Indian Patents. 279169:A process for preparation of stable aluminum phosphate nanoparticles

Title of Invention	A process for preparation of stable aluminum phosphate nanoparticles
Abstract	The instant invention claims stable aluminium phosphate nanoparticles and a process for preparation thereof.

Full Text	THE PATENTS ACT, 1970 COMPLETE SPECIFICATION Section 10 "Aluminium Phosphate Nanoparticles." Serum Institute of India Ltd, an Indian company, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028 Maharashtra India. The following specification particularly describes the nature of this invention and the manner in which it is to be performed: \\Vulkan\Patent Data\daleep\Year 2008\App!icnlion\Conventional phase\March.doc Aluminium Phosphate Nanoparticles FIELD OF INVENTION The present invention relates to aluminium phosphate nanoparticles and method of preparation thereof. BACKGROUND OF THE INVENTION Adjuvants are defined as substances used in combination with a specific antigen to enhance the immune response. An effective vaccine usually requires an adjuvant to stimulate the immune response. The only adjuvants approved for use in currently licensed vaccines are the aluminium-containing adjuvants. Aluminium hydroxide and aluminium phosphate are the most commonly used adjuvants. In 1926, the adjuvant effect of aluminium compounds was described by Glenny et. al. (Glenny AT, Pope CG, Waddington H, Wallace U. Immunological Notes XVII to XXIV. J. Pathol. 29, 31-40, 1926) . Scholtz et. al. in 1984 demonstrated that pure aluminium phosphate can be prepared using equimolar amounts of aluminium chloride and trisodium phosphate. Although such adjuvants are widely used today, aluminium phosphate adjuvants can be difficult to manufacture with reproducible properties as the experimental conditions like temperature, concentration and even rate of addition of chemicals reagents strongly influence the properties of the gel prepared. The important properties that are affected are particle size distribution, electric charge and hydrated colloid nature of the precipitate formed. These properties then affect the adsorption of antigen and thus the immunogenicity of the antigen. Subcutaneous administration of vaccines is considered as the most appropriate route for delivering an antigen to secondary lymphoid organs such as lymph nodes where adaptive immunity is initiated. It is known that the fate of the particles following subcutaneous administration depends on the size of the particles. Large particles, having a size greater than 1 micron are unable to access and drain into the lymphatics and may not be phagocytosed. readily and they are retained at the injection site until they degrade to a sufficiently small size. On the contrary, small particles having a size less than 1 micron enter lymphatic capillaries, which form a one-way drainage system connected to the lymphatic vessels, lymph nodes and lymphatic ducts, respectively. Particles with a diameter less than 100 nm enter the lymphatic capillaries through gaps between the lymphatic endothelial cells while particles having diameter between 100 nm to 1 micron are phagocytosed by antigen representing cells such as dendritic cells which subsequently passage into the lymphatic capillaries. Nanoparticles of adjuvants are considered to be the most promising system for delivery of antigen to the draining lymph nodes following subcutaneous administration. Nanoparticles are defined as particles with diameter ranging from 1 to 1000 nm. Nanometer size particles were used as carrier particles in the prior art. See 5178882, 5219577, 5306508, 5334394, 5460830, 5462750 and 5464634. The paricles described herein are in the range of 10 to 200 nm size range and are difficult to make with a degree of consistency. Calcium phosphate particles have been under investigation as an alternative to aluminium-containing adjuvants in parenteral vaccines. Calcium Phosphate particles (>1000nm) as vaccine adjuvants are unsuitable due to inferior adjuvanting activity and possess a rough and oblong morphology. (Goto et. al., Vaccine, vol.15, no. 12-13, 1364-1371, 1997). US 6355271 discusses a method for preparing one or more particles of calcium phosphate having diameters between about 3 00 nm to about 4000 nm, comprising reacting a soluble calcium salt with a soluble phosphate salt, wherein the reaction comprises: (a) mixing an aqueous solution of calcium chloride with an aqueous solution of sodium citrate to form a mixture, (b) adding an aqueous solution of sodium phosphate to the mixture to form a solution, (c) stirring the solution until particles of the desired size and comprising calcium phosphate are obtained and co-crystallized with antigenic material. The particles of size more than 1000 nm may not be phagocytosed and therefore are not suitable as adjuvants. WO 2007/098186 discloses a composition prepared by combining