RU2790024C2 - Organically modified mineral microparticles, their production methods, and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to the field of production and use of mineral microparticles. A method for the production of organically derivatized mineral microparticles is proposed, which includes provision of a solution of one or more organic polyphosphates, provision of mineral microparticles, suspension of the specified mineral microparticles in the specified solution of one or more organic polyphosphates, and mixing of the resulting suspension. At the same time, mixing of the suspension is carried out until the absence of change in a zeta potential of organically derivatized mineral microparticles for more than 10% during a period of time of 5 minutes, with determination, by means of analysis, of reaction product samples isolated with an interval of 5 minutes ± 10 seconds. Mineral microparticles are selected from microparticles of aluminum phosphate, amorphous aluminum hydroxyphosphate, and/or potassium phosphate, and the specified mineral microparticles have a size in the range from 0.01 mcm to 10 mcm. Organically derivatized mineral microparticles obtained by the specified method and the use of the specified particles as biomolecule delivery systems, in particular, as vaccine adjuvants or in blood fractionation are also disclosed.

EFFECT: invention provides simplification of a method for the production of organically derivatized mineral microparticles, increase in the yield of a reaction on a surface of particles, which leads to greater coverage of the surface, wherein obtained microparticles have increased thermal resistance and improved adsorption capacity relatively to antigens.

14 cl, 18 dwg, 17 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to the field of mineral microparticles consisting of aluminum hydroxide, aluminum phosphate or calcium phosphate, or mixtures thereof, for use as a system for the delivery or adsorption of biomolecules, for example, for use as vaccine adjuvants. More specifically, the present invention provides organically derivatized aluminum hydroxide, aluminum phosphate, or calcium phosphate microparticles, their uses, and methods for their preparation.

BACKGROUND OF THE INVENTION

Mineral adjuvants such as aluminum adjuvants including aluminum phosphate, aluminum hydroxide and calcium phosphate have been successfully used for decades to enhance the immune response to killed, inactivated and subunit vaccine antigens. Aluminum-containing adjuvants are currently the most widely used adjuvants in both veterinary and human vaccines. This has generated a lot of interest in improving the effectiveness of these mineral-based adjuvants. More effective adjuvants and vaccines mean potentially simplified and reduced vaccination interventions, which is a way to potentially reduce the burden on patients, medical staff, and improve the cost-benefit ratio.

An important aspect of the effectiveness of adjuvants depends on the delivery and presentation of antigens to the cellular or humoral branches of the immune system in the draining lymph node. The amount or loading of antigen adsorbed by the adjuvant (expressed in terms of weight) is one important parameter because it determines the likelihood that some antigen molecules together with the adjuvant will be recognized as foreign material and induce an appropriate immune response. Achieving higher doses of antigen per unit of adjuvants is potentially desirable as it allows for lower adjuvant burden in the case of the same dose of antigen, which can reduce manufacturing costs. The strength and nature of the binding of the antigen to the adjuvant is another important parameter because it determines the likelihood that the antigen will be presented in the form of particles (bound to adjuvant particles) instead of being released in free form into the surrounding physiological environment. This may be especially true in the case of small soluble peptides with high diffusion constants, in fact peptides as vaccines are notoriously known to induce poor immunogenicity and require adjuvants ((Li, W., Joshi, M. D., Singhania, S., Ramsey, K. H., & Murthy, A. K. (2014) Peptide vaccine: progress and challenges Vaccines, 2(3), 515-536.]). recognition by antigen-presenting cells, which limits the effectiveness of the vaccine.However, studies have shown that too strong adsorption of antigens on aluminum-containing adjuvants can lead to a decrease in the effectiveness of the vaccine due to insufficient processing and presentation of adsorbed antigens by antigen-presenting cells (Hansen B. et al., Relationship between the strength of antigen adsorption to an aluminum-containing adjuvant and the immune response, Vaccine, 2007). Thus, the vaccine community and industry are under pressure to better control the way antigens are best delivered in combination with adjuvants. A possible key to this problem is the ability to design adjuvants adapted to the physicochemical characteristics of the antigen to be delivered.

Another aspect of adjuvant effectiveness may be related to particle size. For example, Morefield et al. (Morefield GL et al., Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro, Vaccine, 2005) and Li et al. (Li X. et al., Aluminum hydroxide nanoparticles show a stronger vaccine adjuvant activity than traditional aluminum hydroxide micro-particles, J. Control. Release, 2014) have found that smaller aluminum-containing particles work better than larger particles, in particular for inducing antigen-specific humoral responses, since smaller particles particles can be transported to nearby afferent lymph nodes. Many particle size reduction methods are currently available, such as sonication, high pressure shearing, filtration, homogenization, milling, microfluidization, precipitation, or recrystallization. In addition, synthetic protocols are well known which result in particles of a certain size. Adjustment of synthesis parameters such as stoichiometric ratio of Al ³⁺ and PO ₄ ^3- (or OH) and pH can potentially lead to different particle sizes (Burrell LS et al., Aluminum phosphate adjuvants prepared by precipitation at constant pH. Part I: composition and structure, Vaccine, 2000; Burrell LS et al., Aluminum phosphate adjuvants prepared by precipitation at constant pH, Part II: physicochemical properties, Vaccine, 2000).

However, there remains a need for improved methods that are less time consuming and costly, that provide better control of particle size uniformity, and that produce mineral microparticles with optimal antigen binding properties.

SUMMARY OF THE PRESENT INVENTION

In the simultaneously pending international application PCT/EP 2017/076232, the authors of the present invention describe how to stir a suspension of mineral microparticles of certain di- or trivalent metal salts selected from aluminum phosphate, amorphous aluminum hydroxyphosphate and/or calcium phosphate, or mixtures thereof, in an aqueous solution of one or more inorganic polyphosphates, for example, a solution of an inorganic polyphosphate, causes a modification of these mineral microparticles, which leads both to a significant change in their nominal electrostatic potential, and to a decrease in particle size.

Without wishing to be bound by any theory, these modifications are believed to be caused by a substitution reaction, or exchange of ligands, hydroxide or orthophosphate groups by inorganic polyphosphate ions on the surface of said mineral microparticles.

The present inventors at that time discovered that a similar ligand displacement or exchange reaction could be achieved using organic polyphosphates of general formula 1A or 1B:

where n is an integer in the range from 0 to 5 and m is an integer in the range from 2 to 10, and where R _a denotes organic substituents selected from adenosine and other nucleosides, thiamines, carbohydrates and isoprenes, and R _b denotes organic substituents selected from inositols and other cyclites, as well as carbohydrates.

Thus, organic polyphosphates in accordance with the present invention can be considered as "polyphosphates" on the basis that they contain at least one polyphosphate group -O-PO ₂ -(-O-PO ₂ -) _n -O-PO ₃ , where n is an integer ranging from 0 to 5, or contain two to ten phosphate groups, -O-PO ₃ .

As examples of organic polyphosphates of formula 1A, mention may be made of adenosine and other nucleoside phosphates such as ATP and ADP, thiamine phosphates such as thiamine triphosphate and α-diphosphate, carbohydrate polyphosphates such as α-D-ribose-5-triphosphate and L-ascorbic acid-2 -triphosphate, pterin phosphates and isoprenoid phosphates such as geranyl diphosphate.

As examples of organic polyphosphates of formula 1B, inositol phosphate and other cyclite phosphates such as inositol bisphosphate (IP2), inosittris phosphate (IP3), inositol tetraphosphate (IP4), inositolpentakisphosphate (IP5) and inositol hexaphosphate (IP6), also known as phytic acid or phytate ( in the form of salt). Further examples of formula 1B include carbohydrate phosphates such as glucose-1,6-bisphosphate, fructose-1,6-bisphosphate, fructose-2,6-bisphosphate, ribulose-1,5-bisphosphate, 2-deoxy-D-ribose- 1,5-bisphosphate and 1,3- and 2,3-diphosphoglycerate.

The present invention disclosed here provides unexpected advantages over the disclosure of co-pending International Application PCT/EP 2017/076232. The use of organic polyphosphates of general formula 1A or 1B leads to an increase in the yield of the reaction on the surface of the particles, which leads to a greater coverage of the surface. As a consequence, there is a greater change in the nominal electrostatic potential of the microparticles than before, and it seems easier to control the decrease in particle size, since the stability of the resulting smaller organically derivatized mineral micro- and nanoparticles seems to be higher. Secondly, and most importantly, the use of organic polyphosphates, especially of general formula 1B, results in a higher thermal stability of the final modified mineral microparticles than can be achieved with the inorganic polyphosphates described in the co-pending international application PCT/EP 2017/076232, which important for autoclaving/sterilization. Finally, the adsorption capacity for antigens, as shown by the model hen egg lysozyme (HEL) antigen, of the organically derivatized mineral microparticles according to the present invention is higher than that observed in the microparticles modified with inorganic polyphosphates described in the co-pending international application PCT/EP 2017/076232. The present inventors believe that this is probably due to the chelating effect of positively charged residues in proteins and polypeptides such as lysine and arginine on the organic polyphosphates of general formula 1B in particular, resulting in stronger adsorption to the surface of the particles.

Organic polyphosphates of general formula 1A may have lower heat resistance than polyphosphates of general formula 1B. Nucleotides (examples of organic polyphosphates of general formula 1A) are biological multifunctional molecules that play a central role in energy metabolism, DNA synthesis and signaling in cells. They consist of a nucleoside moiety with a mono-, di-, or triphosphate group at the 5' position of the -ose moiety. Due to the affinity of phosphates for aluminum-containing gels, nucleotides such as ATP are adsorbed on aluminum-containing adjuvants and therefore can be used to change the electrostatic potential of the surface of the adjuvant particles. Similarly, aluminum-containing adjuvant particles can be used to transport and deliver nucleotides into cells. However, aluminum-containing adjuvants are typically autoclaved, which is incompatible with the chemical integrity of the nucleotides. Under the high temperature and pressure conditions encountered in autoclaving, hydrolysis of the phosphorus anhydride bonds of the inorganic polyphosphate chain of ADP or ATP will occur and the physical and chemical properties of these molecules will be lost or adversely altered. As an alternative to autoclaving, solutions of concentrated ATP, ADP, or AMP up to 100 mM in water can be sterilized by filtration through a 0.22 µm or 0.10 µm membrane and the sterilized solutions loaded into autoclaved aluminum-containing adjuvant particles to the appropriate final concentration, for example, from 0.5 mM to 2 mM, which eliminates the need for autoclaving.

The reaction with organic polyphosphates of formula 1A or formula 1B has been carried out with certain divalent or trivalent metal salts selected from aluminum phosphate, aluminum hydroxide, amorphous aluminum hydroxyphosphate and/or calcium phosphate, but is applicable to a wide range of other mineral microparticles.

The displacement reaction, or ligand exchange, can be carried out over a wide temperature range that is compatible with the aqueous reaction medium.

Throughout this application, the term "unmodified mineral microparticle" will refer to the starting material for said displacement/ligand exchange reaction, while the term "organically derivatized mineral microparticle" will refer to the result or product of said displacement/ligand exchange reaction.

In FIG. 6A shows the proposed reaction between a mineral microparticle starting material (here: aluminum phosphate) and an organic polyphosphate solution that occurs on the surface of the microparticle. As used herein, aluminum phosphate (adjuvants) is essentially aluminum hydroxyphosphate in which some of the hydroxyl groups (Al-OH) have been replaced by orthophosphate groups (Al-OPO ₃ , denoted by Pi). The reaction occurs primarily between available Al-OH groups and dissolved organic polyphosphate ions, and over time leads to a gradual more complete coverage of the surface of the aluminum phosphate particles with organic polyphosphate groups (denoted nP _org ). The substitution reaction presumably also involves orthophosphate groups; those. so that the available orthophosphate groups are to some extent replaced by organic polyphosphate groups. However, hydroxyl groups are expected to be better leaving groups than orthophosphate groups and are therefore preferentially substituted.

The starting material of mineral microparticles actually consists of clusters/aggregates of smaller crystallites, as seen by electron microscopy in FIG. 6C. It is believed that the crystallites are held together, at least in part, by hydrogen bonds between the hydroxyl groups located on the particles. As soon as hydroxyl groups begin to exchange for organic polyphosphate groups, a decrease in the number of hydrogen bonds weakens the cohesion of crystallites. At the same time, the growing number of closely spaced organic polyphosphate groups with their multiple negative charge leads to further destabilization of the crystallite cluster and its eventual disaggregation (Fig. 6B) as seen by electron microscopy (Fig. 6C).

However, compared to the inorganic polyphosphate-modified mineral microparticles described in co-pending international application PCT/EP 2017/076232, the stability of the obtained organically derivatized mineral microparticles of the present invention is increased. Firstly, the phosphoester bond in the type 1B molecule has a much higher activation energy than the phosphoric anhydride bond between two orthophosphates (in the molecule of general formula 1A), which makes it possible to perform heat treatment for sterilization without hydrolyzing most of the phosphate groups and, therefore, preserving properties in the form of modification by such organic polyphosphates. Secondly, and without being bound by any theory, the present inventors believe that the increased stability of the particles in suspension (colloidal) is due, at least in part, to the increased steric hindrance created by the organic phosphate groups that have themselves attached to the surface of the mineral microparticle. The organic residue (R _a or R _b ) of organic phosphate groups is much larger than the inorganic polyphosphate ions discussed in PCT/EP 2017/076232. Therefore, after some of the original hydroxyl or orthophosphate groups located on the surface of the mineral microparticles have been replaced by organic polyphosphate groups, access to the remaining hydroxyl or orthophosphate groups becomes effectively blocked for further replacement.

In addition, the authors of the present invention found that organically derivatized mineral microparticles have increased ability to bind positively charged biomolecules or biomolecules with positive charge sites, more specifically antigen-binding properties. This property is presumably due to the increased attraction of positively charged biomolecules, such as antigens, to the surface of said organically derivatized mineral microparticles, resulting in increased adsorption capacity and/or binding strength, ie. increasing the association constant. In view of this, the organically derivatized mineral microparticles according to the present invention can, for example, be used as improved vaccine adjuvants.

