (IgG) antibody, for example IgG1, IgG2, IgG3 or IgG4, an IgA, isotype M (IgM), isotype D (IgD) or isotype E (IgE), optionally comprising one or more mutations in its Fc part. The recombinant antibody may be a blocking or neutralizing antibody. The recombinant antibody according to the invention can be directed against any therapeutic target of interest. It may be an antibody against a membrane receptor, a cytokine, an interleukin, a hormone, a toxin, an enzyme, an enzyme co-factor, a viral or bacterial protein, a growth factor, a pathogen, a plasma protein, a cellular protein, a cell core protein, DNA or RNA. By way of illustrative and non-limiting example, it may be an anti-RhD, anti-CD20, anti-TNFcc, anti-CD137, anti-HBs, anti-tetanus toxin or anti-CMV antibody. .

By way of example of references describing the production of recombinant antibodies in a transgenic milk, mention may be made of the article by Pollock et al. (Journal of Immunological Methods., 1999, 231: 147-157) or the international application WO 2014125374 in the name of the Applicant which describes the production of an anti-TNFcc antibody in a transgenic animal. A particularly preferred antibody is an anti-TNFcc antibody such as adalimumab.

For the purposes of the invention, the term "transgenic milk" means a milk obtained from a transgenic animal, that is to say from an animal which has been genetically modified so that produce a recombinant protein of interest in its milk. For the purposes of the invention, a "transgenic milk" includes a transgenic milk that has optionally undergone freezing / thawing and / or a sterilization or pasteurization step.

"Protein solution derived from or obtained from said milk or transgenic milk", also hereinafter referred to as "solution obtained from said milk", is understood to mean a solution which comprises the recombinant protein which it is desired to purify and which has been obtained from a transgenic milk by carrying out one or more treatment or purification steps, such as fractionation, viral inactivation and / or supplementation steps. Protein solutions derived from said milk include, but are not limited to, milk in which one or more endogenous components have been at least partially removed, for example skim milk, partially clarified milk, but also virally inactivated milk or sterilization, a milk whose density has been adjusted, for example by dilution, a diluted milk, for example by adding a buffer solution, a condensed milk, a milk supplemented with a preserving agent, an antioxidant or any other agent , for example an enzyme, a viral inactivation agent, or a milk whose pH and / or conductivity constants have been modified, for example by adding an acid, a base and / or an electrolyte.

The solution obtained from a transgenic milk comprises one or more contaminants, including endogenous milk proteins and / or milk lipids. By way of example, said solution or said milk may comprise at least 5 g / l, or even at least 10 g / l, of endogenous proteins, in particular contaminating caseins. In certain embodiments, the weight ratio "recombinant protein / total proteins" in the milk solution is less than 0.8, or even less than 0.7 or 0.6, for example less than 0.5, or even less than 0.5. 0.4 and even less than 0.3. The mass quantity of "total proteins" can be determined by total protein assay techniques such as the Biuret technique, BCA (protein assay with BiCinchoninic acid, Bradford, Coomassie blue, determination of Kjeldahl organic nitrogen, uptake in UV or IR, preferably by the BCA method The mass quantity of the "recombinant protein" can be determined by specific immunological techniques such as ELISA, EIA, RIA, Nephelemetry or Specific biological and / or biochemical such as an enzyme assay, a cell assay, a specific binding assay by surface plasmon resonance (example on BiaCore) Preferably, the mass quantity of recombinant protein is determined by an ELISA test.

"Total proteins" include the recombinant protein to be purified as well as the contaminating proteins, in particular caseins and whey proteins.

Preferably, the solution obtained from the transgenic milk is in the form of a colloidal solution.

For the sake of brevity, the term "milk solution" refers to both "transgenic milk" and "the protein solution obtained from transgenic milk". The term "skimmed milk" means a milk whose lipids, in particular in the form of fat globules, have been at least partially eliminated, for example by ultrafiltration, depth filtration, or centrifugation. For the purposes of the invention, the term "partially eliminated an entity present in a solution" means reducing the amount of this entity by at least 5%, preferably by at least 10%, for example by at least 20%, 30%, 40%, 50%, 60%, or at least 70%, or even at least 80% or 90%, of the initial quantity of that entity in the solution

For the purposes of the present invention, the term "poly (diallyldimethylammonium salt)" (hereinafter pDADMA salt) is understood to mean a diallyldimethylammonium polymer, that is to say comprising the following unit:

wherein n is the number of monomer units and X ^" is a counteranion, preferably n is greater than or equal to 100, preferably less than or equal to 5000.

The polymers of pDADMA are generally presented as a population of polymers.

Preferably, the pDADMA salt has a mean degree of polymerization, i.e., an average number of monomer units per polymer chain, ranging from 500 to 4000, preferably from 1000 to 3000.

X ^" is preferably chosen from pharmaceutically acceptable anions, for example X ^" may be chosen from the following anions: a halide, in particular a chloride or a bromide, a sulphate, a bisulfate, an acetate, a maleate, a bisulfate, phosphate, carbonate or combinations thereof.

Preferably, X ^" is a halide, in particular Cl ^" .

A particularly preferred poly (diallyldimethylammonium) salt within the meaning of the invention is poly (diallyldimethylammonium chloride), hereinafter referred to as pDADMAC.

Suitable pDADMAC typically has an average molecular weight of 70,000 g. mol ^"1 to 700,000 g. mol ^{" 1} , preferably from 100,000 to 500,000 g. mol ^"1 , for example from 200,000 to 350,000 g.mol ^-1 . The clarification step is carried out under conditions making it possible to flocculate or even to precipitate, at least partially, one or more constituents of the milk while maintaining in the soluble state at least a part of the recombinant protein, for example the antibody , initially present in the starting solution.

The term "flocculating" is understood to mean the agglomeration of particles suspended in the milk solution, such as fat globules and casein micelles, so as to generate larger particles called flocculates or flocs.

The term "milk constituent" means any endogenous component of milk, such as casein, whey proteins such as β-lactoglobulin, lactalbumin, albumin or lactoferrin, milk lipids, especially triglycerides. Preferably, the clarification step makes it possible to eliminate, at least partially, the casein micelles and the lipids in the form of fat globules present initially in said milk or said solution.