one or more oils, one or more non-ionic: surfactants, optionally one or more sterols, aluminium salt and an aqueous phase. The aluminium phosphate particles prepared therein range from 50 nm to 5000 nm and are stabilized by the surfactants added therein. WO 2008/009592 discloses a method of preparing nanoparticles of metal phosphates with desired particle size, size distribution and purity wherein the method involves atomization of the solution in a reactor in excess of 100 deg C wherein the said temperature is selected in such a way that the organic acid and the water evaporate from the solution to produce nanoscale particles. Such process seems to be hazardous and teadious. The instant invention overcomes the drawbacks of. the prior art and provides a method for production of nanoparticles of aluminium phosphate with particle diameter less than 1000 nm, preferably 10 to 600 nm. SUMMARY OF THE INVENTION The instant invention relates to stable nanoparticles of Aluminium Phosphate and a process for preparation of thereof. The instant invention provides nanoparticles of aluminium phosphate with particle diameter in the range of 10 to 600 nanometer (nm). The instant invention also provides a process for preparation of aluminium phosphate nanoparticles comprising a.adjusting pH of the aluminium phosphate gel to a pH above 6, preferably between 6.5 to 10 b.passing the solution through high pressure homogenizer to get desired particle size c.optional stabilization of the nanoparticles at a pH in the range of 6 to 8, preferably at pH 7.0 The instant invention has following advantages over prior art: 1. Stability of the nanoparticles 2.Simple, robust and repeatable process 3.Process does not involve use of chemicals or high temperature 4.No additional stabilizer like surfactants, glycols or oils required 5.The robust nanoparticles afforded are stored at physiological pH hence can be used as adjuvant without additional processing DESCRIPTION OF FIGURES Figure 1: Particle size statistics report by intensity of nanoparticles immediately after preparation Figure 2: Particle size statistics report by intensity of nanoparticles one month after preparation DETAILED DESCRIPTION OF THE INVENTION The first embodiment of the instant invention is the stable nanoparticles of aluminium phosphate with particle diameter less than 1000 nm. The second embodiment of the instant invention is the process for preparation of the stable nanoparticles of aluminium phosphate with particle diameter less than 1000 run. Specifically, the stable nanoparticles of aluminium phosphate are of diameter less than 600 nm, preferably between 10 to 600 nm. The particle size or mean particle size of the nanoparticles can be controlled and the particle size distribution can be restricted to a desired level. The process of preparation of aluminium phosphate nanoparticles is a simple and robust process which produces the particles of desired diameter. The process produced nanoparticles with high degree of consistency in terms of properties of the particles. The aluminium phosphate gel used for preparation of the nanoparticles can be prepared in situ or aluminium phosphate powder can be dissolved in suitable buffer before size reduction. The nanoparticles of the instant invention can be prepared from aluminium phosphate gel or precipitate. The pH of the solution can be adjusted to a desired pH in the range of 6 to 10. The preferred pH is around 7.0. The particles can be prepared by treating the gel with either a homogenizer or a sonicator. Appropriate technical parameters e.g. pressure, frequency, speed or amplitude of the homogenizer or sonicator can be adjusted to produce particles of desired size. The particles thus obtained can be stabilized in a suitable buffer. The preferred buffer is phosphate buffer of pH 7.0. The buffer can be the buffer used to formulate the final vaccine. The following Examples further illustrate the essential features of the present invention. However, it will be apparent to those skilled in the art that the specific chemicals, apparatus, instruments, conditions and methods used in the Examples do not limit the scope of the present invention. Example 1 Preparation of Aluminium Phosphate Gel Slowly add 5 L solution of trisodium phosphate (16.9%) with 5 L solution of aluminium chloride (10.74%) with continuous stirring. Stirred the above mixture, for approximately 2 hours, to allow gel or precipitate formation. Diluted the suspension to around 50 L using water for injection (WFI). Allowed the aluminium phosphate gel to settle. Decanted the supernatant and replaced with WFI. The step was repeated till the supernatant is free of chloride ions. Example 2 Preparation of Aluminium Phosphate Nanoparticles by homogenization Aluminium phosphate gel suspension, either from example 1, prepared from aluminum phosphate powder or prepared in situ, was adjusted to pH 