Accordingly, in a first aspect, there is provided herein a method for producing organically derivatized mineral microparticles, which comprises stirring a suspension of mineral microparticle starting material in a solution of one or more organic polyphosphates, said mineral microparticles being selected from aluminum phosphate, aluminum hydroxide, amorphous aluminum hydroxyphosphate and/or phosphate microparticles. calcium or mixtures thereof, and moreover, the specified organic polyphosphate is selected from compounds of formula 1A or 1B:

where n is an integer in the range from 0 to 5 and m is an integer in the range from 2 to 10, and where R _a denotes organic substituents selected from adenosine and other nucleosides, thiamines, carbohydrates and isoprenes, and R _b denotes organic substituents selected from inositols and other cyclites, as well as carbohydrates.

Stirring is continued for a time sufficient to allow a reaction to take place on the surface of the mineral microparticle, whereby the available hydroxide and to some extent also the orthophosphate groups are exchanged, i.e. replaced by organic polyphosphate ions. The displacement reactions can be allowed to proceed for a shorter or longer period to cause partial or more complete replacement of the ions.

The above chemical reaction can be described as a ligand exchange or ligand replacement reaction by which hydroxide anions located on the surface of the raw material of the mineral microparticles are exchanged for organic polyphosphate ions. Since the phosphate groups in organic polyphosphate ions have a much greater affinity for Al(III) than the hydroxide groups, the equilibrium shifts to the right (eg: AdjuPhos+nPorg → AdjuPhos-nPorg+OH ^- ), in accordance with Le Chatelier's principle.

The ligand exchange/displacement reaction described above generally occurs in an aqueous reaction medium and can be carried out in the temperature range from about 0 to about 100 degrees Celsius (°C), for example, from 5 to 95°C, or from 10 to 90° C or 15 to 85°C or 20 to 80°C or 25 to 75°C or 30 to 70°C or 40 to 60°C. The reaction is conveniently carried out at ambient temperature or at about 20°C ± 5-10°C. The displacement reaction or ligand exchange can also be carried out at moderate pressure at temperatures above >100°C.

During the ligand exchange/displacement reaction, the particle size decreases compared to the size of the original (unmodified) particles. The size reduction depends on how far the substitution reaction is allowed to proceed, reaction rates and reaction time. Reaction rates are complex because they depend on parameters such as the specific starting material, the type of organic polyphosphate, the initial concentration of organic polyphosphates, the pH of the bulk solvent, the reaction temperature, and the interfacial effects (at the interface between the particle surface and the bulk solvent).

The progress of the reaction can be monitored either in real time by in situ measurement or offline by sampling and subsequent analysis of the collected sample. Thus, no inventive work is required for the skilled person to establish the correct reaction time for a particular combination of mineral microparticle starting material with organic polyphosphates. For example, the use of ion chromatography would quantify the amount of organic polyphosphate ions disappearing from the bulk solution as a function of time and provide an indirect determination of the amount of such ions that are adsorbed on the surface of aluminum phosphate or calcium phosphate particles. Alternatively, direct measurement of organic polyphosphate ions adsorbed on the surface of the particles can be carried out using sensitive electrochemical methods such as electrochemical scanning tunneling microscopy (STM), electrochemical permeability of quartz crystals (EQCA), or even by detecting changes in mass, using a sensitive quartz microbalance (QCM). According to the electric double layer theory, changes in the electrokinetic potential (zeta potential) of the particles also reflect the adsorption of polyphosphates on the Helmholtz plane in the form of the surface of the particles.

An appropriate procedure for monitoring the ligand exchange/displacement reaction described above thus involves monitoring the change in zeta potential with time or with concentration of the organic polyphosphate, since a highly sensitive surface electrostatic charge will reflect chemical modifications to the surface of the particle. Additional methods for monitoring particle surface modification include, but are not limited to, Raman scattering and infrared absorption spectroscopy, which detects specific chemical signatures of adsorbed organic polyphosphate, secondary ion mass spectroscopy, which monitors secondary ions (from organic polyphosphates) desorbed from particle surfaces at irradiation with ion beams, or elemental analysis, such as energy dispersive X-ray scattering, to detect the presence of carbon from the modifier in particles that are initially devoid of carbon. The ligand exchange/substitution reaction can be stopped when the desired zeta value is reached by removing the organically derivatized mineral microparticles from the organic polyphosphate solution and washing them with water. Alternatively, the reaction can be stopped if there is no change in zeta potential of more than 10%, preferably no more than 5%, over a period of 5-10 minutes, such as 5 minutes, 7.5 minutes or 10 minutes.

For convenience, the above-described ligand exchange/displacement reaction involving mineral microparticles in an organic polyphosphate solution will hereinafter and throughout this patent application be referred to as "equilibration" or "equilibration step".

In a second aspect, the present invention provides organically derivatized mineral microparticles that can be obtained by the equilibration process according to the first aspect.

In specific embodiments, said balancing results in partial or complete replacement of phosphate ions or hydroxide ions that are located on the surface of the microparticles with organic polyphosphate ions.

In other specific embodiments, said balancing is carried out using organic polyphosphate ions of general structure 1A.

In preferred embodiments, said equilibration is carried out using organic polyphosphate ions of general structure 1B.

In a particularly preferred embodiment, said equilibration is carried out using phytic acid/IP-6/inositol hexaphosphate or a salt thereof.

In specific embodiments, said balancing said mineral microparticles using an organic polyphosphate increases the nominal electrostatic potential of said mineral microparticles.

In particular embodiments, said balancing said mineral microparticles using an organic polyphosphate reduces the size of said mineral microparticles.

In specific embodiments, said organic polyphosphate solution comprises a negatively charged organic polyphosphate salt, preferably said solution having a physiological pH.

In another embodiment, said organic polyphosphate solution comprises a mixture of salts of two or more negatively charged organic polyphosphates.

In particularly preferred embodiments, said organic polyphosphate solution comprises inositol hexaphosphate (phytic acid/IP-6).

In other specific embodiments, said balancing step is performed

i. at ambient temperature or at approximately 20°C ± 5-10°C,

ii. within a reaction period of at least 2 minutes, and/or

iii. with an initial concentration of organic polyphosphates of not less than 0.1 mM and not more than 20 mM, and/or

iv. at pH values between pH 4.0 and pH 7.5.

In particular embodiments, said mineral microparticle starting material has

i. a nominal size of not less than 0.1 µm and not more than 5 µm when measured in a colloidal suspension by DLS or laser diffraction, and/or

ii. a zeta potential of not less than -20 mV and not more than -30 mV for aluminum phosphate when measured at pH 7.0 in distilled water.

iii. a zeta potential of not less than +10 and not more than +20 mV for aluminum hydroxide when measured at pH 7.0 in distilled water.

iv. a zeta potential of not less than -10 mV and not more than -20 mV for calcium phosphate when measured at pH 7.0 in distilled water.

Also provided herein are organically derivatized mineral microparticles obtained by balancing mineral microparticles selected from a list consisting of aluminum hydroxide microparticles, aluminum phosphate microparticles, amorphous aluminum hydroxyphosphate microparticles and/or calcium phosphate, wherein at least a portion of the hydroxide ions located on the surface of these mineral microparticles, is replaced by organic polyphosphate ions.

In specific embodiments, the organically derivatized mineral microparticles of the present invention have

(i) a nominal zeta potential of at least -35 mV for aluminum phosphate when measured at pH 7.0 in distilled water,

(ii) a zeta potential of not less than +10 and not more than +20 mV for aluminum hydroxide when measured at pH 7.0 in distilled water.

(iii) a nominal zeta potential of at least -40 mV for calcium phosphate when measured at pH 7.0 in distilled water,

(iv) a nominal size of not less than 0.01 µm and not more than 2 µm,

(v) a stoichiometric ratio of Al to P equal to 1.2 +/- 0.15 to 1,

(vi) a stoichiometric ratio of Ca to P equal to 1.7 +/- 0.2, and/or

(vii) the maximum coverage of the surface of said microparticles with organic polyphosphate ions, leading to the maximum stoichiometric ratio nP _org /Al=0.05.

In other specific embodiments, the organically derivatized mineral microparticles are provided as a slurry or suspension in a sterilized, buffered aqueous saline solution.

In still other specific embodiments, the organically derivatized mineral microparticles are provided as a spray freeze-dried powder (Wanning et al., Pharmaceutical spray freeze drying. International Journal of Pharmaceutics, 2015, 488:136-153), ready to be reconstituted in sterile water. .

In another specific embodiment, the organically derivatized mineral microparticles are provided as a dry lyophilized formulation, optionally containing trehalose as a stabilizing excipient.

In specific embodiments, said organically derivatized mineral microparticles according to the present invention have enhanced biomolecular binding abilities compared to the mineral microparticle starting material, wherein said biomolecule preferably has an opposite charge to that of said modified microparticle, or wherein said biomolecule is neutral when said the modified microparticle is neutral.

In specific embodiments, said biomolecule is an antigen.

It also provides for the use in medicine of these organically derivatized mineral microparticles in accordance with the present invention.

In specific embodiments, these organically derivatized mineral microparticles according to the present invention are used as delivery or adsorption systems for biomolecules.

In specific embodiments, said biomolecule delivery system is a vaccine adjuvant.

In specific embodiments, said organically derivatized mineral microparticles according to the present invention are used as vaccine adjuvants in vaccines.

In specific embodiments, said organically derivatized mineral microparticles according to the present invention are used in blood fractionation, preferably as biomolecule adsorption systems.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Adsorption capacity for hen egg lysozyme (HEL) of Adju-Phos® adjuvant particles treated with IP6.

In FIG. 1 shows the amount of HEL adsorbed on Adju-Phos® particles as a function of the concentration of HEL in suspensions. It appears that treatment of Adju-Phos® particles with IP6 increases the adsorption capacity of the particles compared to treatment with metahexaphosphate (m6Pi), with a maximum capacity of 1.4 mg HEL/mg Al ³⁺ equivalents in the case of IP6 compared to 1.1 mg HEL/mg Al ³⁺ equivalents in the case of m6Pi and ~0.8 mg HEL/mg Al ³⁺ equivalents in the case of orthophosphate (Pi). In addition, these results show that the excellent HEL adsorption capacity of IP6-treated Adju-Phos® particles is achieved even after autoclaving as the Adju-Phos sterilization process.

Fig. 2: Effect of inositol hexaphosphate (IP6) on the zeta potential of calcium phosphate adjuvant particles.

In FIG. Figure 2 shows the dependence of the zeta potential of calcium phosphate particles on the concentration of orthophosphate (Pi), meta-hexaphosphate (m6Pi), and inositol hexaphosphate (IP6). Treatment with any phosphate results in an increase in the zeta potential of the calcium phosphate particles, suggesting an adsorption event of phosphate ions on the surface of the particles. This concentration dependence of the zeta potential resembles a Langmuir-type adsorption isotherm, which suggests saturation of the binding sites. While orthophosphate results in an increase from approximately -5 mV to -20 mV, the m6Pi and IP6 polyphosphates show a sharper increase in zeta potential to approximately -50 mV and -60 mV, respectively.

Figure 3: Adsorption capacity for hen egg lysozyme of calcium phosphate particles treated with inositol hexaphosphate (IP6).

In FIG. 3 shows the amount of HEL adsorbed on calcium phosphate particles as a function of the concentration of HEL in suspensions. It appears that treatment of calcium phosphate particles with IP6 increases the adsorption capacity of the particles compared to treatment with metahexaphosphate, with a maximum capacity of ~180 µg/mg Ca ²⁺ equivalents in IP6 compared to ~120 µg/mg Ca ²⁺ equivalents in in the case of m6Pi and ~45 µg/mg Ca ²⁺ equivalents in the case of Pi.

Fig. 4: Adsorption capacity for adenosine, adenosine 5'-monophosphate (AMP) and adenosine 5'-triphosphate (ATP) of Adju-Phos® particles.

Fig. 4 clearly demonstrates how the adsorption capacity for ATP is increased by at least 5 times compared to AMP and unphosphorylated adenosine. It can be concluded that the presence of a condensed triphosphate group in ATP rather than a monophosphate group in AMP is responsible for this effect. The mechanisms by which the triphosphate group increases adsorption capacity for adenosine remain to be explored. It can be assumed that the presence of additional hydroxyl groups in the polyphosphate backbone statistically increases the likelihood of binding to Adju-Phos® via ligand exchange, or that these reactive hydroxyls are less sterically hindered in ATP. Alternatively, the reactivity of the terminal hydroxyl groups in the triphosphate chain of ATP is higher than in the case of AMP, or that the phosphate at the α-position in ATP becomes more reactive due to the action of pyrophosphate (phosphates at the β- and γ-positions) as a leaving group.

Fig. 5: Effect of inositol hexaphosphate (IP6) concentration on the ζ potential of Adju-Phos.

In FIG. 5 shows the same pattern as observed with other phosphate and polyphosphate ions when, in the first phase, the ζ potential increases from -43 mV to -60 mV in response to the adsorption of inositol hexaphosphate (IP6) on the surface of Adju-Phos® particles, and when, in the second phase, the ζ potential decreases from -60 mV to -35 mV, most likely as a result of an increase in ionic strength, leading to Coulomb charge shielding and contraction of the electrostatic double layer. Thus, in this experiment, it is found that the optimal balance is achieved in the case of concentration=5 mm IP6.

Fig. 6A and 6B. Hypothetical substitution reaction on the surface of aluminum hydroxyphosphate particles and dissolved organic polyphosphate ions.

In FIG. 6A shows how mineral microparticles (eg, aluminum hydroxide) react with organic polyphosphate to form organically derivatized mineral microparticles in which some of the hydroxy groups have been replaced by organic polyphosphate groups.

In FIG. 6B shows how organically derivatized mineral microparticles eventually begin to disintegrate due to (presumably) charge-to-charge repulsion.

Fig. 6C. Electron micrographs (TEM) of Adju-Phos® (top) and Adju-PhosZP (bottom) particles.