Typically, the pDADMA salt may be added to the milk solution, either in pure form or in the form of a solution. The addition may optionally be carried out with stirring, at a temperature of between 2 ° C. and 40 ° C. or at a higher temperature, for example at a temperature of 40 ° C. to 60 ° C. The addition of the pDADMA salt can be fractionated or spread over time, for example over a period of minutes to an hour.

After adding the pDADMA salt, the milk solution may be incubated at a temperature of between 2 ° C. and 60 ° C., the time required to obtain the flocculate, typically for a few minutes to a few hours, for example 10 minutes to 24 hours. Advantageously, the addition and incubation temperature is in a range from 20 ° C to 50 ° C, preferably from 30 ° C to 50 ° C. A particular preferred temperature is in the range of 35 ° C to 50 ° C. An appropriate addition and incubation temperature is, for example, 37 ° C. ± 1 ° C. or 45 ° C. ± 1 ° C.

The duration of the incubation step can range from 3 hours to 22 hours, for example from 6 hours to 12 hours. The incubation step can be carried out with stirring, or in the absence of any agitation. In some embodiments, after adding the pDADMA salt, the medium may be homogenized by stirring for a few minutes to a few hours, or even for about 1 to 2 hours, at the desired incubation temperature. The incubation is then continued without agitation, at the desired temperature for the time necessary for good clarification, typically for 3 hours to 18 hours.

The amount of pDADMA salt to be added in the clarification step depends on the composition of the milk solution. Generally, a final salt concentration of pDADMA in the range of 0.01 g / L to 20.0 g / L is sufficient to obtain a satisfactory clarification. Preferably, the concentration of the pDADMA salt is in the range of from 0.5 g / L to 15.0 g / L, for example 1.0 g / L to 10.0 g / L. A final concentration of the adapted pDADMA salt may range from 1.0 g / L to 5.0 g / L, for example in a range of from 2 g / L to 5 g / L, or even in a range from ranging from 3.0 g / L to 4.0 g / L, in particular for pDADMAC. The final salt concentration of pDADMA can be adapted according to the protein concentration and the density of the milk. In general, the salt concentration of pDADMA is less than or equal to 4 g / L.

The inventors have shown that the initial pH of the milk solution (ie transgenic milk or the solution obtained from the milk) has no significant impact on the salt capacity of pDADMA to precipitate casein micelles and fat globules. PDADMA is generally able to flocculate casein micelles and milk fat globules over a pH range of 5.0 to 11.0; in particular pH 6.0 to 10.0. The initial pH of the solution can therefore be chosen according to the solubility properties of the recombinant protein that one wishes to purify. For example, an initial pH of from 6.0 to 10.0, typically from 6.5 to 9.5 is suitable for purifying a recombinant immunoglobulin from a transgenic milk.

In a preferred embodiment, the pDADMA salt is added as an aqueous solution typically having a pDADMA salt concentration of 50 g / L to 450 g / L, e.g. 100 g / L to 400 g. / L. Such solutions, particularly aqueous solutions of pDADMAC, are commercially available.

As explained above, the addition of pDADMA salt causes the flocculation of casein micelles and milk lipids, the recombinant protein remaining mostly in soluble form, in the aqueous phase. The mixture thus obtained can be directly used in a subsequent purification step. Nevertheless, in order to improve the overall efficiency of the purification process, it is preferable to remove the flocculate formed subsequent to the addition of the pDADMA salt before carrying out the subsequent purification steps.

Thus, in a preferred embodiment, the clarification step further comprises a separation step for removing the formed floc.

In other words, the clarification step can include:

adding a poly (diallyldimethylammonium) salt to the milk solution to form a flocculate, and

removing the flocculate thus formed, so as to recover the solution comprising the recombinant protein. The flocculate removal step may be preceded by a step of incubating the milk solution as previously described. The separation of the flocculate and the aqueous phase comprising the recombinant protein can be carried out by any separation method known to those skilled in the art. For example, one can use a conventional liquid / solid separation technique of the food industry such as dewatering or mechanical pressing. If necessary, the separation step may be carried out by centrifugation and / or filtration, for example by tangential filtration, for example tangential microfiltration, deep filtration and combinations thereof.

For the purposes of the invention, the term "deep filtration" is understood to mean a filtration process in which the filter bed in its entirety is used to trap particles suspended in the fluid. The fluid thus passes through the filtration bed in its entirety, the particles being trapped on the surface of the filtration bed and in the voids and / or pores of the filtration bed. Deep filtration may be performed on matrices based on cellulose fibers, regenerated cellulose, polypropylene, or combinations thereof. These filtration matrices may comprise inorganic compounds such as perlite, diatomites, for example Kieselguhr, and fumed silica. By way of example, the matrix used for the depth filtration may comprise cellulose, propylene, perlite and kieselguhr. The porosity of the matrix may be in a range from 10 μιη to 100 μιη, for example from 20 μιη to 60 μιη.

As mentioned above, it is also possible to use a tangential filtration step in the clarification step according to the invention. In some embodiments, this tangential filtration step is performed in combination, and in particular downstream of a flocculant removal step selected from a pressing step, a dewatering step and a depth filtration step. In other embodiments, the tangential filtration step is the only step used to remove the floc. All membranes adapted to tangential filtration, in particular ceramic mineral membranes, can be used in the process according to the invention. The ceramic membrane may have a porosity threshold ranging from 0.1 to 1.5 μιη, for example 0.2 to 1.4 μιη, preferably 0.2 μιη. Such a ceramic membrane makes it possible to collect a filtrate that is advantageously free of microorganisms.