7.0 using trisodium phosphate. The gel was subjected to high pressure homogenization at 40000 psi for 15 cycles. The average particle diameter was found to be -150 nm. The nanoparticles thus obtained were stabilized using 10 mM phosphate buffer with pH 7.0. The gel was kept at 2 to 8 degrees temperature. Stability of the nanoparticles was checked after one month. The average particle size (diameter) along with the particle size distribution was substantially stable. Example 3 Preparation of Aluminium Phosphate Nanoparticles by sonication . Aluminium phosphate gel suspension, either from example 1, prepared from aluminum phosphate powder or prepared in situ, was adjusted to PH 7.0 using trisodium phosphate. The gel was subjected to sonication using at 40 amplitude with 3 second pulse rate for 1 hour. The nanoparticles thus obtained were stabilized using 10 mM phosphate buffer with pH 7.0. The gel was kept at 2 to 8 degrees temperature. The average particle diameter was found to be 250 nm... CLAIMS: We claim, 1.Stable aluminium phosphate nanoparticles. 2.Stable aluminium phosphate nanoparticles of claim 1, wherein the particle size (diameter) is less than 1000 nm. 3.Stable aluminium phosphate nanoparticles of claim 2, wherein the particle size (diameter) is in the range of 10 to 600 nm. 4.A process for preparation of stable aluminum phosphate nanoparticles comprising a.Preparation of aluminium phosphate gel b.Adjusting pH of the aluminium phosphate gel c.Subjecting the aluminium phosphate gel to size reduction d.Affording aluminium phosphate nanoparticles of desired size e.Optionally suspending the nanoparticles in a suitable buffer 5.The process of claim 4a, wherein the aluminium phosphate gel can be prepared i) in situ, ii)by suspending aluminium phosphate powder in suitable solvent or iii) by treatment of equimolar aluminium chloride with trisodium phosphate to effect aluminium phosphate gel formation, followed by chloride removal, if required. 6.The process of claim 4b, wherein the pH of the aluminium phosphate gel is adjusted in the range of 6 to 10. 7.A process of claim 6, wherein the pH is adjusted to 7.0 8.A process of claim 4c, wherein the size reduction is effected by a technique selected from homogenization or sonication. 9.A process of claim 8, wherein the size reduction technique employed is high pressure homogenization. 10.A process of claim 4d, wherein the nanoparticles of desired size ranging from 10 to 600 nm is obtained by size reduction technique. 11.A process of claim 4e, wherein the nanoparticles prepared in 4c or 4d are suspended in a suitable buffer, preferably in phosphate buffer of pH 7.0 12.A process of claim 4e, wherein the buffer is the buffer used to formulate the antigen or vaccine. 13.The stable nanoparticles of claim 1 to 3 prepared by a process of claims 4 to 11. 14.Use of the nanoparticles of instant invention as adjuvant. Of Anand And Anand Advocates Attorney for the Applicant CLAIMS: We claim, 1.Stable aluminium phosphate nanoparticles. 2.Stable aluminium phosphate nanoparticles of claim 1, wherein the particle size (diameter) is less than 1000 nm. 3.Stable aluminium phosphate nanoparticles of claim 2, wherein the particle size (diameter) is in the range of 10 to 600 nm. 4.A process for preparation of stable aluminum phosphate nanoparticles comprising a.Preparation of aluminium phosphate gel b.Adjusting pH of the aluminium phosphate gel c.Subjecting the aluminium phosphate gel to size reduction d.Affording aluminium phosphate nanoparticles of desired size e.Optionally suspending the nanoparticles in a suitable buffer 5.The process of claim 4a, wherein the aluminium phosphate gel can be prepared i) in situ, ii)by suspending aluminium phosphate powder in suitable solvent or iii) by treatment of equimolar aluminium chloride with trisodium phosphate to effect aluminium phosphate gel formation, followed by chloride removal, if required. 6.The process of claim 4b, wherein the pH of the aluminium phosphate gel is adjusted in the range of 6 to 10. 7.A process of claim 6, wherein the pH is adjusted to 7.0 8.A process of claim 4c, wherein the size reduction is effected by a technique selected from homogenization or sonication. 9.A process of claim 8, wherein the size reduction technique employed is high pressure homogenization. 10.A process of claim 4d, wherein the nanoparticles of desired size ranging from 10 to 600 nm is obtained by size reduction technique. 11.A process of claim 4e, wherein the nanoparticles prepared in 4c or 4d are suspended in a suitable buffer, preferably in phosphate buffer of pH 7.0 12.A process of claim 4e, wherein the buffer is the buffer used to formulate the antigen or vaccine. 13.The stable nanoparticles of claim 1 to 3 prepared by a process of claims 4 to 11. 14.Use of the nanoparticles of instant invention as adjuvant. Of Anand And Anand Advocates Attorney for the Applicant