In FIG. 6C compares the microscopic morphology of unmodified Adju-Phos® particles (upper image) with that of Adju-PhosZP modified with 2 mM Na-IP6. The Adju-Phos® particles have an average distribution data size of ~2.5 µm and are composed of aggregates of small lamellar crystallites of ~20 nm. Upon treatment with IP6, microscopic Adju-Phos® particles partially disaggregate, as seen in the lower image as individual ~20 nm crystallites.

Fig. 7. Adsorption capacity for adenosine, adenosine 5'-monophosphate (AMP), and adenosine 5'-triphosphate (ATP) of Adju-Phos particles.

In FIG. 7 shows how the adsorption capacity for ATP increases by at least 2 times compared to AMP and unphosphorylated adenosine. It can be concluded that the presence of a condensed triphosphate group in ATP rather than a monophosphate group in AMP is responsible for this effect. The mechanisms by which the triphosphate group increases adsorption capacity for adenosine remain to be explored. It can be assumed that the presence of additional hydroxyl groups in the polyphosphate backbone statistically increases the likelihood of binding to Adju-Phos® via ligand exchange, or that these reactive hydroxyls are less sterically hindered in ATP. Alternatively, the reactivity of the terminal hydroxyl groups in the triphosphate chain of ATP is higher than in the case of AMP, or that the phosphate at the α-position in ATP becomes more reactive due to the action of pyrophosphate (phosphates at the β- and γ-positions) as a leaving group.

Fig. 8. Adsorption capacity for adenosine, adenosine 5'-monophosphate (AMP) and adenosine 5'-triphosphate (ATP) of Alhydrogel particles.

In FIG. 8 shows how ATP and AMP, both phosphorylated forms of adenosine, are highly adsorbed on Alhydrogel compared to unphosphorylated adenosine. There is no apparent difference between the adsorption capacities for ATP and AMP, indicating that the presence of the triphosphate chain in ATP does not appear to provide any additional improvement over the monophosphate group in AMP. This is in contrast to Adju-Phos, whose adsorption capacity for ATP is higher than that for AMP for the same concentration range. Aluminum hydroxide, due to its nature, has a much higher proportion of hydroxide groups (per mole of aluminum) than Adju-Phos (aluminum hydroxyphosphate), which can undergo a ligand exchange reaction with phosphates. Therefore, Alhydrogel is expected to have a greater adsorption capacity than Adju-Phos for phosphorylated molecules such as ATP or AMP. The reactivity of Alhydrogel towards AMP and ATP is the same or similar since it can only involve the terminal phosphate group with little difference in reactivity.

Fig. 9. Effect of concentration of adenosine-5'-triphosphate (ATP) and adenosine-5'-monophosphate (AMP) on the ζ-potential of Adju-Phos.

In FIG. 9 shows that the ζ potential of Adju-Phos increases (in absolute terms) from -40 mV to -48 mV as the ATP concentration increases to 1 mM. A further increase in the ATP concentration from 1 mM to 6 mM does not lead to a further increase in the ζ potential, which indicates that the saturation of ATP adsorption sites is achieved within 1 mM. Unlike ATP, treatment with AMP resulted in a decrease in the ζ potential (in absolute terms) from -40 mV to -35 mV. This difference between ATP and AMP can be explained by the greater adsorption capacity of Adju-Phos for ATP than for AMP (Fig. 1) and the higher negative charges that ATP has (triphosphate instead of monophosphate). No significant changes in ζ-potential were observed for Adju-Phos treated with non-phosphorylated adenosine.

Fig. 10. Effect of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP) concentrations on the height of the Adju-Phos precipitate layer.

Fig. 10 is a photograph of homogenized suspensions in plastic spectrophotometric cuvettes showing the effect of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP) concentration on Adju-Phos precipitate layer height. ATP concentrations from left to right: 0.0 mM, 0.5 mM, 1.0 mM, 2.0 mM, 3.0 mM, 4.0 mM and 5.0 mM.

Fig. 11. Effect of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP) concentrations on the height of the Adju-Phos sediment layer.

In FIG. 11 shows that the height of the Adju-Phos precipitate layer, measured after 48 hours at rest, decreases with ATP concentration from 16 mm to 9.5 mm. The most dramatic decrease in layer height occurs from 0.0 to 1.0 mM ATP, which corresponds to the concentration range in which the Adju-Phos ζ potential is most elevated (Fig. 9), suggesting that the two parameters are related. Interestingly, AMP treatment has the opposite effect, with the height of the Adju-Phos sediment layer increasing in a concentration dependent manner. It appears that the triphosphate chain in adenosine 5'-triphosphate induces packing of the Adju-Phos particles, despite the increased ζ potential, due to which electrostatic repulsive forces are expected to separate the particles from each other. A similar picture is observed with inorganic polyphosphates and phytate (inositol hexaphosphate).

Fig. 12. Adsorption capacity towards adenosine, adenosine-5'-monophosphate (AMP) and adenosine-5'-triphosphate (ATP) of calcium phosphate particles.

In FIG. 12 shows that calcium phosphate adsorbs ATP, AMP, and adenosine, and that adsorption capacity increases with concentration, apparently linearly, with little sign of saturation (within the range of nucleotide concentrations used in this experiment). ATP and AMP also appear to be adsorbed in greater amounts than adenosine, suggesting that phosphate groups are responsible for the higher adsorption capacity. It can be hypothesized that the presence of phosphate groups in AMP and ATP increases calcium phosphate adsorption via ligand exchange with hydroxide ions in the calcium phosphate matrix, since the calcium phosphate adjuvant used in this experiment is a (water-insoluble) hydrated calcium salt. and phosphate with an unknown stoichiometric number of hydroxides.

Fig. 13. Effect of the concentration of adenosine-5'-triphosphate (ATP) and adenosine-5'-monophosphate (AMP) on the ζ-potential of calcium phosphate.

In FIG. 13 shows that the ζ potential of calcium phosphate increases (in absolute terms) from -5 mV to -40 mV with increasing ATP concentration to 1 mM. This sharp increase in the ζ-potential is defined as specific adsorption on the particle surface - the Stern layer (according to the double electrostatic layer model). A further increase in the ATP concentration from 1 mM to 6 mM leads only to a slight increase in the ζ potential, which indicates that the saturation of ATP adsorption sites is achieved within 1 mM. In contrast to ATP, treatment with AMP leads only to a slight increase in the ζ potential (in absolute values) from -5 mV to -10 mV, which also indicates specific adsorption on the particle surface. Since the adsorption capacity for ATP and AMP of calcium phosphate seems to be the same (Fig. 3), the marked difference in ζ potential between ATP and AMP can be explained by the higher negative charge that ATP has (triphosphate instead of monophosphate). In the case of calcium phosphate treated with non-phosphorylated adenosine, no significant changes in the ζ-potential were observed, which indicates the absence of specific adsorption on the particle surface - the Stern layer. This result indicates that adsorption of adenosine to calcium phosphate (FIG. 3) may be non-specific, resulting from mass transfer and entrapment into the gel matrix. The adsorption of ATP and AMP is apparently specific, since changes in the ζ potential indicate a chemisorption process, which changes the resulting electrostatic charge of calcium phosphate particles.

Fig. 14. Photograph of homogenized suspensions in a plastic spectrophotometric cuvette showing the effect of the concentration of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP) on the height of the calcium phosphate precipitate layer. ATP concentrations from left to right: 0.0 mM, 1.0 mM, 2.0 mM, 3.0 mM, 4.0 mM and 5.0 mM.

Fig. 15. Effect of concentrations of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP) on the height of the calcium phosphate precipitate layer.

In FIG. 15 shows that the height of the calcium phosphate precipitate layer, measured after 48 hours at rest, decreases as a function of ATP concentration from 11 mm to 6 mm. The most dramatic decrease in layer height occurs from 0.0 to 2.0 mM ATP, which corresponds to the concentration range in which the ζ-potential of calcium phosphate is most elevated (Fig. 13), suggesting that these two parameters are related. Interestingly, AMP treatment does not change the height of the calcium phosphate precipitate layer. It appears that the triphosphate chain in adenosine 5'-triphosphate induces packing of the calcium phosphate particles, despite the increased ζ potential, due to which electrostatic repulsive forces are expected to separate the particles from each other. A similar picture is observed with inorganic polyphosphates and phytate (inositol hexaphosphate).

Fig. 16. Effect of serial dilutions of Adju-Phos® initially treated with sodium inositol hexaphosphate.

In FIG. 16 shows that when diluted with 5 mM imidazole at pH 7.0, the ζ potential of untreated Adju-Phos decreases (in absolute terms) from -35 mV to -22 mV over a dilution range of 5-fold to 320-fold, respectively. In contrast, Adju-Phos initially treated with 0.2 mM Na-IP6 showed higher ζ-potential values from -47 mV to -33 mV, following a trend parallel to that observed for untreated Adju-Phos.

In the case of untreated Adju-Phos, the decrease in ζ-potential as a function of dilution could be explained by a decrease in electrostatic field strength, since the average distance between particles increases with dilution. This indicates that at a low dilution factor, the particles are at a close distance, and their mutual electrostatic field interacts (overlaps) and appears to be stronger (higher ζ-potential). As the dilution increases, the distance between the particles increases and the apparent strength of the electrostatic field decreases (lower ζ-potential). This effect is also observed in the case of Adju-PhosZP. However, the difference in higher ζ potential between Adju-PhosZP and Adju-Phos is a constant value=about -12 mV.

If IP6 were only reversibly adsorbed on Adju-Phos, one would expect the ζ potential of Adju-PhosZP at high dilution fold to reach that of Adju-Phos®, but this is not the case. A 320-fold dilution of stock Adju-Phos® treated with 0.2 mM IP6 would result in a final IP6 concentration of 0.6 μM. When Adju-Phos® is treated with IP6 concentrations below 10 μM, no change in the ζ potential is observed. Thus, the result shown in Fig. 16 strongly indicates that some of the original IP6 is strongly adsorbed on Adju-Phos and has irreversibly altered its ζ potential.

Fig. 17. Effect of serial dilutions of Adju-Phos® initially treated with ATP.

Fig. 17 shows that when diluted with 5 mM imidazole at pH 7.0, the ζ potential of regular (untreated) Adju-Phos® decreases (in absolute terms) from -41 mV to -27 mV over a dilution range of 5-fold to 320-fold , respectively. Adju-Phos® initially treated with 1.0 mM ATP showed slightly higher ζ-potential values from -44 mV to -30 mV, following a trend parallel to that observed for untreated Adju-Phos®.

Compared to Adju-Phos IP6 treatment (experiment no. 12, Fig. 16), ATP-treated Adju-Phos also shows an increase in ζ-potential regardless of the dilution factor, although the difference in ζ-potential is only approximately -3 mV ( compared to -12 mV for IP6). Following the same reasoning as in the case of experiment no. 12, this result indicates a strong adsorption of ATP on Adju-Phos.

Fig. 18. Effect of serial dilutions of calcium phosphate initially treated with IP6 or ATP.

In FIG. 18 shows that when diluted with 5 mM imidazole at pH 7.0, the ζ potential of raw calcium phosphate increases (in absolute terms) from -5 mV to -10 mV over a dilution range of 5-fold to 320-fold, respectively. In contrast, calcium phosphate initially treated with 0.5 mM Na-IP6 exhibits higher ζ-potential values from -26 mV to -23 mV. Similarly, calcium phosphate initially treated with 1.0 mM ATP shows higher ζ-potential values from -16 mV to -22 mV.

If IP6 or ATP were only reversibly adsorbed on calcium phosphate, one would expect that the ζ potential of calcium phosphate at high dilution ratios would reach that of calcium phosphate, but this is not the case. A 320-fold dilution of the original calcium phosphate treated with either 0.5 mM IP6 or 1.0 mM ATP will result in a final IP6 concentration of 1.5 μM IP6 or 3.0 μM ATP, respectively. When calcium phosphate is treated with IP6 or ATP concentrations below 10 μM, no change in the ζ potential is observed. Thus, the result shown in Fig. 18 convincingly shows that part of the original IP6 or ATP is strongly adsorbed on calcium phosphate and has irreversibly changed its ζ potential.

DEFINITIONS

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as they would normally be understood by a person of ordinary skill in the field to which this invention pertains. While any methods and materials similar or equivalent to those described herein may be used in the practice of testing the present invention, the preferred methods and materials are now described.

In this description and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

As used herein, the terms "comprising (including)", "comprises (includes)", and "consisting of" are synonymous with the terms "comprising", "includes" or "comprising", "comprises" and are inclusive or open and do not exclude additional, non-listed members, elements, or method steps.

The terms "comprising (including)", "comprises (includes)" and "consisting of" also include the term "consisting of".

The term "approximately" as used herein in relation to a measurable value such as a parameter, quantity, duration of time, etc. is intended to cover variations of +/- 10% or less, preferably +/- 5% or less, more preferably +/- 1% or less, and even more preferably +/- 0.1% or less of the specified value, if such variations are suitable for implementation in the disclosed invention. It should be understood that the value itself to which the modifier "about" refers is also specifically and preferably reported.

Specifying numeric ranges using endpoints includes all numbers and fractions in the corresponding ranges, as well as the specified endpoints.

In the following passages, various aspects or embodiments of the present invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless explicitly stated otherwise. In particular, any feature indicated as being preferred or useful may be combined with any other feature or features indicated as being preferred or useful.

Reference in this specification to "one embodiment", "an embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the terms "in one embodiment" or "in an embodiment" in various places in this specification does not necessarily refer, but may refer, to the same embodiment. In addition, specific features, structures, or characteristics may be combined in any suitable manner, as will be apparent to one skilled in the art from this disclosure, in one or more embodiments.

The term "aluminum phosphate" as used herein refers to amorphous aluminum hydroxyphosphate (Shirodkar S. et al., Aluminum compounds used as adjuvants in vaccines. Pharmaceutical Research, 1990, 7:1282-1288) in which some of the hydroxyl groups of the aluminum hydroxide are replaced by phosphate groups. . The disordered amorphous state is responsible for the large surface area and high adsorption capacity. It is not a stoichiometric compound and its composition depends on the precipitation method and conditions. Preferably the Al:P atomic ratio is 1.2±0.15-1.

