EP2658864A1 - Procede d'immobilisation de ligands nucleiques - Google Patents
Procede d'immobilisation de ligands nucleiquesInfo
- Publication number
- EP2658864A1 EP2658864A1 EP11813452.7A EP11813452A EP2658864A1 EP 2658864 A1 EP2658864 A1 EP 2658864A1 EP 11813452 A EP11813452 A EP 11813452A EP 2658864 A1 EP2658864 A1 EP 2658864A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- nucleic
- support
- affinity
- coupling
- solid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6437—Coagulation factor VIIa (3.4.21.21)
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3204—Inorganic carriers, supports or substrates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3206—Organic carriers, supports or substrates
- B01J20/3208—Polymeric carriers, supports or substrates
- B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
- B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
- B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
- B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3278—Polymers being grafted on the carrier
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
Definitions
- the invention relates to the field of the purification of substances of interest using affinity supports usable on an industrial scale, in particular for obtaining purified substances of medical interest,
- affinity chromatography supports are used to purify substances subsequently used as drug active ingredients.
- 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 vis-à-vis 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.
- the immunoaffinity supports allow the purification of large quantities of the target substance, which makes their use compatible with the technical and economic requirements of the manufacture of drugs.
- the immunoaffinity supports have disadvantages when they are used for the purification of substances of medical interest, particularly because of (1) 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.
- 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 strong 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. In fact, biotin and avidin or streptavidin are sensitive to treatments likely to induce protein denaturation.
- the biotin / streptavidin complex dissociates at low temperature, especially in nonionic aqueous solutions or at low saline concentration.
- 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 possibility Regenerating and sanitizing 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.
- nucleic aptamers possessing a reactive amino group on a cyanogen bromide-activated sepharose support has been described (Madru et al., 2009, Anal Chem, Vol 81: 7081-7086).
- the grafting of periodate-oxidized RNA gametes to adipic acid dihydrazide-activated agarose support has also been described (Caputi et al., 1999, The EMBO Journal, Vol 18 (14): 4060- 4067).
- Nucleic aptamer grafting techniques are also known by means of bi-functional coupling agents such as SIAB (Rehder et al, Electrophoresis, Vol 22 (17): 3759).
- the state of the art also discloses covalent coupling techniques of nucleic acids, among others aptamers, on solid supports of silica or agarose type comprising carboxylic acid groups pre-activated with N-hydroxysuccylmid (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).
- NHS N-hydroxysuccylmid
- the affinity support obtained by Goss et al. has a grafting rate of poly (dThs) of 0.5 ⁇ per g of silica, which is very low given the number of carboxylate groups per g of silica gel (500 ⁇ per g of silica).
- the present invention relates to a method for immobilizing nucleic acids comprising at least one reactive amine function, by grafting on a solid support having on its surface activated carboxylic acid groups, said method comprising a step of coupling said nucleic acids to said solid support activated at a pH below 6.
- 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:
- nucleic ligand comprising at least one primary amino function
- 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.
- the present invention also relates to a method for 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 an affinity support as defined above, in order to form a complex between (i) the nucleic acids grafted onto said support and (ii) said target ligand and
- step b) releasing said target ligand from the complex formed in step a) and recovering said purified target ligand.
- FIG. 1 illustrates a chromatographic profile obtained by passing a composition comprising 100 g of transgenic human factor VII on a solid support of the invention onto which human anti-FVII DNA aptamers have been grafted.
- Peak # 1 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 No. 3 corresponds to the human FVII contained in the regeneration eluent.
- 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 of transgenic human factor VII onto a solid support of the invention onto which human anti-FVII DNA aptamers have been 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.
- FIG. 3 illustrates a chromatographic profile obtained by passing a composition comprising 1000 g of transgenic human factor VII on a solid support of the invention onto which human anti-FVII DNA aptamers have been 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.
- FIG. 4 illustrates a chromatographic profile obtained by passing a composition comprising 200 ⁇ g of transgenic human factor VII on a solid support of the invention onto which human anti-FVII DNA aptamers have been grafted, said carrier 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.
- FIG. 5 illustrates a chromatographic profile obtained by passing a composition comprising 1000 ⁇ g 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 action.
- 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. 6 illustrates a chromatographic profile obtained by passing a composition comprising 2.7 mg of transgenic human factor VII onto a solid support of the invention onto which human anti-FVII DNA aptamers have been grafted under the following coupling conditions.
- 48h 5 ° C, pH 4.2.
- Point # 1 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.
- FIG. 7 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 following coupling conditions. 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.
- FIG. 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 into the following coupling conditions; 2h, 5 ° C, pH 4.2.
- Point # 1 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.
- FIG. 9 illustrates a graphical chromato 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 coupling conditions.
- Ih TA (room temperature), pH 4.2.
- Point # 1 indicates the timing of human Factor VII injection.
- Peak # 2 (at about 75 min) corresponds to the human FVII contained in the elution fraction.
- On the abscissa the time, expressed in minutes.
- the present invention provides novel affinity carriers comprising immobilized nucleic acids, as well as methods for their preparation.