Full Text

THE PATENTS ACT, 1970
COMPLETE SPECIFICATION
Section 10
"Aluminium Phosphate Nanoparticles."
Serum Institute of India Ltd, an Indian company, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028 Maharashtra India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:
\\Vulkan\Patent Data\daleep\Year 2008\App!icnlion\Conventional phase\March.doc

Aluminium Phosphate Nanoparticles
FIELD OF INVENTION
The present invention relates to aluminium phosphate nanoparticles and method of preparation thereof.
BACKGROUND OF THE INVENTION
Adjuvants are defined as substances used in combination with a specific antigen to enhance the immune response. An effective vaccine usually requires an adjuvant to stimulate the immune response. The only adjuvants approved for use in currently licensed vaccines are the aluminium-containing adjuvants. Aluminium hydroxide and aluminium phosphate are the most commonly used adjuvants. In 1926, the adjuvant effect of aluminium compounds was described by Glenny et. al. (Glenny AT, Pope CG, Waddington H, Wallace U. Immunological Notes XVII to XXIV. J. Pathol. 29, 31-40, 1926) . Scholtz et. al. in 1984 demonstrated that pure aluminium phosphate can be prepared using equimolar amounts of aluminium chloride and trisodium phosphate. Although such adjuvants are widely used today, aluminium phosphate adjuvants can be difficult to manufacture with reproducible properties as the experimental conditions like temperature, concentration and even rate of addition of chemicals reagents strongly influence the properties of the gel prepared. The important properties that are affected are particle size distribution, electric charge and hydrated colloid nature of the precipitate formed. These properties then affect the adsorption of antigen and thus the immunogenicity of the antigen.

