FR2653034A1 - Process for the preparation of a regiospecific porous particulate product, products obtained and application especially to chromatographic separations - Google Patents

Abstract

The invention relates to a process for the preparation of a regiospecific porous particulate product in which each porous particle has on its internal surface (3) specific physicochemical properties different from those of its external surface (2). The process consists (a) in grafting onto the particles an especially hydrophobic (5) ligand over all the internal and external surfaces, (b) in filling the pores by means of an enzymatically hydrolysable liquid protective compound, (c) in enzymatically hydrolysing the protective compound present at the external surface of the particles by means of an enzyme with a size greater than the size of the pores, (d) in carrying out an attack, especially chemical, on the particles by means of an agent which is reactive with respect to the ligand and immiscible with the protective compound with a view to selectively removing the ligand from the external surface, (e) in removing the protective compound still present in the particles, in particular by washing by means of a volatile liquid and (f), if appropriate, in grafting a hydrophilic ligand onto the external surface. The product thus prepared has a very marked regiospecific nature and can in particular be used for carrying out chromatographic separations of proteins.

Description

 The invention relates to a method for preparing a porous regiospecific particulate product.

By "regiospecific" means a product in which each porous particle has on its internal surface specific physicochemical properties different from those of its external surface; the internal surface of a particle is constituted by the surface of the pores which extend in its internal volume, while its external surface is constituted by the peripheral surface of the particle, which forms the interface with the external medium. The invention extends to particulate products prepared by carrying out the process and to applications of the process for the preparation of chromatographic separation products.

 Regiospecific porous particulate products have interesting applications for extracting, separating or purifying certain compounds present in mixtures of compounds of heterogeneous sizes. The internal surface of the particles can in fact be adapted to exert a retention on the compounds of small sizes to be trapped, while their external surface is adapted so as not to interact with the compounds of large sizes. This avoids parasitic fixations of large compounds on the external surface, thereby eliminating, on the one hand, the irreversible clogging phenomena which appear with non-regiospecific particulate products, on the other hand, the denaturation of a certain proportion of large compounds (due to interaction with the external surface).

The preparation of the above-mentioned porous particulate products consists in grafting retentive ligands onto inert porous particles; these ligands are found both on the internal surface and on the external surface, so that there appears a technical difficulty in making the product regiospecific. Currently, two methods exist to resolve this difficulty. One of these methods, called "by shield effect (shîeld)" is described in the following publication: "DARYL J. GISCH, BRIANT. HUNTER and BINYAMIN
FEIBUSH, Journal of Chromatogr. Biomed. Appl. 433, 1988, 264, 268 ". It consists, after grafting of the retentive ligands (generally hydrophobic), in coating each particle by means of a non-retentive layer (generally hydrophilic) to mask the reactivity of the ligands attached to the external surface. However, this layer modifies the pore size of the starting particles and the particulate product obtained only has the property of preventing the penetration of proteins inside the particle and cannot be used to separate proteins from each other as a result of the absence of pores of defined sizes at the level of the coating layer.

The other method, of wider application, is described in US Patent 4,544,483 (PINKERTON); it consists in grafting a ligand of a particular nature onto the surfaces of the particles, this ligand being a peptide ligand capable of being hydrolyzable by a peptidase; the particles are then treated using peptidases of sizes larger than those of the pores so as to hydrolyze and eliminate only the ligands on the external surface, those on the internal surface not accessible by the enzymes remaining intact. However, by its very principle, this process limits the nature of the retentive ligands which can be used which are necessarily peptide ligands; in chromatographic applications, these ligands are unusual and lead to behaviors which are difficult to predict and to optimize (parasitic reactions, partial ionic character, etc.). In addition, peptide ligands use expensive synthetic methods, a cost much higher than the synthesis of ligands usually used in chromatography, such as octadecyls which correspond to 80 y analytical applications; one of the serious consequences of this high cost is that it limits the PINKERTON process to analytical applications and excludes preparative applications of current products. In addition, in protein chromatography, mixtures frequently contain small proteases which would denature the ligand from the inner surface if they accessed it, so that the process
PINKERTON must be limited, for this type of application, to very low porosities (less than 10 nanometers), preventing proteases from accessing the internal surfaces. It should also be noted that in the case of very irregular external surfaces, hydrolysis by peptidase is incomplete and leaves retentive ligands on certain sites of said surfaces.