The inventors have shown that in certain cases, the implementation of a sophisticated separation step, such as depth filtration or tangential filtration, was not necessary to optimally remove the flocculate. The use of a technique of simple separation such as dewatering, pressing or vacuum pumping through a membrane, sintered or fabric, for example, a woven fabric such as a nylon fabric does not detract from the clarity and purity of the Recombinant protein solution harvested. Thus, in some embodiments, the flocculate removal step is performed:

by mechanical pressing: The mechanical pressing can be carried out, for example, using a cloth envelope such as a nylon cloth. The nylon fabric can be positioned in a pressure-resistant support, for example made of stainless steel, and allowing the clarified liquid to be collected, or

- Draining: the dewatering can also be achieved using a preferably nylon fabric positioned in a support, for example composite, allowing the clarified liquid to pass freely through said support by simple gravity. The porosity of the fabric used for dewatering is typically less than 50 μιη, for example less than 30 μιη.

The clarification step according to the invention is generally characterized by a recombinant protein yield of at least 60%, preferably at least 70%.

Typically, the implementation of the clarification step makes it possible to reduce by at least a factor of 10, preferably a factor of 100, or even a factor of 1000, the turbidity of the solution. Preferably, as illustrated in the examples, the turbidity is determined spectrophotometrically at the absorbance wavelength of 400 nm. Turbidity can also be measured by light diffraction for example in a turbidimeter, an apparatus for normalizing turbidity in NTU (normal turbidity unit).

The clarification step generally makes it possible to reduce the turbidity of the solution to a value measured at absorbance at 400 nm less than 0.8, preferably less than 0.5, more preferably less than 0.4, for example less than 0.3 or less than 0.2. The clarification step may also make it possible to reduce the amount of casein by at least 20%, preferably by at least 30%, for example by at least 40%, 50%, 60%, 70%, 80%. %, still at least 90% relative to the initial amount of casein present in the solution, before the implementation of the clarification step.

In the context of the present invention, the protein contents, for example recombinant protein or casein, can be determined by nephelemetry and ELISA.

Alternatively or additionally, the clarification step may make it possible to reduce the amount of lipids, in particular triglycerides, by at least 20%, preferably by at least 30%, for example by at least 40%, 50% or more. %, 60%, 70%, 80%, still at least 90% relative to the initial amount of lipids present in the solution, before the implementation of the clarification step. The amount of lipids may for example be measured by colorimetric assay.

The method for purifying a recombinant protein according to the invention may comprise one or more additional steps that may be performed upstream or downstream of the clarification step by adding the pDADMA salt.

By way of example, the method according to the invention may comprise one or more steps chosen from the group consisting of:

a freeze / thaw or defrost step (for example, if the milk solution is supplied in frozen form),

a step of removing or inactivating a pathogen, for example a sterilization step, a pasteurization, and / or a viral inactivation

a skimming step,

a step of adjusting one or more parameters of the milk solution such as pH, conductivity, osmolarity, density or the total protein concentration,

This or these steps being performed preferably upstream of the clarification step.

The term "skimming step" means a step intended to eliminate, at least partially, the lipids in the form of suspension possibly present in the starting solution. This skimming step can be carried out by centrifugation. If necessary, the skimming step can be carried out by lipid decantation, that is to say by allowing the milk to rest sufficiently to obtain two phases. The relative density of the cream (milk lipids) is lower than that of water, the lipid layer rises to the surface, which allows skim milk to be harvested in the low phase.

In a particular embodiment, the method according to the invention does not include a step of skimming, decantation and / or clarification carried out upstream of the clarification step by adding pDADMA. In other words, the clarification step according to the invention is carried out on a transgenic milk which has not been skimmed, decanted or clarified.

Thus, the clarification step by addition of a pDADMA salt can be carried out on a transgenic raw milk that has undergone, optionally, one or more treatments, preferably physical, such as freezing / thawing or sterilization, for example pasteurization. It may also be a virally inactivated transgenic milk, for example by adding a suitable agent such as a detergent, a milk supplemented with a preservative, an antioxidant or any other diluent, or another milk whose pH and / or conductivity constants have been modified, for example, by adding an acid, a base and / or an electrolyte.

In a particular embodiment, the clarification step by adding pDADMAC is the only step of clarification of the process.

By way of example, the method according to the invention may comprise the following steps:

i. optionally, a step of sterilizing the transgenic milk or the solution obtained from said milk,

ii. optionally, a step of adjusting a parameter of the milk solution, in particular its pH, and

iii. a clarification step by adding a pDADMA salt as described above, said process preferably not comprising a clarification, decantation and / or skimming step upstream of step (iii) ). In a further or alternative embodiment, the purification method according to the invention may comprise one or more steps aimed at increasing the purity of the recombinant antibody. These steps are preferably performed downstream of the clarification step. These steps can eliminate residual traces of lipids or caseins. Alternatively or additionally, these steps may make it possible to eliminate one or more other contaminants present in the milk solution, such as certain whey proteins (β-lactoglobulin, -lactalbumin, etc.).

Thus, the process according to the invention may comprise one or more chromatography and / or filtration steps aimed at eliminating or reducing one or more contaminants. It can be microfiltration, ultrafiltration, tangential ultrafiltration, microfiltration, diafiltration, reverse phase chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, cation exchange resin chromatography, anion exchange chromatography, affinity chromatography, multimodal chromatography, size exclusion chromatography, and combinations thereof. of these.

Preferably, the additional purification step (s) is / are selected from the group consisting of cation exchange resin chromatography, anion exchange chromatography, affinity chromatography, multimodal chromatography, and the like. their highly salt-tolerant versions. A particularly preferred technique is multimodal chromatography.

The term "multimodal chromatography" (also called "mixed mode chromatography") means a chromatography process that uses several types of interactions between the stationary phase (or resin) and the compounds to be separated. By way of example, multimodal chromatography can be based on the implementation of hydrophobic and electrostatic interactions, the stationary phase then having ion exchange properties and properties specific to hydrophobic interaction resins. Other types of interaction may occur, for example by hydrogen bonding or via thiol functions.

Resins suitable for multimodal chromatography are well known to those skilled in the art. Typically, these are resins comprising on their surface a ligand capable of establishing several types of interactions, such as 4-mercapto-ethyl-pyridine and benzyl-methyl-ethanolamine. By way of example, for the purification of a recombinant antibody, it is possible to use a commercial resin such as the MEP Hypercel, HEA Hypercel or PPA Hypercel resins marketed by Millipore or else the Capto-MMC or Capto-Adheres resins marketed by GE. Healthcare.