The surface of the aluminum phosphate mentioned here consists of Al-OH and Al-OPO ₃ groups. The isoelectric point (IEP) varies from 9.4 to 4.5 depending on the degree of substitution with phosphate groups. Commercial aluminum phosphate adjuvants have IEP values ranging from 4.5 to 5.5.

As used herein, the term "aluminum hydroxide" refers to aluminum oxyhydroxide, which is a crystalline stoichiometric compound.

As used herein, the term "calcium phosphate" refers to a non-hydroxyapatite form of calcium phosphate or a composite material containing primarily a non-hydroxyapatite form of calcium phosphate. Calcium phosphate can be represented as the formula Ca ₃ (PO ₄ ) ₂ or as non-stoichiometric hydroxyapatite, Ca _10-x (HPO ₄ ) _x (PO ₄ ) _6-x (OH) _2-x , where x is an integer from 0 to 2 (Jiang D. et al., Structure and adsorption properties of commercial calcium phosphate adjuvant, Vaccine, 2004, 23:693-698). For example, calcium phosphate may refer to a composite material consisting of brushite (CaHPO ₄ ⋅ 2 H ₂ O) and calcium phosphate (Ca ₃ (PO ₄ ) ₂ ), which may be represented by the formula [Ca ₃ (PO ₄ ) ₂ ] _x ⋅ [CaHPO ₄ ⋅ 2 H ₂ O] _y , where the amount of calcium phosphate (x) is greater than the amount of brushite (y), or where x>y. More specifically, calcium phosphate may refer to a composite material consisting of brushite (CaHPO ₄ ⋅ 2 H ₂ O) in which the Ca/P weight ratio is approximately 1.29 and the non-hydroxyapatite form of calcium phosphate (Ca ₃ (PO ₄ ) ₂ ) , in which the weight ratio Ca/P is equal to 1.94. Preferably the atomic ratio Ca:P is 1.7±0.2-1.

The term "microparticles" as used herein refers to particles with a nominal size of not less than 0.01 µm and not more than 10 µm, not more than 5 µm, or not more than 2 µm. The starting material microparticles preferably have a nominal size of not less than 0.1 µm and not more than 5 µm. Further, when said microparticle is an aluminum phosphate microparticle, the starting material microparticle may have a nominal ζ potential of not less than -10 mV and not more than -20 mV when measured in distilled water.

As used herein, the term Pi or P _i will mean inorganic phosphate. When the fused inorganic phosphates are cyclic, "m" (meta) is added, as in the case of m6Pi. For example, diphosphate=2Pi and metahexaphosphate=m6Pi.

As used herein, the term "organic polyphosphate" will mean an organic molecule substituted with at least two phosphate groups (-O-PO ₃ ), or an organic molecule substituted with at least one polyphosphate group -O-PO ₂ -(-O-PO ₂ -) _n -O-PO ₃ where n is an integer ranging from 0 to 5:

As used herein, the term nPorg means an organic polyphosphate with n=number of phosphate groups.

As used herein, the term "polyphosphate" refers to polymers of condensed phosphate or phosphoric acid, more preferably selected from a list including diphosphate, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, meta-triphosphate, meta-hexaphosphate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a further development of the invention disclosed in the simultaneously considered international application PCT/EP 2017/076232, which relates to a method for obtaining modified mineral microparticles, including the suspension of mineral microparticles of some salts of di- or trivalent metals, selected from aluminum phosphate, amorphous aluminum hydroxyphosphate and/or calcium phosphate, or mixtures thereof, in an aqueous solution of one or more inorganic polyphosphates, which causes said mineral microparticles to be modified, both in terms of a significant change in their nominal electrostatic potential, and in terms of reducing the particle size.

The present invention now discloses organically derivatized mineral microparticles containing aluminum phosphate, amorphous aluminum hydroxyphosphate, aluminum hydroxide and/or calcium phosphate, prepared in a similar manner using a solution of one or more organic polyphosphates, for example a solution of one organic polyphosphate, in a preparation process. It has been found that the binding abilities of biomolecules, preferably when said biomolecule is a vaccine antigen, of microparticles of aluminum phosphate, amorphous aluminum hydroxyphosphate, aluminum hydroxide and/or calcium phosphate, are significantly improved by treating said microparticles with organic polyphosphates compared to the aforementioned treatment with inorganic polyphosphates described in the simultaneously pending international application PCT/EP 2017/076232.

In particular, it was found that the proposed replacement of phosphate ions or hydroxide ions with organic polyphosphate ions increases the numerical value of the ζ potential of said microparticles, thereby increasing the strength of the electrostatic potential, for example, to attract biomolecules, preferably antigens, and/or the binding force with the surface of these microparticles. In addition, as described in the simultaneously considered international application PCT/EP 2017/076232, it was found that the substitution of the initial anions in the microparticles of aluminum phosphate, amorphous aluminum hydroxyphosphate, aluminum hydroxide and/or calcium phosphate leads to destabilization of the material superstructure and its decomposition. As a consequence, the treatment of such microparticles with inorganic polyphosphates leads to a decrease in the nominal particle size.

It has now been established that the modifications described in co-pending international application PCT/EP 2017/076232, including the reduction of the nominal size of the microparticles of aluminum phosphate, amorphous aluminum hydroxyphosphate, aluminum hydroxide and/or calcium phosphate, can also be achieved using organic polyphosphates, described here.

However, reactions using organic polyphosphates disclosed herein are easier to control, especially with regard to size reduction, which will be described in detail elsewhere in this specification. Moreover, the reaction products using organic polyphosphates described herein generally have higher thermal stability than those observed for the reaction products using inorganic polyphosphates described in co-pending international application PCT/EP 2017/076232.

Commercially available microparticles of aluminum phosphate, amorphous aluminum hydroxyphosphate, aluminum hydroxide and/or calcium phosphate can conveniently be used in the process of the present invention.

Accordingly, a first aspect of the present invention relates to a process for producing organically derivatized mineral microparticles which comprises the step of balancing said mineral microparticles with a solution of one or more organic polyphosphates, for example a solution of one organic polyphosphate, said mineral microparticles being selected from a list consisting of aluminum phosphate microparticles. , microparticles of amorphous aluminum hydroxyphosphate, microparticles of aluminum hydroxide and/or microparticles of calcium phosphate, and moreover, the specified organic polyphosphate is selected from compounds of formula 1A or 1B:

where n is an integer in the range from 0 to 5 and m is an integer in the range from 2 to 10, and where R _a denotes organic substituents selected from adenosine and other nucleosides, thiamines, carbohydrates and isoprenes, and R _b denotes organic substituents selected from inositols and other cyclites, as well as carbohydrates.

The reactions of organic polyphosphates of formula 1A and 1B with mineral microparticles have been studied separately, and each shows significant advantages over the same reaction with inorganic polyphosphates.

Formula 1A organic polyphosphates with a short fused polyphosphate group, such as triphosphates (i.e. for which n=1, exemplified here by ATP), appear to react much better with hydroxyl groups on the surface of aluminum phosphate particles than the analogous monophosphate. (AMP). Without wishing to be bound by any theory, the inventors of the present invention assume that the phosphoanhydride in ATP is more energy intensive and therefore more nucleophilic. Another reason may be the additional negative charges in the ATP molecule compared to AMP.

Organic polyphosphates of formula 1B are advantageous in terms of increasing the zeta potential of the particles, which again leads to an increase in the adsorption of antigens. This is especially true in the case of organic polyphosphates derived from inositols and other cyclites. Inositol hexaphosphate (IP6), for example, has a formal charge of 12 at neutral pH compared to 6 for hexametaphosphate (m6Pi). Thus, the charge density will be 100% higher in case of IP6 than in case of m6Pi. In addition, the phosphoester bond in IP6 has been experimentally shown to be more resistant to hydrolysis than the phosphoanhydride in m6Pi, indicating that IP6-based adjuvants withstand autoclaving better than m6Pi-based adjuvants.

In a preferred embodiment, the one or more organic polyphosphates are selected from compounds having the general structure 1B. In another preferred embodiment, the one or more organic polyphosphates are selected from inositol phosphate and other cyclite phosphates in their various isomeric and/or enantiomeric forms, e.g. IP6), also known as phytic acid or phytate (as a salt).

More preferably, the one or more organic polyphosphates include inositol triphosphate (IP3) or inositol hexaphosphate (IP6).

Most preferably, the present invention relates to a process for the preparation of organically derivatized mineral microparticles which comprises the step of balancing said mineral microparticles with a solution of inositol hexaphosphate (IP6), also known as phytic acid, or a salt (phytate) thereof, preferably sodium phytate.

In another preferred embodiment, the mineral microparticles are calcium phosphate microparticles.

The organically derivatized microparticles preferably have a nominal size of not less than 0.01 µm and not more than 1 µm, preferably from 0.1 to 0.5 µm, and a nominal ζ-potential as defined elsewhere in the specification. The microparticles may have a variety of shapes and may be, for example, spherical, conical, ellipsoidal, irregularly shaped, cylindrical or cubic. In addition, all microparticles in a population of microparticles may not have the same size or shape. The term "modified" or "modifying" as used herein refers to altering, altering or making changes, either minor or fundamental, to the shape or nature of an object, preferably with the aim of creating an improved (modified) version of said object. For example, organically derivatized mineral microparticles described herein may have full or partial replacement of phosphate ions or hydroxide ions, which are located on the surface of the original material of the original unmodified microparticles, organic polyphosphate ions, a change in surface charge, a change in ζ-potential, a change in the ability to the binding of biomolecules and adsorption of biomolecules and/or change in size compared to the source material of the mineral microparticles before balancing with a solution of one or more organic polyphosphates. Preferably, when said surface charge is numerically higher, said ζ-potential is numerically higher, said biomolecule binding and biomolecule adsorption capabilities are increased and/or said nominal size is reduced compared to the mineral microparticle starting material prior to equilibration with the organic polyphosphate solution. In preferred embodiments, said biomolecule is an antigen. In addition, organically derivatized mineral microparticles may have specific binding sites for chelation of basic amino acid residues on the surface of peptides, proteins, or polypeptides, such as biomolecules, that are not present in the original mineral microparticles.

In specific embodiments, balancing said mineral microparticles with an organic polyphosphate solution changes the nominal electrostatic potential of said mineral microparticles.

As used herein, the terms "electrostatic potential", "electrical potential", or "V" refer to the general meaning of that term as understood by one of ordinary skill in the art, and in particular to the potential energy of a charged object such as a proton, electron, or ion. , at a specific location next to the molecule and can be defined as the energy per unit charge (q) (V=U/q). The electrostatic potential can be represented in units of joules/coulombs or volts. The electrostatic potential can be used to predict and/or calculate the energies required to move charges from, for example, one potential V ₁ to another potential V ₂ .

The term "zeta potential" or "ζ potential" as used herein describes the relative electrical charge of microparticles suspended in a liquid. More specifically, the ζ-potential refers to an intermediate electrical potential at a certain distance from the physical surface of the particle, the boundary of the so-called diffuse layer (the so-called glide plane), where ions are in equilibrium between the attractive electrostatic field of the particle surface and the surrounding liquid (e.g., solvent). Accordingly, the ζ-potential describes the electric potential at a certain distance from the physical surface of the particle, where the charge of said particle no longer affects the surrounding liquid. The ζ potential is typically in the range +100 mV to -100 mV and can be measured with a Zetasizer nano ZS (Malvern Instruments Inc.) in electrokinetic mode, preferably at 25° C. and/or in deionized water. It applies to microparticles in liquid that the higher the nominal ζ potential, the higher the stability in terms of reducing the tendency to settle in suspension. For example, microparticles with a ζ potential greater than +25 mV or less than -25 mV generally have a high degree of stability. Changing the ζ potential of the mineral microparticles by balancing said microparticles with an organic polyphosphate solution as described herein can be an increase, decrease, reversal and/or neutralization of the ζ potential of said microparticles. An increase or decrease in the nominal ζ-potential, as described herein, refers to an increase or decrease in the absolute value of the nominal ζ-potential, regardless of the symbol ((+) or (-)) in front of it. For example, when the initial nominal ζ potential is -10 mV, the increase in the specified nominal ζ potential may be -15 mV, -20 mV, -25 mV, -30 mV, etc., while the decrease in the specified nominal ζ -potential can be -5 mV, -3 mV, -1 mV, 0 mV, etc. Another example, when the initial nominal ζ potential is +10 mV, the increase in the specified nominal ζ potential can be +15 mV, +25 mV, +50 mV, +100 mV, etc., while the decrease in the specified nominal The ζ potential can be +5 mV, +3 mV, +1 mV, 0 mV, etc. Changing any nominal ζ potential to 0 mV can also be referred to as neutralizing the ζ potential. Inverting the nominal ζ-potential means changing the charge, for example a nominal ζ-potential of plus 10 mV can be inverted to minus 10 mV.

The type of change (e.g., increase, decrease, reversion, neutralization) of the ζ potential of the mineral microparticles by balancing said microparticles with a polyphosphate solution, such as an organic polyphosphate solution as described here, depends on the type of mineral microparticle and its initial charge and/or ζ- potential. For example, replacing hydroxide ions with polyphosphate ions on the surface of positively charged aluminum hydroxide can lead to an inversion of the ζ potential of aluminum hydroxide microparticles, while replacing phosphate ions with organic polyphosphate ions on the surface of negatively charged aluminum phosphate or calcium phosphate can lead to an increase in the absolute value of the ζ-potential of aluminum phosphate microparticles or calcium phosphate microparticles, respectively.

In particular embodiments, said mineral microparticle starting material has

(i) a zeta potential of not less than -20 and not more than -30 mV for aluminum phosphate when measured at pH 7.0 in distilled water.

(ii) a zeta potential of not less than +10 and not more than +20 mV for aluminum hydroxide when measured at pH 7.0 in distilled water.

(iii) a zeta potential of not less than -10 and not more than -20 mV for calcium phosphate when measured at pH 7.0 in distilled water.