 The invention proposes to provide a new process for the preparation of porous regiospecific particulate products.

 An essential objective of the invention is to allow the use of very varied types of ligands and in particular the conventional chromatographic ligands (for example ligands with C 4, C 8, C 18 hydrocarbon chains) in order to eliminate the drawbacks of the PINKERTON process due to the peptide ligand.

 Another objective is to extend the porosity range of the particulate products obtained, whatever the application.

 Another objective is to obtain a porous particulate product with a very marked regiospecific character, that is to say in which the internal and external surfaces have very different physicochemical properties (designated hereafter by "pit for the properties of surfaces internal and by "Pe" for those of external surfaces).

To this end, the preparation process targeted by the invention is of the type consisting in (a) grafting onto particles of calibrated porosity a ligand capable of penetrating into the pores of said particles and of fixing themselves on all of their surfaces in view to give them physicochemical properties (Pi); the process according to the present invention is characterized in that, after said grafting:
(b) the pores of the particles are filled with a liquid protective compound, enzymatically hydrolysable and having an affinity for the ligand allowing it to penetrate into the pores and occupy the available volume,
(c) an enzymatic hydrolysis of the protective compound present on the external surface of the particles is carried out by means of an enzyme of size larger than the pore size in order to selectively remove the protective compound on said external surface,
(d) an attack on the particles is carried out by means of a reactive agent with respect to the ligand and immiscible with the protective compound with a view to selectively eliminating the ligand from the external surface,
(e) and the protective compound still present in the particles is eliminated.

 Thus in the invention, the de-grafting is carried out by a chemical attack in particular which can be applied to any type of ligand; the internal ligands are preserved by the protective compound, which remains confined in the internal volume after the action of the enzymatic hydrolysis intended to rid the external surfaces of said protective compound. It should be noted that this enzymatic hydrolysis targets the protective compound and is therefore suitable for it (unlike the PINKERTON process where the enzymatic hydrolysis targets the ligand itself with the defects already developed).

 The process of the invention is therefore compatible with the prior grafting of any ligand and in particular of conventional hydrophobic ligands: methyl, propyl, butyl, hexyl, octyl, octadecyl, phenyl, cyanopropyl, these ligands being preferably grafted onto particles with porous silicic backbone, which give siloxane bonds with them. The grafting of these ligands can be carried out on site during the implementation of the process; it is also possible to use particles previously grafted by the manufacturer, the process (be) being implemented on said grafted particles (the grafting of the ligand is in any case a necessary prior operation, whether it is carried out on site or by a previous operation).

 In the case of the above-mentioned ligands, the protective compound may be chosen from the following group, triglyceride wrinkle, per-ester of mono or droligosaccharide, hydrophobic polypeptide or their acylated derivatives; the enzymatic hydrolysis is then carried out by means of a hydrolase adapted to the protective compound used: lipase, esterase or peptidase. The chemical attack can be carried out by means of an acid solution immiscible with the protective compound, in particular acid solution of pH less than 1, the solvent of which has been chosen immiscible with the protective compound.

 The method of the invention eliminates all the defects relating to the use of peptide ligands of the PINKERTON method and in particular their high cost. It preserves the porosity of the starting particles since the protective effect does not lead to modifications of the porosity, this will be chosen according to the application, in particular pore diameter equal to or less than 30 nanometers for the chromatographic separation of the proteins.

 It should be emphasized that the enzymatic hydrolysis of the protective compound can be carried out both with natural enzymes and with enzymes polymerized or fixed on macromolecules; the polymerization and fixing techniques are known in themselves and make it possible to obtain the desired sizes for the enzymes, in particular large enzymes in the case of large pores. In the case of pores with a diameter greater than 10 nanometers, use will in particular be made of enzymes polymerized or fixed on a macromolecule, having a molecular weight greater than 80,000 preventing them from penetrating into said pores.