- a first object of the invention is a method of immobilizing nucleic acids having at least one reactive amine function on a solid support having activated carboxylic acid functions.
- nucleic acid or “nucleic ligand” is intended to mean a compound comprising a polymer of nucleotides or polynucleotide, that is to say of ribonucleotides and / or of deoxyribonucleotides, optionally chemically modified, having a length ranging from From 5 to 10,000 nucleotides in length, preferably from 5 to 1,000 nucleotides in length, and more preferably from 5 to 120 nucleotides in length.
- a nucleic acid conventionally includes polyribonucleotides (AR) 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 base nitrogen selected from adenine, guanine, thymine, cytosine, puracil and their chemical analogues.
- a nucleotide may to be modified both on its osidic part and on its nitrogenous base by methods well known to those skilled in the art.
- patent US5958691 describes aptamers exhibiting a chemical modification at the level of one or more nucleotides.
- a nucleic acid is a polymer of nucleotides, ribonucleotides or deoxyribonucleotides.
- a nucleic acid consists essentially of a polymer of nucleotides and comprises a non-nucleotide portion, said non-nucleotide portion being preferably of reduced length, compared to the length of the nucleotide portion, for example a length linear less than the length occupied by a chain of five nucleotides, ribonucleotides or deoxyribonucleotides.
- a nucleic acid comprises a reactive amine function when said nucleic acid has an amino function accessible to the solvent and capable of reacting with a suitable reactive group carried by another molecular entity.
- the reactive amino functions include, in particular, primary amines. This primary amine is distinct from the aromatic amines carried by the purine or pyrimidine nucleotide nuclei.
- 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.
- these are nucleic acids that have been modified by the addition of an amino function at their 3 'end or at their 5' end.
- 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 of synthesizing a polynucleotide.
- the reactive amino function and the 5 'or 3' end of the nucleic acid are separated by a spacer chain.
- a nucleic acid may 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.
- 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 nucleic acid. , but is valvably bonded to a non-nucleotide portion of said acid nucleotide, 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.
- nucleic acid also referred to hereinafter as “nucleic ligand”
- nucleic ligand comprises:
- polynucleotide consisting of a sequence of ribonucleotides and / or deoxyribonucleotides, optionally chemically modified, and
- B optionally, a non-nucleotide portion, preferably a spacer chain,
- Said ligand or nucleic acid further comprises a reactive amine function, said reactive amine function being able to be attached either to a 3 'or 5' end of the polynucleotide or optionally to the spacer chain.
- the polynucleotide is at least modified at the 3 'or 5' nucleotide to introduce the reactive amine function directly or via a non-nucleotide part, in particular a spacer chain.
- the nucleic acid may comprise a chemical entity additional to those previously mentioned, for example a fluorophore or a chromophore.
- 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.
- the target molecules include molecules of AR, DNA, chemical molecules of organic or mineral nature, peptides and proteins that are human, animal, plant, viral or bacterial.
- nucleic acid-based affinity supports can be prepared by chemical grafting of said nucleic acids onto 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.
- 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.
- 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 obtaining an excellent grafting efficiency, close to 100%, on the preactivated support with NHS.
- the Applicant has shown that contacting the support thus grafted 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 use in industrial purification processes, said processes generally comprising drastic washing steps and / or or microbial inactivation.
- the technical solution that was finally developed according to the invention consisted in carrying out the coupling reaction of the nucleic acids on the support pre-activated with N-hydroxysuccinimide under pH conditions of less than 6.
- 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 year. 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 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.
- 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 support grafted with nucleic aptamers capable of binding to human factor VII (FVIIh). It is shown that the ability of said anti-FVHh nucleic aptamers is intact after grafting under acidic pH conditions, at low temperature or at room temperature, on the NHS pre-activated 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 ability of the -failed to discriminate (i) the active forms of the correctly gamma-carboxylated Gla domain Factor VII from the (ii) non-active forms of human Factor VII.
- the combined characteristics of high selectivity with respect to a target ligand and high capture capacity of said target ligand illustrate the compatibility of an affinity support obtained according to the method of the invention with a 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 coupling step co ects said nucleic acids on said solid support at a pH below 6.
- the method of immobilizing the nucleic ligands comprising at least one reactive amine function on a solid support comprises the following steps:
- nucleic ligand comprising at least one reactive amino function
- the coupling step c) allows the creation of amide bonds between the solid support and the nucleic ligands, each amide bond resulting from the reaction between an activated carboxylic acid function of the support and a primary amine function present at the nucleic ligand.
- the coupling conditions at a pH below 6 include coupling conditions at a pH below 5.5, 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.
- the pH of the coupling step is in a range of 3 to 6, 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, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4 , 7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5, 4, a pH of 5.5, a pH of 5.6, a pH of 5.7, a pH of 5.8 and a pH of 5.9.
- the pH of the coupling step is less than 4.5.
- the pH of the coupling reaction is in the range of 3.5 to 4.5.
- the coupling step can be carried out at a pH of about 4.2.
- the coupling is carried out in the presence of an aqueous buffered medium having a pH below 6.
- the buffered medium can be prepared from any type of acid and / or. weak bases, insofar as the weak acid (s) and base (s) used are not likely to react during the coupling reaction. As illustrated in the examples, it may be an aqueous solution of sodium acetate.