Subcutaneous administration of vaccines is considered as the most appropriate route for delivering an antigen to secondary lymphoid organs such as lymph nodes where adaptive immunity is initiated. It is known that the fate of the particles following subcutaneous administration depends on the size of the particles. Large particles, having a size greater than 1 micron are unable to access and drain into the lymphatics and may not be phagocytosed. readily and they are retained at the injection site until they degrade to a sufficiently small size. On the contrary, small particles having a size less than 1 micron enter lymphatic capillaries, which form a one-way drainage system connected to the lymphatic vessels, lymph nodes and lymphatic ducts, respectively. Particles with a diameter less than 100 nm enter the lymphatic capillaries through gaps between the lymphatic endothelial cells while particles having diameter between 100 nm to 1 micron are phagocytosed by antigen representing cells such as dendritic cells which subsequently passage into the lymphatic capillaries.
Nanoparticles of adjuvants are considered to be the most promising system for delivery of antigen to the draining lymph nodes following subcutaneous administration. Nanoparticles are defined as particles with diameter ranging from 1 to 1000 nm.
Nanometer size particles were used as carrier particles in the prior art. See 5178882, 5219577, 5306508, 5334394, 5460830, 5462750 and 5464634. The paricles described herein are in the range of 10 to 200 nm size range and are difficult to make with a degree of consistency.

Calcium phosphate particles have been under investigation as an alternative to aluminium-containing adjuvants in parenteral vaccines. Calcium Phosphate particles (>1000nm) as vaccine adjuvants are unsuitable due to inferior adjuvanting activity and possess a rough and oblong morphology. (Goto et. al., Vaccine, vol.15, no. 12-13, 1364-1371, 1997).
US 6355271 discusses a method for preparing one or more particles of calcium phosphate having diameters between about 3 00 nm to about 4000 nm, comprising reacting a soluble calcium salt with a soluble phosphate salt, wherein the reaction comprises: (a) mixing an aqueous solution of calcium chloride with an aqueous solution of sodium citrate to form a mixture, (b) adding an aqueous solution of sodium phosphate to the mixture to form a solution, (c) stirring the solution until particles of the desired size and comprising calcium phosphate are obtained and co-crystallized with antigenic material. The particles of size more than 1000 nm may not be phagocytosed and therefore are not suitable as adjuvants.
WO 2007/098186 discloses a composition prepared by combining one or more oils, one or more non-ionic: surfactants, optionally one or more sterols, aluminium salt and an aqueous phase. The aluminium phosphate particles prepared therein range from 50 nm to 5000 nm and are stabilized by the surfactants added therein.
WO 2008/009592 discloses a method of preparing nanoparticles of metal phosphates with desired particle

size, size distribution and purity wherein the method involves atomization of the solution in a reactor in excess of 100 deg C wherein the said temperature is selected in such a way that the organic acid and the water evaporate from the solution to produce nanoscale particles. Such process seems to be hazardous and teadious.
The instant invention overcomes the drawbacks of. the prior art and provides a method for production of nanoparticles of aluminium phosphate with particle diameter less than 1000 nm, preferably 10 to 600 nm.
SUMMARY OF THE INVENTION
The instant invention relates to stable nanoparticles of Aluminium Phosphate and a process for preparation of thereof.
The instant invention provides nanoparticles of aluminium phosphate with particle diameter in the range of 10 to 600 nanometer (nm).
The instant invention also provides a process for preparation of aluminium phosphate nanoparticles comprising
a.adjusting pH of the aluminium phosphate gel to a pH
above 6, preferably between 6.5 to 10
b.passing the solution through high pressure homogenizer
to get desired particle size
c.optional stabilization of the nanoparticles at a pH in
the range of 6 to 8, preferably at pH 7.0
The instant invention has following advantages over prior art:
1. Stability of the nanoparticles

2.Simple, robust and repeatable process
3.Process does not involve use of chemicals or high temperature
4.No additional stabilizer like surfactants, glycols or oils required
5.The robust nanoparticles afforded are stored at physiological pH hence can be used as adjuvant without additional processing
DESCRIPTION OF FIGURES
Figure 1: Particle size statistics report by intensity of nanoparticles immediately after preparation
Figure 2: Particle size statistics report by intensity of nanoparticles one month after preparation
DETAILED DESCRIPTION OF THE INVENTION
The first embodiment of the instant invention is the stable nanoparticles of aluminium phosphate with particle diameter less than 1000 nm.
The second embodiment of the instant invention is the process for preparation of the stable nanoparticles of aluminium phosphate with particle diameter less than 1000 run.
Specifically, the stable nanoparticles of aluminium phosphate are of diameter less than 600 nm, preferably between 10 to 600 nm. The particle size or mean particle size of the nanoparticles can be controlled and the particle size distribution can be restricted to a desired level. The process of preparation of aluminium phosphate nanoparticles is a simple and robust process which produces