 In addition, the chemical attack on the external surfaces completely rid of the protective compound leads to a total elimination of the external ligands and therefore to a very marked regiospecific character. The specific physicochemical properties (Pe) of the external surfaces can be adjusted either by choosing starting particles which already have these properties on their surfaces, or by a subsequent grafting of another ligand on the external surface; it should be noted that this subsequent grafting can be complete since all the sites are available (the other liqand having been completely eliminated on the external surfaces).

 Furthermore, the removal of the protective compound still present inside the particles can easily be carried out by several processes known in themselves, and in particular by washing with a volatile liquid having an affinity for the ligand to less equal to that of said protective compound, said liquid then being eliminated by evaporation. In the case of the protective compounds mentioned above, it is possible in particular to choose as washing liquid a compound from the following group alkane, organochlorine, ketone, ether, volatile alcohol.

 The process of the invention is applicable in all cases where it is advantageous to have a porous regiospecific particulate product, and in particular for the preparation of chromatographic separation product making it possible to separate two groups of compounds which have different sizes . In this case, the method is implemented, on the one hand, by choosing particles having a calibrated porosity to allow the penetration of a group of compounds and to prohibit that of the other group, said particles having the physicochemical property ( Pe) not to interact with the compounds to be separated and, on the other hand, by grafting onto these particles a ligand having the physicochemical (Pi) property of retaining the compounds to be separated.

 The process of the invention makes it possible to manufacture particulate products which are particularly well suited for the chromatographic separation of proteins: for example separation in a plasma of proteins of large sizes from molecules of small sizes in order to isolate the latter in particular for an analytical purpose. , or purification of proteins of large sizes from a plasma (in particular Ig.M.) vis-à-vis other proteins and other compounds of smaller sizes.

The invention extends to certain porous regiospecific products prepared by the process of the invention and which do not make it possible to obtain the previous processes; these products are composed of porous silicic particles characterized
by the presence on their internal surface of a hydrophobic ligand having at least one of the following groups: methyl, propyl, butyl, hexyl, octyl, octadecyl, phenyl, cyanopropyl, in order to present the property (Pi) of retaining the hydrophobic compounds,
by the presence on their external surface of a hydrophilic ligand, in particular glyceropropyl, in order to present the property (Pe) of not interacting with the hydrophobic compounds.

 These products can have a wide porosity range in order to apply to separations of molecules of very varied sizes. In the case of a separation of large proteins, a pore diameter equal to or less than 30 nanometers will generally be chosen as a function of the desired cut point.

The description which follows provides several examples of implementation of the process for preparing the invention and examples of application; the implementation of Example 1 is illustrated by the accompanying drawings; on these drawings
FIGS. 1 to 12 illustrate the steps of the preparation process according to the invention,
FIGS. 13 and 14 are chromatograms obtained at the end of the separations described respectively in Example 2 and in Example 4.

EXAMPLE 1
Preparation of a bifunctionalized C18 and diol regiospecific porous product
a) Grafting of octadecyl ligands on silica particles
5 g of "NucléosilR" silica are used (particle diameter 15-25 micrometers, pore diameter 100 Angstrom, "Macherey-Nagel n0 712-32"); a particle is symbolized in Figure 1 with its pores 1, its external surface 2, its internal surface 3; these surfaces originally carry silanol groups 4.

 These particles, dried beforehand for 15 h at 1100 ° C., are suspended in 50 ml of anhydrous toluene containing 0.75 g of dimethyloctadecylchlorosilane and 1 ml of anhydrous pyridine. After 24 h of reaction at 1100 ° C. (reflux of toluene) and with stirring (magnetic stirring 500 rpm), the silica is filtered, washed successively with 30 ml of toluene and 30 ml of ethyl ether and then dried for 15 h at 450 vs.