- 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.
- 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.
- 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 1-hydroxybenzotriazole (HOBt), HOAt, N-hydroxysuccinimide or one of their derivatives.
- HOBt 1-hydroxybenzotriazole
- HOAt 1-hydroxybenzotriazole
- N-hydroxysuccinimide N-hydroxysuccinimide
- the carboxylic acid groups of the support have been activated by reaction with N-hydroxysuccinimide or one of its derivatives such as N-hydroxysulfosuccinimide.
- Activation by NHS or sulfc-NHS has the advantage of generating activated esters reactive with primary amines but also sufficiently stable to allow 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 methodologies 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 functions with a view to the subsequent formation of an amide bond.
- a suitable chemical agent allowing the activation of the carboxylic acid functions with a view to the subsequent formation of an amide bond.
- Solid supports having "activated carboxylic acid” functions may be of any type. These supports include the supports conventionally used for chromato graphy, including silica and agarose 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, cellulose derivatives, or synthetic polymers such as polyamides, trisacryl, methacrylate derivatives, polystyrene derivatives, and methacrylate derivatives. polyacrylamides, or supports such as silica supports (especially porous glass supports) or alumina, on the surface of which are present activated carboxylic acid groups.
- 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 supports 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.
- said solid support can be obtained by reacting a commercial gel having free carboxylic acid functional groups with N-hydroxysuccinimide (NHS), optionally presence of a carbodiimide such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).
- NHS N-hydroxysuccinimide
- EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
- 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 non-nucleotide portion.
- non-nucleotide portion being preferably bound to the polynucleotide.
- 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.
- the reactive amine is an aliphatic primary amine, which means that the amine function is not directly linked to an aromatic group.
- the nucleic ligand is a polynucleotide 5 to 120 nucleotides in length comprising at least one amine function reactive at its 3 'or 5' end.
- 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 polynucleotide or spacer chain.
- the spacer chain is preferably bonded to the 5 'end or the end
- the nucleic ligand comprises a polynucleotide and a spacer chain, wherein said spacer chain comprising at one of its ends an amine function and being connected by its second end to the end, 5 ' 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 portion of the nucleic ligand and to reduce the steric hindrance.
- the spacer chain can be of any type.
- the examples illustrate in particular the implementation of the process 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 1-amino-3,6,9-trioxaundecan-1 yl called in the following Hydrophilic or non-specific oligonucleotide
- the spacer chain does not comprise ionizable groups other than primary amine functions or secondary amine functions.
- the spacer chain does not comprise groups or bonds sensitive to alkaline pH or to oxidation or reduction reactions.
- 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 selected 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 type polyethylene glycol, for example Hexaethylene glycol (HEG) or a 1,1-amino-3,6,9-trioxaundecan-III, hereinafter referred to as hydrophilic Ci or a nonspecific oligonucleotide substituted by a primary amine function
- the spacer chain may be introduced according to methods well known to those skilled in the art, in particular as a final step in the chemical synthesis of the polynucleotide.
- 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".
- the coupling step can be performed indifferently at low temperature and at room temperature.
- the implementation of the reaction at room temperature does not induce a decrease in the yield of the reaction.
- 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.
- the coupling step is carried out at a temperature ranging from 0 ° C. to 50 ° C.
- the coupling step may be carried out at a temperature ranging from 0 ° C to 35 ° C.
- 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 carried out at low temperature, typically at a temperature ranging from 0 ° C. to 8 ° C., if the reagents at stake - in particular the nucleic ligands - have chemical groups that are sensitive, in particular, to hydrolysis.
- the coupling step can be carried out at a pH ranging from 3.5 to 4.5, at room temperature and over a period of about one hour.
- the coupling reaction can be completed by placing the pre-activated carrier / nucleic acid combination under conditions of alkaline pH for a given duration.
- the coupling step of the method of the invention itself comprises the following two steps:
- 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.
- the final phase of coupling at alkaline pH is carried out at a pH of at least 7.5, which includes the pH of at least 8, and at least 8.5.
- the examples illustrate the completion of step c2) at a pH of about 9
- Step c2) can be carried out at room temperature or at a low temperature.
- 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.
- the coupling reaction is followed by a step of neutralizing d) or blocking the unreacted activated carboxylic acid groups during the coupling step itself.
- the blocking of the activated and unreacted carboxylic acid functions can be achieved by incubation of the grafted support with a blocking solution comprising 0.5 M ethanolamine, 0.5 M NaCl at pH 8.3. or with a 0.1M Tris-HCl 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 can be carried out for example for a period of 2 h 30 at the temperature of 4 ° C, as described in the examples.
- 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 ready-to-use affinity support.
- the washing step (s) can be carried out with a buffer solution of 0.1 M Tris-HCl at a pH ranging from 8 to 9, or with a buffer solution of 0.1 M acetate, 0.5 M NaCl at a pH of 4 to 5, as is illustrated in the examples.
- a washing step comprises successively (i) washing with a 0.1 M 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.
- a plurality of washing steps are carried out, for example 3 washing steps, as illustrated in the examples.