the particles of desired diameter. The process produced nanoparticles with high degree of consistency in terms of properties of the particles.
The aluminium phosphate gel used for preparation of the nanoparticles can be prepared in situ or aluminium phosphate powder can be dissolved in suitable buffer before size reduction.
The nanoparticles of the instant invention can be prepared from aluminium phosphate gel or precipitate. The pH of the solution can be adjusted to a desired pH in the range of 6 to 10. The preferred pH is around 7.0. The particles can be prepared by treating the gel with either a homogenizer or a sonicator. Appropriate technical parameters e.g. pressure, frequency, speed or amplitude of the homogenizer or sonicator can be adjusted to produce particles of desired size. The particles thus obtained can be stabilized in a suitable buffer. The preferred buffer is phosphate buffer of pH 7.0. The buffer can be the buffer used to formulate the final vaccine.
The following Examples further illustrate the essential features of the present invention. However, it will be apparent to those skilled in the art that the specific chemicals, apparatus, instruments, conditions and methods used in the Examples do not limit the scope of the present invention.
Example 1
Preparation of Aluminium Phosphate Gel

Slowly add 5 L solution of trisodium phosphate (16.9%) with 5 L solution of aluminium chloride (10.74%) with continuous stirring. Stirred the above mixture, for approximately 2 hours, to allow gel or precipitate formation. Diluted the suspension to around 50 L using water for injection (WFI). Allowed the aluminium phosphate gel to settle. Decanted the supernatant and replaced with WFI. The step was repeated till the supernatant is free of chloride ions.
Example 2
Preparation of Aluminium Phosphate Nanoparticles by homogenization
Aluminium phosphate gel suspension, either from example 1, prepared from aluminum phosphate powder or prepared in situ, was adjusted to pH 7.0 using trisodium phosphate. The gel was subjected to high pressure homogenization at 40000 psi for 15 cycles. The average particle diameter was found to be -150 nm. The nanoparticles thus obtained were stabilized using 10 mM phosphate buffer with pH 7.0. The gel was kept at 2 to 8 degrees temperature. Stability of the nanoparticles was checked after one month. The average particle size (diameter) along with the particle size distribution was substantially stable.
Example 3
Preparation of Aluminium Phosphate Nanoparticles by sonication .

Aluminium phosphate gel suspension, either from example 1, prepared from aluminum phosphate powder or prepared in situ, was adjusted to PH 7.0 using trisodium phosphate. The gel was subjected to sonication using at 40 amplitude with 3 second pulse rate for 1 hour. The nanoparticles thus obtained were stabilized using 10 mM phosphate buffer with pH 7.0. The gel was kept at 2 to 8 degrees temperature. The average particle diameter was found to be 250 nm...

CLAIMS:
We claim,
1.Stable aluminium phosphate nanoparticles.
2.Stable aluminium phosphate nanoparticles of claim 1, wherein the particle size (diameter) is less than 1000 nm.
3.Stable aluminium phosphate nanoparticles of claim 2, wherein the particle size (diameter) is in the range of 10 to 600 nm.
4.A process for preparation of stable aluminum phosphate nanoparticles comprising
a.Preparation of aluminium phosphate gel
b.Adjusting pH of the aluminium phosphate gel
c.Subjecting the aluminium phosphate gel to size
reduction
d.Affording aluminium phosphate nanoparticles of
desired size
e.Optionally suspending the nanoparticles in a
suitable buffer
5.The process of claim 4a, wherein the aluminium phosphate gel can be prepared i) in situ, ii)by suspending aluminium phosphate powder in suitable solvent or iii) by treatment of equimolar aluminium chloride with trisodium phosphate to effect aluminium phosphate gel formation, followed by chloride removal, if required.
6.The process of claim 4b, wherein the pH of the aluminium phosphate gel is adjusted in the range of 6 to 10.
7.A process of claim 6, wherein the pH is adjusted to 7.0

