WO2010136992A1

WO2010136992A1 - Method for purifying and restoring a recombinant protein

Info

Publication number: WO2010136992A1
Application number: PCT/IB2010/052368
Authority: WO
Inventors: Simon Crelier; Rémy DUFRESNE
Original assignee: Fondation The Ark; Brennan, Gary
Priority date: 2009-05-29
Filing date: 2010-05-27
Publication date: 2010-12-02
Also published as: CH701129A2

Abstract

The invention relates to a method for purifying and restoring a protein. The invention specifically relates to a method for purifying a recombinant protein built-up in the form of insoluble inclusion bodies by prokaryotic or eukaryotic cells. The method particularly comprises a restoring step, and makes it possible to carry out said step at a high protein concentration.

Description

Process for purification and renaturation of a recombinant protein

The invention relates to a method for purifying and renaturing a protein. It relates more particularly to a process for purifying a recombinant protein accumulated in the form of insoluble inclusion bodies by prokaryotic or eukaryotic cells. This process comprises in particular a renaturation step, and makes it possible to drive the latter at a high protein concentration.

There are a very large number of expression systems (host cell / gene vector combination) specific for each protein or family of recombinant proteins. The choice of the expression system is essentially guided by the modifications that the protein must undergo to be biologically active.

Until the mid-1990s, the bacterium, and more specifically Escherichia coli (E. coli), was the host of choice for producing a recombinant protein. E. coli has been extensively studied since the 1960s, so it is one of the best known organisms today. All of this knowledge in biochemistry, genetics and molecular biology has been exploited to express proteins in large quantities. This bacterium has several interesting properties with regard to the expression of proteins: it is easy to handle, it grows quickly in relatively inexpensive media and the laboratory strains are harmless. In addition all molecular biology laboratories use E. coli for other experiments (cloning, sequence ...). This facility has made it the most popular expression system. On the other hand, the production of recombinant proteins in the bacteria frequently leads to inactive proteins, aggregated in a solid form called the inclusion body. These are dense and can be found either in the cytoplasm or in the periplasmic space of the cell.

Currently, with the development of cell culture technologies, approximately 70-80% of the proteins of interest are produced in animal cells. Most human or humanized recombinant antibodies are produced in such host cells (Brekke & Leset, 2003). Indeed, despite a much slower growth rate and a great fragility especially with respect to agitation, animal cells have the property of glycosylating the proteins they synthesize. As a post-translational modification, this glycosylation is necessary for the biological activity of molecules such as therapeutic antibodies.

To restore their function, proteins must adopt a precise three-dimensional conformation. Native state, acquired during cellular refolding. However, many recombinant proteins are not produced in their native state, but are aggregated in a mostly biologically inactive Elat that ^is called ^inclusion bodies. The aggregation phenomenon described above leads to a significant reduction in productivity and efficiency for these expression systems, since it makes additional processing steps necessary in the process. Moreover, contrary to what is commonly accepted, inclusion body formation ^is not ^the prerogative of bacterial systems but is also observed in some eukaryotic cells.

These inclusion bodies, however, have the advantage of representing a concentrated and relatively pure form of the target molecule. In addition, the denatured and insoluble form of the proteins that compose them makes them less susceptible to degradation by the proteases released during the cell lysing step.

This mode of production is therefore frequently encountered in industrial developments. Known examples relate, for example, to certain processes for the manufacture of somatropin (bovine growth hormone), insulin, erythropoietin (EPO) or TPA (Tissue Plasminogen Activator), a thrombolytic agent. But in practice, any recombinant protein expressed in a bacterium such as E. coli is likely to form inclusion bodies there if its level of expression is sufficiently high. This problem is therefore not specific to certain protein families. These inclusion bodies, granules of aggregated proteins sometimes visible in optical microscopy, greatly facilitate the purification of the protein. By cons they must then be solubilized, usually in the presence of a chaotropic agent, which causes their almost complete denaturation. This is why the solubilization must imperatively be followed by a folding step (or renaturation) in vitro. This last step is empirical, requires a tedious focus specific for each protein, and its yield is often low. Thus the main difficulty of this kind of process is to convert an inactive and insoluble protein into an active and soluble protein with the highest possible yield and in the most economical way possible. Another important point is that no variation of the process should occur during industrial scale-up; this process must be easy to automate and be generic for a wide range of similar proteins so that the technology does not have to be reinvented each time. Figure 1 shows a sequence of commonly used steps for isolation of inclusion bodies, their solubilization and renaturation of the target protein.

International Patent Application WO9202540, filed in the name of

BioEurope describes precisely one of these purification methods, with however a different approach to that described in FIG. 1. This application describes a process for the purification of a secreted protein in the form of inclusion bodies that are insoluble by a bacterium, characterized in that (a) after harvesting the microbial cells, the pellet ("peilet") or solid fraction combining the above-mentioned cell fragments and inclusion bodies is harvested; b) the pellet is suspended in a solution; of hydrosubstantially hydrophilic substance. c) the mixture of the sodium particles of the cuiot and the solution of water-soluble substance is injected dropwise into a solution of crystallization. for example a solution containing cations muitivaients, thus making encapsulation of solid particles of the pellet in the gelled substance, d) the encapsulated peels are suspended in a solution of dissolution containing at least one chaotropic agent and optionally a reducing agent, e) the denatured and purified protein is obtained in solution, cellular debris and other insoluble compounds remaining trapped in the gelled matrix. This approach makes it possible to separate This is a simple and effective way of incorporating (ie the product of interest) the cell fragments, an important step that is difficult to master but often obscured in the descriptions of the literature. A major drawback of this approach lies in the fact that other compounds will be solubilized at the same time as the inclusion bodies, thus reducing the purity of the product thus recovered relative to the direct insulation of solid inclusion bodies. . Renaturation should therefore be done in the presence of more or less significant amounts of contaminants, which can have a negative impact on renaturation yields. Moreover, this international application does not go beyond the solubilization of the inclusion bodies, and does not in any way deal with the means of obtaining a native protein from the denatured material, except by the conventional methods of dilution.

The protein is then renatured by removing denaturants in its environment; this is done by diluting the medium used for the dissolution of the inclusion bodies. On the other hand, many proteins can not be corrected in this way and the result obtained does not always allow effective subsequent use of these recombinant proteins. Indeed, it is known that the protemes obtained by this kind of process are very good. degree of purification sufficient for certain experiments (in particular for immunizations) However, other uses, therapeutic in particular, require obtaining a purified solubie protein and having covered its original three-dimensional conformation

International Patent Application WO9402625, filed in the name of Immunex Corporation, also does not provide an efficient and reliable renaturation method. Indeed, this application discloses a method of isolating a recombinant polypeptide produced as a component of an insoluble fraction in a transformed host cell, which comprises entrapping the host cells (before or after the lysis of these cells). ci) in a porous matrix; to break, if it had not already been done, the membrane of the host cells to allow the diffusion of the soluble proteins contained in said cells; washing the trapped host cells to extract soluble proteins, while leaving the insoluble fraction in the porous matrix; and to bring the insoluble fraction into contact with porous matrix with a chaotropic agent to isolate the recombinant polypeptide from the insoluble fraction. Again, aspects related to the renaturation and the yield of this last step are not described in the invention.

The two patent applications cited above, while using similar materials and approach, essentially only treat the facilitated solubilization of a recombinant protein from inclusion bodies, while maintaining solid residues. prisoners in a hydrogel-like matrix. The renaturation phase of the recombinant protein thus solubilized, however critical and most often limiting in terms of costs and time, is hardly addressed.

The most frequently used method for renaturation is dilution, which consists of diluting the soluble but denatured protein with a renaturation buffer. This is a simple method, but one that can lead to difficulties in commercial applications. Indeed, it requires the manipulation of large volumes of buffer for dilution, which necessitates a subsequent concentration step and increases the cost of the process [De Bernardez Clark, 1998]. Dilution is necessary, however, for refolding solution because high concentrations of protein promote the town, characterized by kinetic ^2nd order {Schlegl et al., 2005), to the detriment of the parallel process and annealing competitor following as for him a kinetics of ^1st order.

Different approaches have been proposed to allow the renaturation of high concentration proteins. Among them are techniques where it is sought to reduce the mobility of the protein during the process; this must make it possible to limit the phenomenon of aggregation, which implies that the protein molecules meet. This category includes the approaches described by Ferré et al. (2005), Middelberg (2002), or Lanckriet & Middelberg (2004). The protein is generally adsorbed on a chromatographic column, which limits its mobility, while the denaturing agent is removed. A major drawback of this approach is that the denatured protein, even if it carries the same overall charge, is often not adsorbed in the same way on the stationary phase as the native protein. One possible solution to this problem is to use protein bearing a tag (commonly called "tag" in English) of type (His) 6 for example, and adsorbing them on an affinity column of "IMAC" type (Immobilized Metal Affinity Chromatography) carrying transition metal ions such as that Cu ² , Co ² , Ni ² or Zn ²⁺ and for which Histidine has a high affinity (Hutchinson & Chase, 2006). Other affinity markers such as intein, glutathione-S-transferase, MBP (maltose binding protein) or calmodulin binding peptide (CBP) are also used, with their respective advantages and disadvantages. The (His) 6 for example has, by its small size, the property of being functional both under native and denaturing conditions. In another approach, high hydrostatic pressures are used to promote folding of the recombinant proteins to their native structure even at high concentrations. The principle and examples of applications of this technology developed and operated by BaroFold, Inc. are described in publications (Randolph et al., 1999 and 2002) as well as in a series of patent applications (AU 2007202306, WO 2007062174). Although this method has proved its effectiveness in many applications, the equipment to reach pressures of several kbar are however extremely expensive and the residence time in the machine can reach 24 hours.

Therefore, in view of the state of the art described above, there is still a need for a large-scale efficient method of renaturing a recombinant protein, preferably at high concentration.

The invention described hereinafter makes it possible to work on a large scale, with a high concentration of proteins, with inexpensive materials, under easily reproducible conditions and with a high yield. The use of high protein concentrations thus makes it possible to reduce the volumes and thus to facilitate their handling. The present invention relates primarily to a method for purifying and renaturing a recombinant protein accumulated in the form of an insoluble inclusion body by prokaryotic or eukaryotic cells, characterized in that: a) the inclusion bodies are harvested under in the form of a pellet after lysis of the prokaryotic or eukaryotic cells and centrifugation, b) the pellet is put in the presence of a denaturing solution containing at least one denaturing agent and / or a reducing agent and / or a detergent agent, in order to dissolve the proteins contained in the pellet at high concentration, c) dissolving a gelling polymer in the denaturing solution, d) contacting the denaturing solution comprising the gelling polymer with a bath causing gelling of the dissolved gelling polymer, leading thus to the encapsulation of the denatured proteins and the denaturing agent and / or the reducing agent and / or the detergent in the s gelled solution, e) the denaturing agent and / or the reducing agent and / or the detergent (which are molecules of relatively low molecular weight) are allowed to diffuse through the solution thus gelled comprising the denaturated proteins encapsulated much faster than the latter, thus resulting in a progressive renaturation of these denatured proteins while limiting their aggregation (due to a greatly reduced mobility), f) the capsules containing the renatured protein are placed in the presence of a solution allowing the dissolution of the gel, g) the renaturated and purified protein is obtained in solution at a high concentration

The invention also relates to a kit and an apparatus for carrying out the method for purifying and renaturing a recombinant protein accumulated in the form of an insoluble inclusion body by prokaryotic or eukaryotic cells.