- the method of immobilizing nucleic acids according to the invention can also be defined as a process comprising the following steps:
- nucleic acid comprising at least one primary amine function
- the method for immobilizing nucleic acids according to the invention can also be defined as a process comprising the following steps:
- nucleic acid comprising at least one primary amine function
- step c) itself comprises the following two steps:
- the method for immobilizing 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.
- 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.
- the method for preparing an affinity support according to the invention comprises the following steps:
- nucleic ligand comprising at least one primary amino function
- Step c) of the method may comprise steps c1) and c2) as defined above.
- the method may further comprise steps d) and e) previously described.
- the method and the method according to the invention are particularly suitable for the preparation of an affinity support intended for the purification of one or more target molecules, in particular by chromatography.
- the solid support is a support adapted to the implementation of a chromatography, filtration or solid phase extraction process.
- the solid support is suitable for use as a stationary phase in a chromatography, or filtration or solid phase extraction process.
- 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 can 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 gels and their derivatives and polystyrene and their derivatives.
- silica gels and polysaccharide gels such as agarose gels, dextran gels and their derivatives and also acrylamide gels and their derivatives, methacrylate gels and their derivatives and polystyrene and their derivatives.
- 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
- 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 a 1-amino-3,6,9-trioxaundecanyl-lyl, hereinafter referred to as hydrophilic or a non-specific oligonucleotide
- the solid support is selected from agarose gels and their derivatives
- the nucleic ligand is a polynucleotide of 5 to 120 nucleotides linked at its 5 'end to a spacer chain selected from polyethylene glycol C4-C20, the reactive amine function preferably being carried by the spacer chain.
- the solid support is selected from agarose gels and their derivatives and
- the nucleic ligand is a polynucleotide of 5 to 120 nucleotides linked at its 5 'end to a spacer chain chosen from C4-C20 unary 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 / washing / elution / lossless washing cycles. significant of its selective and quantitative retention properties 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.
- 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. M, or even by treatment with a solution of NaOH having a final concentration of 1 M, that is to say a final concentration of NaOH much higher than that which is usually used during the sanitization steps, and this during a very long period of time (100 hours) also much longer than the duration of a conventional sanitization step which is generally a few minutes.
- the chromato graphic properties of an affinity support according to the invention are not altered by treatment with a solution having a final concentration of propylene glycol of 50% (vol / vol).
- the results of the examples illustrate the ability of an affinity support according to the invention to achieve perfectly reproducible quantitative purification steps of a target ligand, and this under industrial conditions of use. conventional, which are generally deleterious to known affinity carriers, including for immunoaffinity carriers.
- affinity carriers according to the invention therefore constitute purification tools that are both reliable and reproducible, stable over time, and do not require repeated maintenance operations.
- the affinity supports of the invention because of the numerous technical advantages they provide, allow the implementation of purification processes of a target molecule at moderate costs.
- the examples describe obtaining an affinity support of the invention by grafting nucleic aptamers, and illustratively by grafting DNA aptamers.
- the nucleic ligand is a nucleic aptamer.
- nucleic aptamer or aptamer means a single-stranded nucleic acid which binds specifically 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 RNA aptamers and DNA aptamers.
- aptamer 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 mechanisms essentially distinct from hybridization. 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. Because of their oligonucleotide nature, aptamers possess low immunogenicity and high resistance to stringent physicochemical conditions (presence of urea, DMSO, very acidic or very basic pH, use of organic solvents or high temperature ) allowing various sanitization strategies in the context of use as an affinity ligand. Moreover, their selectivity is important. Finally, the production of aptamers involves relatively limited costs.
- a "nucleic aptamer" used for grafting to the pre-activated solid support may comprise, by definition, a non-nucleotide or nucleotide portion, for example a chain non-nucleotide spacer, 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.
- a nucleic aptamer may have the following formula (I):
- n is an index of 1 or 0, where 0 means that the aptamer does not comprise a free spacer chain and 1 signifies that the aptamer comprises a spacer chain.
- N3 ⁇ 4 means the reactive amino function used for grafting onto the solid support pre-activated by NHS groups
- [NUCL] means a nucleic acid specifically binding to a target molecule, said nucleic acid comprising from 5 to 120 nucleotides.
- 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 acid nucleic acid and molecules of the target ligand capable of being brought into contact therewith.
- the spacer chain is preferably bonded to the 5 'end or the 3' end of [NUCL] nucleic acid.
- 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, Cl 2 or a hydrophilic chain which may be of polyethylene glycol type, for example Hexaethylene glycol (HEG) or a 1-amino-3,6,9-trioxaundecanyl-lyl referred to in the following Cl 1 hydrophilic or non-specific oligonucleotide.
- HOG Hexaethylene glycol
- Cl 1 hydrophilic or 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.
- the spacer chain may be composite and include the sequence of HEG and an oligonucleotide, for example an oligo (dT).
- an oligonucleotide for example an oligo (dT).