In specific embodiments, balancing aluminum phosphate and/or calcium phosphate microparticles with an organic polyphosphate solution, as described herein, increases the electrostatic potential strength and/or the absolute value of the ζ potential of said microparticles. Preferably said ζ potential is increased to at least -20 mV, at least -25 mV, at least -30 mV, at least -35 mV, at least -40 mV, at least -50 mV, at least -60 mV, at least -70 mV, at least -80 mV or at least -90 mV when measured in distilled water, preferably at least -40 mV, at least -50 mV, at least -60 mV or at least -70 mV, more preferably at least -50 mV.

In specific embodiments, equilibrating the aluminum hydroxide microparticles with an organic polyphosphate solution as described herein cancels or reverses the nominal electrostatic potential of said microparticles and/or the nominal ζ potential of said microparticles.

In specific embodiments, the implementation of the balancing of mineral microparticles, described here, leads to a decrease in the size of these microparticles. The obtained size of organically derivatized mineral microparticles, as a rule, depends on the initial size of the microparticles of the starting material and on the duration of the equilibration period.

In particular embodiments, said mineral starting material microparticles have a nominal size of at least 0.1 µm and at most 5 µm when measured in a colloidal suspension by dynamic light scattering (DLS) or laser diffraction.

In specific embodiments, the methods described herein can provide organically derivatized mineral microparticles having a size of no greater than 1 μm, no greater than 0.5 μm, no greater than 0.2 μm, or no greater than 0.1 μm, no greater than 0.05 μm, no more than 0.02 µm, preferably not more than 0.2 µm. For example, the procedure described here for equilibrating aluminum phosphate microparticles over a period of up to 40 hours can result in a reduction in nominal size=2 µm for the starting material particles to a nominal size=0.2 µm for the resulting organically derivatized mineral microparticles. The organically derivatized microparticles described herein may occur as amorphous aggregates of smaller crystallites or smaller amorphous aggregates with the same crystallite size.

In other specific embodiments, the methods described herein can provide organically derivatized mineral microparticles having a size smaller than the parent material particles, such as at least about 10% less, or at least about 20%, or at least about 30% less. , or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared with the particle size of the starting material, i.e. mineral microparticles of the source material with which comparison is made.

The term "equilibrating", "equilibrating" or "equilibrating" as used herein refers to the act of bringing mineral microparticles, such as microparticles of aluminum phosphate, amorphous aluminum hydroxyphosphate, aluminum hydroxide, and/or calcium phosphate, into contact with a solution of one or more organic polyphosphates for a certain period of time at a certain temperature and ensure the flow of matter and/or energy between the mineral microparticles and the surrounding solution of one or more organic polyphosphates until the microparticles no longer tend to change further with time (for example, until there is no change in the ζ-potential ). It is assumed that during this period of time on the surface of these mineral microparticles there is a substitution reaction, or exchange of ligands, hydroxide or orthophosphate groups for organic polyphosphate ions. This substitution reaction, or ligand exchange, continues until equilibrium is reached.

The balancing step typically involves mixing the mineral microparticles with a solution of one or more organic polyphosphates. Non-limiting examples of devices that can be used to perform mixing are oscillating platforms, a rotating carousel, vortex mixers, mixers (eg screw, belt or paddle) or mixers (eg screw, belt or paddle). In addition, the mineral microparticles can be washed before adding one or more organic polyphosphates to the solution. Preferably, the washing is carried out in deionized water. The length of the equilibration period depends on the type of mineral microparticles (for example, aluminum phosphate, amorphous aluminum hydroxyphosphate, aluminum hydroxide and/or calcium phosphate) and/or the purpose of the equilibration (for example, changing the size, electrostatic potential and/or ζ-potential of said microparticle) and can vary from at least 1 minute to at least 50 hours. As a rule, to change the electrostatic potential and/or ζ-potential of mineral microparticles, as described here, requires a shorter period of equilibrium compared to the change in size. Preferably, the equilibration period for changing the electrostatic potential and/or ζ potential of the mineral microparticles is at most one hour, while the equilibration period for changing the size of the mineral microparticles is at least one hour. More preferably, the equilibration period for changing the electrostatic potential and/or ζ potential of the mineral microparticles is at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, even more preferably at least 10 minutes, and an equilibration period for resizing mineral microparticles is at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 25 hours, at least 30 hours, at least 35 hours, at least 40 hours , at least 45 hours, at least 50 hours, even more preferably at least 40 hours. In particular embodiments, said equilibration period is performed at room temperature. Optionally, either the solution of one or more organic polyphosphates can be removed from the resulting organically derivatized mineral microparticles, or the resulting organically derivatized mineral microparticles can be separated from the solution of one or more organic polyphosphates after an equilibration period. Non-limiting examples of methods to achieve this separation are filtration, centrifugation, optically induced dielectrophoretic (ODEP) forces, buffer exchange, washing, or other methods known to those skilled in the art.

In a second aspect, the present invention provides organically derivatized mineral microparticles that can be obtained by the equilibration process according to the first aspect.

The microparticles produced by the method of the present invention are difficult to describe in precise, objective terms due to the chemical reactions, surface changes, and size reduction described above. The measured zeta potential changes significantly during the ligand exchange/substitution reaction and should be considered as a result of these chemical reactions, surface changes and size reduction. However, experience shows that organically derivatized mineral microparticles are repeatedly obtained with almost identical characteristics / zeta potential, following the described procedure. Thus, organically derivatized mineral microparticles are most accurately described as the product of this equilibration process.

As mentioned above, it is believed that organic polyphosphates react with the aluminum phosphate or calcium phosphate surface by exchanging ligands with hydroxide ions ionically bonded to the metal cation. The measurement of changes in the zeta potential of the particles is a direct evidence of the adsorption of ions at the interface between the physical surface of the particle and the main volume of the solvent (the so-called Helmholtz plane). In the case of negatively charged organic polyphosphates, adsorption on a negatively charged surface is unfavorable due to electrostatic repulsion. However, the experimental data presented here show that the zeta potential of aluminum phosphate increases upon treatment with organic polyphosphates, demonstrating specific adsorption of organic polyphosphates on aluminum phosphate particles. In addition, the data show that increased zeta potential values of aluminum phosphate particles treated with organic polyphosphates (compared to untreated particles or particles treated with orthophosphate) are maintained after extensive washing of the particles with deionized water. The data indicate that organic polyphosphates bind strongly to the surface of the particles, which confirms the fact of ligand exchange.

Therefore, tracking the change in the zeta potential of particles as a function of time gives the kinetics of adsorption of organic polyphosphates on the particle surface (Helmholtz plane). However, this method does not provide ligand exchange kinetics in place of hydroxide ions. Ligand exchange rates for Al(III) are among the slowest recorded in aluminum(III) hydrates (Martin R.B., The chemistry of aluminum as related to biology and medicine. Clinical Chemistry, 1986, 32:1797-806). However, in the case of aluminum phosphate, where Al(III) mainly forms a complex with phosphate, the kinetics of ligand exchange instead of hydroxide ions in Al(III) is unknown.

Direct evidence of ligand exchange using spectroscopy is expected to be less difficult than for a similar reaction using inorganic polyphosphates due to the presence of carbon in these molecules and the presence of phosphoester bonds, giving distinct unmasking features in analytical spectroscopy such as the Raman spectrum or IR, chemical revealing features not originally present in the original unmodified particles. Inorganic polyphosphates composed of phosphoric acid polymers show the characteristic features of orthophosphate already present in the aluminum phosphate or calcium phosphate particles. It is expected that the spectrum of Raman scattering of light (Raman spectrum) from the surface of a particle modified with organic polyphosphates will differ from the spectrum for the original unmodified particles. Moreover, the hydroxide ions released from the ligand exchange reaction will lead to an alkalization of the solution, and pH versus time measurements can therefore also reveal the kinetics of the ligand exchange.

In specific embodiments, the process described here begins with commercially available (unmodified) microparticles of aluminum phosphate, amorphous aluminum hydroxyphosphate, aluminum hydroxide and/or calcium phosphate, which are subsequently modified. Preferably said aluminum phosphate microparticles as starting material are AdjuPhos® microparticles and said unmodified aluminum hydroxide microparticles (i.e. starting material) are Alhydrogel® microparticles, more preferably 2% AdjuPhos® or 2% Alhydrogel® microparticles, respectively. Preferably said calcium phosphate microparticles as starting material are non-hydroxyapatite microparticles. In further specific embodiments, the implementation of the microparticles of aluminum phosphate or calcium phosphate may contain a proportion of hydroxyl groups. In even further specific embodiments, the aluminum phosphate or calcium phosphate microparticles may contain small proportions of other chemical compounds or elements such as sodium chloride (e.g. 0.8% - 1.0% w/w), chloride (e.g. ≤0, 33% w/w), nitrogen (e.g. ≤0.05% w/w), nitrate (e.g. ≤100 ppm), sulfate (e.g. ≤0.1% w/w), iron (e.g. ≤15 ppm), arsenic (eg ≤1 ppm), heavy metals (eg ≤20 ppm), ammonium (eg ≤50 ppm).

In specific embodiments, methods comprising the step of equilibrating said microparticles of aluminum hydroxide, aluminum phosphate, amorphous aluminum hydroxyphosphate and/or calcium phosphate with said solution of one or more organic polyphosphates as described herein result in partial or complete replacement of hydroxide ions or phosphate ions, which are located on the surface of microparticles, organic polyphosphate ions.

The term "substitution" as used herein refers to the replacement of one anion with another in a solid matrix, also referred to as ligand exchange. Due to their opposite charges, cations and anions will be attracted to each other due to the Coulomb forces they can exert mutually on each other. As the interatomic distance decreases, it will become short enough for an electron to move from the valence shell of the anion to the valence shell of the cation, thereby forming an ionic (electrovalent) bond. For comparison, in aluminum hydroxide, aluminum (Al ³⁺ ) is in ionic bond with the hydroxide (OH). Ionic bonds, unlike covalent bonds, are not localized. The maximum coverage of the surface of microparticles of aluminum phosphate, aluminum hydroxide or calcium phosphate with organic polyphosphate ions can range from 0.2% to 0.8% (w/w), assuming that the microparticles have a simple spherical shape and a monolayer surface coverage of organic polyphosphate ions.

The term "surface" as used herein refers to the outer boundary of a three-dimensional structure, such as a microparticle, protein, peptide, polypeptide, biomolecule, or antigen, that is available to interact with solvents (eg, water) and solutes (eg, ions) from the surrounding liquid medium. (e.g., liquid buffer), also referred to as the solvent-accessible surface. The solvent accessible surface of the mineral microparticles described herein can be determined or extrapolated from binding isotherms.

In specific embodiments, balancing said mineral microparticles with a solution of one or more organic polyphosphates, as described herein, increases biomolecular binding abilities and/or biomolecule adsorption capacity of said mineral microparticles. Biomolecule binding capacity and/or biomolecule adsorption capacity of said organic derivatized mineral microparticles selected from a list consisting of aluminum phosphate microparticles, amorphous aluminum hydroxyphosphate, aluminum hydroxide and/or calcium phosphate may be at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, at least 5.5 times, at least 6 times higher biomolecular binding capacity and/or adsorption capacity relative to biomolecules of starting materials selected from microparticles of aluminum phosphate, aluminum hydroxide or calcium phosphate. Preferably, said biomolecules are antigens.

The term "bind", "interact", "specifically bind" or "specifically interact" as used throughout this specification means that an agent binds or affects one or more desired molecules or analytes, to a large extent excluding other molecules that are random or unrelated, and possibly to a large extent excluding other molecules that are structurally related. The term "bind", "interact", "specifically bind" or "specifically interact" does not necessarily require that the agent binds exclusively to its intended target(s). For example, an agent can be said to specifically bind to a target(s) of interest if its affinity for that intended target(s) under binding conditions is greater than at least about 2-fold, preferably at least about 5, more preferably at least at least about 10 times, even more preferably at least about 25 times, even more preferably at least about 50 times, and even more preferably at least about 100 times, than its affinity for the non-target molecule.

The binding or interaction between the agent and its intended target(s) may be non-covalent (i.e., mediated by non-covalent forces such as, for example, ionic interactions, hydrogen bridges, dipolar interactions, van der Waals interactions, and the like. ). Preferably, the agent can bind or interact with its intended target(s) with an affinity or association constant (K _A ) of such binding K _A ≥1×10 ⁶ M ^-1 , more preferably K _A ≥1×10 ⁷ M ^-1 , even more preferably K _A ≥1×10 ⁸ M ^-1 , even more preferably K _A ≥1×10 ⁹ M ^-1 , and even more preferably K _A ≥1×10 ¹⁰ M ^-1 or K _A ≥1×10 ¹¹ M ^-1 where K _A =[A_T]/[A][T]=k _a /k _d , where A is the agent, T is the target, k _a is the absorption rate, and k _d is the desorption rate. Determination of K _A can be performed using methods known in the art, such as, for example, the use of equilibrium dialysis and Scatchard plot analysis.

To increase the intensity of the electrostatic potential responsible for the attraction of biomolecules (and binding force) to the surface of microparticles, it is preferable that the absolute value of the ζ-potential of the modified microparticles be as high as possible. In addition, since a higher surface to weight ratio results in higher potential biomolecule loadings per unit weight of adjuvant, the size of the modified microparticles is in the submicron range, preferably in the nanometer range. Preferably said biomolecule is a vaccine antigen.

The first general mechanism by which soluble ionic particles can be adsorbed to particle surfaces is through long-range electrostatic attractive forces. These forces are recognized as the most important factor in the adsorption of biomolecules, such as antigens, on the surface of microparticles (eg, adjuvant microparticles). By following this mechanism, they will cause biomolecules, such as antigens, to be captured by the attractive electrostatic field of the microparticle, the aforementioned diffuse layer, with freedom to diffuse within the diffuse layer, as well as in and out of the main volume of the solvent. As the biomolecules approach the surface of the particle, other short acting forces such as van der Waals forces or dipole-dipole interactions (hydrogen bonds) may take place. Solvent effects (hydrophobic effect) can also add to the mechanisms and overall strength of the binding of the biomolecule to the surface of the particle.