8.A process of claim 4c, wherein the size reduction is effected by a technique selected from homogenization or sonication.
9.A process of claim 8, wherein the size reduction technique employed is high pressure homogenization.

10.A process of claim 4d, wherein the nanoparticles of desired size ranging from 10 to 600 nm is obtained by size reduction technique.
11.A process of claim 4e, wherein the nanoparticles prepared in 4c or 4d are suspended in a suitable buffer, preferably in phosphate buffer of pH 7.0
12.A process of claim 4e, wherein the buffer is the buffer used to formulate the antigen or vaccine.
13.The stable nanoparticles of claim 1 to 3 prepared by a process of claims 4 to 11.
14.Use of the nanoparticles of instant invention as adjuvant.
Of Anand And Anand Advocates Attorney for the Applicant

CLAIMS:
We claim,
1.Stable aluminium phosphate nanoparticles.
2.Stable aluminium phosphate nanoparticles of claim 1, wherein the particle size (diameter) is less than 1000 nm.
3.Stable aluminium phosphate nanoparticles of claim 2, wherein the particle size (diameter) is in the range of 10 to 600 nm.
4.A process for preparation of stable aluminum phosphate nanoparticles comprising
a.Preparation of aluminium phosphate gel
b.Adjusting pH of the aluminium phosphate gel
c.Subjecting the aluminium phosphate gel to size
reduction
d.Affording aluminium phosphate nanoparticles of
desired size
e.Optionally suspending the nanoparticles in a
suitable buffer
5.The process of claim 4a, wherein the aluminium phosphate gel can be prepared i) in situ, ii)by suspending aluminium phosphate powder in suitable solvent or iii) by treatment of equimolar aluminium chloride with trisodium phosphate to effect aluminium phosphate gel formation, followed by chloride removal, if required.
6.The process of claim 4b, wherein the pH of the aluminium phosphate gel is adjusted in the range of 6 to 10.
7.A process of claim 6, wherein the pH is adjusted to 7.0

8.A process of claim 4c, wherein the size reduction is effected by a technique selected from homogenization or sonication.
9.A process of claim 8, wherein the size reduction technique employed is high pressure homogenization.

10.A process of claim 4d, wherein the nanoparticles of desired size ranging from 10 to 600 nm is obtained by size reduction technique.
11.A process of claim 4e, wherein the nanoparticles prepared in 4c or 4d are suspended in a suitable buffer, preferably in phosphate buffer of pH 7.0
12.A process of claim 4e, wherein the buffer is the buffer used to formulate the antigen or vaccine.
13.The stable nanoparticles of claim 1 to 3 prepared by a process of claims 4 to 11.
14.Use of the nanoparticles of instant invention as adjuvant.
Of Anand And Anand Advocates Attorney for the Applicant

Documents:

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Patent Number

279169

Indian Patent Application Number

1040/MUM/2008

PG Journal Number

02/2017

Publication Date

13-Jan-2017

Grant Date

13-Jan-2017

Date of Filing

08-May-2008

Name of Patentee

SERUM INSTITUTE OF INDIA PRIVATE LIMITED

Applicant Address

212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411028, Maharashtra India.

Inventors:

#	Inventor's Name	Inventor's Address
1	KAPRE Subhash v,	Serum Institute of India Ltd., 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411028 Maharashtra India.
2	SINGH Sanjay K.	Serum Institute of India Ltd., of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411028 Maharashtra India.

PCT International Classification Number

A61K9/51; C01B25/36

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA