FR2981651A1 - Immobilizing nucleic acid ligands including a reactive amine function, by grafting on an activated solid substrate, including a step of coupling the nucleic acids on the activated solid substrate - Google Patents

Abstract

Immobilizing nucleic acid ligands including at least one reactive amine function, by grafting on an activated solid substrate, including a step of coupling the nucleic acids on the activated solid substrate having a pH of less than 6. Independent claims are included for: (1) immobilizing nucleic acid ligands comprising at least a primary amine function on a solid support, comprising providing a solid support on its surface of active carboxylic acid groups, providing a nucleic acid ligand comprising at least one primary amine function, and coupling the nucleic acid with active carboxylic acid groups present at the surface of the solid support under conditions of pH of less than 6; (2) preparing an affinity support, comprising implementing the immobilization process; (3) the solid support of affinity obtained by the preparation process; (4) a complex resulting from the interaction of a nucleic acid ligand and a target molecule, where the complex is formed on the surface of a solid support; and (5) purifying a target molecule with affinity support, comprising contacting a composition to be purified comprising the target molecule with a solid affinity support to form a complex between nucleic acid ligands grafted on the support and the target molecule, and releasing the target molecule from the complex and recovering the purified target molecule.

Description

FIELD OF THE INVENTION The invention relates to the field of the purification of substances of interest using affinity supports that can be used on an industrial scale, in particular for obtaining purified substances of medical interest. PRIOR ART There is a recurring need for affinity chromatography supports which allow the selective enrichment of a starting material into a substance of interest. In the medical field, affinity chromatography supports are used to purify substances subsequently used as drug active ingredients. Primarily, immunoaffinity chromatography supports are used on which antibodies are immobilized. The immunoaffinity supports are suitable for purification of substances of medical interest on an industrial scale because they have a good retention capacity and a high selectivity with respect to their target molecule. Such immunoaffinity carriers can be regenerated at the end of the purification processes without substantially altering their retention capacity or their selectivity, allowing their use over a long period of time. In addition, because of their high retention capacity, immunoaffinity carriers allow the purification of large quantities of the target substance, making their use compatible with the technical and economic requirements of drug manufacture. However, immunoaffinity carriers have drawbacks when they are used for the purification of substances of medical interest, particularly because of (i) the release of immunogenic protein fragments derived from the immobilized antibodies during the elution phase of the target substance previously retained and (ii) the fragility of the antibodies with respect to elution conditions and periodic treatments of antibacterial and anti-viral sanitization. Various studies have been undertaken to find alternatives to known immunoaffinity carriers. A limited number of studies relate to the use of aptamers as affinity ligands for the purification of target substances, including target proteins. For example, the work of Romig et al. (J. Chromatogra, B Biomed Sei Appl, 1999, 731 (2): 275-84) which relates to the purification of L-selectin on a chromatographic support on which anti-L-selectin DNA aptamers have been immobilized. via the streptavidin-biotin couple. It has long been known that the affinity and specificity of aptamers for their target molecule can be as high as that of antibodies. Moreover, the 5 aptamers can. be obtained by chemical synthesis, their manufacturing cost is much lower than that of antibodies. Nevertheless, the use of aptamer affinity carriers for the purification of target substances is currently marginal despite the many economic and technical advantages that aptamers provide as affmity ligands. To our knowledge, at the present time, no industrial process for purifying a target substance, including a target protein, comprises a step based on the use of an affinity support. aptamers. One reason that can be advanced to explain this lack of use of aptamers in industrial scale purification processes is the difficulty in obtaining affinity supports on which the aptamers are stably and quantitatively fixed. The main aptamer immobilization technique described in the state of the art is based on the use of the biotin-streptavidin or biotin-avidin pair. This technique takes advantage of the selectivity and high affinity of biotin for its avidin or streptavidin ligand as well as the stability of the non-covalent complex resulting from their association. This type of affinity support, however, has technical limitations resulting from the protein nature of the coupling agents used and the non-covalent nature of the bond formed between the support and the aptamers. Indeed, biotin and avidin or streptavidin are sensitive to treatments likely to induce protein denaturation. Furthermore, the biotin / streptavidin complex dissociates at low temperature, especially in nonionic aqueous solutions or at low saline concentration. For these reasons, the affinity supports on which nucleic aptamers immobilized as ligands via the biotin / streptavidin complex are not suitable for use in purification processes, in particular industrial processes, for which the The ability to regenerate and sanitize the affinity media between each purification cycle is paramount.

The state of the art also describes several techniques allowing the covalent grafting of nucleic acids onto solid supports, essentially with the aim of having new tools for the implementation of analytical methods. The grafting of nucleic aptamers possessing a reactive amine group on a cyanogen bromide-activated sepharose support has been described (Madru et al., 2009, Anal Chem, Vol 81: 7081-7086). The grafting of oxidized RNAs with periodate on an adipic acid dihydrazide activated agarose support (Caputi et al., 1999, The EMBO Journal, Vol 18 (14): 40604067) has also been described. . Nucleic aptamer grafting techniques are also known via bi-functional coupling agents - such as SIAB (Rehder et al., Electrophoresis, Vol 22 (17) 3759) State of the art also discloses covalent coupling techniques of nucleic acids, inter alia aptamers, onto solid supports of silica or agarose type comprising carboxylic acid groups pre-activated with N-hydroxysuccinimide (NHS) (Goss et al., 1990, J Chromatogr, Vol 508: 279-287, Larson et al., 1992, Nucleic Acids Research, Vol 20 (13): 3525, Allerson et al., 2003, RNA, Vol 9: 364-374). . These coupling techniques have consisted of direct application of the techniques conventionally used for coupling proteins to solid supports, Goss et al. (1990, J Chromatogr, Vol 508: 279-287) have thus described the grafting of a 5'-aminoethyl-poly (dT) 18 oligonucleotide on a macroporous support of silica pre-activated with NHS in a phosphate buffer of sodium at p11 = 7.4. The affinity support obtained by Goss et al. has a degree of grafting of poly (dT) 18 of 0.5 ktmol per g of silica, which is very low considering the number of carboxylate groups per g of silica gel (500 pmol per g of silica). In other words, only 0.1% of the carboxylate groups present on the surface of the support are linked via an amide bond to a poly (dT) is oligonucleotide. Goss et al. succeeded in capturing a small amount of a mixture of oligo- (dA) 12-is model nucleic acids with the affinity support thus obtained, and then eluting them successively in a salinity gradient elution buffer. . In their article published in 2003, Allerson et al. (2003, RNA, Vol.9: 364-374) lamented that chromatography based on the use of nucleic acids as an affinity ligand is rarely used for protein purification on an industrial scale due to the low capacity and / or low stability of the affinity media obtained to date. To the knowledge of these authors, the known coupling methods allow at most to immobilize only a few nanomoles of RNA per milliliter of support. According to a method analogous to that of Goss et al., Allerson et al. (2003, RNA, Vol 9 364-374) have described an attempt to couple nucleic aptamines, directed against an IRP1 or IRP2 regulatory protein, on an NHS pre-activated agarose support. Allerson et al. referred to the "agarose carrier manufacturer's recommendations" for carrying out this coupling and thus performed the coupling reaction at a basic pH of 8.3. It is specified that the manufacturer recommends, even today, to perform the coupling of a ligand on a support pre-activated by the NHS at a pH of between 6 and 9 (see for example the instruction document no. 5000-14 AC from GE Healthcare for the NHS-activated Sepharose 4 Fast Flow Gel)). Allerson et al. have themselves found that the coupling reaction of RNA aptamers having a primary amine function with a support preactivated by NHS provides a very low coupling efficiency, of the order of 2%, regardless of the duration of the reaction. the coupling reaction or the type of buffer solution used, without being able to overcome this technical disadvantage. Allerson et al. (2003, Supra) finally turned away from this grafting technique in favor of a multi-step grafting technique comprising (i) the introduction of alkyl-thiol functions on the support, (ii) the introduction of the group 5 iodoacetamide at the 5 'position of the aptamers via the bifunctional agent Su1fo-SIAB and (iii) a coupling step proper between the thiol functions of the support and the iodoacetamide groups of the aptamers so as to create a thiol bond. In the same way, Ruta et al. (2008, Anal Bioanal .. Chem., Vol 390 1051-1057) have shown that the stationary phase resulting from the grafting of DNA aptamers against D-adenosine on an NHS-activated silica support showed binding capacity for very low D-adenosine. There is therefore a need in the state of the art for novel processes for the preparation of affinity media, especially affinity media suitable for the industrial scale purification of substances of medical interest.