The present inventors have found that by increasing the nominal negative electrostatic potential of aluminum phosphate microparticles by balancing said microparticles in a solution of one or more organic polyphosphates, such as a solution of one organic polyphosphate as described herein, the strength of this electrostatic field is also increased, which in turn turn, increased the ability to bind biomolecules, such as antigenic proteins, with the opposite (positive) charge. Furthermore, without wishing to be bound by any theory, the present inventors speculate that the replacement of hydroxides with organic polyphosphates on the surface of positively charged aluminum hydroxide may result in a ζ potential inversion, which allows the adsorption of positively charged biomolecules, such as antigenic proteins, which are normally are electrostatically repelled by aluminum hydroxide microparticles.

S., Amazing stability of the arginine-phosphate electrostatic interaction, Journal of Proteome Research, 2005), и это свойство используется в случае некоторых применений (Fokkens M. et al., A molecular tweezer for lysine and arginine, Journal of the American Chemical Society, 2005; Schug K. A. et al., Noncovalent binding between guanidinium and anionic groups: focus on biological-and synthetic-based arginine/guanidinium interactions with phosph[on]ate and sulf[on]ate residues, Chemical Reviews, 2005). Авторы настоящего изобретения, кроме того, предполагают, что этот тип взаимодействия может происходить на поверхности фосфатсодержащих микрочастиц (например, используемых в качестве адъювантов), таких как фосфаты алюминия или кальция, при соответствующих значениях pH и ионной силе основного объема растворителя. Кроме того, введение органический полифосфат-ионов на поверхность минеральных микрочастиц, как здесь описано, может улучшить этот механизм связывания таким образом, что благодаря своей полимерной природе и конформационной гибкости органические полифосфаты могут хелатировать положительно заряженные аминокислотные остатки, предпочтительно боковые цепи лизина или аргигина. В результате способность к связыванию или сила связывания для таких биомолекул, как антигены, представляющих такие основные остатки на своей поверхности, может быть значительно улучшена за счет хелатных эффектов. Интересно, что в случае гидроксида алюминия, который является положительно заряженным, в качестве сравнения, замещение некоторых гидроксид-ионов органический полифосфат-ионами может создавать некоторые сайты для специфической адсорбции положительно заряженных биомолекул, таких как антигены, которые, как ожидается в противном случае, будут демонстрировать слабое сродство к гидроксиду алюминия из-за отталкивающих заряд-зарядовых взаимодействий при значениях pH, обычно используемых в случае применений систем доставки или систем биомолекул, таких как производство вакцин.Without wishing to be bound by any theory, the present inventors believe that the second mechanism by which biomolecules such as antigens can bind to the surface of mineral microparticles as described herein lies in more specific interactions involving the formation of ionic bonds between positively charged amino acids. on the surface of the protein as a biomolecule, preferably with lysine and arginine, and negatively charged organic phosphate groups on the surface of microparticles (eg adjuvant microparticles). The affinity of phosphate groups for positively charged amino acid residues in proteins, especially lysine and arginine, is well documented in biochemistry for protein kinases and phosphatases, with the phosphoryl group of nucleotides transiently participating in ion pairing with lysine or arginine residues of the catalytic center of the enzyme (Mavri J. and Vogel MJ, Ion pair formation of phosphorylated amino acids and lysine and arginine side chains: A theoretical study, Proteins Structure Function and Bioinformatics, 1996). In some specific cases of protein-protein interactions, it has been found that this type of ionic bond can be as strong as a covalent bond (Woods AS and

S., Amazing stability of the arginine-phosphate electrostatic interaction, Journal of Proteome Research, 2005), and this property is used in some applications (Fokkens M. et al., A molecular tweezer for lysine and arginine, Journal of the American Chemical Society, 2005; Schug KA et al., Noncovalent binding between guanidinium and anionic groups: focus on biological-and synthetic-based arginine/guanidinium interactions with phosph[on]ate and sulf[on]ate residues, Chemical Reviews, 2005). The present inventors further contemplate that this type of interaction can occur on the surface of phosphate-containing microparticles (eg, used as adjuvants), such as aluminum or calcium phosphates, at appropriate pH and bulk solvent ionic strength. In addition, the introduction of organic polyphosphate ions onto the surface of mineral microparticles as described herein can improve this binding mechanism such that, due to their polymeric nature and conformational flexibility, organic polyphosphates can chelate positively charged amino acid residues, preferably lysine or argigin side chains. As a result, the binding capacity or binding strength for biomolecules such as antigens presenting such basic residues on their surface can be greatly improved by chelating effects. Interestingly, in the case of aluminum hydroxide, which is positively charged, by comparison, substitution of some hydroxide ions for organic polyphosphate ions may create some sites for the specific adsorption of positively charged biomolecules, such as antigens, that would otherwise be expected to be exhibit a low affinity for aluminum hydroxide due to repulsive charge-charge interactions at pH values commonly used in delivery systems or biomolecular systems applications such as vaccine production.

Another aspect of the present invention relates to organically derivatized mineral microparticles selected from the list consisting of microparticles of aluminum phosphate, amorphous aluminum hydroxyphosphate, aluminum hydroxide and/or calcium phosphate, in which orthophosphate ions located on the surface of the microparticles of aluminum phosphate or calcium phosphate, or hydroxide ions located on the surface of aluminum hydroxide microparticles are partially or completely replaced by organic polyphosphate ions.

In specific embodiments, the organically derivatized mineral microparticles described herein have

(i) a nominal ζ potential of at least -40 mV for aluminum phosphate when measured at pH 7.0 in distilled water, or

(ii) a nominal ζ potential of at least -20 mV for aluminum hydroxide when measured at pH 7.0 in distilled water, or

(iii) a nominal ζ potential of at least -40 mV for calcium phosphate when measured at pH 7.0 in distilled water.

In specific embodiments, the modified aluminum phosphate microparticles have a nominal ζ potential of at least -20 mV, at least -25 mV, at least -30 mV, at least -35 mV, at least -40 mV, at least -50 mV, at least -60 mV, at least -70 mV, at least -80 mV or at least -90 mV when measured in distilled water, preferably at least -40 mV, at least at least -50 mV, at least -60 mV, at least -70 mV, more preferably at least -50 mV.

In specific embodiments, the organically derivatized mineral microparticles selected from the list consisting of aluminum phosphate, amorphous aluminum hydroxyphosphate, aluminum hydroxide and/or calcium phosphate microparticles according to the present invention have a nominal size of at least 0.01 µm and at most 2 µm, not more than 1 µm, not more than 0.5 µm, not more than 0.2 µm, not more than 0.1 µm, not more than 0.05 µm, not more than 0.02 µm, preferably not more than 2 µm.

In specific embodiments, the organically derivatized aluminum phosphate of the present invention has a stoichiometric ratio of Al to P of 1.2 +/- 0.15 to 1.

In specific embodiments, the organically derivatized calcium phosphate of the present invention has a stoichiometric Ca to P ratio of 1.7 +/- 0.20 to 1.

In specific embodiments, the organically derivatized mineral microparticles described herein may have a maximum surface coverage of said microparticles with organic polyphosphate ions from 0.2 to 0.8% by weight.

As noted above, mineral adjuvants including aluminum phosphate, aluminum hydroxide, and calcium phosphate have been used successfully in vaccine formulation for decades to enhance the immune response to killed, inactivated, and subunit antigens.

In specific embodiments, the organically derivatized mineral microparticles described herein have enhanced biomolecular binding abilities compared to aluminum hydroxide, aluminum phosphate, amorphous aluminum hydroxyphosphate and/or calcium phosphate microparticles as a starting material, wherein said biomolecule preferably has a charge, opposite to the charge of said modified microparticle, or wherein said biomolecule is neutral when said modified microparticle is neutral. For example, organically derivatized aluminum phosphate microparticles are negatively charged and preferentially bind to positively charged biomolecules.

As used herein, the term "biomolecules" is intended to include ingredients or agents that are derived from living organisms by purification or synthesis and that may be biologically active. These terms also include diagnostics as well as so-called "cosmeceuticals". Diagnostic agents include, for example, fluorescent proteins (eg, green fluorescent protein or GFP) or radiolabeled molecules. Cosmeceuticals include active ingredients that affect the appearance of an individual, such as the skin, hair, lips, and eyes, and include anti-wrinkle agents and complexion-improving agents. In these applications, the modified microparticles described herein are preferably applied externally. Active pharmaceutical ingredients (also called drugs or drugs) are of particular interest and form a subset of biomolecules.

Biomolecules can include small molecules (eg, having a molecular weight of less than about 1500), synthetic or natural, such as monosaccharides, disaccharides, trisaccharides, oligosaccharides, peptides, nucleic acids, and analogs and derivatives of nucleic acids; or large molecules, including plasmids, vectors, polysaccharides, biological macromolecules, e.g. larger peptides (polypeptides), proteins, peptide analogs and their derivatives, peptidomimetics, nucleic acid-based molecules (e.g. DNA, RNA, mRNA, tRNA, mRNA , miRNA, microRNA or any other DNA or RNA-like molecules), polynucleotides, oligonucleotides, enzymes, antibiotics, extracts derived from biological materials such as bacteria, plants, fungi or animal cells or tissues, therapeutic agents, prophylactic agents, diagnostic tools, imaging tools, aptamers (including aptamers in the form of oligonucleotide or protein molecules).

In one embodiment, the biomolecules are water soluble, in particular water soluble active pharmaceutical ingredients. Such ingredients may be class I or III of the Biopharmaceutical Classification System (BCS), which categorizes drug substances into four classes: class I - high permeability, high solubility; class II - high permeability, low solubility; class III - low permeability, high solubility; class IV - low permeability, low solubility. Water-soluble drugs can also be defined by the amount of water (g) required to dissolve 1 g of a compound, water-soluble drugs being those that meet the following solubility criteria: 10-30 g ("soluble"); 30-100 g ("hardly soluble"); 100-1000 g ("poorly soluble"); 1000-10000 g ("very slightly soluble" or "poorly soluble"); or particularly soluble, sparingly soluble and sparingly soluble drugs.

In another embodiment, the biomolecules may be antibodies or antibody fragments. The term "antibody" is intended to include monoclonal antibodies, polyclonal antibodies, and polyspecific antibodies (eg, bispecific antibodies). Antibody fragments include a portion of an antibody, typically its antigen-binding or variable region. Examples of antibody fragments include Fab-, Fab'-, F(ab') ₂ - and Fv fragments; diabody; linear antibodies; single chain antibody molecules; polyspecific antibodies formed from antibody fragments.

In preferred embodiments, the biomolecules may be antigens that are capable of inducing an immune response in the host. Accordingly, in preferred embodiments, the organically derivatized mineral microparticles described herein have increased antigen-binding abilities compared to the microparticles of the starting material: aluminum phosphate, amorphous aluminum hydroxyphosphate, aluminum hydroxide and/or calcium phosphate, and preferably said antigen has an opposite charge the charge of said modified microparticle, or wherein said antigen is neutral when said modified microparticle is neutral. For example, modified aluminum phosphate microparticles are negatively charged and preferentially bind to positively charged antigens.

The term "host organism" generally refers to animals, preferably vertebrates, including birds, humans, and non-human mammals such as mice, rats, hamsters, guinea pigs, pigs, cows, horses, sheep, goats, dogs , cats or primates.

In specific embodiments, the organically derivatized mineral microparticles described herein may have increased adsorption capacities for antigens as a result of their increased antigen-binding abilities. Such increased adsorption capacities for antigens will make it possible to create combination vaccines that can contain antigens from a greater number of infectious agents than those available in the prior art. Adsorption capacities for adjuvant proteins can be determined using a number of analytical methods. For example, comparing the protein content in the aqueous phase of an antigen solution before and after adsorption to an adjuvant (Lindblad E., Aluminum compounds for use in vaccines, Immunology and Cell Biology, 2004, 82:497-505), or in the case where an antibody is available, specific for the desired antigen, adsorption, adsorption capacity for protein can be determined using immunoprecipitation techniques using either quantitative immunoelectrophoresis or simple radial immunodiffusion. Without the use of an antibody, it can be checked spectrophotometrically (Lindblad E., Aluminum compounds for use in vaccines, Immunology and Cell Biology, 2004, 82:497-505).

As used herein, the term “adsorption” refers to physical adsorption (eg, by van der Waals force) or chemisorption (eg, by covalent or ionic bonding) in which a bond is established between the surface of microparticles and a biomolecule (eg, antigen and/or or organic molecule).

In specific embodiments, the ability to bind biomolecules in these organically derivatized mineral microparticles selected from the list consisting of microparticles of aluminum hydroxide, aluminum phosphate, amorphous aluminum hydroxyphosphate and/or calcium phosphate, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, at least 5.5 times, at least 6 times higher the ability to bind biomolecules in the microparticles of the source material: aluminum hydroxide, aluminum phosphate, amorphous aluminum hydroxyphosphate and/or calcium phosphate, preferably when said biomolecule is an antigen. The ratio of adsorbed biomolecule (e.g., antigen) to microparticles of aluminum hydroxide (mg/g), aluminum phosphate (mg/g), amorphous aluminum hydroxyphosphate (mg/g), or calcium phosphate (mg/g) depends on the combination of the type of modified microparticle and the nature biomolecules (for example, an antigen). For example, the ratio of biomolecule (e.g. antigen) to mineral microparticle (mg/g) may be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, according to at least 9, at least 10, at least 11, at least 12, preferably at least 12.

The organically derivatized mineral microparticles selected from the list consisting of aluminum hydroxide, aluminum phosphate, amorphous aluminum hydroxyphosphate and/or calcium phosphate microparticles according to the present invention may have improved physicochemical properties, i. increased colloidal stability or reduced aggregation, which may be due to an increase in electrostatic repulsion between particles; and/or increased adsorption and binding of biomolecules, preferably when said biomolecule is an antigen.

In addition, another aspect of the present invention is the use of these organically derivatized mineral microparticles described herein in medicine.

In specific embodiments, the "drug" may be a human and/or veterinary drug.

In specific embodiments, the organically derivatized mineral microparticles described herein can be used as biomolecule delivery or adsorption systems, preferably said biomolecule delivery system being a vaccine adjuvant.

In specific embodiments, said organically derivatized mineral microparticles described herein can be used in vaccines, preferably as biomolecule delivery systems, more preferably as vaccine adjuvants.

In specific embodiments, these organically derivatized mineral microparticles described herein can be used to manufacture a vaccine.

In other specific embodiments, organically derivatized mineral microparticles derived from a starting material selected from aluminum hydroxide, aluminum phosphate, amorphous aluminum hydroxyphosphate, and/or calcium phosphate may be used to manufacture a vaccine adjuvant or vaccine component, and/or may be used in the manufacture of a vaccine. .

Modified mineral microparticles, to the surface of which antigens are bound, as described here, can be used to induce the production of antibodies, such as polyclonal antibodies, in animals. This is achieved by injecting said mineral microparticles, to the surface of which antigens are bound, into laboratory or farm animals in order to induce high levels of expression of antigen-specific antibodies in serum, which can then be isolated from the animal. Polyclonal antibodies can be isolated directly from serum, while monoclonal antibodies are obtained by fusing antibody-secreting spleen cells of immunized mice with immortalized myeloma cells to create monoclonal hybridoma cell lines that express specific antibodies in cell culture supernatant.

Therefore, another aspect of the present invention is the use of organic derivatized mineral microparticles in accordance with the present invention for the production of antibodies.

Mineral microparticles capable of binding biomolecules and/or contaminants (e.g., arsenic, chromium, nitrate, calcium, radium, uranium, fluoride) can be used as adsorption systems for biomolecules, for example, in the purification, separation and decontamination of aqueous and other solutions containing ions (for example, as ion exchangers). For example, positively charged mineral microparticles may be able to bind negatively charged albumin and may therefore be used to remove albumin from blood samples. In another example, charged mineral microparticles can be used to selectively enrich acidic or basic proteins in a variety of samples. On an industrial scale, purification, separation and decontamination is often carried out using sorption columns (eg, ion exchange chromatography).

Accordingly, the organically derivatized mineral microparticles described herein can be used as adsorption systems for biomolecules in purification, separation, and decontamination processes.

In specific embodiments, the organically derivatized mineral microparticles described herein can be used to remove unwanted proteins during blood fractionation.

The present invention is further illustrated by the following non-limiting examples.

EXAMPLES

Experiment #1

Adsorption of model hen egg lysozyme (HEL) antigen was performed using Adju-Phos® particles treated with inositol hexaphosphate (IP6) and compared with metahexaphosphate (m6Pi), orthophosphate (Pi) and water as controls. Adju-Phos® at a concentration of 20 g/l was treated by adding IP6 or m6Pi to a final concentration of 5 mM, and the pH was again adjusted to pH 5.0, and the suspension was left for 12 hours with gentle agitation to equilibrate and to ensure that no precipitation of particles occurred. . The Adju-Phos® particles were then thoroughly washed with ultrapure water to remove excess ions and autoclaved. The pH of the suspension was adjusted to 7.0 before carrying out the HEL adsorption.

The adsorption capacity of the particles was determined as a function of the HEL concentration. Serial dilutions were made from a 2 mM HEL stock solution and added respectively to fixed volumes of calcium phosphate particles, e.g., final concentrations ranged from 10 μM to 120 μM, and the final concentration of calcium phosphate particles was 4 mg/ml. The mixtures were left to equilibrate for 30 minutes at room temperature with stirring to ensure that sedimentation of the particles did not occur. The amount of adsorbed HEL was determined from the amount remaining in the supernatant after particle settling. The results are shown in FIG. 1.

Experiment #2

Effect of inositol-6-phosphate (I6P) concentration on the zeta potential of calcium phosphate particles. Calcium phosphate adjuvant particles (Brenntag-Biosector, Denmark) were thoroughly washed with ultrapure water (6 μS/cm) before being treated for 60 minutes at room temperature with various concentrations of orthophosphate (Pi), metahexaphosphate (m6Pi) or inositol hexaphosphate (IP6) . Zeta potential measurements were performed using a Zetasizer Nano ZS (Malvern, UK) on 10-fold dilutions of the initial suspension of washed particles in the corresponding phosphate solutions. The results are shown in FIG. 2.

Experiment #3

Adsorption of the model hen egg lysozyme (HEL) antigen was performed using calcium phosphate particles treated with 0.2 mM phosphates to determine the adsorption capacity of the particles as a function of HEL concentration. Serial dilutions were made from a 2 mM HEL stock solution and added respectively to fixed volumes of calcium phosphate particles, e.g., final concentrations ranged from 10 μM to 120 μM, and the final concentration of calcium phosphate particles was 4 mg/ml. The mixtures were left to equilibrate for 30 minutes at room temperature with stirring to ensure that sedimentation of the particles did not occur. The amount of adsorbed HEL was determined from the amount remaining in the supernatant after particle settling. The results are shown in FIG. 3.

Experiment #4

The influence of the degree of phosphorylation of adenosine-containing nucleotides on adsorption to Adju-Phos® was investigated by comparing adenosine triphosphate (ATP) with adenosine monophosphate (AMP) and adenosine (A). Adju-Phos® at a concentration of 1% (w/v) was adjusted to pH 7.0 and mixed with solutions A, AMP and ATP at concentrations from 0.1 mM to 2.5 mM and left to equilibrate for 1 hour at room temperature with stirring to avoid precipitation of Adju-Phos®. The particles were then centrifuged and the absorbance of the supernatants was measured at 259 nm. The amounts of adsorbed adenosines were calculated from the difference between the initial concentrations and the final concentrations after adsorption, using the molar extinction coefficient = 15400 cm ^-1 M ^-1 . The results are shown in FIG. 4.

Experiment #5

The effect of inositol hexaphosphate (IP6) concentration on the zeta potential of Adju-Phos® particles was investigated. The Adju-Phos® particles were diluted to 0.1% (w/v) with IP6 solutions ranging from 0.2 mM to 40 mM, adjusting the pH to 7.0. After 60 min of equilibration at room temperature, the ζ potential was recorded using DLS (Zetasizer Nano ZS, Malvern Instruments). The results are shown in FIG. 5.

Experiment #6

The adsorption of adenosine-containing nucleotides on Adju-Phos and Alhydrogel was studied by comparing adenosine triphosphate (ATP) with adenosine monophosphate (AMP) and adenosine (A). Adju-Phos or Alhydrogel at a concentration of 1% (w/v) was buffered at pH 7.0 with 5 mM imidazole and mixed with solutions of A, AMP and ATP at concentrations from 0.2 to 6.0 mM and left to equilibrate for 2 hours at room temperature with stirring to avoid precipitation of adjuvant particles. The adjuvant particles were centrifuged and the absorbance at 259 nm (maximum absorbance for adenine) of the supernatants was measured with a spectrophotometer. The number of adsorbed nucleotides was calculated from the difference between the initial concentrations and the final concentrations after adsorption, using the coefficient of molar extinction = 15400 cm ^-1 M ^-1 for adenine. The results are shown in FIG. 7 and FIG. 8.

Experiment #7

The effect of adenosine-containing nucleotides (ATP and AMP) on the electrostatic ζ potential of Adju-Phos was determined using dynamic light scattering (Zeta-Sizer, Nano series, Malvern). Adju-Phos at a concentration of 0.2% (w/v) in 5 mM imidazole buffer at pH 7.0 was mixed with solutions of ATP or AMP at concentrations from 0.2 to 4.0 mM and left to equilibrate for 2 hours at room temperature with stirring to avoid precipitation of adjuvant particles. Samples were transferred to capillary cells (DTS1060, Malvern) and measurements were taken in triplicate at 25° C. after 2 minutes of temperature equilibration. The results are shown in FIG. 9.

Experiment #8

The effect of adenosine-containing nucleotides (ATP and AMP) on Adju-Phos settling and layer height was investigated. When Adju-Phos is treated with polyphosphates under the same pH and percentage salt conditions, the packing density of the particulate material increases, resulting in a decrease in bed height. The higher packing density of the Adju-Phos pellet makes it difficult to resuspend the adjuvant particles in the main solvent phase, which can be a burden for vaccine manufacturers. Therefore, tracking the packing density of the modified Adju-Phos is an important parameter for downstream applications.

To 1 ml of 2% (w/v) Adju-Phos in 5 mM imidazole buffer at pH 7.0 was added concentrated solutions of ATP or AMP to achieve final concentrations ranging from 0.2 mM to 5.0 mM. The homogenized suspension (with vigorous shaking) was transferred to a plastic spectrophotometric cuvette and left to settle for 48 hours until a clear phase separation was observed between the particle layer and the liquid above. The layer height was measured depending on the concentration of nucleotides. Under these conditions, the height of the total suspension volume in the cuvette was constant and equal to 22 mm. The results are shown in FIG. 10 and FIG. eleven.

Experiment No. 9 Adsorption of adenosine-containing nucleotides on calcium phosphate

The adsorption of adenosine-containing nucleotides to calcium phosphate was studied by comparing adenosine triphosphate (ATP) with adenosine monophosphate (AMP) and adenosine (A). Calcium phosphate at a concentration of 1% (w/v) was buffered at pH 7.0 with 5 mM imidazole and mixed with solutions A, AMP and ATP at concentrations from 0.2 mM to 6.0 mM and left to equilibrate for 2 hours at room temperature with stirring to avoid precipitation of adjuvant particles. The adjuvant particles were centrifuged and the absorbance at 259 nm (maximum absorbance for adenine) of the supernatants was measured with a spectrophotometer. The number of adsorbed nucleotides was calculated from the difference between the initial concentrations and the final concentrations after adsorption, using the coefficient of molar extinction = 15400 cm ^-1 M ^-1 for adenine. The results are shown in FIG. 12.

Experiment No. 10 The effect of adenosine-containing nucleotides on the electrostatic ζ-potential of calcium phosphate

The effect of adenosine-containing nucleotides (ATP and AMP) on the electrostatic ζ potential of calcium phosphate was determined using dynamic light scattering (Zeta-Sizer, Nano series, Malvern). Adju-Phos at a concentration of 0.2% (w/v) in 5 mM imidazole buffer at pH 7.0 was mixed with ATP or AMP solutions from 0.2 mM to 4.0 mM and left to equilibrate for 2 hours at room temperature with stirring to avoid precipitation of adjuvant particles. Samples were transferred to capillary cells (DTS1060, Malvern) and measurements were taken in triplicate at 25° C. after 2 minutes of temperature equilibration. The results are shown in FIG. 13.

Experiment #11 Effect of adenosine-containing nucleotides on calcium phosphate precipitation.

The effect of adenosine-containing nucleotides (ATP and AMP) on calcium phosphate precipitation and layer height was determined. When calcium phosphate is treated with polyphosphates under the same pH and percentage salt conditions, the packing density of the particulate material increases, resulting in a decrease in bed height. The higher packing density of the calcium phosphate precipitate makes it difficult for adjuvant particles to be resuspended in the main solvent phase, which can be a burden for vaccine manufacturers. Therefore, tracking the packing density of the modified calcium phosphate is an important parameter for downstream applications.

To 1 ml of 2% (w/v) calcium phosphate in 5 mM imidazole buffer at pH 7.0 was added concentrated solutions of ATP or AMP to achieve final concentrations ranging from 0.5 mM to 5.0 mM. The homogenized suspension (with vigorous shaking) was transferred to a plastic spectrophotometric cuvette and left to settle for 48 hours until a clear phase separation was observed between the particle layer and the liquid above. The layer height was measured depending on the concentration of nucleotides. Under these conditions, the height of the total suspension volume in the cuvette was constant and equal to 22 mm. The results are shown in FIG. 14 and FIG. 15.

Experiment #12 IP6 Irreversible Adsorption Test on Adju-Phos® Particles

To test for irreversible adsorption of IP6 to Adju-Phos® particles (e.g. via ligand exchange), Adju-Phos at 2% (w/v) was treated with 0.2 mM Na-IP6 for 24 hours in 5 mM imidazole buffer at pH 7.0. This corresponds to the minimum IP6 concentration at which the Adju-Phos ζ potential reaches its maximum value in order to limit the excess of free, unadsorbed IP6. The resulting Adju-PhosZP (modified IP6) was serially diluted with 5 mM imidazole buffer at pH 7.0 to maintain constant pH and ionic strength conditions. ζ-potential was recorded depending on the dilution factor and compared with conventional (untreated) Adju-Phos®. The results are shown in FIG. 16.

Experiment #13 Irreversible ATP Adsorption Test on Adju-Phos® Particles

To test for the irreversible adsorption of ATP to Adju-Phos® particles (e.g., via ligand exchange), an experiment similar to Experiment No. 12 was performed. Adju-Phos at a concentration of 2% (w/v) was treated with 1.0 mM ATP for 24 hours in 5 mM imidazole buffer at pH 7.0. The resulting ATP-modified Adju-Phos was serially diluted with 5 mM imidazole buffer at pH 7.0 to maintain constant pH and ionic strength conditions. ζ-potential was recorded depending on the dilution factor and compared with conventional (untreated) Adju-Phos®. The results are shown in FIG. 17.

Experiment #14 Irreversible Adsorption Test of IP6 and ATP on Calcium Phosphate

To test for irreversible adsorption of IP6 or ATP to calcium phosphate particles (e.g. via ligand exchange), an experiment similar to Experiment Nos. 13 and 14 was performed. Calcium phosphate at a concentration of 2% (w/v) was treated with either 0.5 mM IP6 , or 1.0 mm ATP for 24 hours in 5 mm imidazole buffer at pH 7.0. The resulting calcium phosphate modified with either IP6 or ATP was serially diluted with 5 mM imidazole buffer at pH 7.0 to maintain constant pH and ionic strength conditions. The ζ-potential was recorded depending on the dilution factor and compared with conventional (untreated) calcium phosphate. The results are shown in FIG. 18.