- the examples illustrate the production of affinity supports as defined above by grafting the pre-activated support with NHS with three distinct anti-FVTI nucleotide aptamers comprising spacer chains of a similarly distinct nature, namely, a hydrophobic chain consisting of a C6 alkyl chain, a hydrophobic chain consisting of a C1 2 alkyl chain, and a Cl.1-TFA hydrophilic chain.
- the results of the examples show that the process for obtaining a carrier of The affinity which is described in the present description can be used, whatever the identity or the type of nucleic acid of interest to be grafted.
- the methods and method according to the invention allow the preparation of affinity supports which are distinguished from known affinity supports by their high density of nucleic acids immobilized on their surface. This high density of nucleic ligands stems directly from the specific method of obtaining the affinity supports.
- 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.
- the process according to the invention makes it possible to prepare affinity chromatography gels presenting 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 0.2 ⁇ / ml nucleic ligands at 0.5 ⁇ / ml whereas the initial density in activated carboxylic acid functions of the chromatography gels that the it is commercially available generally between 5 ⁇ / ml to 25 ⁇ / ml of gel.
- 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% more preferably at least 1% and more preferably at least 2% of said activated carboxylic functions.
- 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 ⁇ / 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.
- 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.
- Another object of the present invention is an affinity support obtainable according to the method and method described hereinbefore.
- the present invention relates to a solid affinity support on which nucleic ligands are immobilized by an amide bond and wherein at least carboxyl functions initially present on its surface are derivatized by a nucleic ligand.
- amide linkage binding 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.
- 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 bond, said affinity support being a chromatography gel having a nucleic acid density of at least 0.005 ⁇ / ml. of gel, which includes at least 0.01 ⁇ / ml, 0.05 ⁇ / ml, 0.1 ⁇ / ml, 0.15 ⁇ ⁇ / ml, 0.2 ⁇ / ⁇ , 0.25 ⁇ / ⁇ , 0.3 ⁇ / ⁇ , 0,35 ⁇ / ml and at least 0,38 ⁇ / ml of gel.
- the density of nucleic ligands is at most 10 ⁇ / ml, which encompasses at most 5 ⁇ / ml, at most 1 ⁇ / ⁇ and at most 0,5 ⁇ ⁇ / ⁇ .
- the affinity supports according to the invention may have any of the properties described above for solid supports or affinity supports.
- 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 and silica gels and their derivatives.
- the affinity support can be a highly crosslinked agarose gel on which the nucleic ligands are immobilized.
- 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.
- the affinity support according to the invention is characterized in that the nucleic ligands are aptamers of formula (II) presented above.
- the solid affinity support according to the invention may be represented by the following formula (III):
- - [SUP] represents the solid support of the affinity support - [SPACjn and [NUCL] are as previously defined.
- - H- (SPAC) n - UCL represents a nucleic aptamer in which:
- ⁇ n is index of 0 or 1
- ⁇ SPAC represents a spacer chain
- NUCL represents a nucleic acid specifically binding 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 acid 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.
- the support according to the invention can be used as a stationary phase in filtrafion, chromatography or solid phase extraction steps.
- the present invention is also related to a method for purifying a target molecule with an affinity support as defined above, comprising the following steps:
- composition to be purified comprising a target molecule of interest with an affinity support as defined in the present description, in order to form a complex between (i) the nucleic acids grafted onto said support and (ii) said target ligand and
- step b) releasing said target molecule from the complex formed in step a) and recovering said purified target molecule.
- 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.
- step a ') of a washing buffer having a high hydrophobicity, in particular a high concentration of propylene glycol makes it possible to effectively remove the non-ionically bound substances. specific to the affinity support without simultaneously detectably affecting the binding of the target ligand to the affinity support.
- 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.
- 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.
- a wash buffer used in step a ') of the process has a final propylene glycol content of at most 50%.
- a washing buffer used in step a ') of the process has a final propylene glycol content of between 20% and 50%, preferably between 30% and 50%.
- the washing buffer used in step a ') contains both NaCl and propylene glycol as described in the examples.
- step b) is performed by contacting the affinity support with an elution buffer containing a divalent ion chelating agent, preferably EDTA.
- the elution buffer may contain a final concentration of EDTA of at least 1 mM and at most 30 mM.
- At least 1 mM includes at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM.
- 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, 12 or 11 mM .
- 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, is used for the regeneration of the affinity support.
- target molecule refers to a molecule capable of to link specifically to the aptamer.
- ⁇ can be nucleic acids, proteins or organic or mineral substances. Proteins can be any type of protein, and in particular, plasma proteins.
- 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 macroglobulin, antichymotry sine, 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, Cls, C2 C3, C4, C4bP, C5, C6, Clq, C8, C9, Carboxypeptidase N, Ceruloplasmin, Factor B, Factor D, Factor H, Factor I, Factor ⁇ , Factor V, Factor VII, Factor Vlla, 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
- plasma proteins include coagulation proteins, that is, plasma proteins involved in the chain of cascade reactions resulting in the formation of a blood clot.
- Coagulation proteins include Factor I (fibrinogen), Factor II (prothrombin), Factor V (proaccelerin), Factor VII (proconvertin), Factor VIII (antihemophilic factor A), Factor IX (factor Antihemophilic B), Factor X (Stuart Factor), Factor XI (Rosenthal Factor or PTA), Factor XII (Hageman Factor), Factor XIII (Fibrin Stabilizing Factor or FSF), PK (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 the tissue factor pathway inhibitor (TFPI), or tissue factors.