In a preferred embodiment, the method according to the invention comprises at least one multimodal chromatography step. This multimodal chromatography step can be carried out under conditions that make it possible to selectively capture the recombinant protein at the surface of the stationary phase, the contaminants (other proteins and / or lipids) present in the solution not being or very little retained on the stationary phase, under the load conditions used. The recombinant protein can then be unhooked, and thus recovered from the stationary phase by modifying the properties of the elution solution, for example by modifying the pH. If necessary, this multimodal chromatography step can be carried out under "negative" conditions in which the recombinant protein is not retained during charging, while the contaminants are. The recombinant protein is therefore recoverable directly in the feed effluent.

In the context of the purification of a recombinant antibody, it is preferable that the pH of the clarified solution is between 5 and 10, typically at a pH of about 6-9 for the implementation of the multimodal chromatography, in particular. particularly for resins having as ligand 4-mercapto-ethyl-pyridine and benzyl-methyl-ethanol amine. As illustrated in the examples, the recombinant antibodies present in the clarified solution strongly adsorb to the 4-mercapto-ethyl-pyridine-based resin and can then be eluted by decreasing the pH of the elution solution to value less than 5, for example pH 4. The resin based on benzyl-methyl-ethanol amine allows, for its part, the implementation of a "negative" multimodal chromato graphy, the recombinant antibodies present in the clarified solution not being retained on the resin for a pH of about 7, unlike protein contaminants that are strongly adsorbed.

In another additional or alternative embodiment, the method according to the invention may comprise one or more additional steps aimed at improving the sanitary quality of the final recombinant protein composition, that is to say aimed at eliminating and / or inactivate any pathogens present in the milk solution. These pathogens include bacteria, viruses as well as unconventional pathogens such as prions or endotoxins. The step of inactivation and / or elimination of possible pathogens may be carried out according to any of the techniques known to those skilled in the art. This step of inactivation and / or removal of the pathogens may be chosen from the group consisting of UV irradiation, gamma irradiation, a heat treatment, for example a pasteurization, a treatment with a chemical agent, for example a solvent and / or detergent, sterilizing nanofiltration, and combinations thereof. Sterilizing nanofiltration generally refers to filtration through a filter having a pore size of less than 80 nm, preferably 15 to 50 nm. The nanofiltration can be carried out on a single filter or on several filters in series of decreasing porosity.

In some embodiments, the method for purifying a recombinant protein according to the invention comprises one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ) the following characteristics:

the milk solution is a transgenic milk,

the recombinant protein is a recombinant antibody,

the pDADMA salt has an average degree of polymerization ranging from 500 to 4000; the pDADMA salt is pDADMAC, said pDADMAC preferably having an average molecular weight ranging from 70,000 gmol ¹ to 700,000 gmol ¹ , for example from 100,000 to 500,000 g.mol ^"1 ,

the clarification step comprises incubating the milk solution, after adding the pDADMA salt,

in the clarification step, the addition of the pDADMA salt and / or the incubation of the solution is carried out at a temperature of 30 ° C to 60 ° C, in the precipitation step, the final pDADMA salt concentration is 1.0 g / L to 10.0 g / L, preferably 1.0 g / L to 5.0 g / L, more preferably preferred from 2.0 g / L to 5.0 g / L, in the clarification step, the formed flocculate is removed by a method selected from mechanical pressing and dewatering, preferably using a web woven nylon, - the method comprises at least one chromatography step, preferably at least one multimodal chromatography step, carried out downstream of the clarification step by addition of pDADMA salt,

the method comprises at least one purification step selected from microfiltration, ultrafiltration, tangential ultrafiltration, microfiltration, diafiltration, reverse phase chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, cations, anion exchange chromatography, and affinity chromatography, size exclusion chromatography.

the method comprises a step of removing a pathogen carried out downstream of the clarification step by adding the pDADMA salt,

the process optionally comprises a pasteurization step carried out upstream of the clarification step by addition of the pDADMA salt,

the clarification step by addition of pDADMA salt is the only step of clarification of the process,

the process does not include a skimming step performed upstream of the clarification step by adding the pDADMA salt.

It will be understood that the method according to the invention may comprise one or more additional steps, to those mentioned above. The method may comprise a step of recovering the purified recombinant protein, or a purified recombinant protein concentrate.

The method may also comprise steps for modifying or adjusting the protein concentration, the conductivity or the pH of the solution before carrying out a purification step such as chromatography. By way of example, the process according to the invention may comprise one or more steps of adding an acid, a base or a buffer, or else a dialysis, a reverse osmosis, a concentration step, or a dilution step.

The method according to the invention may also comprise one or more steps for conditioning the recombinant protein or the recombinant protein concentrate. obtained at the end of the purification steps, in particular a step of formulating the recombinant protein with one or more excipients, preferably one or more stabilizing agents.

As additional examples, particularly in the context of the purification of a recombinant antibody from a transgenic milk, the method according to the invention may comprise a step for removing endogenous immunoglobulins which have been secreted into the body. milk, that is to say immunoglobulins other than the recombinant antibody possibly present in the milk. The elimination of endogenous immunoglobulins can be carried out by affinity chromatography, for example by using an affinity resin capable of selectively retaining the recombinant antibody, the recombinant antibody then being recovered by elution, or an affinity resin capable of selectively retain endogenous immunoglobulins. By way of example, the affinity matrix may comprise a ligand capable of selectively binding endogenous immunoglobulins, this ligand possibly being an antibody or an antibody fragment.

^■ Sample purification method according to the invention

According to a further aspect, the invention relates to a method for purifying a recombinant protein, preferably a recombinant antibody, present in a transgenic milk or in a protein solution obtained from said milk, said method comprising any one of following combinations of steps:

Combination 1

at. a step of clarifying the milk solution by adding a salt of pDADMA, preferably pDADMAC,

b. at least one purification step selected from the group consisting of multimodal chromatography, ultrafiltration, tangential ultrafiltration, microfiltration, diafiltration, reverse phase chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, resin chromatography cation exchange, anion exchange chromatography, affinity chromatography, size exclusion chromatography and combinations thereof, and

vs. at least one step of inactivation and / or elimination of residual pathogens.