Experiment No. 15. Method for large-scale production of Adju-PhosZP® (Adju-Phos® modified with sodium inositol hexaphosphate)

After precipitation and fixation of 45 kg salt in the form of aluminum (III) phosphate hydrate in a volume of 2000 liters of pure water in the reaction tank, 50 liters of a freshly prepared solution of 20 mm dodecanatrium inositol hexaphosphate (Na-IP6) in pure water is added to the suspension at a flow rate of 5 ml / min and with constant stirring. After adding the entire volume of the Na-IP6 solution, the suspension is left under stirring for another two hours. The zeta potential is measured at regular intervals from the start of the addition of the Na-IP6 solution to monitor the adsorption of phytate on the aluminum phosphate particles. As a rule, a stable value of the zeta potential is reached within the first two hours after the addition of 20 mm Na-IP6 solution. Then 5.84 kg of NaCl is added to the suspension to control the ionic strength as well as the apparent density of the aluminum phosphate particles. The suspension is then transferred to an autoclave tank where Na-IP6 treated Adju-Phos is sterilized at 121° C. for 30 minutes at a pressure of 1.3 bar. After cooling, the suspension is packaged under aseptic conditions in plastic containers for transportation.

Experiment No. 16 Comparison of the adjuvant effect of IP6-modified Adju-Phos® (Adju-PhosZP®) with conventional Adju-Phos®.

The adjuvant effect of IP6-modified Adju-Phos® (Adju-PhosZP®) was compared in vivo with conventional Adju-Phos® and free (no adjuvant) antigen control in two different models using Balb/C female mice and hen egg lysozyme ( HEL) as an antigen. Experiment set up using Majgaard Jensen, O. et al. “On the effect of Al(OH) ₃ as an immunological adjuvant” APMIS 96, 257-264 (1988).

Model 1

In the case of model 1, an amount of HEL is administered subcutaneously that closely matches the adsorption capacity of Adju-PhosZP®. This value can be obtained from FIG. 1 above. Thus, saturation of Adju-PhosZP® and conventional Adju-Phos® is achieved (FIG. 1) at a concentration of hen egg lysozyme = 125 μM, at which adsorption capacities of 1.4 and 0.5 mg HEL/mg Al are observed, respectively. , for two adjuvants.

For in vivo studies, two different doses of adjuvant are used: 250 µg Al and 500 µg Al.

Based on an adsorption capacity of 1.4 mg HEL/mg Al, the following amounts of HEL should be used for Adju-PhosZP® in a model 1 system:

In the case of a dose of 250 µg, the quantity = 350 µg HEL (250 µg * 1.4) is used, and

In the case of a dose of 500 µg, the amount used is 700 µg HEL (500 µg * 1.4).

These amounts, 350 µg and 700 µg HEL, are then tested in three different ways: adsorbed on ¹⁾ Adju-PhosZP®, ²⁾ adsorbed on regular Adju-Phos®, and ³⁾ used without adjuvant, i.e. not adsorbed.

Similarly, based on an adsorption capacity of 0.5 mg HEL/mg Al, the following amounts of HEL should be used for regular Adju-Phos® in a model 2 system:

In the case of a dose of 250 µg, the quantity = 125 µg HEL (250 µg * 0.5) is used, and

In the case of a dose of 500 µg, the amount used is 250 µg HEL (500 µg * 0.5).

Under the conditions of model 1, the amount of HEL used will be completely adsorbed by the amount of Adju-PhosZP®, but only partially by the amount of conventional Adju-Phos® due to the lower adsorption capacity of this adjuvant. Thus, for a dose of 250 µg, the injection will contain (350-125) µg = 225 µg of unbound antigen using conventional Adju-Phos®. In the case of a dose of 500 µg, there will be (700-250) µg = 450 µg of unbound antigen using conventional Adju-Phos®. For the control group, all HEL administered will, by definition, be unbound.

Model 2

In the case of model 2, an amount of HEL is injected subcutaneously that closely matches the adsorption capacity of conventional Adju-Phos®. Saturation values are calculated according to model 1 above. Again, two different doses of adjuvant are used: 250 µg and 500 µg Al, so in the case of a dose of 250 µg, use the amount = 125 µg HEL (250 µg * 0.5), and in the case of a dose of 500 µg, use the amount = 250 mcg HEL (500 mcg * 0.5). These amounts, 150 and 300 µg HEL, are again tested in three different ways: adsorbed on ¹⁾ Adju-PhosZP®, ²⁾ adsorbed on regular Adju-Phos®, and ³⁾ used without adjuvant, i.e. not adsorbed.

Under model 2 conditions, the amount of HEL used will be completely adsorbed by the amount of Adju-PhosZP® as well as the amount of regular Adju-Phos®. However, under these conditions, the entire capacity of Adju-PhosZP® is not fully utilized. This means that the administered dose of Adju-PhosZP® will have a free capacity which can be represented as an equivalent amount of HEL. Thus, in the case of a dose of 250 µg, the free adsorption capacity of Adju-PhosZP® will be (350-125) µg = 225 µg HEL. In the case of a dose of 500 µg, the free adsorption capacity of Adju-PhosZP® will be (700-250) µg = 450 µg HEL. In the case of the control group, by definition, there will be no free adsorption capacity.

Study:

Each of the two models (1 or 2) contains 3 study groups, each containing 6-8 female Balb/C mice. They are injected subcutaneously with 200 µl containing the following amounts of antigen ("AG") and adjuvant (Adju-PhosZP®, designated "ZP", and conventional Adju-Phos®, designated "AP")

250 µg Al Model 1 Model 2 Antigen only (AG) 350 mcg HEL 125 mcg HEL AG + adjuvant ZP 350 mcg HEL 125 mcg HEL AG + AP adjuvant 350 mcg HEL 125 mcg HEL 500 µg Al Model 1 Model 2 Antigen only (AG) 700 mcg HEL 250 mcg HEL AG + adjuvant ZP 700 mcg HEL 250 mcg HEL AG + AP adjuvant 700 mcg HEL 250 mcg HEL

Anti-HEL antibodies (Ab=IgG) are analyzed after the final bleed in mice 21 days post-vaccination using a serum antibody ELISA assay as described above. A higher humoral response is observed for the AG+ZP combination than for AG+AP. In the case of model 2, there is also a higher humoral response for the AG+ZP combination than for AG+AP, despite the same amount of injected antigen, indicating that the free adsorption capacity of Adju-PhosZP® in the model 2 system may have a positive effect. in the case of similar doses of antigen.

Experiment No. 17 Comparison of the adjuvant effect of IP6-modified calcium phosphate (IP6-CAPO) with conventional calcium phosphate (CAPO).

This experiment is carried out similarly to experiment No. 15 described above. Saturation of IP6-CAPO and conventional CAPO (see FIG. 3) is achieved at adsorption capacities of 175 μg HEL/mg Ca and 40 μg HEL/mg Ca, respectively. In the case of these values for saturation, the following amounts of antigen (HEL) for injection can be calculated similarly to Experiment 15 above:

250 mcg Ca Model 1 Model 2 Antigen only (AG) 44 mcg HEL 10 mcg HEL AG + adjuvant IP6-CAPO 44 mcg HEL 10 mcg HEL AG + CAPO adjuvant 44 mcg HEL 10 mcg HEL 500 mcg Ca Model 1 Model 2 Antigen only (AG) 88 mcg HEL 20 mcg HEL AG + adjuvant IP6-CAPO 88 mcg HEL 20 mcg HEL AG + CAPO adjuvant 88 mcg HEL 20 mcg HEL

For model 1: in the case of the above amounts, there will be an unbound CAPO antigen*. In the case of an administered dose of Ca of 250 μg, the unbound amount of antigen would be (44-10) μg = 34 μg. In the case of an administered dose of Ca of 500 μg, the unbound amount of antigen would be 88-20=68 μg.

For Model 2: In the case of the above quantities, there will be a free adsorption capacity* IP6-CAPO. In the case of an administered dose of Ca of 250 μg, it would be 44-10=34 μg of antigen, and in the case of an administered dose of Ca of 500 μg, it would be 88-20=68 μg of antigen.

Study:

An in vivo study of two models (1 or 2) was carried out as in experiment 15 described above.

Each model contains 3 study groups, each containing 6-8 female Balb/C mice. They are injected subcutaneously with 200 µl containing the following amounts of antigen ("AG") and adjuvant (IP6-CAPO, designated "CAPO*", and conventional calcium phosphate, designated "CAPO"):

250 mcg Ca Model 1 Model 2 Antigen only (AG) 44 mcg HEL 10 mcg HEL AG + CAPO* adjuvant 44 mcg HEL 10 mcg HEL AG + CAPO adjuvant 44 mcg HEL 10 mcg HEL 500 mcg Ca Model 1 Model 2 Antigen only (AG) 88 mcg HEL 20 mcg HEL AG + CAPO* adjuvant 88 mcg HEL 20 mcg HEL AG + CAPO adjuvant 88 mcg HEL 20 mcg HEL

Anti-HEL antibodies (Ab=IgG) are analyzed after the final bleed in mice 21 days post-vaccination using a serum antibody ELISA assay as described above. A higher humoral response is observed for the AG+CAPO* combination than for AG+CAPO. In the case of model 2, a higher humoral response is also observed for the AG+CAPO* combination than for AG+CAPO, despite the same amount of injected antigen, indicating that the free adsorption capacity of IP6-modified calcium phosphate (IP6-CAPO, CAPO *) in the model 2 system can have a positive effect in case of similar doses of antigen.

Claims (44)

1. Method for obtaining organically derivatized mineral microparticles, which includes a) providing a solution of one or more organic polyphosphates, b) providing mineral microparticles, c) suspending said mineral microparticles in said solution of one or more organic polyphosphates, d) mixing the resulting slurry, where said mixing is continued until there is no change in the zeta potential of organically derivatized mineral microparticles by more than 10% over a period of 5 minutes, with determination by analysis of samples of the reaction product isolated at intervals of 5 minutes ± 10 seconds, wherein said mineral microparticles are selected from aluminum phosphate microparticles, amorphous aluminum hydroxyphosphate and/or calcium phosphate, and said mineral microparticles have a size ranging from 0.01 µm to 10 µm, and wherein said organic polyphosphate is selected from compounds of formula 1A or 1B:

where n is an integer in the range from 1 to 5, R _a denotes organic substituents selected from adenosine;

where m is an integer in the range from 2 to 10, and where R _a denotes organic substituents selected from adenosine and other nucleosides, and R _b denotes organic substituents selected from inositol. 2. The method of claim 1 wherein the solution of one or more organic polyphosphates comprises inositol phosphate selected from inositol bisphosphate (IP2), inosittrisphosphate (IP3), inosittetraphosphate (IP4) and inositpentakisphosphate (IP5) inositol hexaphosphate (IP6). 3. The method according to claim 1 or 2, wherein the solution of one or more organic polyphosphates comprises inositol hexaphosphate (IP6) or a salt thereof. 4. The method according to any one of paragraphs. 1-3, in which step d) is continued until there is no change in the zeta potential of organically derivatized mineral microparticles by no more than 5% over a period of 5 minutes, with determination by analysis of samples of the reaction product isolated at intervals of 5 minutes ± 10 seconds. 5. The method according to any one of paragraphs. 1-4, wherein the mineral microparticle starting material comprises calcium phosphate microparticles. 6. The method according to any one of paragraphs. 1-4, in which the starting material of mineral microparticles contains aluminum. 7. Organically derivatized mineral microparticles obtained by the method according to any one of paragraphs. 1-6 which includes a) providing a solution of one or more organic polyphosphates, b) providing mineral microparticles, c) suspending said mineral microparticles in said solution of one or more organic polyphosphates, d) mixing the resulting slurry, where said mixing is continued until there is no change in the zeta potential of organically derivatized mineral microparticles by more than 10% over a period of 5 minutes, with determination by analysis of samples of the reaction product isolated at intervals of 5 minutes ± 10 seconds, wherein said mineral microparticles are selected from aluminum phosphate microparticles, amorphous aluminum hydroxyphosphate and/or calcium phosphate, and said mineral microparticles have a size ranging from 0.01 µm to 10 µm, and wherein said organic polyphosphate is selected from compounds of formula 1A or 1B:

where n is an integer in the range from 1 to 5, R _a denotes organic substituents selected from adenosine;

where m is an integer in the range from 2 to 10, and where R _a denotes organic substituents selected from adenosine and other nucleosides, and R _b denotes organic substituents selected from inositol. 8. Organically derivatized calcium phosphate microparticles obtained by the method according to paragraphs. 1-5. 9. Organically derivatized mineral microparticles according to any one of claims 7 to 8, characterized in that they have a nominal zeta potential, measured at pH 7.0 in distilled water, which is at least -35 mV in the case of microparticles obtained from aluminum phosphate or hydroxyphosphate, or at least 40 mV in the case of microparticles derived from calcium phosphate. 10. Organically derivatized mineral microparticles for use in medicine, obtained by the method according to any one of paragraphs. 1-6 which includes: a) providing a solution of one or more organic polyphosphates, b) providing mineral microparticles, c) suspending said mineral microparticles in said solution of one or more organic polyphosphates, d) stirring the resulting suspension, wherein said mineral microparticles are selected from aluminum phosphate microparticles, amorphous aluminum hydroxyphosphate and/or calcium phosphate, said mineral microparticles having a size ranging from 0.01 µm to 10 µm, and said organic polyphosphate is selected from compounds of formula 1A or 1B:

where n is an integer in the range from 1 to 5, R _a denotes organic substituents selected from adenosine;

where m is an integer in the range from 2 to 10, and where R _a denotes organic substituents selected from adenosine and other nucleosides, and R _b denotes organic substituents selected from inositol. 11. The use of organic derivatized mineral microparticles according to any one of paragraphs. 7-9 as delivery or adsorption systems for biomolecules. 12. The use of organic derivatized mineral microparticles according to claim 10 as biomolecule delivery systems, said biomolecule delivery system being a vaccine adjuvant. 13. The use of organic derivatized mineral microparticles according to claim 10 as vaccine adjuvants, in vaccines. 14. The use of organic derivatized mineral microparticles according to claim 10 in blood fractionation.

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