- Factor I fibrinogen
- 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 ), the postman XII (Hageman Factor), Factor XIII (Fibrin Stabilizing Factor or FSF) and PK (Prekalicrin).
- the plasma protein consists of a natural or recombinant human plasma protein.
- the plasma protein is the factor
- an aptamer comprising the Mapt 2CS polynucleotide of sequence SEQ ID No. 1 comprising at its 5 'end a hydrophilic C 11 chain (11-amino-3, 6, 9, -trioxaundecan-1 yl)
- an aptamer comprising the "Mapt 1.2" polynucleotide of sequence SEQ ID No. 2 linked at its 5 'end to a spacer chain composed of 12 methylenes (CH 2) (Cl spacer) 2) and linked at its 3 'end to an oligo-dT
- the supernatant was recovered and the amount of non-grafted aptamers was assayed.
- the gel “NHS Activated Sepharose 4 fast flow (GE)” is a crosslinked agarose gel having on its surface functional carboxylic acid functions 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 ⁇ / ml of gel.
- the supernatant of the reaction medium does not comprise a detectable amount of nucleic aptamer, that is to say, under the analysis conditions used, comprises a quantity aptamer less than 0.08 mg / ml
- Example 2 Use of an affinity support for the purification of recombinant human factor VIII produced in the milk of transgenic rabbits.
- FVII-TG a purified recombinant human FVII factor composition produced in the milk of transgenic rabbits
- the FVI 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 rM NaCl and between 3.2 and 4 mM MgCl 2
- Buffer used for chromatography 50mM Tris / 50mM NaCl / 10mM CaCl 2 /
- 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 (100 ⁇ g and 200 ⁇ 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 ⁇ g (FIG. 4) and 1000 ⁇ g (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.
- the loading capacity (or in other words the retention capacity) of the affinity supports was evaluated by injection of a recombinant human FVII composition into the "Tp5" buffer (Tris 50 mM, 1M CaCl 2 , pH 7.5).
- Tables 1 and 2 below show the performance of the Mapt 2CS-PEG (C1 1) and Mapt 2.2CS-PEG (C11) aptamer grafting reaction on the pre-activated NHS Activated Sepharose 4 fast flow gel (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 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 support capacity of the support and therefore loss of aptamer functionality for the high amounts of grafted aptamers.
- affinity supports having a grafted aptamer density of greater than ômg / ml and having a loading capacity for FVII greater than 8 mg of human recombinant FVII per mL of gel.
- Table 3 shows the load capacity of the affinity supports as a function of the number of aptamers grafted onto their surface.
- the amount of non-grafted aptamers was measured at the column outlet after the grafting step.
- the results show that the grafting yield varies from 0% to 10% maximum, whatever the grafting conditions described in the Table 1 that were used. Contrary to what Goss et al. (supra), high salinity is not sufficient to increase the coupling efficiency.
- the amount of ungrafted aptamer at the column outlet after the grafting step was measured.
- the results show that the grafting yield, whatever the grafting conditions described above, varies from 0% to 10% at most.
- the implementation of the coupling step in the presence of an acidic pH makes it possible to increase significantly coupling the aptamers to the pre-activated 3 medium without impairing the binding ability of said aptamers to their target protein.
- a Mapt 2CS-PEG (Ci 1) aptamer solution at a concentration of 2 g / L in the grafting buffer was prepared.
- reaction medium was added 1 mL of borate buffer at 200 mM pH 9 with stirring and incubated for 3 hours with stirring at 4 ° C.
- the supernatant was removed for final determination of the supernatant.
- the pH is a crucial parameter for the implementation of the coupling reaction according to the invention.
- 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.
- the use at room 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 spacer level of the aptamers makes it possible to only to obtain a grafting yield close to 100% but also a maintenance of the functional integrity of the aptamers.
- aptamers comprising a DNA polynucleotide and aptamers comprising an RNA polynucleotide were used respectively.
- the polynucleotide is linked to a spacer chain by its 5 'end while for the other aptamers, the polynucleotide is linked to a spacer chain by its 3' end.
- aptamers having 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 spacer chain Ci I described for the preparation of the first affinity support disclosed in paragraph "a)" of Example 1, said aptamer being designated “Mapt2CS oligoS (5 'Hydrophilic Amine Cl 1)" in Table 6; the aptamer comprising the "Mapt 2 CS" DNA polynucleotide of sequence SEQ ID No.
- the aptamer comprising the "Mapt2 CS" DNA polynucleotide of sequence SEQ ID No. 1 comprising at its 5 'end the hydrophobic Cl 2 spacer chain described for the preparation of the second affinity support disclosed in paragraph "b" of the 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. 1 comprising at its 3 'end the hydrophobic C6 spacer chain described for the preparation of the third affinity support disclosed in paragraph "c" of the example 1, 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 (3'-dT-5'), said aptamer being designated "Mapt RNA Anti FD has 5 'Amine C1 hydrophilic" in Table 6.