4.5 g of modified octadecyl silica (C18) are obtained, as symbolized in FIG. 2. The hydrophobic octadecyle 5 ligands are fixed to the internal and external surfaces by siloxane bonds.

b) Filling the pores of silica C18 with the protective compound
The grafted C18 silica previously obtained (1 g) is dispersed in a solution of glycerol tridecanoate (1.5 g) in 10 ml of chloroform. The chloroform is evaporated on a rotary evaporator. As symbolized in Figure 3, each particle is soaked in glycerol tridecanoate 6 which fills its pores and covers its outer surface.

c) Enzymatic hydrolysis
50 ml of 0.2 M tris-maleate buffer are added to the residue previously obtained, then the suspension is sonicated for 15 minutes in the bath to obtain good dispersion.

The suspension is then placed in an enclosure thermostatically controlled at 450 C and stirred at 500 rpm. The pH is maintained at 7 by means of a pH stat which allows, thanks to a recording, to monitor the progress of the reaction.

100 mg of lipase extracted from Candida cvlindracae and having an enzymatic activity of 2 units per mg are then added. One enzymatic unit corresponds to the quantity of enzyme necessary to produce one micromole of fatty acid per minute at 370 ° C. at pH7. The C. cylindracae lipase, symbolized at 7 in FIG. 4, has a molecular weight of approximately 67,000 and therefore does not have the possibility of penetrating inside the pores of C18 silica. When no more fatty acid production is observed (the quantity of fatty acid produced corresponds to the hydrolysis of 900 mg of triglyceride) the silica is decanted, washed with 10 ml of water and then with 2 X 10 ml of a Methanol / H2O mixture, 50/50 (V / V). A particle is obtained, the external surface 2 of which is completely free of the protective compound as symbolized in FIG. 5.

d) Chemical attack
The silica is then resuspended in a Methanol / H2SO4 2N, 50/50 (V / V) mixture (as symbolized in FIG. 6) and is kept stirred by means of a plate stirrer at 370 C for 24 hours. The particles obtained are symbolized in Figure 7, their outer surface being completely free of ligands.

e) Removal of the protective compound
The hydrolyzed silica is then decanted, washed with 3 X 20 ml of Methanol then with 2 X 30 ml of chloroform and 2 X 30 ml of hexane (e). The solvent is symbolized at 8 in Figures 8 and 9 which illustrate the dissolution of the protective compound. After evaporation of the solvent, 0.8 g of regioselectively hydrolyzed silica C18 is obtained. The particles are symbolized in FIG. 10: they carry hydrophobic ligands only on their internal surface, all the sites on the external surface being available. At the macroscopic level, this silica no longer has the character of non-wettability of silica
C18 of departure. On the contrary, it disperses perfectly in water.

f) Attachment of diol grafts on the external surface
1 g of regioselectively hydrolyzed silica is suspended in 20 ml of an aqueous solution of glycidoxypropyltrimethoxysilane adjusted to pH 3.5. The mixture is heated to 900 ° C. with stirring for 2 hours. The silica is then filtered and washed with 20 ml of water, 20 ml of acetone and 20 ml of ethyl ether successively. The moderately acidic conditions used during coupling are sufficient to convert the oxirane to glycol. The particles obtained are symbolized in Figure 11; they are characterized by the presence on their internal surface of hydrophobic ligands 5
C18 and the presence on their outer surface of hydrophilic ligands 9 diol, giving them a very marked regiospecific character. This character was demonstrated by retention measures, on the one hand, of naphthalene (small hydrophobic molecule) which enters the pores and is retained in a similar manner to what happens with a known C18 silica, on the other hand, proteins of sizes larger than those of the pores (molecular weight 67,000) which are not retained by the external surface (measurement "Break Through").

EXAMPLE 2
chromatographic separation using the product obtained in Example 1
Chromatography is carried out of a mixture of two active ingredients, nesdonal (0.1 mg / ml) and phenobarbital (0.3 mg / ml) and a plasma protein serum bovine albumin (BSA) (5 mg / ml). 10 of the above mixture is injected into a 30 × 4.6 mm column filled with the product C18 / diol prepared above. This chromatography is symbolized in FIG. 12 (albumin in 10 and small molecules in 11). Mobile phase: methanol / acetate buffer 0, 1M pH 5, 40/60. Flow rate 1 ml / minute, UV detection 265 nm. We observe on the corresponding chromatogram (Figure 13) a very significant peak of bovine albumin eluted before t (retention time corresponding to the dead volume) followed by the peaks corresponding to nesdonal and phenobarbital . The retention of these compounds is similar on the C18 / diol phase to that on a conventional C18 phase. All the albumin was eluted, without interaction with the porous product.