Combination 2 at. a step of clarifying the milk solution by adding a salt of pDADMA, preferably pDADMAC,

b. at least one step of multimodal chromato graphy,

vs. optionally, one or more additional purification steps selected from the group consisting of ultrafiltration, tangential ultrafiltration, microfiltration, diafiltration, reverse phase chromato graphy, hydrophobic interaction chromatography, hydroxyapatite chromatography, resin chromatography cation exchange, anion exchange chromatography, affinity chromatography, size exclusion chromatography and combinations thereof, and

d. at least one step of inactivation and / or elimination of residual pathogens.

Combination 3

at. pasteurizing the transgenic milk or said solution,

vs. the implementation of a multimodal chromatography step on the clarified solution obtained in step b, possibly after adjusting the pH or the salinity of said solution,

d. optionally, carrying out one or more additional purification steps on the recombinant protein solution resulting from step c, preferably chosen from the group consisting of ultrafiltration, tangential ultrafiltration, microfiltration, diafiltration, reverse phase chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, cation exchange resin chromatography, anion exchange chromatography, affinity chromatography, size exclusion chromatography, and combinations of these, and

e. at least one step of inactivation and / or elimination of residual pathogens, preferably by sterilizing nanofiltration.

Combination 4

at. providing a transgenic milk, said recombinant protein being a recombinant antibody,

b. pasteurization of the transgenic milk, preferably at a temperature of 45 ° C to 65 ° C, vs. a step of clarifying pasteurized transgenic milk by adding pDADMAC, said step preferably comprising:

a step of adding pDADMAC to the transgenic milk so as to await a final pDADMAC concentration of 1.0 g / L at 5.0 g / L, preferably 2.0 g / L at 5.0 g / L ,

a step of incubating said transgenic milk after adding pDADMAC, preferably at a temperature of between 30 ° C. and 60 ° C., and

a step of separating the formed floc and the liquid phase, preferably by a technique selected from tangential filtration, depth filtration, pressing and dewatering, more preferably by pressing or dewatering, for example through a woven fabric nylon,

d. a multimodal chromatography step carried out on the clarified solution obtained in step c, said multimodal chromatography being carried out, preferably on a resin having as ligand 4-mercapto-ethyl-pyridine or benzyl-methylethanolamine and for a charge pH of 6 to 9,

e. optionally, at least one chromatography step selected from: hydrophobic interaction chromatography, hydroxyapatite chromatography, cation exchange resin chromatography, anion exchange chromatography, size exclusion chromatography,

f. an ultrafiltration step, preferably on a polyethersufone membrane

boy Wut. a step of inactivation and / or elimination of the residual pathogens, preferably by sterilizing nanofiltration, and

h. recovering the purified recombinant protein, or a purified protein concentrate.

Combination 5

b. a multimodal chromatography step which is carried out under conditions for which the recombinant protein is not absorbed on the matrix at the charge pH and is recovered in the effluent,

vs. a second multimodal chromatography step which is carried out under conditions for which the recombinant protein is absorbed on the resin at the pH of charge and is then eluted by changing the pH of the elution solution, step b. can be performed upstream or downstream of step c.

Combination 6

at. providing a transgenic milk or a protein solution obtained from said milk, wherein the recombinant protein is a recombinant antibody,

b. a step of clarifying the milk solution by adding a salt of pDADMA, preferably pDADMAC,

vs. a first step of multimodal chromatography carried out on a resin having, as ligand, benzylmethylethanolamine, at a pH of between 6 and 9, in which the unabsorbed recombinant antibody is collected in the effluent,

d. a second stage of multimodal chromatography carried out on a resin having 4-mercapto-ethyl-pyridine as ligand, with a charge pH of between 6 and 9, in which the recombinant antibody is absorbed on the resin and is then eluted by acidification eluent at a pH below 5, preferably at pH 4.

Combination 7

vs. a first step of multimodal chromatography performed on a resin having as a ligand 4-mercapto-ethyl-pyridine, with a charge pH of between 6 and 9, in which the recombinant antibody is absorbed on the resin and is then eluted by acidification eluent at a pH below 5, preferably at pH 4,

d. a second stage of multimodal chromatography carried out on a resin having, as ligand, benzylmethylethanolamine, at a pH of between 6 and 9, in which the unabsorbed recombinant antibody is collected in the effluent.

It goes without saying that the method according to the invention may comprise one or more steps additional to those listed in each combination. It goes without saying that the various steps of the method, in particular the clarification step may be implemented according to any of the embodiments described above, unless otherwise indicated in the combination considered. ^■ Additional objects according to the invention

According to a further aspect, the invention also relates to a recombinant protein or a recombinant protein concentrate that can be obtained by the purification method as described above. Said recombinant protein or said concentrate are preferably suitable for use in human or veterinary therapy. An additional object according to the invention is a pharmaceutical composition comprising a recombinant protein purified according to the method described above in combination with one or more excipients. Said pharmaceutical composition may be in liquid or solid form, for example freeze-dried.

The invention also relates to a method for preparing a pharmaceutical composition comprising a recombinant protein, comprising:

i. Providing a purified recombinant protein by the method according to the invention, and ii. The mixture of the recombinant protein with one or more pharmaceutically acceptable excipients.

In some embodiments, the method of preparing a pharmaceutical composition comprises:

i. The implementation of the purification method according to the invention, so as to obtain a purified recombinant protein, and

ii. The mixture of the purified recombinant protein with one or more pharmaceutically acceptable excipients.

Those skilled in the art will be able to choose the excipient (s) to be combined with the recombinant protein depending on the dosage form and the desired route of administration. For this purpose, those skilled in the art may refer to the following reference works: Pharmaceutical Formulation Development of Peptides and Proteins (S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis, 2000), Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilkins, Twenty First Edition, 2005) and Handbook of Pharmaceuticals Excipients, American Pharmaceutical Association (2009).