- An affinity support was also prepared on which was grafted an aptamer comprising the "Maptl.2 CSO" DNA polynucleotide of sequence SEQ ID No. 5 comprising at its 5 'end the hydrophilic spacer chain Ci I described for the preparation of the first affinity support disclosed in paragraph "a)" of Example 1, said aptamer being designated “Mapt 1.2CSO oligoS (5'Amin Ci l hydrophilic)".
- the results for the latter aptamer are not shown in Table 6 below.
- the aptamer RNA sequence SEQ ID No. 5 was grafted under the following conditions:
- RNA aptamer 300 ⁇ g were incubated with 500 ⁇ l of gel at the final gel concentration of 0.6 g / l. the grafting reaction was carried out for 2.5 hours at 17 ° C. (RT) and at pH 4.2.
- reaction was stopped by neutralization with a 200 mM borate buffer for 2.5 hours at 17 ° C and pH 9.
- the residual amount of non-grafted aptamers in the supernatant was measured for graft yield, agarose gel electrophoresis, and GelRed® staining (3.5%), and then compared with a standard range of known quantities of the aptamer RNA loaded on other tracks of the same electrophoresis gel.
- 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.
- 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 of MgCl 2
- Buffer used for chromatography 10mM Tris 50mM / CaCl 2 , pH 7.5
- FIGS. 6 to 9 and of Table 7 above show that almost all of the human factor VIII is retained on the affinity support at the time of the passage of the composition to be purified, with the exception of the affinity support.
- Example 7 it is shown that affinity supports as defined in the present description retain their capacity for retention and elution of the target protein, even after many cycles of use, under conditions of implementation.
- graft support No. 1 an aptamer comprising the Mapt 2CS polynucleotide of sequence SEQ ID No. 1 comprising at its 5 'end a hydrophilic Cl chain (11-amino-3,6,9-trioxaundecanyl),
- graft support No. 2 an aptamer comprising the Mapt 2.2CS polynucleotide of sequence SEQ ID No. 3 comprising at its 5 'end a hydrophilic Cl 1 chain (11-amino-3,6,9-trioxaundecanyl); , and
- graft support No. 3 an aptamer comprising the "Mapt 2.2 CS" polynucleotide of sequence SEQ ID No. 3 linked at its 5 'end to a spacer chain composed of 6 methylene (CH 2) (C 6 spacer)
- the grafting yields were respectively 100% (grafting support No. 1), 93% (grafting support No. 2) and 87% (grafting support No. 3).
- the theoretical static capacity of the prepared affinity media that is, the amount of human F VII that should be retained on the affinity media if each of the grafted aptamers bound a human F VII molecule, was, respectively, 8.7 mg per carrier (carrier # 1), 17.3 mg / mL (carrier # 2) and 16.3 mg / mL (carrier # 3). 7.2. Operating conditions of the process for purifying human FVII.
- a FVII-TG composition enriched in human FVII was used, the final concentration of FVII-TG being about 50,000 ppm, said composition comprising a high proportion of FVII forms of -gla (inactive forms of FVII) and said composition having a specific activity FVII of about 0.4 (activity expressed as amidolytic activity / antigen).
- washing with the washing buffer (eight times the volume of the grafted support),
- the three supports were determined to have a dynamic retention capacity of human FVIIa of about 10 mg of human FVIIa per milliliter of grafted support, which represents between 53% and 58% of the theoretical maximum static capacity calculated in paragraph 7.1. above. These results show that the respective aptamers grafted on the three supports are widely accessible and functional.
- the following two biological media have been tested: (i) a human blood plasma cryosurnant and (ii) a clarified milk solution, the milk being a source of human FVII, for example when human FVII is produced in the human body. milk from transgenic animals for the gene encoding human FVII.
- the affinity support No. 1 prepared as described in paragraph 7.1. was contacted with (i) human plasma cryosurnant or (ii) clarified milk for a period of 100 hours. After washing to remove the tested biological medium, the ability of each of the three affinity carriers to purify the human FVII was measured. The results are shown in Table 10 below.