Figure 1 represents a conventional scheme of isolation, solubilization and renaturation of recombinant proteins accumulated in a host cell in the form of inclusion bodies. Figure 2 shows the release kinetics of guanidiniun chloride (GdnCl) and ovalbumin out of 3 mm diameter beads prepared with different concentrations of alginate. Temperature = 22 ⁰ C.

Figure 3 shows the release kinetics of guanidiniun chloride

(GdnCl) and ovalbumin out of 5 mm diameter beads prepared with different concentrations of alginate. Temperature = 22 ⁰ C.

Figure 4 shows the influence of stirring rate on the kinetics of release of guanidinium chloride from 5 mm diameter beads prepared with 2% alginate. Temperature = 22 ⁰ C.

By recombinant protein is meant a protein produced by cells (eukaryotes or prokaryotes) whose DNA (deoxyribonucleic acid) has been modified by genetic recombination. The use of an expression vector (generally a plasmid or a virus for eukaryotic vectors), acting as the genetic carrier of the gene of interest encoding the desired protein is the most often used means for modify the DNA of the cells used for the production of the recombinant protein. Any protein capable of being produced by cells (eukaryotic or prokaryotic) and whose DNA (deoxyribonucleic acid) has been modified by genetic recombination is concerned by this invention.

By protein is meant a macromolecule composed of one or more chain (s) (or sequence (s)) of amino acids linked together by peptide bonds. In general, we speak of protein when the chain contains more than 50 amino acids. In the opposite case, we speak of peptides and polypeptides. Therefore, the present invention also contemplates a method of purifying and renaturing a recombinant protein of less than 50 amino acids or peptides (as defined above) accumulated as an inclusion body insoluble by cells. prokaryotes or eukaryotes.

In general, the recombinant protein is produced by prokaryotic or eukaryotic host cells. Prokaryotic cells lack a cell nucleus and are generally unicellular. They include bacteria, cyanobacteria, archaebacteria. Their genetic material is diffuse circular deoxyribonucleic acid (DNA or DNA) in the cytoplasm of the cell. Prokaryotic cells are considered more evolutionarily primitive and generally have fewer genes than eukaryotic cells.

Eukaryotic cells, in turn, are characterized primarily by the fact that they have a nucleus composed of a double membrane surrounding the DNA. These cells include all the organisms included in four great kingdoms of the living world: animals, fungi, plants and protists.

For the time being, there is no absolute rule making it possible to predict the stability, solubility, activity and conformation of a recombinant protein (heterologous) expressed by a eukaryotic or prokaryotic E. coli cell. The main problem encountered is the formation of inclusion bodies insoluble in the cytoplasm of these host cells. Indeed, in E. coli, the important expression of heterologous proteins often leads to the appearance of granules called insoluble inclusion bodies which are visible by phase contrast microscopy. They generally contain the overexpressed recombinant protein as well as nucleic acids and some bacterial proteins. These insoluble inclusion bodies can be quite easily isolated thus making it possible to obtain the recombinant protein with a good degree of purification, which is sufficient for certain experiments (in particular for conventional immunizations). However other approaches such as therapeutic treatments with peptides or antibodies, require the production of a purified soluble protein and being in a native conformation, or as close as possible to this conformation.

Indeed, the recombinant protein e & l produced in the form of insoluble inclusion bodies Classically we will abofd concentrate buoaryofes cells or prokaryotos from the culture medium of these ooϋuios, conîπfugaîion or ultrafiltration. The present invention thus advantageously relates to the renaturation of all recombinant proteins (hormones, growth factors, antibodies, etc.) which tend to, or are likely to form, inclusion bodies during known purification processes. Generally, the more protein is insoluble (because of its conformation) and more it will tend to form inclusion bodies in prokaryotic or eukaryotic cells.

Lysis of eukaryotic or prokaryotic cells is then achieved by disrupting the plasma membrane of these cells by the action of a physical, chemical or biological agent, thereby leading to death of the cell.

Preferably, mechanical milling (eg in a ball mill, a high pressure homogenizer or a "french press") or sonication (ultrasonic treatment) but chemical agents such as detergents (anionic) will be used. cationic, neutral), organic solvents, strong bases or acids or enzymatic preparations are also conceivable.

Step a) The inclusion bodies are then reooled by centrifugation. During this digestion, the bumage contains the biotic constituents of the bacterium. However, the particulate fraction thus recovered, oncoro, called cuiot, contains in addition to the insoluble inclusion phases containing the enzymes. recombinant, non-shed cells. cell debris and protein contaminants, solubias or not

Optionally, the pellet is washed, prior to the dissolution step, with an aqueous buffer solution to remove soluble contaminants. Buffer solutions commonly used are, depending on the desired pH, of the acetate, citrate, phosphate, tris or glycine type and any other buffer which a person skilled in the art will deem useful to use.

Step b): This pellet is then placed in the presence of a denaturing solution containing at least one denaturing agent and / or a reducing agent and / or a detergent agent, in order to dissolve the proteins contained in the pellet. The denaturing solution comprises at least one denaturing agent selected from chaotropic agents known to those skilled in the art such as urea or guanidinium chloride, these examples not being limiting. The role of this denaturing agent is to cause the dissociation of proteins by altering their spatial configuration or their conformation. The solution may also contain at least one reducing agent, selected from the non-limiting group comprising β-mercaptoethanol, glycine, dithiothreitol (DTT), dithioeryhtritol (DTE) or glutathione. The main function of this agent is to open up the maintenance of cysteine residues in reduced form, thereby minimizing the risk of non-native intramolecular disulfide bond formation or the formation of undesirable disulfide bridges between protein molecules. A preferred agent is glutathione. Commonly used concentrations are low, usually between 0.2 and 10 mM, but preferably between 0.5 and 5 mM. The concentrations commonly used for DTT are higher, of the order of 10 mM (usually between 5 and 50 mM).