The excipient or excipients present in the compositions according to the invention may be chosen from diluents, cryoprotective agents and / or lyoprotectants, stabilizing agents, antioxidants, pH-regulating agents, buffering agents and surfactants. detergent agents, etc. By way of example of excipients, mention may be made of sugars (sucrose, trehalose, glucose, lactose, maltose, etc.), polyols (mannitol, sorbitol), amino acids (glycine, arginine, histidine, alanine, methionine), polysorbates (Tween 20 or Tween 80 for example), polyoxamers, polyethylene glycol, monothioglycerol, glutathione, citric acid, ascorbic acid, metabisulphite, sodium, and sodium sulfite, carbonate salts, phosphate, citrate, acetate, borate, etc.

The pharmaceutical composition according to the invention is preferably intended for parenteral administration, for example intravenous, subcutaneous, intradermal or intramuscular. A particularly preferred route of administration is the intravenous route.

The pharmaceutical composition and the pharmaceutical adjuvant according to the invention may be in any form, for example in the form of a powder, a suspension, a solution, an emulsion or a lyophilizate, preferably intended to be administered by injection to the patient.

Finally, the Applicant has also shown that the clarification step by adding a salt of pDADMA such as pDADMAC had a viral elimination and / or inactivation effect, in particular with respect to non-enveloped viruses such as than the PPV virus.

For the purposes of the invention, it is considered that there is a viral elimination or inactivation effect, when a viral reduction of a factor greater than 1 log is observed _.

Thus, an additional object according to the invention is the use of a salt of pDADMA, in particular pDADMAC for inactivation or viral elimination. Preferably, the pDADMA salt is used for the elimination and / or viral inactivation of a biological solution comprising a protein of interest, for example a cell medium, a cell lysate, a fermentation medium, a biological fluid and the products derived from said solutions.

In some embodiments, the pDADMA salt is used as a viral elimination and / or inactivation agent in the purification of a protein from a biological fluid or a product derived from a biological fluid. The biological fluid can be of any type. It may be, for example, blood, a blood plasma, including a human blood plasma, or a milk, for example a transgenic milk.

In some embodiments, the pDADMA salt is used as a viral elimination and / or inactivation agent in a method for purifying a recombinant protein from a transgenic milk or a milk derived product. transgenic.

In a further or alternative embodiment, the pDADMA salt is used both as a viral elimination and / or inactivation agent and as a clarifying agent, preferably for the purification of a protein of interest from a transgenic milk or a product derived from said transgenic milk.

The present invention also relates to a process for the elimination and / or viral inactivation of a biological solution comprising a protein of interest, said method comprising a step of incubating said solution with a pDADMA salt, preferably pDADMAC. This incubation step can be carried out under the conditions described above for the clarification step with a salt of pDADMA according to the invention. Typically, the final concentration of the pDADMA salt is less than 20 g / l, preferably less than 5 g / l, for example from 1 g / l to 4 g / l. The incubation can be carried out at a temperature ranging from 30 ° C to 60 ° C. The duration of the incubation can vary from a few minutes to a few hours, typically from 1h to 20h.

The following examples are intended to illustrate more fully the invention without limiting its scope.

Examples

Example 1 Evaluation of Several Chemical Agents for the Clarification of a Transgenic Milk

Transgenic milk

It is a transgenic milk from transgenic goats. The recombinant therapeutic protein secreted in milk is an anti-TNF-cc antibody.

The milk was collected and frozen for storage for purification.

- Evaluation of pDADMAC and comparative trials PDADMAC was evaluated as a flocculant. The pDADMAC stock solution is a commercial solution provided by Merck-Millipore. PDADMAC has an average molecular weight of 100,000 to 500,000 gmol ¹ . By way of comparison, supposedly clarifying agents described in the literature for the purification of proteins were tested under the conditions indicated below.

Operating mode

Each clarifying agent was tested as follows:

The transgenic milk (5 g) was thawed and brought to the desired incubation temperature (4 ° C, 25 ° C or 37 ° C). The pH of the milk was adjusted to pH 9.5. For each agent tested, a suitable amount of the stock aqueous solution was added to obtain the desired concentration (see table below). After addition of the clarifying agent, the milk was incubated for 20 hours, at the desired incubation temperature. At the end of the incubation, the supernatant samples were taken for analysis.

Turbidity of the supernatant was determined by absorbance at 400 nm. The clarity of the supernatant corresponds to the inverse of the turbidity (ratio = 1 / turbidity).

The recombinant antibody content (mg / l) was determined by nephelemetry (total anti-Ig.) The protein purity of recombinant antibody was determined by the relative measurement of the relative density of the immunoglobulin bands after electrophoretic migration in SDS PAGE according to the Laemmli technique.

Results

The incubation temperature at 37 ° C showed the best results, regardless of the clarifying agent considered. The addition of pDADMAC resulted in very clear phase separation: formation of a white sediment flocculation and a clear supernatant was observed. An analogous result has been observed for acidic EDTA. On the other hand, agents such as CaCl ₂ , sodium citrate or disodium EDTA did not make it possible to obtain a clear phase. .

The supernatant assays for each agent tested are shown in Figures 1A-1C for the incubation temperature of 37 ° C. These analyzes confirm that pDADMAC is the most effective agent in terms of clarification (FIG. 1A) and final purity of recombinant antibody (FIG. 1C). As illustrated in FIG. 1B, pDADMAC also makes it possible to recover, in the supernatant, a significant proportion of the recombinant antibodies initially present in the transgenic milk.

Example 2: Optimization of the clarification step

The following conditions have been tested:

The procedure is similar to that used in Example 1. Each clarifying agent was tested as follows:

The transgenic milk (100g) was thawed and adjusted to the incubation temperature of 37 ° C. The thawed milk was pasteurized at 60 ° C for 6 hours to neutralize any bacteria present in the milk and prevent bacterial growth during incubation with the clarifying agent. The pH of the milk has been adjusted to the desired value. For pDADMAC, the initial pH of the milk was the desired pH, no pH adjustment was performed. For each agent tested, a suitable amount of the corresponding stock aqueous solution was added to the pasteurized transgenic milk. After adding the agent clarifying and homogenizing, the milk was incubated for 20 hours without stirring, at 37 ° C. At the end of the incubation, the supernatant samples were taken for analysis.