EXAMPLE 3 Preparation of a bifunctional reqiospecific t porous product from silica 5 C4, 300 Angström
a) Silica C4 was not synthesized but was obtained from the company "Macherey-Nagei"("Nucleosil 300-5C4 ref 71262").

 This large pore silica has a cutoff point of 250,000 daltons for proteins.

 b) Filling the pores of silica C4 with the protective agent.

 1 g of C4 silica is dispersed in a solution of glycerol tridecanoate (2 g) in 20 ml of chloroform which is evaporated on a rotary evaporator.

c) Enzymatic hydrolysis
C. cylindracae lipase having a molecular weight of 67,000 therefore has the possibility of penetrating inside the pores of the product. To keep it outside, a prior polymerization is carried out.

Lipase polymerization
C. cylindracae
1 g of lipase is dissolved in 50 ml of a 5 a solution of glutaraldehyde in 0.1 M phosphate buffer at pH 8. After overnight at room temperature, the unblocked sites are saturated with a glycine solution (0, 1 M final concentration). The reaction mixture is dialyzed and then purified by chromatography on a Nucleosil 300-70H gel permeation column. The fractions corresponding to the dead volume of the column and containing the lipase aggregates of size larger than that of the pores of the support are recovered, dialyzed and lyophilized. 200 mg of polymerized enzyme are obtained having an enzymatic activity of 1 unit / mg.

Hydrolysis
50 ml of 0.2 M trismaleate buffer is added to the residue. The mixture is sonicated for 15 min in the bath, then placed in a stirred chamber, heated to 450 ° C. and whose pH is maintained at 7 by a pH stat. 200 mg of polymerized lipase obtained above are added and left to act until no more fatty acid production is observed. The C4 silica is then decanted, washed with water (20 ml) then with a Methanol / H2O mixture, 50/50 (v / v), (2 x 20 ml).

d) Chemical attack
The silica C4 is then resuspended in a mixture of Methanol / H2SO4 2N, 50/50 (v / v) and stirred on a plate stirrer at 370 C for 24 hours. The silica is then decanted, washed with 20 ml of water, 2 x 20 ml of Methanol, 2 x 20 ml of chloroform and 2 x 20 mI of hexane then dried. 0.8 g of hydrolyzed silica C4 is obtained which is regrafted diol according to the same protocol as above (f).

EXAMPLE 4
Chromatography of a mixture of two plasma proteins of different molecular weight, BSA (67,000) and Ig.M. (900,000)
Chromatography is carried out on a 30 x 4.6 mm column filled with product prepared in Example 3, in an acetate buffer pH 5, 0.1 M with UV detection at 280 nm and a flow rate of 1 ml / minute . 10 l of a solution of BSA (5 mg / ml) and of Ig.M are injected. (0.1 mg / ml).

Only one peak corresponding to Ig.M is observed on the chromatogram (FIG. 14). and eluted before all the BSA is trapped on the support.

Claims (13)