The present invention relates to a method for immobilizing nucleic acids comprising at least one reactive amine function, by grafting onto a solid support having on its surface activated carboxylic acid groups, said process comprising a step of coupling said nucleic acids to said activated solid support at a pH below 5. According to an alternative definition, the present invention relates to a method for immobilizing nucleic ligands comprising at least one primary amine function on a solid support comprising the following steps: a ) providing a solid support comprising on its surface activated carboxylic acid groups, b) providing a nucleic ligand comprising at least one primary amine function, and c) coupling said nucleic acid with the activated carboxylic acid groups present on the surface of said solid support under pH conditions below 5 having heard that the order of steps a) and b) is indifferent. In a preferred embodiment, the activated carboxylic acid groups are obtained by reaction with N-hydroxysuccinimide or a derivative thereof. The invention also relates to a method for preparing an affinity support comprising the implementation of the immobilization method as defined above. Another object of the invention is a solid affinity support obtainable by the method of preparation as defined above as well as its use in methods of purification or detection of a target protein. An additional object is a complex resulting from the interaction of a nucleic ligand and a target molecule, said complex being formed on the surface of a solid support as defined above. Finally, the present invention also relates to a method of purifying a target ligand with an affinity support, comprising the following steps: a) contacting a composition to be purified comprising a target ligand of interest with a carrier of affinity as defined above, to form a complex between (i) the nucleic acids grafted on said support and (ii) said target ligand and b) releasing said target ligand from the complex formed in step a) and recovering said purified target ligand. DESCRIPTION OF THE FIGURES FIG. 1 illustrates a chromatographic profile obtained by passing a composition comprising 100 ng of transgenic human factor VII on a solid support of the invention onto which human anti-FVII DNA aptamers have been grafted. Peak rg.Ù1 corresponds to a fraction of human FVII not retained on the affinity support. Peak # 2 corresponds to the human FVII contained in the elution fraction. Peak # 3 corresponds to the human FVII contained in Regeneration Panant. On the abscissa: the time, expressed in minutes. On the ordinate: the absorbance, expressed in units of Optical Density at the wavelength of 280 nanometers. FIG. 2 illustrates a chromatographic profile obtained by passing a composition comprising 200 μg of transgenic human factor VII on a solid support of the invention onto which human α-FVII DNA aptamers have been grafted. Peak No. 2 corresponds to the human FVII contained in the elution fraction. Peak No. 3 corresponds to the human FVII contained in the regeneration effluent. On the abscissa: the time, expressed in minutes. On the ordinate: the absorbance, expressed in units of Optical Density at the wavelength of 280 nanometers. Figure 3 illustrates a chromatographic profile obtained by passing a composition comprising 1000 μg of transgenic human Factor VII on a solid support of the invention onto which human anti-FV1.1 DNA aptamers were grafted. Peak # 2 corresponds to the human FVII contained in the elution fraction. Peak No. 3 corresponds to the human FVII contained in the regeneration effluent. On the abscissa: the time, expressed in minutes. On the ordinate: the absorbance, expressed in units of Optical Density at the wavelength of 280 nanometers. FIG. 4 illustrates a chromatographic profile obtained by passing a composition comprising 200 μg of transgenic human factor VII on a solid support of the invention onto which human anti-FVII DNA aptamers have been grafted, said support having been previously submitted. treatment with a sanitizing solution comprising 0.5 M NaOH. Peak # 2 corresponds to the human FVII contained in the elution fraction. Peak No. 3 corresponds to the human FVII contained in the regeneration effluent. On the abscissa: the time, expressed in minutes. On the ordinate: the absorbance, expressed in units of Optical Density at the Wavelength of 280 nanometers. FIG. 5 illustrates a chromatographic profile obtained by passing a composition comprising 1000 ng of transgenic human factor VII on a solid support of the invention onto which human anti-FVII DNA aptamers have been grafted, said support having been previously subjected to treatment with a sanitizing solution comprising 0.5 M NaOH. Peak # 2 corresponds. to the human FVII contained in the elution fraction. Peak No. 3 corresponds to the human FVII contained in the regeneration effluent. On the abscissa: the time, expressed in minutes. On the ordinate: the absorbance, expressed in units of Optical Density at the wavelength of 280 nanometers. Figure 6 illustrates a chromatographic profile obtained by passing a composition comprising 2.7 mg of transgenic human Factor VII on a solid support of the invention onto which human anti-FVII DNA aptamers were grafted under the coupling conditions. following: 48h, 5 ° C, pH 4.2. Point # 1 (at about 45 min) indicates the timing of human Factor VII injection. Peak # 2 (at about 70 min) corresponds to the human FVII contained in the elution fraction. On the abscissa: the time, 20 expressed in minutes. On the ordinate: the absorbance, expressed in units of Optical Density at the wavelength of 280 nanometers. Figure 7 illustrates a chromatographic profile obtained by passage of a composition comprising 2.7 mg of transgenic human Factor VII on a solid support of the invention onto which human anti-FVII DNA aptamers were grafted under the coupling conditions. following: 48h, 5 ° C, pH 3.8. Point # 1 (at about 35 min) indicates the timing of Human Factor VII injection. Peak # 2 (at about 70 min) corresponds to the human FVII contained in the elution fraction. On the abscissa: the time, expressed in minutes. On the ordinate: the absorbance, expressed in units of Optical Density at the wavelength of 280 nanometers. Figure 8 illustrates a chromatographic profile obtained by passing a composition comprising 2.7 mg of transgenic human Factor VII on a solid support of the invention onto which human anti-FVII DNA aptamers have been grafted under the conditions of the invention. following coupling 2h, 5 ° C, pH 4.2. Point # 1 (at about 35 min) indicates the timing of Human Factor VII injection. Peak # 2 (at about 70 min) corresponds to the human FVII contained in the elution fraction. In. abscissa: the time, expressed in minutes. On the ordinate: the absorbance, expressed in units of Optical Density at the wavelength of 280 nanometers. FIG. 9 illustrates a chromatographic profile obtained by passing a composition comprising 2.7 mg of transgenic human factor VII on a solid support of the invention onto which human DNA anti-FVII aptamers have been grafted under the following coupling conditions. : 1h, RT (room temperature), pH 4.2. Point # 1 (at about 40 min) indicates the timing of human Factor VII injection. Peak # 2 (at about 75 min) corresponds to the human FVII contained in the elution fraction, abscissa: time, expressed in minutes. On the ordinate: the absorbance, expressed in units of Optical Density at the wavelength of 280 nanometers. DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel affinity carriers comprising immobilized nucleic acids, as well. as processes for their preparation. A first object of the invention is a method of immobilizing nucleic acids having at least one amine functional group reactive with a solid support having acetic acid carboxylic functions. The term "nucleic acid" or "nucleic ligand" is understood to mean, according to the invention, a compound comprising a polymer of nucleotides or polynucleotide, that is to say of ribonucleotides and / or of deoxyribonucleotides possibly chemically modified, having a length ranging from 5 to 10,000 nucleotides in length, preferably 5 to 1000 nucleotides in length, and more preferably 5 to 120 nucleotides in length. A nucleic acid conventionally includes polyribonucleotides (RNA) and polydeoxyribonucleotides (DNA), where appropriate chemically modified. A nucleotide is composed of (i) a phosphate group (mono-, di- or tri-) or an analogue, (ii) a sugar selected from ribose, deoxyribose and their chemical analogues and (iii) a nitrogen base selected from adenine, guanine, thymine, cytosine, uracil and their chemical analogues. Within the meaning of the invention, a nucleotide can be modified both on its osidic part and on its nitrogenous base by methods well known to those skilled in the art. By way of example, reference may be made to US Pat. No. 5,958,691, which describes aptamers exhibiting a chemical modification at the level of one or more nucleotides. In some embodiments, a nucleic acid is a polymer of nucleotides, ribonucleotides or deoxyribonucleotides. In other embodiments, a nucleic acid consists essentially of a polymer of nucleotides and comprises a non-nucleotide portion, said non-nucleofide portion being preferably of reduced length, compared to the length of the nucleotide portion, for example a length which is less than the length occupied by a chain of five nucleotides, ribonucleotides or deoxyribonucleotides. According to the invention, a nucleic acid comprises a reactive amine function when said nucleic acid has an amine functional group accessible to the solvent and capable of reacting with a reactive group. appropriate carried by another molecular entity. The reactive amine functions include, in particular, primary amines. This primary amine is distinct from the aromatic amines carried by the purine or pyrimidine nucleotide nuclei. Such nucleic acids are well known in the state of the art and are conventionally used for their chemical coupling to supports or to marker substances. Conventionally, these are nucleic acids that have been modified by the addition of an amine function at their 3 'end or at their 5' end. Most frequently, the amine function is added to the 5 'end of the nucleic acid where its incorporation is easier as the final step of a method for synthesizing a polynucleotide. In some embodiments, the reactive amine function and the 5 'or 3' end of the nucleic acid are separated by a spacer chain. According to the invention, a nucleic acid can comprise a reactive amine function at its 3 'or 5' end, which means that the reactive amine function is coupled to the nucleotide portion of said nucleic acid. According to other embodiments, a nucleic acid may comprise a reactive amine function "on the side" of its 3 'or 5' end, which means that said amine function is not coupled directly to the nucleotide portion of said acid nucleic acid, but is covalently linked to a non-nucleotide portion of said nucleotide acid, for example a non-nucleotide spacer chain which is interposed between said reactive amine function and said end of the nucleotide portion of said nucleic acid. To summarize, within the meaning of the invention, a "nucleic acid", also referred to in the following as "nucleic ligand", comprises: n a polynucleotide, said polynucleotide consisting of a sequence of ribonucleotides and / or deoxyribonucleotides which may be chemically modified and optionally, a non-nucleotide moiety, preferably a spacer chain, said ligand or nucleic acid further comprises a reactive amine function, wherein said reactive amine function may be attached to either a 3 'or a 5' end of the polynucleotide or optionally on the spacer chain. In general, the polynucleotide is at least modified at the 3 'or 5' nucleotide to introduce the reactive amine function directly or via a non-nucleotide moiety, particularly a spacer chain. The nucleic acid may comprise a chemical entity additional to those previously mentioned, for example a fluorophore or a chromophore. In general, the nucleic acid according to the invention is a ligand, that is to say that it is capable of binding specifically to one or more target molecules. We will therefore speak indifferently in what follows of "nucleic ligand" or nucleic acid ". The target molecules include RNA molecules, DNA molecules, organic or inorganic chemical molecules, peptides and proteins, whether human, animal, plant, viral or bacterial. The applicant has shown that it is possible to prepare affinity supports on which are immobilized nucleic acids (or nucleic ligands), said affinity supports being used in preparative techniques for purifying substances of therapeutic interest, because they possess to. both a good selective retention capacity of the target substances, an excellent resistance to the regeneration treatments and their use does not entail any risk of introduction of potentially toxic or immunogenic substances into the resulting purified preparation. It has been shown according to the invention that nucleic acid-based affinity carriers can be prepared by chemical grafting of said nucleic acids to a solid support comprising activated carboxylic acid functions, under specific grafting conditions allowing both a high grafting efficiency and the maintenance of the structural and functional integrity of the grafted nucleic acids. The Applicant has shown that, contrary to what the article by Goss et al. (1990, Supra) the coupling technique of a ligand comprising a reactive amine function on a pre-activated NHS support, which is commonly used for the coupling of proteins on a support, can not be directly transposed for grafting. nucleic acids on a support. The results of the examples show in particular that by using the conventional coupling method carried out at a pH of between 6 and 9 with a variety of distinct nucleic acids, a grafting yield ranging from 0% to 10% maximum is obtained. The results of the examples confirm those obtained by Allerson et al (2003, Supra), who had opted in the end for another coupling technique. In order to find alternative coupling methods to the known methods, the Applicant has tested NHS-supported support coupling conditions in which the anionic charges carried by the nucleic acids are masked by the addition of a source of monovalent or divalent ions, in this case by addition of Mg2 +, Ca2 + and / or Na + ions. The results of the examples show that the neutralization of the anionic charges carried by the nucleic acids and / or the increase of the ionic strength by addition of divalent or monovalent cations does not make it possible to increase the grafting yield which remains included in a range, ranging from 0% to 10%. It is also shown that the neutralization of the anionic charges carried by the nucleic acids by the addition of ionized macromolecules, such as Polybrene®, leads to an impossibility of grafting these nucleic acids. Also, the Applicant has tested the effectiveness of a coupling using nucleic acids in which the reactive amine function and the 5 'end of the polynucleotide are separated from each other by a positively charged spacer chain. The spacer chain consisted of a polyamide comprising at least one tertiary amine function. The results, not shown in the examples, showed that an excellent grafting efficiency, close to 100%, was obtained on the pre-activated support with NHS. On the other hand, the Applicant has shown that bringing the support thus grafted into contact with a solution at an alkaline pH of about 9-10 alters the structure of the spacer chain and causes the nucleic acid to dislodge the support. Such a coupling technique, which allows an excellent coupling efficiency, thus provides an affinity support which has proved unsuitable for implementation in industrial purification processes, said processes generally comprising drastic washing steps and / or or microbial inactivation. The technical solution which was finally developed according to the invention consisted in carrying out the nucleic acid coupling reaction on the N-hydroxysuccinimide pre-activated support, under pH conditions of less than 5. The technical solution of the invention is quite surprising because at an acidic pH, the person skilled in the art would normally have expected that the coupling reaction would not take place at all, or at least take place in such a weak proportion that a very low grafting yield is obtained because of the low reactivity of the primary amines and the degradation by hydrolysis of the nucleic acids generally observed at acidic pHs Gold, it has been shown in the examples that the realization of the step coupling a nucleic acid on a support pre-activated with NHS, under pH conditions of less than 5, makes it possible to obtain a grafted support with a grafting yield of at least 70%. The grafting yield is even about 100% when the coupling reaction is conducted at pH below 4.5. It is equally surprising that it is shown that performing the coupling step at an acidic pH does not affect the functional integrity of the nucleic acids, whereas the high sensitivity of the nucleic acids to the conditions of Acidic pH is well known to those skilled in the art. Even more surprisingly, the Applicant has shown that this coupling reaction - which leads to the formation of an amide bond between the solid support and the nucleic acid - is very rapid, this reaction being generally completed in less than one hour, regardless of the reaction temperature. Moreover, the Applicant has shown that the implementation of the reaction at low temperature is not a prerequisite for preserving the functional integrity of the grafted ligands. In other words, the immobilization method of the nucleic ligands according to the invention can be carried out independently at low temperature or at room temperature. It has also been shown in the examples that the nucleic acids grafted on the support retain their chemical and physical integrity, since their functionality is intact. This aspect is illustrated in the examples by a grafted support with as soon as nucleic aptamers able to bind to human factor VII (FVIIh). It is shown that the ability of said anti-FVIIII nucleic aptamers is intact after grafting under acidic pH conditions, at low temperature or at room temperature, onto the pre-activated NHS support. The Applicant has also shown that when a nucleic aptamer capable of selectively binding to forms of active human factor VII comprising a correctly gamma-carboxylated Gla domain is used for grafting, the grafted aptamer retains the aptitude of the aptamer. non-grafted to discriminate (0 the active forms of the correctly gamma-carboxylated Gla domain Factor VII of the (ii) non-active forms of human Factor VII.