Supernatant turbidity, recombinant antibody content, protein purity of recombinant antibody were determined as described in Example 1.

Results

PDADMAC was once again the most effective clarifying agent, even in the absence of any adjustment of the initial pH of the solution. Indeed, pDADMAC at a final concentration of 0.1% (w / v) gave a supernatant with a very high purity of recombinant antibody and clarity. The use of a higher concentration of pDADMAC, namely 0.3% (w / v) or 3 g / L, gave equally satisfactory results in terms of purity and antibody content in the supernatant.

Figure 2 shows the electrophoresis gel obtained in SDS PAGE (staining with Coomasie blue) for the supernatants obtained for the different conditions tested. For the supernatants obtained with pDADMAC, the electrophoresis gels have a revelation of the caseins practically nonexistent, which attests the elimination of caseins in the flocculate. On the other hand, the protein bands corresponding to immunoglobulins are clearly visible. Such a profile is not observable for supernatant gels obtained with CaCl ₂ , sodium citrate or sodium EDTA, for which the casein revelation remains high. It should be noted, however, that EDTA in acid form has allowed a good elimination of caseins. Nevertheless, the amount of immunoglobulin recovered in the supernatant is lower than that obtained with pDADMAC.

Example 3: Evaluation of the supernatant collection methods

The addition of pDADMAC effectively precipitates casein micelles and fat globules while generating an almost clear supernatant enriched with recombinant antibody. The supernatant has a clarity such that it is not necessary to subject it to a complex step of deep filtration. Several liquid / solid separation techniques have been tested. The inventors have shown that the "pressing liquid" that is to say the liquid phase obtained by pressing the flocculate had an SDS PAGE profile equivalent to that of the clear supernatant, in particular in terms of protein purity immunoglobulin.

Such a result led the inventors to test two simple solid / liquid separation techniques, namely: Mechanical pressing using a nylon cloth and a porous support with vacuum harvesting by a vacuum pump

Draining through a nylon fabric with a porosity of 25μιη, by gravity.

The filtrates thus obtained have a total protein content of 13.3 g / l, with the following composition determined by SDS PAGE: 45% immunoglobulins, 0% caseins and 55% other milk proteins.

The total yield of the clarification step (addition of pDADMAC followed by removal of the flocculate by draining or pressing) in immunoglobulins is between 75% and 90%, depending on the experiment carried out.

As shown in Example 4, the filtrates from the clarification step have a concentration of recombinant antibodies, a conductivity (less than 20 mS / cm) and a pH of about 6.5 allowing the implementation directly from a multimodal chromatography step, without prior adjustment of their composition.

Example 4 Example of Implementation of a Method According to the Invention

Figure 3 shows an example of a method according to the invention.

- Operating mode

The thawed crude transgenic milk (100 g) was subjected to a pasteurization step by heating at 60 ° C for 10 hours. The milk was then brought back to a temperature of 37 ° C. An aqueous solution of pDADMAC at 10% (w / v) was added to the pasteurized milk so as to reach a final content of 0.3% (w / v). The transgenic milk was then incubated for 20 hours at 37 ° C. No pH adjustment of the transgenic milk was performed prior to the addition of pDADMAC. The recombinant antibody-rich supernatant was separated from the flocculate formed subsequent to the addition of pDADMAC by gravity. The supernatant thus harvested was then subjected to a multimodal chromatography step, without prior adjustment of its pH, its conductivity or its protein concentration. Two types of multimodal chromatography were tested, namely multimodal chromatography by adsorption of Ig on MEP Hypercel resin and multimodal chromatography by adsorption of non-Ig proteins on Capto Adhere resin. The recombinant antibody solution from the multimodal chromatography step was purified x times. The antibody solution can then be concentrated by ultrafiltration on a polyethersulfone membrane. ^■ multimodal chromatography on MEP Hypercel resin (ligand: 4-mercapto-ethylpyridine)

Operating conditions tested:

Adsorption: at pH 6.5

- Washes: at a conductivity lower than 5 mS / cm

- Elution: at pH 4.0

At pH 6.5, the recombinant antibody is strongly absorbed on the resin, while the contaminants are not retained. The recombinant antibody is recovered by elution with the citric acid solution at pH 4.0.

^■ Multimodal chromatography on Capto Adhere resin (ligand: benzyl-methylethanolamine)

Operating conditions tested: Adsorption: at pH 7.0

At pH 7.0 the recombinant antibody is weakly retained on the resin, whereas the contaminants are. The recombinant antibody is recovered in the non-retained charge eluent fraction. Multimodal chromatography thus works in negative for the recombinant antibody.

- Results

The results obtained by combining a clarification step with pDADMAC followed by multimodal chromatography are satisfactory. Both multimodal chromatography techniques tested offer good yields of recombinant antibody (around 70-80%).

MEP Hypercel resin allows excellent adsorption of recombinant antibodies (100% in this example of charge), leading to a final yield of about 70%. Depending on the charge applied, a contaminating protein, casein, can be detected in small amounts in SDS-PAGE in the purified antibody solution by multimodal chromatography on MEP Hypercel resin.

The Capto-Adhere resin allows the implementation of a negative chromatography, the contaminants of the milk being strongly retained on the resin during the charge while the recombinant antibody is directly eluted at pH 7. According to the applied load, quantities Residuals of two contaminating proteins can be detected in SDS-PAGE in the purified antibody solution by multimodal chromatography on Capto-Adhere resin. The combination of a clarification step with pDADMAC followed by chromatography multimodal resin with benzyl-methyl-ethanolamine as ligand completely eliminates caseins and β-lactoglobulin, as well as the fat globules of milk while having an overall yield, on both steps, recombinant antibody of the order of 60%. Example 5: Robustness of the clarification step with pDADMAC as a function of the protein composition of the starting transgenic milk.

The method of clarification according to the invention has been implemented on two transgenic milks having different protein compositions. These are goat transgenic milks expressing a human antibody. An anti-TNFcc antibody was expressed in milk No. 1. An anti-HER2 / neu receptor antibody was expressed in milk No. 2.

These milks have the following characteristics:

For each milk, a clarification step using pDADMAC was performed according to a protocol similar to that described in Example 2.

The transgenic milk (100 g) was thawed and pasteurized at 60 ° C for 6 hours to neutralize any bacteria present in the milk and prevent bacterial growth during incubation with the clarifying agent.

PDADMAC was added to give a final concentration of about 2 g / L or 4 g / L in solution. After adding pDADMAC and stirring for 1 h 30 min at 45 ° C, the milk was incubated for 6 h to 12 h without shaking at 45 ° C. At the end of the incubation, supernatant samples were taken for analysis.

The conditions of the procedure are summarized in the table below:

The table below shows the composition of raw milks and supernatants obtained at the end of the clarification step.

The clarification step with pDADMAC allows a significant enrichment of immunoglobulin while effectively eliminating caseins, and this independently of the initial contents of caseins and recombinant immunoglobulin of the raw milk of departure. The yield of recombinant antibody after removal of the flocculate is at least 50% when the supernatant is collected by gravity (by dripping) and at least 70% when rinsing the flocculate is carried out after dewatering with a volume of solution adapted.

Example 6: Effect of inactivation / viral elimination of the treatment step with pDADMAC

1. Protocol

A volume of transgenic milk (4 g / L) was thawed. The temperature of the thawed product was between 21 ° C and 22 ° C. The thawed milk was then overloaded with 5% (v / v) model virus (PPV). A sample was taken to quantify the amount of virus at this stage. A volume equivalent to 3% (v / v) of a 10% pDADMAC solution was then added to the volume of milk overloaded with the model virus (ie a final concentration of pDADMAC of about 3 g / L). A sample was taken to quantify the amount of virus at this stage. The solution was then mixed for 1 hour at 45 ° C and incubated for 20 hours at 45 ° C. A sample was taken to quantify the amount of virus at this stage.

2. Results

PPV is a representative virus for small, non-enveloped viruses. It is a DNA genome virus, 18-24 nm in size, highly resistant to physicochemical treatments. chemical. The viral reduction is a factor of 2.61 ± 0.34 log ₁₀ after incubation of the transgenic milk with pDADMAC. This result is mainly the action of pDADMAC treatment on this non-enveloped virus.

Claims

A method for purifying a recombinant protein from a transgenic milk or a protein solution obtained from a transgenic milk, comprising a step of clarifying with a poly (diallyldimethylammonium) salt preferably poly (diallyldimethylammonium chloride) (pDADMAC).

2. Method according to claim 1 wherein the recombinant protein is selected from hormones, cytokines, proteins involved in the immune response, antibodies, and coagulation factors and inhibitors, the recombinant protein being preferably an antibody .

The purification method according to claim 1 or 2, said clarification step comprising:

adding the poly (diallyldimethylammonium) salt to said transgenic milk or to said solution so as to form a liquid phase comprising the recombinant protein, and a flocculate, and

4. The purification method according to claim 3, the final content of the poly (diallyldimethylammonium) salt in the milk or the solution is in a range from 0.01 g / L to 20.0 g / L.

5. A purification method according to any one of claims 3 or 4, said milk or said solution is incubated at a temperature of 20 ° C to 60 ° C, after the addition of the salt of poly (diallyldimethylammonium).

6. Purification process according to any one of claims 3-5, wherein the separation of the liquid phase and the flocculate is carried out by a method selected from mechanical pressing or dewatering.

7. Purification method according to any one of claims 1 to 6, wherein the clarification step is performed on a crude transgenic milk having optionally undergone one or more treatments selected from freezing, thawing, and pasteurization.

8. Purification method according to any one of the preceding claims, said method comprising at least one additional purification step, preferably selected from the group consisting of ultrafiltration, tangential ultrafiltration, microfiltration, diafiltration, chromato graphy. inverse phase, hydrophobic interaction chromato graphy, hydroxyapatite chromato graphy, cation exchange resin chromatography, anion exchange chromatography, affinity chromatography, multimodal chromatography, size exclusion chromatography, and the combinations of these.

9. Purification process according to any one of the preceding claims, said method comprising at least one multimodal chromatography step.

The purification method according to any one of the preceding claims, further comprising a step of removing and / or inactivating residual pathogens.

11. Purification process according to any one of the preceding claims comprising one, more or all of the following characteristics:

the milk solution is a crude transgenic milk, and / or

the recombinant protein is a human, humanized or chimeric antibody, and / or the pDADMA salt is a pDADMAC having a molecular weight of between 100,000 and 500,000 g.mol- ¹ , and / or

the process comprises at least one purification step chosen from microfiltration, ultrafiltration, tangential ultrafiltration, microfiltration, diafiltration, reverse phase chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, resin exchange chromatography, of cations, anion exchange chromatography, and affinity chromatography, size exclusion chromatography, and / or

the method comprises a pasteurization step carried out before the clarification step, and / or

the step of clarification by adding the salt of pDADMA is the only step of clarification of the process, and / or

The purification method according to any one of claims 1 to 6, comprising the combination of the following steps:

at. pasteurizing the transgenic milk or said solution,

vs. the implementation of a multimodal chromatography step on the clarified solution obtained in step c, possibly after adjusting the pH or the conductivity of said solution,

d. optionally, the implementation of one or more additional purification steps on the recombinant protein solution resulting from step d., preferably chosen from the group consisting of ultrafiltration, tangential ultrafiltration and microfiltration. , diafiltration, reverse phase chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, cation exchange resin chromatography, anion exchange chromatography, affinity chromatography, size exclusion chromatography and the combinations thereof, and

A method of preparing a pharmaceutical composition comprising a recombinant protein, said method comprising:

providing a recombinant protein by carrying out a purification method as defined in any one of claims 1 to 12, and

14. Use of a poly (diallyldimethylammonium) salt, preferably pDADMAC, as a clarifying agent for the purification of a therapeutic protein from a transgenic milk, or a protein solution obtained from transgenic milk.