 1 / - Process for the preparation of a porous regiospecific particulate product, in which each porous particle has on its internal surface specific physicochemical properties (Pi) different from those of its external surface, consisting of (a) grafting onto particles with porosity calibrated a ligand able to penetrate into the pores of said particles and to be fixed on all of their surfaces in order to give them the physicochemical properties (Pi), characterized in that, after said grafting  (b) filling the pores of the part-icules by means of a liquid protective compound, enzymatically ment hydrolyzable and having an affinity for the ligand allowing it to penetrate into the pores and to occupy the available volume,  (c) an enzymatic hydrolysis of the protective compound present on the external surface of the particles is carried out by means of an enzyme of size larger than the size of the pores in order to selectively eliminate the protective compound on said external surface,  (d) an attack on the particles is carried out by means of a reactive agent with respect to the ligand and immiscible with the protective compound with a view to selectively eliminating the ligand from the external surface,  (e) and the protective compound still present in the particles is removed.  2 / - Process according to claim 1 for preparing porous particles having on their external surface specific physicochemical properties (P) different from those (Pi) of their internal surface, in which after selective elimination of the ligand (d)  (f) another ligand is grafted onto the external surface of the particles in order to give the latter specific properties (Pe)  3 / - Process according to claim 1 for preparing porous particles having on their external surface specific physicochemical properties (Pe) different from those (Pi) of their external surface, in which one chooses starting particles having on the whole of their surfaces the specific properties (Pe)  4 / - Method according to claim 2 characterized in that  (a) particles with a silicic porous skeleton are chosen and a hydrophobic ligand capable of giving siloxane bonds with the silica is grafted onto it,  (b) the pores of the silicic particles are filled with a hydrophobic protective compound capable of breaking down into sub-elements having a weaker hydrophobic character,  (c) the enzymatic hydrolysis of the protective compound present on the external surface of the particles is carried out by means of a hydrolase with a view to breaking it down into its subelements,  (d) a chemical attack on the particles is carried out by means of an acid solution, immiscible with the hydrophobic protective compound.  5 / - Method according to claim 4, characterized in that  (a) a hydrophobic ligand having at least one of the following groups is grafted onto the silicic particles: methyl, propyl, butyl, hexyl, octyl, octadecyl, phenyl, cyanopropyl,  (b) the pores of the particles are filled with a protective compound of the group following triglyceride, per-ester of mono or oligosaccharide, hydrophobic polypeptide or their acylated derivatives,  (c) hydrolysis is carried out using a hydrolase adapted to the protective compound: lipase, esterase, peptidase,  (d) the chemical attack is carried out using a strong acid solution of pH less than 1, the solvent of said solution being chosen to be immiscible with the protective compound.  6 / - Method according to one of claims 1 to 5 for the preparation of a particulate product of porosity greater than 10 nanometers, characterized in that (c) the enzymatic hydrolysis is carried out by means of a polymerized enzyme or fixed on a macromolecule in order to give it a molecular weight greater than 80,000.  7 / - Method according to one of claims 1 to 6, characterized in that:  (e) the protective compound is eliminated by washing with a liquid-volatile liquid having an affinity for the ligand at least equal to that of said protective compound, said liquid then being eliminated by evaporation.  8 / - Process according to claims 6 and 7 taken together, characterized in that (e) the protective compound is removed by washing with a liquid from the following group: alkane, organochlorine, ketone, ether, volatile alcohol.  9 / - Application of the method according to one of claims 1 to 8 for the preparation of a chromatographic separation product for separating two groups of compounds having different sizes, in which (a) we choose particles having a porosity calibrated to allow the penetration of a group of compounds and to prohibit that of the other group, said particles having the physicochemical (Pe) property of not interacting with the compounds to be separated and a ligand having the physicochemical property (Pi) of retaining the compounds to be separated.  10 / - Application according to claim 9 for the preparation of a chromatographic separation product for separating in a plasma molecules of small sizes from proteins of large sizes in order to isolate said molecules of small sizes.  11 / - Application according to claim 9 for the preparation of a chromatographic separation product for purifying proteins of large sizes from a plasma with respect to other proteins and other compounds of smaller sizes.  12 / - Regiospecific porous particulate product prepared by implementing the method according to claim 6, comprising porous silicic particles characterized  by the presence on their internal surface of a hydrophobic ligand having at least one of the following groups: methyl, propyl, butyl, hexyl, octyl, octadecyl, phenyl, cyanopropyl, in order to present the property (Pi) of retaining the hydrophobic compounds,  by the presence on their external surface of a hydrophilic ligand, in particular glyceropropyl, in order to present the property (Pe) of not interacting with the hydrophobic compounds.  13 / - Porous particulate product according to claim 12 for the chromatographic separation of proteins, in which the pores of the particles are of diameter equal to or less than 30 nanometers.