It has also been shown that the specific coupling conditions disclosed in this specification are suitable for grafting any type of nucleic acid, i.e., both DNA nucleic acids and RNA nucleic acids. In addition, the examples show that the process of the invention results in obtaining affinity supports having a high density of grafted nucleic acids and consequently allows the preparation of affinity supports possessing high capacity for capturing nucleic acid. Target ligands, usable on an industrial scale. To the knowledge of the Applicant, such supports have never been described in the state of the art. The combined characteristics of high target ligand selectivity and high capture capacity of said target ligand illustrate the compatibility of an affinity support obtained according to the method of the invention with use in a step of purification of target ligands on an industrial scale. It goes without saying that the method according to the present invention also allows the preparation of affinity supports for the detection of a target molecule. The present invention more precisely relates to a method for immobilizing nucleic ligands comprising at least one reactive amine function, by grafting on a solid support having activated carboxylic acid groups, said method comprising a step of covalently coupling said nucleic acids. str said solid support at a pH below 5. According to another definition, the method of immobilization of nucleic ligands comprising at least one reactive amine function on a solid support according to the invention comprises the following steps: a) provide a solid support comprising on its surface activated carboxylic acid groups, b) providing a nucleic ligand comprising at least one reactive amine function, and c) coupling said nucleic ligand with the activated carboxylic acid groups present on the surface of said solid support under conditions of pH less than 5. it being understood that the order of the steps a) and b) is indifferent. It goes without saying that the coupling step c) allows the creation of amide bonds between the solid support and the nucleic ligands, each amide linkage resulting from the reaction between an activated carboxylic acid function of the support and a primary amine function present in the level of the nucleic ligand. According to the invention, the coupling conditions at a pH of less than 5 include coupling conditions at a pH of less than 5, less than 4.9, less than 4.8, less than 4.7, less than 4, 6, less than 4.5, less than 4.3. In some embodiments, the pH of the coupling step is in a range of 3 to 5, which includes a pH of 3.0, a pH of 3.1, a pH of 3.2, a pH of pH 3.3, pH 3.4, pH 3.5, pH 3.6, pH 3.7, pH 3.8, pH 3.9, pH of 4.0, a pH of 4.1, a pH of 4.2, a pH of 4.3, a pH of 4.4, a fold of 4.5, a pH of 4.6, a pH of 4.7, a pH of 4.8, a pH of 4.9 and a pH of 5.0. Preferably, the pH of the coupling step is less than 4.5. In some embodiments, the pH of the coupling reaction is in a range. 3.5 to 4.5. As illustrated in the examples, the coupling step can be performed at a pH of about 4.2. Preferably, the coupling is carried out in the presence of an aqueous buffered medium having a pH below 5. The buffered medium can be prepared from any type of acid and / or weak bases, insofar as the acid (s) and weak bases used are not likely to react during the coupling reaction. As illustrated in the examples, it may be an aqueous solution of sodium acetate.

Without wishing to be bound by any theory, the Applicant believes that, at the acid pH conditions used for the coupling reaction, the nucleic acids to be grafted are in linear form, which promotes the accessibility of their solvent-reactive amine group, and in particular promotes the reaction of said reactive amine group with an activated carboxylic acid group accessible on the surface of the solid support.

By "activated carboxylic acid function" or "activated carboxylic acid group" is meant a chemical function derived from the "carboxylic acid" function capable of reacting with a nucleophile. More specifically, the term "activated carboxylic acid function" means a chemical function derived from the "carboxylic acid" function capable of reacting with a primary amine so as to form an amide bond. The functions "activated carboxylic acids" are well known to those skilled in the art and include the functions acid chlorides, mixed anhydrides and esters. In certain embodiments, the activated carboxylic acid functions are in the form of esters resulting from the reaction of said carboxylic acid functions with a compound selected from the group consisting of 14 hydroxybenzotriazole (BOBO, HOAt, N-hydroxysuccinirnide or In a preferred embodiment, the carboxylic acid groups of the support have been activated by reaction with N-hydroxysuccinimide or one of its derivatives such as N-hydroxysulfosuccinimide, which means that the carboxylic acid groups The "solid support" groups correspond to "N-succinimidyl ester" groups, also known as "succinimidyl esters" or "N-hydroxysuccinimide esters", of the following formula (I) where R represents the branching of the solid support at which the ester function is fixed: Activation by NHS or sulo-THS has the advantage of generating reactive activated esters with respect to the primary amines but also stable enough to allow the conditioning and storage of the pre-activated support obtained. Solid supports having "activated carboxylic acid" functions are well known in the state of the art and many of them are commercially available. The solid supports may also be prepared according to methods well known to those skilled in the art, for example by reacting a support initially having carboxylic acid functions on its surface with a suitable chemical agent allowing the activation of the carboxylic acid functional groups. view of the subsequent formation of an amide bond. In particular, it will be possible to refer to the conventional methods for activating the carboxylic acid functions used in peptide synthesis, in particular by the solid route. By way of illustration, it is also possible to use, under the specific coupling conditions prescribed by the invention, the activation methodology by a combination of EDC (1-Ethyl-343-dimethylaminopropyl) carbodiimide hydrochloride) and NHS, although known to those skilled in the art. Solid supports having "activated carboxylic acid" functions may be of any type. These supports include the supports conventionally used for chromatography, including silica and agarose [Al] supports, and which have been treated in order to present on their surface activated carboxylic acid groups. Pre-activated solid supports include dextran, agarose, starch gels, cellulosic derivatives, or synthetic polymers such as polyamides, trisacryl, methacrylate derivatives, polystyrene derivatives, and the like. polyacrylamides, or inorganic supports such as silica supports (especially porous glass supports) or alumina, on the surface of which are present activated carboxylic acid groups. In general, the solid supports on which the nucleic ligands according to the invention can be immobilized include any type of support having the structure and the composition found commonly for filter supports, membranes, etc. Solid carriers include resins, resins or gels for affinity chromatography columns, polymer beads, magnetic beads, paramagnetic beads, filter membrane support materials, and the like. Solid supports also include glass or metal-based materials such as steel, gold, silver, aluminum, copper, silicon, glass and ceramics. Solid supports also include, in particular, polymeric materials, such as polyethylene, polypropylene, polyamide, polyvinylidene fluoride, polyacrylamide derivatives, and combinations thereof.

In particular embodiments for which the carboxylic acid functions of the solid support are activated by NUS, said solid support can be obtained by reacting a commercial gel having free carboxylic acid functions with N-hydroxysuccinimide (NHS), optionally presence of carbodiimide such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).

It is also possible to use a commercial solid support pre-activated with NUS, for example an "N1-15 Activated Sepharose Fast Flow (GE)" gel marketed by General Electric Healtheare (United States), a "HiTrapTM NHS" gel. -Activated "marketed by General Electric Healthcare (United States) or an" NHS-Activated Agarose "gel marketed by the company Thermo Scientific Pierce. As is explained above, a nucleic ligand according to the invention comprises a polynucleotide, that is to say a polymer of nucleotides. The polynucleotide of the nucleic ligand is responsible for a large part of the specific binding properties of said ligand with respect to its target molecule or molecules. It generally comprises from 5 to 120 nucleotides in length. The nucleic ligand may further comprise a portion. non-nucleotide. Said non-nucleotide portion being preferably bound to the polynucleotide. By "non-nucleotide" part is meant a chemical moiety that does not consist essentially of a polynucleotide. This non-nucleotide portion is preferably a spacer chain. The reactive amine of said nucleic ligand is preferably a primary amine present at the 3 'or 5' end of the polynucleotide or, where appropriate, a primary amine present at the non-nucleotide portion. Preferably, the reactive amine is an aliphatic primary amine, which means that the amine function is not directly linked to an aromatic group. Thus, in some embodiments, the nucleic ligand is a polynucleotide 5 to 120 nucleotides in length comprising at least one reactive amine function. its end 3 'or 5'.

In other embodiments, the nucleic ligand comprises (i) a polynucleotide 5 to 120 nucleotides in length and (ii) a spacer chain linked to said polynucleotide, the reactive amine function being attached to a 3 'or 5' end of said polynucleotide or the spacer chain.

The spacer chain is preferably bonded to the 5 'end or the 3' end of the nucleic acid. In a certain embodiment according to the invention, the nucleic ligand comprises a polynucleotide and a spacer chain, said spacer chain comprising at one of these ends an amine function and being linked at its second end to the 5 'end of the polynucleotide. Said spacer chain has the function of physically removing the polynucleotide from the surface of the solid support, which makes it possible to increase the relative mobility of the nucleotide part of the nucleic ligand and to reduce the steric hindrance. The spacer chain can be of any type. The examples illustrate in particular the use of the method according to the invention for a hydrophobic chain consisting of a chain consisting of a hydrophobic chain consisting of a chain composed of 3, 6, 12 or more (for example 18) methylene (CH 2 ) named in the following C3, C6, C12 or a hydrophilic chain which may be of polyethylene glycol type, for example Hexaethylene glycol (HEG) or a: 11-amino-3,6,9-trioxaundecan-ly1 called in the following Hydrophilic C11 or a Non-specific Oligonucleotide Preferably, the spacer chain does not comprise ionizable groups other than primary amine functions or secondary amine functions. In general, the spacer chain does not comprise alkaline pH sensitive groups or bonds or oxidation or reduction reactions. In particular, the spacer chain does not contain a disulfide bond or thiol groups. The spacer chain essentially contains carbon-carbon, carbon-oxygen and carbon-nitrogen bonds. The spacer chain is preferably chosen from the group consisting of: a hydrophobic chain consisting of a chain composed of 3, 6, 12 or more (for example 18) methylene (CH 2) named hereinafter C3, C6, C12 or a hydrophilic chain which may be of polyethylene glycol type, for example Hexaethylene glycol (HEG) or. an 11-amino-3,6,9-trioxa-wadecan-1-yl referred to hereinafter as hydrophilic C 1 1 or a nonspecific oligonucleotide substituted by a primary amine function The spacer chain may be introduced according to methods well known to humans of the art, particularly as a final step in the chemical synthesis of the polynucleotide. In this particular case, the spacer chain can be introduced at the 5 'end of the polynucleotide using a derivative comprising a phosphoramidite function as described in the examples. The general principle of this reaction is shown in Figure 2 of Greco and Tor, Nature Protocols, 2007, 2, 305-316 entitled "Key steps in solid DNA phospharimidite synthesis cycle". It is also possible to introduce a molecule containing a primary amine at the 5 'position of the polynucleotide by coupling a diamine such as ethylene diamine in the presence of EDC and imidazole (see Thermo Technical Data Sheet No.TR0030.5). Scientific). Reference can be made to Hermanson's reference work (Bioconjugate Techniques, 2008, 2nd Edition, Academic Press, San Diego) and in particular Chapter 27, p.970. As illustrated in the examples of the present application, the coupling step can be performed indifferently at low temperature and at room temperature. Remarkably, the implementation of the reaction at room temperature does not induce a decrease in the yield of the reaction. In the same way, the implementation of the reaction at low temperature - typically at a temperature of 5 ° C - does not induce a substantial decrease in the reaction rate. Thus, in certain reaction modes, the coupling step is carried out at a temperature in a range from 0 ° C to 50 ° C. Preferably, the coupling step may be carried out at a temperature ranging from 0 ° C to 35 ° C. Conveniently, the coupling reaction may be carried out at room temperature, i.e. at a temperature of from 15 ° C to 35 ° C, preferably at a temperature of from 15 ° C to 25 ° C. . Nevertheless, the coupling step may be performed at low temperature; typically at a temperature ranging from 0 ° C to 8 ° C, if the reagents involved - in particular the nucleic ligands - have chemical groups sensitive, in particular, to hydrolysis. Since the coupling reaction is particularly rapid, satisfactory progress in the reaction is generally obtained after about an hour or even after a few minutes. By using appropriate kinetic monitoring techniques, one skilled in the art will be able to determine the optimal duration of the reaction. It is the same for the reaction temperature. In general, as illustrated in the examples, the coupling step can be carried out at a pH of from 3.5 to 4.5 at room temperature and for a period of about one hour. Of course; Those skilled in the art may, on the basis of the above general conditions, adapt the conditions of the coupling reaction in order to determine the optimum conditions adapted in each specific case, on the basis of his general knowledge of chemistry. As an illustration, one skilled in the art can easily predict that, in order to obtain a given coupling efficiency, the lowering of the reaction temperature is likely to require an increase in the duration of the coupling step. In certain embodiments of the immobilization method according to the invention, the coupling reaction can be completed by placing the pre-activated carrier / nucleic acid combination under conditions of alkaline pH for a given duration. In these embodiments, the coupling step of the process of the invention itself comprises the following two steps: cl) reacting said nucleic acid with the activated carboxylic acid groups present on the surface of said solid support, under conditions pH of less than 5 and c2) continue the coupling reaction of said nucleic acid with the activated carboxylic acid groups present on the surface of said solid support, under conditions of pH greater than 7.5 Without wishing to be bound by any theory, the Applicant believes that the implementation of substep c2) may, in certain specific cases, induce the nucleic acids immobilized on the support to adopt a suitable conformation. The plaintiff is of the opinion that this step is optional. Advantageously, the final phase of coupling at alkaline pH is carried out at. a pH of at least 7.5, which includes pHs of at least 8, and at least 8.5. The examples illustrate the realization of step e2) at a pH of about 9. Step c2)) can be carried out at room temperature or at a low temperature. By low temperature for the final stage of the coupling reaction is meant a temperature below 15 ° C, including a temperature below 14 ° C, 13 ° C, 12 ° C, 11 ° C, 10 ° C, 9 ° C, 8 ° C, 7 ° C, 6 ° C or 5 ° C. The duration of the final phase of the coupling step is variable. It is between a few minutes and a few hours. In general, the duration of step c2) is less than 9 hours, which includes a duration of less than 8, 7, 6, 5, 4, 3, 2 and 1 hours. The duration of step c2) may be about 8 hours or about 3 hours, as illustrated in the examples. For said final phase of the coupling step, those skilled in the art can easily, on the basis of the above indications, determine the optimum conditions for the combination of pH, temperature and time, on a case-by-case basis. In advantageous embodiments, the coupling reaction is followed by a step of neutralizing d) or blocking the unreacted activated carboxylic acid groups during the coupling step itself. By way of illustration, the blocking of the activated and unreacted carboxylic acid functions can be carried out by incubating the grafted support with a blocking solution comprising 0.5 M ethanolamine and 0.5 M NaCl at pH 8.3. or with a 0.1M TrisHCI blocking solution at pH 8.5, as recommended in particular by the manufacturer and described elsewhere in the examples. The duration of the neutralization or blocking step is advantageously at least one hour at low temperature. It may be carried out for example for a period of 2 hours 30 minutes at a temperature of 4 ° C., as described in the examples. Finally, the method according to the invention comprises at the end of the coupling step c) and / or at the end of the blocking or neutralization step d) one or more washing steps e) of said support under conventional conditions so as to obtain a ready-to-use affinity support. By way of illustration, the washing step (s) may be carried out with a 0.1M Tris-HCl buffer solution at a pH up to. from 8 to 9, or with a buffer solution of 0.1M acetate, 0.5M NaCl at a pH of from 4 to 5, as illustrated in the examples. In some embodiments, a washing step comprises successively (i) washing with a 0.1M Tris-HCl buffer solution at a pH of from 8 to 9, followed by (ii) washing with a buffer solution of 0.1M acetate, 0.5M NaCl at a pH of from 4 to 5. Conventionally, a plurality of washing steps are carried out, for example 3 washing steps, as illustrated in the examples.

In view of the foregoing description, the method of immobilizing nucleic acids according to the invention may also be defined as a process comprising the following steps: a) providing a solid support comprising on its surface activated carboxylic acid groups, preferably by N-hydroxysuccinimide, b) providing a nucleic acid comprising at least one primary amine function, c) coupling said nucleic acid with activated carboxylic acid groups present on the surface of said solid support under conditions of pH less than 5, and d) block the coupling reaction. The method for immobilizing nucleic acids according to the invention can also be defined as a process comprising the following steps: a) providing a solid support comprising, on its surface, activated carboxylic acid groups, preferably N-hydroxysuccinimide, b ) providing a nucleic acid comprising at least one primary amine function; c) coupling said nucleic acid with activated carboxylic acid groups present on the surface of said solid support under conditions of pH less than 5; d) blocking the reaction of. coupling, and e) perform one or more steps of washing the support. As already mentioned above, in some embodiments, step c) itself comprises the following two steps: c) reacting said nucleic acid with the activated carboxylic acid groups, preferably with N- hydroxysuccinirnide, present on the surface of said solid support under pH conditions of less than 5, and c2) continue the coupling reaction of said nucleic acid with the activated carboxylic acid groups present on the surface of said solid support under conditions of pH higher than 7.5 The immobilization method of the nucleic ligands according to the invention finds a direct application for the manufacture of affinity supports intended for the purification or the detection of target molecules, including target proteins. Thus, more generally, the present invention relates to a method for preparing an affinity support comprising the implementation of the method for immobilizing nucleic ligands as defined above. Thus, the method of preparing an affinity support according to the invention comprises the following steps: a) providing a solid support comprising on its surface activated carboxylic acid groups, b) providing a nucleic ligand comprising at least one function primary amine, and c) coupling said nucleic ligand with the activated carboxylic acid groups present on the surface of said solid support under pH conditions of less than 5, it being understood that the order of steps a) and b) is indifferent. Step c) of the method may comprise steps c1) and c2) as defined above. In the same way, the method may further comprise steps d) and e) previously described. As illustrated in the examples, the method and method according to the invention are particularly suitable for the preparation of an affinity support for the purification of one or more target molecules, in particular by. chromatography. Thus, in certain embodiments of the process according to the invention, the solid support is a support adapted to the implementation of a chromatography, filtration or solid phase extraction process. In other words, the solid support is suitable for use as a stationary phase in a chromatography, or filtration or solid phase extraction process. For the implementation of a solid affinity support for solid phase extraction, reference may be made to Madru et al., (Analytical Chemistry, 2009, 81, 7081-7086). Such a solid support may be selected from the group consisting of silica gels and polysaccharide gels such as agarose gels, dextran gels and their derivatives and also acrylamide gels and their derivatives, methacrylate salts and the like. their derivatives and polystyrene surfaces and their derivatives. Thus, in a particular embodiment of the method or method of immobilizing the invention, (i) the solid support comprising on its surface activated carboxylic acid groups is a support selected from the group consisting of silica gels, agarose gels, dextran gels and their derivatives and (ii) the nucleic acid ligand comprising at least one reactive amine is chosen from the group of polynucleotides of 5 to 120 amino acids optionally comprising at their 3 'or 5' end a spacer chain which may be chosen from the group consisting of a hydrophobic chain consisting of a chain composed of 3, 6, 12 or more (for example 18) methylene (CH 2) named hereinafter C3, C6, C12 or a hydrophilic chain which may be of polyethylene glycol type, for example Hexaethylene glycol (HEG) or an 11-amino-3,6,9-trioxau.ndecan-1) 71 hereinafter referred to as hydrophilic C11 or a non-specific oligonucleotide In another In a particular embodiment of the method or method according to the invention, (i) the solid support is selected from agarose gels and their derivatives, and (ii) the nucleic ligand is a 5 to 20 polynucleotide. 120 nucleotides linked at its 5 'end to a spacer chain selected from polyethylene glycol C4-C20, the reactive amine function being preferably carried by the spacer chain. In a further embodiment of the method or method according to the invention, (i) the solid support is selected from agarose gels and their derivatives and (ii) the nucleic ligand is a polynucleotide of 5 to 120 nucleotides bound at its 5 'end to a spacer chain chosen from linear C4-C20 alkyls, the reactive amine function preferably being carried by the spacer chain. The examples show that the affinity supports obtained according to the process of the invention allow the quantitative purification of a target ligand in an extremely selective manner. Also, the results of the examples show that an affinity support obtained according to the process of the invention can be used over a very long period of time, in particular according to a large number of capture / wash / wash cycles without significant loss of its properties. selective and quantitative retention of the target ligand. It is also shown that an affinity support according to the invention retains its selective and quantitative retention properties of the target ligand, even after undergoing the drastic stages of regeneration or antibacterial, anti-fungal or anti-viral sanitization. In particular, it has been shown in the examples that the retention capacity of an affinity support according to the invention for its target protein is not altered by treatment with a solution having a final concentration of NaOH of 0.5. It has also been shown that the chromatographic properties of an affinity support according to the invention are not impaired by treatment with a solution having a final propylene glycol concentration of 50% (vol / vol). In other words, the results of the examples illustrate the ability of the affinity support according to the invention to achieve perfectly reproducible quantitative purification steps of a target ligand, and this under conditions of use. Conventional industrial processes, which are generally deleterious for known affinity carriers, including for immunoaffinity carriers. The technical characteristics of each of the steps of the process of the invention, according to the various definitions above, have already been specified previously in the present description. The examples describe obtaining an affinity support of the invention by grafting nucleic aptamers, and illustratively by grafting DNA aptamers. Thus, in a preferred embodiment of the method or method according to the present invention, the nucleic ligand is a nucleic aptamer. By "nucleic aptamer" or aptamer is meant according to the invention a single-stranded nucleic acid specifically binding to one or more target ligands. Aptamers include those for which complexes can be detected with a single target ligand or with a variety of given target ligands, after a prior step of contacting the respective nucleic and target ligand partners. Aptamers include RN aptamers and DNA aptamers. The term "aptamer" as used herein refers to a single-stranded nucleic acid molecule, DNA or RNA, capable of specifically binding to one or more target ligands, such as a protein. Aptamers bind to their target molecules by essentially distinct mechanisms of rhybridization. Aptamers are generally characterized by a secondary structure including loops and stems. In other words, the active conformation of the aptamers (i.e. the conformation in which the aptamers are able to bind to their target protein) is non-linear. Aptamers generally comprise between 5 and 120 nucleotides and can be selected in vitro by a process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Aptamers have many advantages. Due to their oligonucleotide nature, aptamers possess low immunogenicity and resistance to stringent physicochemical conditions (presence of urea, DMSO, very acidic or very basic pH, use of organic solvents or temperature high) allowing various sanitization strategies in the setting. of use as an affinity ligand. In addition, their selectivity is important. Finally, the production of aptamers involves relatively limited costs. In certain embodiments, a "nucleic aptamer" used for grafting to the pre-activated solid support, according to the method or method defined above, may comprise, by definition, a non-nucleotide or nucleotide portion, for example a non-nucleotide spacer chain, which connects one of the 5 'or 3' ends of the nucleic portion of said aptamer and the reactive amine function used for the chemical grafting of said pre-activated support. In these embodiments, a nucleic aptamer may be of the following formula (I): ## STR2 ## does not include a free spacenr chain and I means that the aptamer comprises a spacer chain. NH 2 means the reactive amine function used for grafting on the pre-activated solid support with NHS groups, [SPAC] means a spacer chain, and [NUCL1 means a nucleic acid binding specifically to a target molecule, said nucleic acid comprising from 5 to 1 nucleotides.

Preferably, the nucleic acid [NUCL] comprises from 10 to 80 nucleotides and even more preferably from 20 to 60 nucleotides. The "spacer chain" designated [SPAC] in the compound of formula (I) may be of any known type. It may be a non-nucleotide compound, an oligonucleotide or a compound comprising one or more non-nucleotide moieties and one or more nucleotide moieties. The spacer chain does not participate, in general, in the binding of the target ligand on the support. Said spacer chain has the function of physically moving the nucleic acid [NUCL] away from the surface of the solid support on which said compound of formula (I) is chemically grafted, which allows a relative mobility of the nucleic acid [NUCL], relative to the surface of said solid support. The spacer chain limits or avoids that steric hindrances, due to an excessive proximity of the solid support to the nucleic portion of the aptamer, interfere with the binding events between said nucleic acid and molecules of the target ligand that may be contact with this. In the compound of formula (II), the spacer chain is preferentially bonded to the 5 'end or the 3' end of the nucleic acid [NUCL]. This spacer construction has the advantage of not immobilizing directly the aptamer on solid support. Preferably, as indicated above, the spacer chain may be a hydrophobic chain consisting of a chain composed of 3, 6, 12 or more (for example 18) methylene (CH 2) named hereinafter C3, C6, C12 or a hydrophilic chain which may be of polyethylene glYcol type, for example Hexaethylene glycol (HEG) or an 11-amino-3,6,9-trioxaundecan-1yl called hydrophilic C11 or a non-specific oligonucleotide. When the spacer chain consists of a nonspecific oligonucleotide, said oligonucleotide advantageously comprises at least 5 nucleotides in length, preferably between 5 and 15 nucleotides in length. In some embodiments, the spacer chain may be composite and include the sequence of HEG and an oligonucleotide, for example an oligo (dT). It will be recalled that the examples illustrate the production of affinity supports as defined above by grafting of the NHS pre-activated support with three distinct anti-FVII nucleic aptamers comprising spacer chains of a similarly distinct nature. that is, a hydrophobic chain consisting of a C6 alkyl chain, a hydrophobic chain consisting of a C12 alkyl chain, and a C11-TFA hydrophilic chain. Thus, the results of the examples show that the process for obtaining a carrier of The affinity described in this specification can be used regardless of the identity or type of nucleic acid of interest to be grafted.

As has been mentioned above, the methods and method according to the invention allow the preparation of affinity supports which are distinguished from the known affinity supports by their high density of nucleic ligands immobilized on their surface. This high density of nucleic ligands stems directly from the specific method of obtaining the affinity supports.

Remarkably, the affinity supports according to the invention are also characterized by a high retention capacity of the target molecule or molecules against which the grafted nucleic acids are directed. In particular, the process according to the invention makes it possible to prepare affinity chromatography gels having a high density of nucleic ligands, it has thus been observed that chromatographic gels obtained by the process according to the invention have a density of ligands. Nucleic acids are 0.2 μmol / ml, while the initial density in activated carboxylic acid functions of the commercially available chromatography gels is generally between 5 pmol / ml gel unioliml. Thus, according to the coupling method of the invention, it is possible to derivatize according to the process of the invention at least 0.01% of the activated carboxylic functions initially present on the surface of the solid support, advantageously at least 0.1% at least 1% and more preferably at least 2% of said activated carboxylic functions. By way of comparison, the grafting levels in affinity chromatography with the antibodies as ligand are often less than 1 mg / ml, that is to say less than 0.06 p.mol / ml, ie less than 0.2% of derivatized carboxylic functions. Derivatization of at least 0.01% of the activated carboxylic functions includes derivatization of at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% , 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1 , 6%, 1.7%, 1.8%, 1.9% and at least 2% of said activated carboxylic functions.

In some embodiments, the percentage of derivatized carboxylic functions is at most 100%, which encompasses at most 50%, 40%, 30%, and at most 25% of said activated carboxylic functions.

Thus, another object of the present invention is an affinity support obtainable according to the method and method described hereinbefore. More generally, the present invention relates to a solid affinity support on which nucleic ligands are immobilized by an amide linkage and wherein at least carboxyl functions initially present on its surface are derivatized by a nucleic ligand. It goes without saying that the amide linkage linking a nucleic ligand to the support results from the reaction of a carboxyl function initially present on the surface of the support with a primary amine function present at the level of the nucleic ligand. In a preferred embodiment, the solid affinity support is a chromatography gel. Another object of the present invention is a solid affinity support on which nucleic ligands are immobilized by an amide linkage, said affinity support being a chromatography gel having a nucleic ligand density of at least 0.005 pinol. of gel, which includes at least 0.01 μmol / ml, 0.05 μmol / l, 0.1 μmol / l, 0.15 lmolol, 0.2 μmol / ml, 0.25 lnol / ml, , 34 mol / ml, 0.35 μmol and at least 0.38 mnol / ml gel. In some embodiments, the density of nucleic ligands is at most 10 .mu.m / ml, which encompasses at most 5 pmol / ml, at most 11mM / rat and at most 0.5. Affinity carriers according to the invention may have any of the properties described above for solid supports or affinity supports.

Thus, in certain embodiments, the affinity support may be a gel that can be used in chromatography, filtration or solid phase extraction selected from the group consisting of agarose, dextran, silica gels and their salts. derivatives. As illustrated in the examples, the affinity support may be a highly crosslinked agarose gel on which the nucleic ligands are immobilized. Preferably, the affinity support is a gel (that is, a stationary phase) for use in affinity chromatography methods. The affinity supports according to the invention may comprise on their surface any type of nucleic ligands as described hereinabove in the present description.

In certain embodiments, the affinity support according to the invention is characterized in that the nucleic ligands are aptamers of formula (II) presented above. In particular embodiments, the solid affinity support according to the invention can be represented by the following formula (III): (SP!% C) n in which: [SUP] represents the solid support of the affinity support [SPAC] n and [NUCL] are as previously defined. In other words, -NFI- (SPAC) .- NUCL represents a nucleic aptamer in which: n is an index of 0 or 1, SPAC represents a spacer chain, and NUCL represents a nucleic acid binding specifically to . a target molecule, said nucleic acid comprising from 5 to 120 nucleotides. Another object according to the invention is a complex formed between a nucleic ligand and a target molecule, said complex being formed on the surface of a solid support as defined above. Said complex results essentially from no-covalent interactions between the target molecule and the nucleic ligand. A further object of the invention relates to the use of an affinity support as described above for the purification or detection of a target molecule. In the context of the purification of a target molecule, the support according to the invention can be used as a stationary phase in filtration, chromatography or solid phase extraction steps. The present invention also relates to a process for purifying a target molecule with an affinity support as defined above, comprising the following steps: a) contacting a composition to be purified comprising a target molecule of interest with an affinity support as defined in the present description, to form a complex between (i) the nucleic acids grafted on said support and (ii) said target ligand and b) release said target molecule from the complex formed in step a) and recovering said purified target molecule.

In a particular embodiment, the method comprises a step a '), step a' following step a) and preceding step b), which consists of a step of washing the affinity support with a buffer washing. It has also been shown in the examples that the use in step a ') of washing buffer having a high hydrophobicity, in particular a high concentration of propylene glycol, makes it possible to effectively eliminate the non-specifically bound substances. affinity support without simultaneously detectably affecting the binding of the target ligand to the affinity support. In step a '), a wash buffer having a final propylene glycol content of at least 20% by volume, based on the total volume of the buffer solution, is preferably used. According to the invention, a wash buffer having a final propylene glycol content of at least 20% includes wash buffers having a final propylene glycol content of at least 25% M, 30%, 35%, 40% , 45%, 50% ,. 55%, or at least 60% by volume, based on the total volume of the buffer solution. Preferably a wash buffer used in step a ') of the process has a final propylene glycol content of at most 50%. Advantageously, a washing buffer used in step a ') of the process has a final content of propylene glycol of between 20% and 50%, preferably between 30% and 50%. According to a particular embodiment, the washing buffer used in step 25 contains both NaCl and propylene glycol as described in the examples. In addition, in some embodiments of the above purification method, step b) is performed by contacting the affinity support with an elution buffer containing a divalent ion chelating agent, preferably EDTA. . As an illustration, the elution buffer may contain a final EDTA concentration of at least 1 mIV1 and at most 30 mM. "At least 1 mM" includes at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 inNI, "not more than 30 mM" includes not more than 29, 28, 27, 26 , 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 11 nuM. 17, 16, 15, 14, 13, 12 Advantageously, for the regeneration of the affinity support, a buffer comprising a mixture of. NaCl and propylene glycol, which may be of the same type as that described above for the washing step. Within the meaning of the present invention and for the various objects described above included in the invention, the term "target molecule" (or "target substance" or "target ligand") as used herein refers to a molecule capable of to link specifically to the aptamer. It can be nucleic acids, proteins or 1.0 organic or mineral substances. Proteins can be any type of protein, and in particular, plasma proteins. By "plasma protein" is meant according to the invention any protein, especially any protein of industrial or therapeutic interest, contained in the blood plasma. Blood plasma proteins include antibodies, albumin, alpha / macroglabulin, antichymotrypsin, antithrombin, antitrypsin, Apo A, Apo B, Apo C, Apo D, Apo E, Apo F. Apo G, beta Xlla, C 1 inhibitor, C-reactive protein, C7, Clr, Cs, C2C3, C4, C4bP, C5, C6, Clq, C8, C9, carboxypeptidase N, ceruloplasmin, Factor B, Factor D, Factor H, Factor I, Factor IX, Factor V, Factor VII, Factor Vila, Factor VIII, Factor X, Factor XI, Factor XII, Factor XIII, Fibrinogen, Fibronectin, Haptoglobin, Hemopexin, Heparin Cofactor II, Histidine Rich GP, IgA, IgD, IgE, IgG, ITI, IgM, Kininase II, Kininogen 1EPM, Lysozyme, PAI 2, PAI I, PCI, Plasmin, Plasmin Inhibitor, Plasminogen, Prealbumin, Prokallikrein, Properdine, Nexin INH Protein, Protein C, Protein S, Z Protein, Prothrombin, TFPI, Thiol Proteinase, Thrombomodulin, Tissue Factor (TF), TPA, Transcolabamine II, Transcortin, Transferrin, Vitronectin, and Factor from Willebrand. In particular, plasma proteins include coagulation proteins, that is, plasma proteins involved in the chain of cascade reactions resulting in the formation of a blood clot. Coagulation proteins include Factor I (fibrinogen), Factor II (prothrombin), Factor V (pro-accelerin), Factor VII (proconvertin), Factor VIII (antihemophilic factor A), Factor IX (anti-hemophilic factor B), Factor X (Stuart Factor), Factor XI (Rosenthal Factor or PTA), Factor XII (Hageman Factor), Factor XIII (Fibrin Stabilizing Factor or FSF), there PK (Prekalicrin ) KHPM (high molecular weight kininogen), tissue thromboplastin, heparin cofactor II (HCII), protein C (PC), thrombomodulin (TM), protein S (PS), von Willebrand factor (Wf) and tissue factor pathway inhibitor (TFPI), or tissue factors. In some embodiments, the plasma protein is an enzymatically active coagulation protein. Enzymatic coagulation proteins include Factor II (prothrombin), Factor VII (proconvertin), Factor IX (antihemophilic factor B), Factor X (Stuart Factor), Factor XI (Rosenthal Factor or PTA), Factor XII (Hageman Factor), Factor XIII. (Fibrin stabilizing factor or FSF) and PK (Prekalicrin). In certain preferred embodiments, the plasma protein consists of a natural or recombinant human plasma protein. In preferred embodiments, the plasma protein is human, natural or recombinant factor VII. The present invention is further illustrated, without being limited, by the following examples. EXAMPLES Example I Obtaining an affinity support Grafting buffer: 100mM sodium acetate, pH 4.2 - Preparation of 1595 g of aptamer 2.5 in grafting buffer or 4 mg aptarnère. For the grafting, the following has been used: a) for producing a first affinity support, an aptamer comprising the Mapt 2CS polynucleotide of sequence SEQ ID No. 1 comprising at its 5 'end a C 1 1 chain hydrophilic (11-amino-3,6,9-trioxaundecan-ly1) b) for the production of a second affinity support, an aptamer comprising the "Mapt 1.2" polynucleotide of sequence SEQ ID No. 2 linked to its end 5 'to a spacer chain composed of 12 methylenes (CH2) (C12 spacer) and linked at its 3' end to an oligo-dT c) for the production of a third affinity support, an aptamer comprising the "Mapt" polynucleotide 2 CS "of sequence SEQ ID No. 1 linked at its 5 'end to a spacer chain composed of 6 methylenes (CH2) (C6 spacer) Preparation of gel lnal comprising NHS-activated carboxylic acid groups, namely the pre-activated gel "NHS Activated Sepharose 4 fast flow (GE)" in real washing with 1 mM HCl and then washing with the grafting buffer. It was verified that the pH in the aptamer preparation in the buffer solution was 4.2. The aptamer preparation was mixed with 1 μl of pre-activated gel. The pre-activated gel was incubated in the presence of the stirring chambers for 48 h (+/- 2H) at 4 ° C. - A reaction volume (7970 of 200 mM borate buffer pH = 9 was added with stirring, then incubated for 8 h with stirring at 4 ° C. 1.5 - The supernatant was recovered and the amount of non-grafted aptamers was assayed - 2 ml of 0.1M Tris-HCl pH 8.5 was added with stirring for 2.5 hours at 4 ° C. to block the coupling reaction - 3 cycles of addition / stirring / elimination of the supernatant comprising: 1) He Tris-HCl 0.1M pH --- 8.5 then 2) 1ml of sodium acetate 0.1M 0.5M NaCl, pH 4.0, to obtain ready-to-use affinity support. For all practical purposes, it is indicated that the "NIIS Activated Sepharose 4 Fast Flow (GE)" gel is a crosslinked agarose gel having, on its surface, carboxylic acid functional groups activated by NHS. The carboxylic acid functions were introduced on the surface of the gel by grafting 6-aminohexanoic acid. This pre-activated agarose gel is described in Technical Instruction Manual No. 71-5000-14 AD dated March 2011 and published by GE Healthcare. The "NHS activated Sepharose 4 Fast Flow" gel exhibits a density of activated carboxylic acid functions ranging from 16 to 23 gmaliml of gel. The results show that, at the end of the coupling reaction, the supernatant of the reaction medium comprises no detectable amount of nucleic aptamer, that is to say, under the analysis conditions used, comprises an amount of The aptamer is less than 0.08 mg / ml. It can be deduced from these results that the grafting yield is 100%, or very close to 100%. e 2 Use of 6-n human factor affinity derived in the milk of transgenic rabbits. Gel: Iml grafted with Mapt 2CS-PEG (C11) prepared as described in Example 1, at 4 mg / ml packed in an XK-16 (GE) column. Injection of a purified recombinant human FVII factor composition, produced in the transgenic rabbit milk (FVH-TG): 100 or 200 or 10001.14 FVII-TG The FVII-TG composition used for the injection is prepared by neutralizing the citrate initially contained in the formulation with CaC12 and modifying the formulation buffer to obtain: between 35 and 40 mM NaCl and between 3.2 and 4 mM MgCl 2 - buffer. used for chromatography: Tris 50n-iM 50mM NaCl / 10mM CaCl2 / MgCl2 4mM - Flow: 0.5m1 / min - 50mM Tris Elution Buffer, 10mM EDTA, pH - 7.4 - Wash Buffer or Regeneration: -NaCl 1M / Propylene glycol 50% - Sanitization solution used between each test: 0.1 or 0.5 M NaOH (1m1) 1M NaCl / 50% Propylene Glycol - (. 2x 1ml) The results are shown in Figures 1-5. Figures 1 and 2 show that almost all human factor VII is retained on the affinity support at the time of passage of the composition to be purified, regardless of the amount of Factor VII contained in the starting composition. It has been estimated for the two amounts of factor VII to be purified (100ptg and 200 .mu.g) that less than 10% of the Factor VII contained in the starting composition is not retained on the affinity support. Figures 1 and 2 also show a narrow elution peak, illustrating the excellent chromatographic properties of the affinity support of the invention. Figure 3 illustrates the chromatographic profile obtained with a starting composition containing 1 mg of human factor VII. The chromatographic profile of FIG. 3 is very similar to those shown in FIGS. 1 and 2, which illustrates the very high retention capacity of a target ligand of the affinity support of the invention. FIGS. 4 and 5 illustrate the chromatographic profiles obtained by purification of a quantity of human factor VII of 200 μg (FIG. 4) and 1000 μl (FIG. 5) on an affinity support prepared as described in example 1 and having underwent drastic sanitization treatment steps with a sanitizing solution comprising a mixture of 0.5 M NaOH and 50% propylene glycol. The chromatographic profiles obtained show the resistance capacity of an affinity support according to the invention to deleterious sanitization treatments.

It is specified that the same chromatographic profile is obtained when carrying out a succession of purifications of transgenic human factor VII and washing and sanitizing steps. This demonstrates the excellent stability of the affinity support, which makes it possible to purify target ligands of interest in an extremely reproducible manner.

EXAMPLE 3 Grafting Yield as a Function of the Amount of Aptamers Used and Loading Capacity of the Affinity Media Obtained The influence of the amount of aptamers on the grafting efficiency and the loading capacity of the affinity supports was studied for aptaptors Mapt 2CS- (hydrophilic C11) and Mapt 2.2CS- (hydrophilic C11). The grafting protocol used is identical to that described in Example 1, the aptaptor Mapt 2.2CS- (C11-hydrophilic) results from the coupling of Mapt 2.2CS sequence SEQ ID No. 3 and 11- (trifiluoroacetamido) - 3,6,9-Trioxaundecan-1-yl - [(2-cyanoethyl) - (N, N-diisopropyl)] phosphoramidite (Link Technologies) followed by the generation of the phosphodiester group by oxidation and elimination of the cyanoethyl group and the subsequent deprotection of the primary amine function present at the end of the spacer chain. Mapt 2.2Cs is directed against factor VII. The loading capacity (or otherwise the retention capacity) of the affinity supports, expressed in mg of FVII per ml of gel, was evaluated by injection of a recombinant human FVII composition into the "Tp5" buffer ( 50 mM Tris, 10 mM CaCl 2, pH 7.5).

Tables I and 2 below show the performance of the grafting reaction of aptaptors Mapt 2CS-PEG (C11) and Mapt 2.2CS-PEG (C11) on the pre-activated gel NHS Activated Sepharose 4 fast flow (GE) Depending on the amount of aptamers involved in the reaction per ml of gel. The yield was determined by quantifying the amount of aptamers present in the supernatant at the end of the quantitative PCR coupling reaction. The final amount of aptamers grafted per ml of gel is also indicated in Tables I and 2 below.