The solution may also contain a detergent agent known to those skilled in the art and which may be selected from the non-limiting group comprising anionic, neutral, cationic or zwitterionic detergents. Preferably the detergent agent will be selected from the non-exhaustive group including Triton X100, CHAPS, dodecyl maltoside, DTAB, CTAB.

These three agents (denaturant, reducer and detergent) can be together in the same solution or alone or in groups of two.

Once the recombinant protein solubilized, it can, if necessary, easily separate by filtration or centrifugation of cell debris and other insoluble particles resulting from the lysis step.

Alternatively, the cells can be lysed and the insoluble solid material recovered (after centrifugation and / or filtration and washing). This solid fraction contains cell debris and inclusion bodies. These can be dissolved by contacting the centrifugation pellet with a denaturing solution containing at least one denaturing agent and / or a reducing agent and / or a detergent agent, in order to dissolve the proteins contained in the high concentration pellet, and isolating by centrifugation and / or filtration the supernatant containing the denatured target molecule,

Step c): In the next step, a gelling polymer is dissolved in the denaturing solution. This dissolution will be performed without generating air bubbles. If necessary, these can be removed for example by centrifugation or by placing the solution under vacuum. Among the gelling polymers most commonly encountered include, among other examples, alginate, carrageenan, chitosan, pectin, starch (modified or not), gelatin, cellulose sulfate, carboxymethylcellulose , xanthan gum, locust bean gum, gellan gum or agar agar.

Apart from these polysaccharides, gelatin and other proteins also have interesting properties vis-à-vis the formation of more or less permeable gels.

Synthetic polymers giving rise to the formation of a hydrogel are also usable, as long as the gel can be broken down at the end of the renaturation process, whether by dissolution or degradation of the polymer.

The so-called smart polymers, which have the property of changing phase according to the temperature are another possibility of confinement of the denatured protein.

Carrageenan (or carrageenan) is a polysaccharide extracted from red algae as a thickening and stabilizing agent in the food industry. It bears the code E407 of the classification of food additives. Carrageenans make it possible to form hot gels (up to 60 ⁰ C) and are therefore of interest compared to traditional animal gelatins.

A more preferred polysaccharide according to the present invention is alginate. Alginate is a polymer formed from two monomers bonded together: mannuronate or mannuronic acid and guluronate or guluronic acid. The proportion and distribution of these two monomers is a major determinant of the physical and chemical properties of alginate.

Alginates can form hard, heat-stable gels used as food additives (E400 to E405) for the reconstruction of food (ham, blue strings, breaded fish ...). Alginate beads can also be used in medicine to encapsulate drugs or fragile biological substances (enzymes, microorganisms, animal or human cells). In general, the concentration of alginate used according to the invention is between 1 and 10%, preferably between 2 and 5%.

These concentrations can also be advantageously applied to other gelling polymers. In general the concentration will be between 1 and 10%, preferably between 2 and 5%.

It will generally be noted that the composition as well as the solubility and viscosity properties of these different gelling polysaccharides depend to a large extent on their origin, the state of the matrix from which they were extracted and the steps in the process of their preparation. manufacturing. Thus the composition (distribution of manuronate and guluronate residues), the molecular weight, the viscosity and the gelling properties of an alginate can vary significantly between several suppliers, or even between several production batches from the same supplier. This is why the concentrations mentioned above are indicative and should be adjusted by those skilled in the art.

The choice and concentration of the encapsulation matrix will be made by those skilled in the art depending on the size and the charge of the protein as well as its activity. For example, better retention can be achieved by encapsulating a positively charged protein (at a pH below its isoelectric point) within a negative charge matrix.

In addition, it is envisaged the possibility of adding within the encapsulation matrix or in the surrounding solutions any compound capable of facilitating or accelerating the renaturation of the target molecule. Many examples are cited in the scientific literature and are known to those skilled in the art. Mention may be made briefly and non-exhaustively of redox systems such as reduced / oxidized glutathione (GSH / GSSG), certain amino acids such as arginine, proline, polyols such as glycerol or polyethylene glycol, sugars such as sucrose or polymers such as heparin, a glycosaminoglycan. Conventional concentrations for glutathione (oxidized or reduced) are of the order of 0.5 to 5 mM, preferably around 1-2 mM. For amino acids such as glycine or proline, concentrations of between 50 mM and 1 M, and preferably around 400 mM, are commonly employed. Glycerol is often used at concentrations in the region of 5%, ammonium sulphate at around 200 mM. Glucose, finally, is often added to the renaturation medium to the extent of a few% (5%, usually).

Step d): The denaturing solution comprising the gelling polymer is then brought into contact with a bath causing the gelling of the dissolved gelling polymer, thus leading to the encapsulation of the denatured proteins and of the denaturing agent and / or of the reducing agent and / or detergent in the solution thus gelled.

In general, the contact between the denaturing solution and the bath causing the gelation of the polymer is carried out by dropping the denaturing solution into the bath, thus causing the polymer to gel. However, any other technique for obtaining capsules or polymer beads is suitable for carrying out the present invention. The diameter of the beads thus obtained may typically be between 1 and 10 mm, but an extrusion in the form of spaghetti or a bulk or other form of gelation is also possible. The tests carried out by the applicant of the present invention have shown that large logs (4-5 mm in diameter) do not constitute a handicap insofar as the denaturing agent can still escape rather quickly and where the The target protein was better retained by 4-5 mm beads than by smaller beads.