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FR1061366A FR2970003B1 (fr) | 2010-12-30 | 2010-12-30 | Procede d'immobilisation d'acides nucleiques |
FR1159604A FR2981651B1 (fr) | 2011-10-24 | 2011-10-24 | Procede d'immobilisation de ligands nucleiques. |
PCT/IB2011/056028 WO2012090183A1 (fr) | 2010-12-30 | 2011-12-30 | Procede d'immobilisation de ligands nucleiques |
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US (1) | US9803184B2 (fr) |
EP (1) | EP2658864A1 (fr) |
JP (1) | JP6178727B2 (fr) |
KR (1) | KR20140057187A (fr) |
CN (1) | CN103403015A (fr) |
AR (1) | AR084757A1 (fr) |
AU (1) | AU2011350672B2 (fr) |
BR (1) | BR112013016704A2 (fr) |
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WO2012090183A1 (fr) | 2010-12-30 | 2012-07-05 | Lfb Biotechnologies | Procede d'immobilisation de ligands nucleiques |
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FR3011250A1 (fr) | 2013-09-30 | 2015-04-03 | Lab Francais Du Fractionnement | Acides nucleiques se liant specifiquement au facteur ix/ixa humain, et utilisations |
FR3021330A1 (fr) | 2014-05-20 | 2015-11-27 | Lab Francais Du Fractionnement | Procede de dosage d'aptameres adn |
GB201503578D0 (en) * | 2015-03-03 | 2015-04-15 | Ge Healthcare Bio Sciences Ab | Sanitization method for affinity chromatography matrices |
PT109480B (pt) * | 2016-06-22 | 2020-03-13 | Inst Superior Tecnico | Polímero de polibenzimidazolo com cadeia espaçadora funcionalizada e seu método de obtenção para remoção de impurezas genotóxicas |
CA3028030A1 (fr) | 2016-07-06 | 2018-01-11 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Aptameres anti-fibrinogene et utilisations associees |
US20190256853A1 (en) * | 2016-07-28 | 2019-08-22 | Laboratoire Francais Du Fractionnement Et Des Biot Echnologies | Method for Obtaining Aptamers |
US20190161756A1 (en) | 2016-07-28 | 2019-05-30 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Anti-Immunoglobulin G Aptamers and Uses Thereof |
CN106512958B (zh) * | 2016-11-22 | 2019-03-08 | 华南师范大学 | 一种核酸适配体修饰壳聚糖纳米纤维的制备方法及应用 |
WO2018109213A1 (fr) | 2016-12-16 | 2018-06-21 | Laboratoire Français Du Fractionnement Et Des Biotechnologies | Aptamères dirigés contre une protéine contenant une chaîne légère kappa et utilisations correspondantes |
GB201714563D0 (en) * | 2017-09-11 | 2017-10-25 | Life Tech As | Coupling methods and compositions |
US11598769B2 (en) * | 2018-07-27 | 2023-03-07 | Massachusetts Institute Of Technology | Oligomerized protein-polymer conjugates |
GB2580384B (en) * | 2019-01-08 | 2021-01-27 | Quantumdx Group Ltd | Oligonucleotide deposition onto polypropylene substrates |
CN109806852B (zh) * | 2019-03-04 | 2020-08-07 | 北京理工大学 | 一种腺嘌呤功能化聚丙二醇的应用 |
WO2023084369A1 (fr) * | 2021-11-12 | 2023-05-19 | 3M Innovative Properties Company | Procédé de traitement d'acides polynucléiques |
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DK641487A (da) * | 1987-12-07 | 1989-06-08 | Gluetech Aps | Fremgangsmaade til modificering af polymeroverflader |
US5215882A (en) * | 1989-11-30 | 1993-06-01 | Ortho Diagnostic Systems, Inc. | Method of immobilizing nucleic acid on a solid surface for use in nucleic acid hybridization assays |
US5660985A (en) | 1990-06-11 | 1997-08-26 | Nexstar Pharmaceuticals, Inc. | High affinity nucleic acid ligands containing modified nucleotides |
US5756291A (en) * | 1992-08-21 | 1998-05-26 | Gilead Sciences, Inc. | Aptamers specific for biomolecules and methods of making |
US6037124A (en) * | 1996-09-27 | 2000-03-14 | Beckman Coulter, Inc. | Carboxylated polyvinylidene fluoride solid supports for the immobilization of biomolecules and methods of use thereof |
US7482119B2 (en) * | 2002-04-01 | 2009-01-27 | Blue Heron Biotechnology, Inc. | Solid phase methods for polynucleotide production |
WO2003097094A1 (fr) * | 2002-05-21 | 2003-11-27 | Anadis Ltd | Methode de prophylaxie d'infections |
ES2375962T3 (es) * | 2003-09-22 | 2012-03-07 | Bioarray Solutions Ltd | Polielectrolito inmovilizado en superficie con múltiples grupos funcionales capaces de unirse covalentemente a biomoléculas. |
JP2005283572A (ja) | 2004-03-04 | 2005-10-13 | Toray Ind Inc | 選択結合性物質固定化担体 |
US20060110594A1 (en) * | 2004-11-24 | 2006-05-25 | Frutos Anthony G | Polymer-coated substrates for binding biomolecules and methods of making and using thereof |
FR2920024B1 (fr) * | 2007-08-14 | 2012-12-14 | Lfb Biotechnologies | Procede de purification ou de detection d'une proteine cible |
US8563527B2 (en) * | 2007-08-20 | 2013-10-22 | Pharmain Corporation | Oligonucleotide core carrier compositions for delivery of nucleic acid-containing therapeutic agents, methods of making and using the same |
US8815611B2 (en) * | 2008-04-10 | 2014-08-26 | Corning Incorporated | Surface for label independent detection and method thereof |
CN103403015A (zh) | 2010-12-30 | 2013-11-20 | 法国血液分割暨生化制品实验室 | 固定核配体的方法 |
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JP6178727B2 (ja) | 2017-08-09 |
AU2011350672A1 (en) | 2013-05-02 |
US9803184B2 (en) | 2017-10-31 |
CA2823030A1 (fr) | 2012-07-05 |
BR112013016704A2 (pt) | 2017-03-14 |
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