Table 1 Influence of the amount of Mapt 2CS aptamer per ml of gel on the efficiency of the coupling reaction and the characteristics of the affinity support obtained Amount of aptamers Efficiency of the coupling reaction Amount of aptamers Amount of aptamers . Percentage of transplant recipients in acid sites. mg / ml gel gel mg / ml gel min / mi gel carboxyl gel gel coupled to a aptamer 0.1 99.97% 0.1 0.007 0.04% 0.2. 99.96% 0.2 0.014 0.07% 0.6 99.95% 0.6 0.042 0.21% 0.8 99.94% 0.8 0.056 .0.29% 1.0 100% 1.0 0.67 0.36% 2.0 100 % 2.0 0.12 0.6% 3.5 100% 3.5. 0.23 1.2% 6.0 97% 5.8 0.39 2% Table 2 Influence of the amount of aptaptin Mapt 2.2 CS per ml of gel on the yield of the coupling reaction and the characteristics of the affinity support obtained Amount of aptamer Coupling reaction efficiency Quantity Amount of aptamers Percentage of aptamers grafted into acid sites mg / ml gel grafted in pmol / ml of carboxyl gel of mg / ml of gel gel coupled to mid aptamer 0.1 100% 0.1 0.007 0.04% 3.5 100% 3.5 0.23 1.2% 6.0 92.6% 5.55 0.37 1.9% Coupling efficiencies obtained for Mapt 2.2Cs-PEG (C11) aptamers and Mapt2Cs-PEG (C11) are between 90% and 100% for all quantities of aptamers tested. Remarkably, it is possible to graft up to 0.4 limol per ml of gel, which corresponds to a percentage of functionalization of the activated carboxylic acid functions present on the surface of the gel by 2%. The loading capacity of the affinity carriers for recombinant human FVII varies linearly with the amount of grafted aptamers per ml of gel. There is therefore no saturation effect of the load capacity of the support and therefore loss of aptamer functionality for the high amounts of grafted aptamers. In view of these results, it may be possible to obtain affinity supports having a grafted aptamer density greater than 6 mg / ml and having a FVII loading capacity greater than 8 mg of human recombinant FVII per ml. of gel. Table 3 below shows the load capacity of the affinity supports as a function of the number of aptamers grafted onto their surface. Table 3: Carrying capacity of affinity supports obtained Mapt 2CS-PEG (C11) Mapt 2.2CS-P F; G (C11) Amount of aptarners 0.1 1.00 3.50 5.80 1.00 3, 50 5,60 grafted in ing / m1 of gel Quantity of aptamers 0.007 007 0.23 039 0.07 0.23 0.37 grafted in mmoliml of gel Load capacity in human FVII expressed in mg of FVIllml of gel 0.05 1.00 3, 60 5,40 1,6 5,2 8,0: xem4e 4: eomative tests of an acid chemistry ucl- solid support pre-activated by troops MIS 4.1. Comparative tests using different pH conditions Comparative tests were carried out on the grafting of the starting pre-activated support used in Example 1, under conditions of neutral or slightly alkaline pH, as described in Table 4 presented below. Table 4 Modification (nature of the species) Amount Conditions: Assay of ampon graft aptamer Assay 1 6, Smg TOPS 100mM, CaC12 10mM, Assay 2 6.5mg iH7.0 (Condition 1) 10PS 100mM, CaC12 10mM 0.5M NaCl, pH7.0 Condition 2) Mapt2 CS Oligo 1 Oligo 1 Oligo 1 5 'amine spacer C6 Assay)) 6, Smg Modification Mapt2 Amount Conditions: (Cs test type ampon aptamère grafting P espaceur) aHCO3 0 , 2M, 0.5M NaCl, pH 8.3 5 'amine Test 4 3.5mg Oligo 4 spacer C12 Condition 3) afIC03 0.2M, 2M NaC1, pH 8. 3'dT Test 3.5mg Condition 4) amine Oligo 5 CI spacer 1 Assay 5 6.5mg OPS 100mM, CaCl2 10m1g, hydrophilic aC1 0.5M pH7.0 (Condition 2) The following protocol was used: iml of gel rinsed with water. c 15m1HCIlmM then 7m1 of grafting buffer. Addition of 6.5 ml (or 3.5 ml) of Mapt2CS at 1 g / l and incubation of the gel with Mapt2CS for 4 hours at room temperature Neutralization of activated sites with a 50 mM Tris buffer pH 7.4 The amount of non-grafted aptamers at the column outlet after the grafting step. The results show that the graft yield ranges from 0% to 10% maximum, regardless of the grafting conditions described in Table 1 that have been used. Contrary to what Goss et al. (supra), high salinity is not sufficient to increase the coupling efficiency. 4.2. Comparative tests by neutralizing the negative charges of the nucleic acids to be grafted by the addition of divalent cations or by addition of NaCl.

The following protocol was used: 1000 rinsed gel with 15m! 1mM HC1 then 700111 of grafting buffer / addition of 650 μl of Mapt2CS (oligo 5) at 130 mg / l and incubation of the gel with Mapt2CS for 4 hours at room temperature - Conditions used: Condition I: Buffer MOPS 92 mM pH 7 200mM CaCl2, MgCl2 20OrnM '^ Condition: 2: MOPS Buffer 92m-vi = 7.0, Ca C12 0 1, Mg 200mM - Condition 3: 0.2M NaHCO 3%, 0.5M NaCl, pH 8.3 - Condition 4: 0.2M NaHCO 3, 2M NaCl, pH 8.3 The amount of non-grafted aptamer was measured at the column outlet after the grafting step. The results show that the grafting yield, whatever the grafting conditions described above, varies from 0% to 10% at most. It results from all the above experiments presented that the implementation of the coupling step in the presence of a basic or neutral pH as recommended in the state of the art or in the instructions for use of pre-activated gels provides an aptamer coupling efficiency on the very low pre-activated gel that is to say less than 10% The increase in ionic strength by using a solution of NaCl a concentration up to at 2M or the use of divalent cation salts likely to mask the negative charges of the aptamers do not make it possible to increase the coupling efficiency. Contrary to what the skilled person would have expected, the implementation of the coupling step in the presence of an acidic pH makes it possible to significantly increase the coupling of the aptamers on the pre-activated support without adversely affect the binding ability of said aptamers to their target protein. EXAMPLE 5 Modulation of implementation parameters for implementation of the cost reaction according to the invention The influence of pH, temperature and reaction time was evaluated in order to determine the parameters controlling the reaction. coupling reaction of the activated carboxylic acid functions of the gel ("NHS-Activate Sepharose 4 fast Flow") with the aliphatic primary amine functions present on the spacer chains of Mapt 2CS-PEG (C11) aptamer. The following protocol was followed: - The grafting buffer was prepared using 100 mM sodium acetate with adjustment to the desired pH except for the implementation of the reaction at pH 8.3 where a buffer Nal-ICC / 3 was prepared. A solution of Mapt 2CS-PEG aptamers (Cl 1) at a concentration of 2 g / L in the grafting buffer was prepared. -1 ml of the aptamer solution was mixed with 1 ml of gel and incubated at the desired temperature, with stirring. The progress of the coupling reaction is followed by quantification of the aptamers present in the supernatant. At the end of the kinetic monitoring of the reaction, the reaction medium was added with 1 ml of 200 mM borate buffer at pH 9 with stirring and then incubated for 3 h with stirring at 4 ° C. The supernatant was removed for final determination of the supernatant. The neutralization of the residual carboxylic acid functions of the gel was carried out by adding 2 mL of 0.1 M Tris-HCl buffer and pH 8.5 while stirring for 2 h 30 at 4 ° C. 3 cycles of addition / stirring / removal of the supernatant, comprising: 1) 0.1M Tris-HCl 0.1M pH = 8.5 and then 2) 1ml of 0.1M sodium acetate 0.5M NaCl, pH - - 4.0 were made. - The supernatant has been removed. The gel thus obtained is stored in Tpl buffer supplemented with 0.2% azide for storage at 4 ° C. The results of the kinetic monitoring of the coupling reaction as a function of temperature, reaction time and pH are shown in Table 5 below: Table 5: Influence of the reaction temperature (RT: room temperature) , pH and reaction time pH T ° C Duration Reaction point (/ 0) Influence of 3.8 5 ° C 48 h 99.8 PH 4.3 5 ° C 48 hr 98.4 4.8 5 ° C 48 h 75.9 5.6 ° C 48 h 64.9 8.3 5 ° C 48 h 10 Influence of temperature and 4.3 5 ° C 24h 99.5 4.3 5 ° C 2h 100 pH PC Duration Reaction point (%) of the reaction time 4.3 5 ° C 4h 1.00 4.3 5 ° C 6h 100 4.3 5 ° C 8h 100 4.3 5 ° C 24h 100 4.3 5 ° C 48 h 97.8 4.3 TA * lh 100 4.3 TA * 2h 100 4.3 TA * 3h 100 4.3 TA * 3h 99.8 As shown in Table 5 above, the pH is a crucial parameter for the implementation of the coupling reaction according to the invention. The implementation of the reaction according to the conditions described in the state of the art, that is to say at a basic pH of 8.3, offers a very low coupling efficiency of at most 10%. The decrease in pH makes it possible to significantly improve the yield of the reaction. In particular, a yield of at least 98% is observed for a pH of about 3.8 to 4.3. Such a result is quite surprising: one skilled in the art would expect a low coupling efficiency at these pHs due to the degradation of the nucleic acids by acid hydrolysis and the decreased reactivity of the nucleic acids. primary amines. In view of these experimental results, it appears that the coupling reaction can be conducted indifferently at low temperature or at room temperature. Remarkably, the affinity supports obtained at pH 3.8 or at room temperature at pH 4.3 exhibit a loading capacity (i.e., a retention capacity) of human recombinant Factor VII equivalent to supports obtained at pH = 4.2 and at 5 ° C. In general, the load capacities obtained are particularly high, which indicates not only a high level of aptamer grafting but also the ability of aptamers to bind specifically to their target protein. In other words, the use at ambient temperature and at a pH below 4.5 of the coupling reaction between the activated carboxylic acid functions of the support and the primary amine functions present at the level of the aptamer spacer. allows not only to obtain a grafting yield close to 100% but also a maintenance of the functional integrity of the aptamers.

Example: Additional Embodiments of Affinity Error Other affinity media were prepared by varying (i) the grafting conditions as well as (ii) the types of aptamers used. 6.1. Types of aptamers grafted lb. Concerning aptamer types, aptamers comprising a polynucleotide DNA and aptamers comprising an RNA polynucleotide were used respectively. In addition, for certain aptamers, the polynucleotide is linked to a spacer chain by its end. whereas for the other aptamers, the polynucleotide is linked to a spacer chain by its 3 'end. Also, aptamers comprising a hydrophilic spacer chain or a hydrophobic spacer chain have been used. The aptamers used in Example 6 are the following: the aptamer comprising the "Mapt 2 CS" DNA polynucleotide of sequence SEQ ID No. 1 comprising at its 5 'end the hydrophilic C11 spacer chain described for the preparation of the first the affinity disclosed in paragraph "a)" of Example 1, said aptamer being designated "Mapt2CS oligo5 (5'Amin C11 hydrophilic)" in Table 6; the aptamer comprising the "Mapt 2 CS" DNA polynucleotide of sequence SEQ ID 1 comprising at its 5 'end the hydrophobic C6 spacer chain described for the preparation of the third affinity support disclosed in paragraph "c" of Example 1 and Comprising at its 3 'end an inverted deoxyribothymidine residue (3'-dT-5'), said aptamer being designated "Mapt2CS oligo2 (5'Amin C6 and 3 'dT) in Table 6; the aptamer comprising the "Mapt2 CS" DNA polynucleotide of sequence SEQ ID No. comprising at its 5 'end the hydrophobic C12 spacer chain described for the preparation of the second affinity support disclosed in paragraph "b" of example 1, said aptamer being designated "Mapt2CS oligo3 (5 'Amine C12)" in Table 6, the aptamer comprising the "Mapt2 CS" DNA polynucleotide of sequence SEQ ID No. I comprising at its 3' end the hydrophobic C6 spacer chain described for the preparation of the third affinity support discloses in paragraph "c" of Example I, said aptamer being designated "Mapt2CS oligo7 (3 'Amine C6)" in Table 6; the aptamer comprising the RNA polynucleotide "Mapt RNA Anti FIXa" of sequence SEQ ID No. 4 comprising at its 5 'end the hydrophilic spacer chain C1 1 described for the preparation of the first affinity support disclosed in paragraph "a)" of Example 1 and comprising at its 3 'end an inverted deoxyribothymidine residue (31-dT-5'), said aptamer being designated as "Anti FIXa RNA Mapta." 5 "Hydrophilic Amine C11" in Table 6. Also prepared was an affinity support on which has been grafted an aptamer comprising the "Mapt1.2 CSO" DNA polynucleotide of sequence SEQ ID No. 5 comprising at its 5 'end the hydrophilic C11 spacer chain described for the preparation of the first affinity support disclosed in paragraph "a)" of Example 1, said aptamer being designated "Mapt1.2CSO oligo5 (5`Amine C11 hydrophilic)". The results for the latter aptamer are not shown in Table 6 below. 6.2. Grafting conditions of Pantamer RNA The aptamer RNA sequence SEQ ID No. 5 was grafted under the following conditions: 300 μg of aptamer RNA were incubated with 500 L of gel, at the final concentration of gel of 0, 6 g / L. the grafting reaction was carried out for 2.5 h at 17 ° C. (TA) and at pH 4.2. the reaction was then stopped by neutralization with a 200 mM borate buffer for 2.5 hours at 17 ° C. and at pH 9. the residual amount of grafted non-grafted aptamers was measured in the supernatant in order to determine the yield of grafting, agarose gel electrophoresis and GelRed® staining (3.5%), then comparison with a standard range of known amounts of the RNA aptamer loaded on other tracks of the same electrophoresis gel.

The results show that the grafting yield was greater than 99%. These results show that the grafting process at a pH below 5 is effective for the grafting of all types of nucleic ligands, that these nucleic ligands comprise a polynucleotide DNA or an RNA polynucleotide (and thus also the nucleic ligands comprising a hybrid polynucleotide DNA / RNA), including ligands comprising a modified base polynucleotide, including a modified base RNA polynucleotide. 6.3. Efficiency of the grafting reaction The coupling reaction was carried out as described in Example 5, except for specific indication as to time, temperature and pH. The results are shown in Table 6 below. Table 6 Grafting conditions of grafting Aptamer grafting * (mg / mL d (time, time, pH) gel) ** 8h; 5 ° C; pH 4.3, 4 100 8h; 5 ° C; pH 3.8 0 1% 4apt2CS olig h; 5 ° C; pH4.3 (5 Amine C 1h, TA, pH 4.3, 400 Hydrophilic) $ P h; 5 ° C; pH, 4,100 Mapt2CS olig (5 'Amine C6 and dT) h; YOUR; pH4.3 1.7 100 Mapt2CS oligo3 2h; YOUR; pH4.3 0.700 Amine C12) Mapt2CS oligo7 ne C6) h; FA; pH4.3 1.8 0 apt1.2CSO oligo 00 h; 5 ° C; pH4, I I * Duration of the coupling reaction expressed in hours; Temperature in degrees Celsius - RT ambient temperature (18 ° C-25 ° C) **. The target grafting rate is achieved for a grafting yield of 100%. The graft yield was measured as described in Example 3. Not Determined The results in Table 6 show that for a given aptamer, 100% graft yield is achieved regardless of the conditions tested. In particular, these results confirm those of Example 4, and more specifically the results of Table 5, which show that a maximum graft yield is obtained, even at pH, very acidic of 3.8. These results also confirm the rapid kinetics of the coupling reaction carried out at room temperature. It is also shown that the neutralization step at pH 9, subsequent to the coupling reaction, is not essential for obtaining a maximum grafting yield, since the grafting rate and yield under these conditions are identical to those observed when the neutralization step is performed (see Table 6 above). The results in Table 6 also show that the hydrophilic or hydrophobic nature of the spacer chain of the aptamer has no influence on the grafting efficiency. It is also shown that grafting is also effective when the hydrophilic or hydrophobic spacer chain is bonded to the 5 'end or the 3' end of the DNA or RNA polynucleotide. 5 'Amin Hydrophilic) Mapt A FIXa Is Hydrophilic 3H7175' 47 C 2h; YOUR; pl: 14.2 1.6 100 It is added that the prior neutralization of the polynucleotide DNA charges by incubation of that with a Polybrenee4 (Hexamethrin broinide, 1,5-Dimethyl-1,5-diazaundecamethylene polymethobromide) renders impossible the realization of the coupling reaction. 6.4. Functionality The functionality of affmity supports on which the "Mapt2CS oligo5" adapter has been immobilized described in §6.1 has been tested. above, respectively in the following temperature, time and pH conditions of the coupling reaction. : - Conditions No. 1: 48h, 5 ° C, pH 4.2; Conditions 2: 48h, 5 ° C, pH 3.8; - Conditions No. 3: 2h, 5 ° C, pH 4.2; and - Conditions No. 4 1h, RT (Ambient Temperature), pH 4.2.

For the test, 500 μl of aptamer-grafted gel were used in each of the conditions 1-4 above, at a final concentration of 4.4 mg / ml. For each of the chromatographic supports, 2.7 mg of a purified recombinant human FVII composition produced in the milk of Tyransgenic rabbits (FVII-TG) was injected.

The FVII-TG composition used for the injection is prepared by neutralizing the citrate initially contained in the formulation with CaCl 2 and modifying the formulation buffer to obtain: between 35 and 40 mM NaCl and between 3.2 and 4 mM MgCl 2 - Buffer used for the chromatography: Tris SOmM 10mM CaCl 2, pH 7.5 - Flow: 0.5 ml / min - Elution Buffer: 50mM Tris, 10mM EDTA, pH - 7.5 The results are shown in FIGS. 9, respectively for the coupling conditions 1 to 4 described above. The results are also shown in Table 7 below.

Table 7 Coupling conditions (time, temperature, pH) fraction of FVII not retained on the fraction column of FV11 eluted (%) (%) n ° 1: 48h; 5 ° C; pH 4.2 5 95 No 2: 48h; 5 ° C; pH 3.8 24 76 No.3: 2h; 5 ° C; pH 4.2 15 85 No. 4: 1H; YOUR; The results of FIGS. 6 to 9 and Table 7 above show that almost all of the human factor VII is retained on the affinity support at the time of the passage of the composition to be purified. exception of the affinity support prepared by grafting under conditions No. 2 (48h, 5 ° C., pH 3.8) for which there is a retention rate of factor VII which is substantial but not maximal.

The results also show that all the human factor VII retained on the affinity support is released during the elution step. These results show that fully functional affinity carriers can be prepared using grafting conditions # 1 and # 3-4, which means that the conditions of temperature and duration of grafting are not absolutely critical to the ability of the affinity support obtained to retain and release the target molecule. In particular, it is observed that the coupling conditions of aptamers for a short time (1 hour) and at high temperature (ambient temperature) (i) allows a grafting rate of 100% of the aptamers and (ii) does not cause any alteration. in the ability of grafted aptamers to bind to the target human factor VII. On the other hand, the poorer results obtained with the affinity support prepared by coupling the aptamers at pH 3.8 show that the pH conditions are important for the maintenance of the integrity of the grafted aptamers, and in particular for the maintenance of the ability of grafted aptamers to bind to the target human factor VII. When the coupling reaction is carried out at pH 3.8, an affinity support is obtained which remains functional to selectively enrich a starting sample of human factor VII. However, such an affinity support can not be validly used in the context of an industrial process for the purification of human factor VII, because of the significant loss of factor VII (more than 20%) and therefore the economically disadvantageous nature of such a method. Table 8: Sequence Listing No. SEQ ID Description SEQ ID No. 1 Aptamer Mapt 2 CS SEQ ID No. 2 Aptamer Mapt 1.2 SEOID No. 3 AtapmètMapt 2.2 CS SEQ ID No. 4 Aptamer Mapt RNA Anti FIXa SEQ ID N ° 5 Aptamer Mapt 1.2 CSO

Claims (15)

REVENDICATIONS1. A method of immobilizing nucleic ligands comprising at least one primary amine function, by grafting onto a solid support having activated carboxylic acid groups, comprising a step of covalently coupling said nucleic acids to said solid support at a pH below 5. 2. A method of immobilizing the nucleic ligands comprising at least one primary amine function on a solid support the following steps: a) providing a solid support comprising, on its surface, activated carboxylic acid groups; b) providing a nucleic ligand comprising at least a primary amine function, and c) coupling said nucleic acid with the activated carboxylic acid groups present on the surface of said solid support under pH conditions of less than 5, provided that the order of steps a) and b) is indifferent: 3. Method according to any one of claims 1 and 2 characterized in that the activated carboxylic acid groups are obtained by reaction with N-hydroxysuccinimide or one of its derivatives. 20 4. Method according to any one of claims 1 to 3 characterized in that the pH of the coupling step is in a range from 3.5 to 4.5. 5. Process according to any one of claims 1 to 4, characterized in that the coupling step is carried out at a temperature ranging from 0 ° C to 35 ° C. 6. Method according to any one of claims 1 to 5 characterized in that the nucleic ligand is a polynucleotide of 5 to 120 nucleotides comprising at least one primary amine function at its 3 'or 5' end. " 7. Method according to any one of claims 1 to 5 characterized in that the nucleic ligand comprises (i) a polynucleotide 5 to 120 nucleotides in length and (ii) a spacer chain linked to said polynucleotide, the primary amine function being attached to a 3 'or 5' end of said polynucleotide or to the spacer chain. 8. Process according to claim 7, characterized in that the spacer chain is chosen from the group of linear C2-C20 alkyls and C2-C20 polyethylene glycols substituted with a primary amine function. 9. Process according to any one of claims 1 to 8, characterized in that the solid support is selected from the group consisting of silica gels, dextran gels, agarose gels and their derivatives. 10. Method according to any one of claims 2 to 9 characterized in that the coupling step e) comprises the following two steps: cl) reacting said nucleic ligand with activated carboxylic acid groups present on the surface of said support solid, under pH conditions below 5 and c2) continue the coupling reaction of said nucleic ligand with the activated carboxylic acid groups present on the surface of said solid support, under conditions of pH greater than 7.5. 15 11. Method according to one of claims I to 10, characterized in that the coupling step is followed by a step d) of blocking the coupling reaction. 12. Method according to one of claims 1 to 10, characterized in that nucleic ligand comprising at least one primary amine function consists of a nucleic aptamer. 20 13. A method of preparing an affinity support comprising the implementation of the immobilization method, as defined in any one of claims 1 to 12. 14, solid affinity support obtained by I. method of preparation as defined in claim 13. 15. Solid affinity support on which nucleic ligands are immobilized by arnidic linkage and in which at least 0.01% of the The carboxyl functions initially present on its surface are derivatized by a nucleic ligand. 16. Solid affinity support on which nucleic ligands are immobilized by amidic linkage, said affinity support being a chromatography gel having a nucleic ligand density of at least 0.005 pinot / ml to 0.4 pmol gel. . Solid affinity support according to claim 16, characterized in that the solid affinity support is selected from the group consisting of agarose gels, dextran gels, silica gels and their derivatives. The solid affinity support according to one of claims 14 to 17, characterized in that the nucleic ligands are nucleic aptamers and in that said solid support has on its surface the following structures of formula (11I) (SPAC) where NUCL represents the nucleic aptamer in which - n is an index of 0 or 1. - SPAC represents a spacer chain, and - NUCL represents a nucleic acid. specifically binding to a target molecule, said nucleic acid comprising from 5 to 120 nucleotides. 19. A complex resulting from the interaction of a nucleic ligand and a target molecule, said complex being formed on the surface of a solid support as defined in any one of claims 14 to 18. 20. Use of an affinity support as defined in any one of claims 14 to 18 for the purification of a target molecule. 21: Use of an affinity support as defined in any one of claims 14 to 18 for the detection of a target molecule 22. A method for purifying a target molecule with an affinity support such as as defined in any one of claims 14 to 18, comprising the steps of: a) contacting a composition to be purified comprising the target molecule of interest with a solid affinity support as defined in one of the claims 14 to 18, to form a complex between (i) nucleic ligands grafted onto said support and (ii) said target molecule andb) releasing said target molecule from the complex formed in step a) and recovering said purified target molecule . 23. Use according to claims 20 and 21 or method according to claim 22 characterized in that the target molecule is a plasma protein.