Preferably, if the gelling polymer is alginate then the bath causing the gelation of the polymer consists of a solution of calcium salts. The most commonly used calcium chloride is between 2 and 10%, even more preferably around 5% but depending on the applications and the method of manufacture of the capsules may be used other calcium derivatives (calcium lactate for example) . Carrageenan can be gelled either by cooling (gel thermal), either by ionic interactions between the polysaccharide and potassium ions (K +). Potassium salts (KCl most often) are used in gelling baths at concentrations between 2 and 10%, even more preferably at around 5%. Chitosan, which is soluble in a slightly acidic medium, can itself be gelled either by a change in pH (the chitosan solution is extruded in a NaOH bath between 0.1 and 1.0 M, preferably around 0.5 M) or at room temperature. using sodium tripolyphosphate. Pectins with a low degree of methylation (low methoxy pectins) can be solidified using calcium solutions at the same concentrations as for alginate. Cellulose sulphate is gelled by complexation with other polyelectrolytes such as poly (diallyldimethylammonium chloride) chloride (p-DADMAC) in equivalent concentrations. Gelatin and agar agar form thermal gels and are solidified by cooling like alginate.

There are many methods for solidifying solutions of these polymers. The choice of the most relevant approach will be conditioned by the stability of the protein to be renatured and will be based on the knowledge and experience of those skilled in the art. In some protocols the solidification of the beads is completed by crosslinking the gels formed using reagents such as glutaraldehyde. This approach is undesirable in the context of the present application, since it involves degradation of these matrices at the end of the process, in order to release the renatured protein.

In general it is the nature of the gelling polymer that will determine that of the bath causing the gelation of the polymer. Also each polymer pair / gelling bath will be precisely determined by the skilled person according to his usual knowledge.

It is the same with the concentration of the component present in the gelling bath; this concentration is directly dependent on the concentration of the gelling polymer and will also be precisely determined by the skilled person according to his usual knowledge. Step e): In the next step, the denaturing agent and / or the reducing agent and / or the detergent agent are allowed to diffuse through the solution thus gelled comprising the encapsulated denatured proteins, thereby causing a progressive renaturation of these denatured proteins while limiting their aggregation. Indeed and surprisingly, the inventors of the present invention have noticed that the use of a gelling polymer to encapsulate the denatured proteins and in solution in the denaturing solution made it possible to obtain a high degree of renaturation of the denatured recombinant protein. while limiting the aggregation of these same proteins.

This progressive renaturation is obtained by a favorable combination of two effects:

1) the denaturing agent and / or the reducing agent and / or the detergent agent, which are molecules of relatively small size, are allowed to diffuse through the gelled matrix, thus reducing the concentration of these substances at values allowing the renaturation of the protein.

2) On the other hand, by strongly limiting the mobility of the protein within the matrix during the renaturation step, it is prevented from forming aggregates, which leads to renaturation yields higher than those observed with any another known technique despite the high concentration of protein.

The diffusion of the denaturing agent and / or the reducing agent and / or the detergent agent through the solution thus gelled comprising the encapsulated denatured proteins is carried out by bringing into contact said encapsulated denatured proteins, generally in the form of capsules or beads, in the presence of water, preferably distilled or a buffer providing the protein pH conditions favorable to its stability. The volume of distilled water is in general 10 to 30 times, preferably 20 times, larger than the volume of beads. A slight agitation can be applied to allow a faster and more homogeneous diffusion. As the denaturing agent is removed, the target molecule will thus be placed in conditions favorable to its renaturation and limiting its tendency to agglomeration. This renaturation process makes it possible to obtain excellent results. The temperature can be maintained at a value known to those skilled in the art and guaranteeing the stability of the protein for the duration of this step.

Step f):

After this step of in silico renaturation, the capsules containing the denatured proteins are put in the presence of a solution allowing the dissolution of the gel.

The latter is selected from solutions of sodium citrate, EDTA or sodium fluoride, are compounds known to those skilled in the art and for complexing calcium, thus leading to the disintegration of the alginate network.

Preferably, a buffered sodium citrate solution is used at one of the following concentrations: 100 mM, 200 mM, 300 mM or 50 mM, with a preference for a concentration of 200 mM.

These concentrations can also be advantageously applied to the other compounds at the base of the solution allowing dissolution of the gel. In general, the concentration of the solution allowing the dissolution of the gel will be between (approximately) 100 mM and 500 mM.

This will be conducted over a period of 30 to 180 minutes (preferably 60 minutes) and at a temperature between 5 and 40 ⁰ C (preferably 10-25 ⁰ C).

In general, it is preferable to carry out this step of dissolving the gel in small volumes of solution, in order to finally obtain a medium to high concentration of recombinant protein which is renatured and purified in the solution.

Step g): The protein is then obtained in renatured and purified form in concentrated solution.

The present invention also comprises an apparatus for carrying out the method of purification and renaturation of an accumulated recombinant protein in the form of an inclusion body that is insoluble by prokaryotic or eukaryotic cells as described above.

The present invention also comprises a kit for carrying out the method of purification and renaturation of a recombinant protein accumulated in the form of an insoluble inclusion body by prokaryotic or eukaryotic cells as described above. This kit may comprise, inter alia, i) the denaturing agent and / or the reducing agent and / or the detergent agent in general in the form of solutions, ii) a gelling polymer, iii) the compounds for the preparation of the bath causing gelling said gelling polymer, iv) compounds for the preparation of a dissolution solution of the gel and, optionally, v) prokaryotic or eukaryotic cells necessary for the expression of the recombinant protein. The kit may also comprise an expression vector (plasmid or other) necessary for the expression of the recombinant protein of interest.

The following examples are intended to demonstrate the feasibility of the present invention and can not limit the invention to these descriptions alone.

The first example below is a proof of concept that demonstrates that a protein entrapped in an alginate matrix diffuses much more slowly than the denaturing agent that is also encapsulated therein. The model chosen for the protein is ovalbumin, with a molecular weight of approximately 45 kDa, and isoelectric point 4.6.

The second example shows a renaturation test of B. amyloliquefaciens α-amylase (molecular weight 55 kDa, isoelectric point around 5.2) at high concentration (10 mg / ml) carried out in two different ways:

In the former, the denaturing agent (guanidinium chloride) was removed by dialysis, the enzyme remaining in solution inside the dialysis chamber.

Amylase activity was measured before denaturation and after dialysis.

• In the second approach the denatured enzyme (10 mg / ml) and the denaturing agent (3.0 M) were encapsulated in an alginate matrix. The The capsules thus formed were immersed in a buffer solution, which allowed rapid removal of the denaturing agent. The beads were then recovered and dissolved by complexation of calcium with citrate. Amylase activity was measured before denaturation and after renaturation treatment.

All the tests described below were carried out at ambient temperature (22 ° C.).

Example 1: Proof of concept - measurements of diffusion rates of BSA and guanidinium chloride out of an alginate matrix.

Chemicals and reagents

Sodium Alginate, Lot # KLTV 45184A, Inotech AG, Basel (Switzerland) Calcium Chloride Dihydrate (CaCl2-2H2O), Fluka Chemicals, Buchs (Switzerland). Guanidinium Chloride, Sigma Aldrich, Buchs (Switzerland) Crystalline Ovalbumin 5x, Fluka Chemicals, Buchs (Switzerland) Tri-sodium Citrate Dihydrate, Panreac Chemicals, Barcelona (Spain)

Description of the test

In a 10 mM Tris buffer at pH 7.0, 10 mg / ml of ovalbumin was dissolved, 3.0 mol / l of guanidinium chloride (GdnCl) and sodium alginate at varying concentrations were dissolved. between 2 and 5%. The solution was then degassed and the air bubbles removed by centrifugation and / or under vacuum.

This mixture was then dripped into a 10 mM Tris buffer solution at pH 7.0 containing 5% CaCl _{2 as} well as ovalbumin and GdnCl at the same concentrations as before (10 mg / ml and 3.0 mol / l). l, respectively) to avoid losses during storage of the balls. Alginate beads with a diameter of about 5 mm were thus formed.

For the beginning of the test, the balls, with a total volume of 10 ml, were recovered and drained with a strainer before being immersed in a container containing 190 ml of Tris buffer solution. mM at pH 7.0. The suspension was stirred with a magnetic bar. The migration of guanidinium chloride and ovalbumin from the alginate matrix to the outer solution was measured from this moment onwards. The dosage of the

GdnCL was done continuously by conductimetry. The concentration of ovalbumin in the liquid was determined by regular sampling and by measuring the absorbance of the solution at 280 nm.

Two sizes of beads as well as different concentrations of alginate and variable stirring speeds were tested in these tests.

Results

The results of the tests described above are shown in FIG. 2. It can be seen that it takes only 20 minutes to eliminate almost all of the denaturing agent contained in beads 3-4 mm in diameter, and that whatever either the alginate concentration used for the manufacture of the beads (between 2 and 5%), Figure 3 shows exactly the same trends for larger beads, 5 mm in diameter. It is also noted for the two sizes of beads that ovalbumin, because of its size, diffuse for its part that much slower out of the matrix of alginate. FIG. 2 also demonstrates the influence of the alginate concentration on the release rate of the components: first observation, a gel with 5% alginate does not seem to retain the denaturing agent more strongly than a gel with 2 % this is not too surprising given the small size of the GdnCl molecule. Ovalbumin, on the other hand, with a molecular weight of 45 kDa, is released much less rapidly from a 5% alginate gel than from a 2% gel. The "tighter" network of the 5% gel therefore retains better the protein studied here.

Other tests, the results of which are reported in FIG. 4, have also shown that the stirring speed had practically no impact on the migration speed of the various components, in particular guanidinium chloride, the outside of the balls. All the above results clearly indicate the transport inside the beads as the limiting step of the process.

These results also demonstrate unequivocally that small diameter beads are not required to carry out the application described here. In Accordingly, the present invention does not require the use of sophisticated technology for the generation of small diameter monodisperse droplets as described in the microencapsulation literature.

The observations reported here thus amply demonstrate the validity of the proposed concept, namely the rapid migration of the denaturing agent out of the alginate beads as well as the retention of the major part of the protein within them during this period. time lapse.

EXAMPLE 2 Renaturation of the high concentration amyloliquefaciens α-amylase - comparison of the method by dialysis and by diffusion of the denaturing agent out of an alginate matrix.

Chemicals and reagents Sodium alginate, Lot # KLTV 45184A, Inotech AG, Basel (Switzerland) Bacillus amyloliquefaciens α-amylase, 839 units / mg _SO , Sigma-Aldrich, Buchs (Switzerland).

Guanidinium Chloride, Sigma Aldrich, Buchs (Switzerland) Calcium Chloride Dihydrate, Fluka Chemicals, Buchs (Switzerland) Tri-Sodium Citrate Dihydrate, Panreac Chemicals, Barcelona (Spain) Phadebas® Kit for Amylase Assay, Magic AB, Lund (Sweden)

Measurement of Guanidinium Chloride Concentration (GdnCl)

The concentration of the denaturing agent was measured by conductimetry as described in Run 1.

Measurement of α-amylase activity.

The activity of α-amylase (and therefore the concentration of native enzyme) was determined during the various tests using a Phadebas® commercial kit marketed by Magie AB, Lund (Sweden). This consists of a modified starch, onto which has been grafted a dye which solubilizes as the substrate is degraded by the amylase. The measurements were carried out according to the following general protocol:

• Three (insoluble) substrate tablets were dispersed in 32 ml of 0.1 M pH 7.0 phosphate buffer also containing 0.5 mM calcium chloride. The suspension was kept stirring to ensure homogeneity.

• 2 ml of suspension were then taken and placed in a test tube. Then 0.5 ml of enzyme solution (at the appropriate dilution) was added before starting the stopwatch and stirring the mixture at room temperature. After 10 minutes of incubation, 0.5 ml of 0.5 M NaOH was added with good stirring in order to stop the reaction.

The resulting suspension was then filtered through a 0.45 μm membrane using a syringe and the absorbance of the supernatant measured at 620 nm (blue color due to the solubilized dye) against a negative control sample where the enzyme solution was replaced by phosphate buffer.

• The difference in absorbance thus measured was, taking into account possible dilutions, directly proportional to the concentration of active enzyme in the sample.

Description of the test

The purpose of this test is to compare the renaturation yields at high concentration of α-amylase obtained either by dialysis or by the method proposed in the present invention.

In the first the concentrated enzyme (10 mg / ml) is in solution and is therefore free to form aggregates. In the second, the enzyme is immobilized in a gel allowing its renaturation but limiting its mobility, so that the aggregation is strongly inhibited.

In the second approach the denatured enzyme (10 mg / ml) and the denaturing agent (3.0 M) were encapsulated in an alginate matrix. The capsules thus formed were immersed in a buffer solution, which allowed rapid removal of the denaturing agent, as demonstrated in Test 1. The beads were then recovered and dissolved by complexation of calcium with citrate. Amylase activity was measured before denaturation and after renaturation treatment.

Denaturation of α-amylase and renaturation by dialysis

About 5 ml of a 10 mg / ml solution of B. amyloliquefaciens α-amylase was denatured at room temperature for 12 h in the presence of 3.0 M guanidinium chloride (GdnCl). The residual activity of the enzyme was measured before and after denaturation. The results are shown in Table 1.

An amount equivalent to 2 ml of the denatured enzyme solution was then transferred to a dialysis cassette with a nominal cutoff of 10 kDa. The cassette was then suspended in 3.0 liters of phosphate buffer stirred with a magnetic bar and left for 12 hours. Once the equilibrium of the concentrations was reached, the denaturing agent was diluted 1 × 50 times, which corresponds to a residual concentration of 2 mM.

It was noted that after a long denaturation of 12 hours, only 8% of relative activity remained. During renaturation by dialysis, a precipitate appeared in the cassette. After 12 hours of dialysis, the sample was recovered, mixed and its activity measured. This proved to be very low, reaching 4% of initial activity. It therefore seems that the aggregation of proteins has caused a significant loss of activity. The problem of dialysis is that the enzyme concentration remains high in the cassette (10 mg / ml). Thus, the tendency to aggregation is strong and renaturation by dialysis appears as an inappropriate method in this case.

The activity of the enzyme was measured at each stage of the experiment and is reported in Table 1 below. It contains the enzyme concentration C _am yiase mg protein per ml, the enzyme activity per unit volume A _VO ι, the specific activity A _sp ec (in units per mg protein) and finally the activity relative A _re ι expressed in% of the activity of the native enzyme. The whole experiment took place at room temperature.

Table 1: α-Amylase Activity at Each Step of Renaturation by Dialysis

Denaturation of α-amylase and renaturation in an alqinate matrix

The aim is to encapsulate the denatured enzyme at high concentration in an alginate matrix in the presence of guanidinium chloride, to allow the latter to rapidly diffuse out of the beads before recovering them and to immerse them in a solution of citrate of sodium to dissolve the gel and recover a concentrated and active enzyme.

200 mg of α-amylase were weighed before being transferred to a 20 ml graduated flask. About 10 ml of 0.1 M phosphate buffer pH 7.0 containing 0.5 mM CaCb- was transferred to the gauge to dissolve the enzyme. After dissolution of the enzyme, 5.732 g of guanidinium chloride (corresponding to 3.0 M when dissolved in 20 ml) was added to the enzyme solution and the volume was supplemented to a total of about 19.5 ml. The mixture was then left at room temperature for 16 hours in order to denature the enzyme as completely as possible.

At the end of the denaturation step, the enzyme solution was transferred to a 50 ml beaker and 0.8 g of alginate added to give a 4% solution of the latter. It was stirred gently with a spatula to dissolve the alginate without generating too much air bubbles. If necessary, these can be effectively removed by centrifugation or by placing the solution under vacuum. Once the alginate had dissolved, a 10 ml syringe was filled with the mixture, which was dropped into 490 ml of a 5% CaCl ₂ solution. The beads thus formed were left for 1 h with gentle stirring. This time is long enough to allow the complete elimination of the denaturing agent and allow the renaturation of the enzyme, while being short enough to ensure good retention of the latter.

The 10 ml of beads were then collected using a strainer and transferred to 90 ml of 500 mM sodium citrate pH 5.0 to dissolve the alginate and recover the soluble and renatured enzyme.

The activity of the thus renatured enzyme was finally measured and compared to the activity recovered after dialysis at the same concentration of 10 mg / ml. The results are shown in Table 2.

Table 2: Renaturation of the enzyme by encapsulation in an alginate matrix

This test shows a renaturation yield of 65%. Under identical experimental conditions, this value is very similar to the yields usually obtained by diluting 50 times a solution containing 1 mg / ml of enzyme and 3.0 M of guanidinium chloride. This result, on the other hand, has been achieved at a concentration of enzyme 50 times higher, thus representing substantial savings for the subsequent purification steps where the enzyme will have to be concentrated again. References

Brekke O H and l_0set G A (2003) New technologies in therapeutic antibody development Current opinion in Pharmacology 3 (5), 544-550

From Bernardez Clark E (1998) Refolding of Recombinant Proteins Current Opinion in Biotechnology 9, 157-163

From Bernardez Clark E (2001) Protein refolding for industal processes Current Opinion in Biotechnology, 12, 202-207

Schlegl R, Tschehessnig A, Necina R, Wandl R, Jungbauer A (2005) Refolding of proteins in a CSTR Chemical Engineering Science 60, 5770-5780

Ferré H, Ruffet E, Nielsen L -L B, Nissen M H, Hobley T J, Thomas O R T, Buus S (2005) Protein Science U, 2141-2153 A Novel Protein Science U, 2141-2153

Middelberg A P J (2002) Preparative Protein Refolding Trends in Biotechnology 20 (10), 437-443

Lancknet H & Middelberg A P J (2004) Continuous Chromatography Protein Refolding Journal of Chromatography A 1022, 103-113

Hutchinson MW & Chase HA (2006) Adsorptive refolding of histidine-tagged glutathione S-transferase using metal affinity chromatography Journal of Chromatography A 1 128 (1-2), 125-132 Randolph TW et al (1999) High pressure fosters protein refolding aggregates at high concentrations, PNAS 96 (23), 13029-13033

Randolph T W et al (2002) High hydrostatic pressure as a tool to study protein aggregation and amyloidosis Biochimica and Biophysica Acta 1595, 224-234

Mondai K, HB Bohidar, Roy RP, Gupta MN (2006) Algmate-chaperoned easy refolding of Chrornobactenurn viscosum hpase Biochimica ei Biophysica Acta 1764 _. , 877-886

Mondai K, Raghava S, Barua B, Varadarajan R, Gupta M N (2007) RoIe of Stimuh- Sensitive Polymers in Protein Refolding r-Amylase and CcdB (Controller of this Division or Death B) as Model Proteins Langmuir 23, 70-75

Claims

1. Process for purifying and renaturing a recombinant protein accumulated in the form of an insoluble inclusion body by prokaryotic or eukaryotic cells, characterized in that: a) the inclusion bodies are harvested after centrifugation in the form of a pellet, optionally with cell debris resulting from the lysis of prokaryotic or eukaryotic cells, b) the pellet is put in the presence of a denaturing solution containing at least one denaturing agent and / or a reducing agent and / or a detergent agent in order to dissolve the proteins contained in the pellet, c) the cell debris is removed by centrifugation if necessary, then the supernatant (the denaturing solution) is recovered and a gelling polymer is dissolved therein, d) the denaturing solution comprising the gelling polymer with a bath causing gelation of the dissolved gelling polymer, thereby leading to the encapsulation of the denatured proteins and denaturing agent and / or reducing agent and / or detergent in the solution thus gelled, e) allowing the denaturing agent and / or the reducing agent and / or the detergent agent to diffuse through the thus gelled solution comprising the encapsulated denatured proteins, thus resulting in a progressive renaturation of these denatured proteins while limiting their aggregation, f) the capsules containing the renatured protein are placed in the presence of a solution allowing the dissolution gel, g) the renatured and purified protein is obtained in solution.

2. purification and renaturation process according to claim 1, characterized in that the lysis of prokaryotic or eukaryotic cells is carried out chemically, mechanically or ultrasonically.

3. purification and renaturation process according to any one of claims 1 or 2, characterized in that step d) is carried out by dropping the denaturing solution comprising the gelling polymer dissolved in the bath causing gelation of said gelling polymer.

4. Process for purification and renaturation according to any one of claims 1, 2 or 3, characterized in that the denaturing agent and / or the reducing agent and / or the detergent agent are allowed to diffuse. to the outside of the gelling polymer beads when they are brought into contact with an aqueous medium, the proteins remaining captive inside because of their larger size.

5. Process for purification and renaturation according to any one of the preceding claims, characterized in that the denaturing agent is selected from the group comprising urea or guanidinium chloride.

6. purification and renaturation process according to any one of the preceding claims, characterized in that the reducing agent is selected from the group comprising β-mercaptoethanol, glycine, dithiothreitol and glutathione.

7. purification and renaturation process according to any one of the preceding claims, characterized in that the detergent agent is selected from the group consisting of anionic, neutral, cationic or zwitterionic detergents.

8. purification and renaturation process according to any one of the preceding claims, characterized in that the detergent agent is selected from the group consisting of Triton X100, CHAPS, dodecyl maltoside, DTAB and CTAB.

9. Process for purification and renaturation according to any one of the preceding claims, characterized in that the three agents (denaturant, reducing agent and detergent) can be together in the same denaturing solution, alone or in groups of two.

10. purification and renaturation process according to any one of the preceding claims, characterized in that the gelling polymer is selected from the group comprising alginate, carrageenan, chitosan, pectin, starch (modified or unmodified), gelatin, cellulose sulfate, carboxymethyl cellulose, xanthan gum, locust bean gum, gellan gum, agar agar, synthetic polymers and polymers intelligent.

11. purification and renaturation process according to claim 10, characterized in that the gelling polymer is alginate at a concentration of between 1 and 10%, preferably between 2 and 5%.

12. Process for purification and renaturation according to claim 11, characterized in that the bath causing the gelling of the alginate consists of a solution of calcium chloride at a concentration of between 2 and 10%, preferably around 5%.

13. purification and renaturation process according to any one of the preceding claims, characterized in that the solution for the dissolution of the gel is selected from the group comprising a solution of sodium citrate, EDTA and sodium fluoride.

14. purification and renaturation process according to any one of the preceding claims, characterized in that the solution for the dissolution of the gel is a buffered solution at a concentration between about 100 mM and 50OmM.

15. purification and renaturation process according to any one of the preceding claims, characterized in that the dissolution is conducted for a period of 30 to 180 minutes and at a temperature between 5 and 40 ⁰ C.

Apparatus for carrying out the method of purifying and renaturing a recombinant protein accumulated as an insoluble inclusion body by prokaryotic or eukaryotic cells according to any one of the preceding claims.

17. A kit for carrying out the method of purifying and renaturing a recombinant protein accumulated in the form of an insoluble inclusion body by prokaryotic or eukaryotic cells according to any one of the preceding claims, the kit comprising ) a denaturing agent and / or a reducing agent and / or a detergent agent in general in the form of solutions, ii) a gelling polymer, iii) compounds for the preparation of the bath causing gelation of said gelling polymer, iv) compounds for the preparation of a dissolution solution of the gel and, optionally, v) prokaryotic or eukaryotic cells necessary for the expression of the recombinant protein.