Method for preparation of aluminium phosphate gel for application in vaccine formulations

Abstract

The present invention relates to an improved process for production of Aluminium phosphate (AlPhos) gel wherein the solutions of aluminium salt and alkaline phosphate salt are added to water by maintaining the pH under stirring to obtain the precipitate, followed by sterilization of the said precipitate and finally obtaining the Aluminum phosphate gel.

Claims

1. A process for preparing an aluminum phosphate gel, comprising: admixing and stirring water and solutions of aluminum salt and alkaline phosphate salt at a pH between 3.0 and 4.0 to obtain the aluminum phosphate gel; and (ii) sterilizing the aluminum phosphate gel of step (i) by exposing the aluminum phosphate gel to a temperature in a range of 120-150 C. for a period of 30 to 90 minutes, wherein the aluminum phosphate gel has particles with a size distribution of d(50) in a range of 3.0 m to 9.0 m and a mean particle size less than 7 m, and wherein the process is devoid of the steps of settling an aluminum phosphate suspension and removal of a supernatant.

2. The process of claim 1, wherein the aluminum salt solution is aluminum chloride and the alkaline phosphate salt solution is trisodium phosphate, wherein concentrations of each of the solutions of aluminum chloride and trisodium phosphate are in the range of 400 and 500 milliMolar (mM), preferably between 480 and 490 mM.

3. The process of claim 2, wherein the admixing and stirring of aluminum chloride solution and trisodium phosphate solution is carried out at least at 400 rpm, preferably between 500 and 600 rpm.

4. The process of claim 1, wherein the admixing and stirring in step (i) is carried out at room temperature or at a temperature between 20 C. and 25 C.

5. The process of claim 1, wherein the admixing and stirring in step (i) is carried out for a period of 10 to 60 minutes, preferably 35 to 55 minutes.

6. The process of claim 1, wherein the aluminum phosphate gel has a particle size distribution wherein d(90) is in a range of <15 m, preferably 5 to 10 m.

7. The process of claim 1, wherein the process is carried out in a closed system.

8. The process of claim 1, wherein the process is carried out at a pH range of 3.2 to 3.5 without addition of any other acid or alkali.

9. The process of claim 1, wherein a supernatant is not formed.

10. A process for preparing an aluminum phosphate gel, comprising: admixing and stirring water and solutions of aluminum chloride and trisodium phosphate for 60 minutes, preferably between 35 to 55 minutes, to form the aluminum phosphate gel, and maintaining a pH of the aluminum phosphate gel between 3.0 and 4.0, preferably between 3.2 and 3.5; and (ii) sterilizing the aluminum phosphate gel of step (i) by exposing the aluminum phosphate gel to a temperature in a range of 120-150 C. for a period of 30 to 90 minutes, wherein the aluminum phosphate gel has particles with a size distribution of d(50) in a range of 3.0 m to 9.0 m and a mean particle size less than 7 m, and wherein the process is devoid of the steps of settling an aluminum phosphate suspension and removal of a supernatant.

11. A method of preparing a vaccine composition comprising adsorbing one or more antigens on the aluminum phosphate gel formed from the process of any one of claims 1-10.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention provides a method of producing aluminium phosphate gel which comprises the steps of adding aluminium salt and alkaline phosphate salt to water by maintaining the pH between 3.0 and 4.0, under stirring to obtain a precipitate.

(2) Aluminium salt, as used according to the present invention, is in the form of Aluminium chloride hexahydrate.

(3) Alkaline phosphate used according to the present invention is Sodium phosphate, preferably, tribasic sodium phosphate or dibasic sodium phosphate.

(4) The pH is maintained by adding specific concentrations of the solutions of aluminium chloride and trisodium phosphate. Preferably, the concentration between 400 and 500 milliMolar (mM) is used, more preferably between 480 and 490 mM is used.

(5) Both aluminium chloride and trisodium phosphate are added to water under stirring for a period of 10 to 60 minutes, preferably, 35 to 55 minutes, with the addition of initial amount of aluminium chloride solution for a period of 1 to 60 seconds, preferably 30 seconds, more preferably 2 to 10 seconds, prior to the addition of sodium phosphate solution, so as to maintain the pH of precipitation between 3.0 and 4.0, preferably between 3.2 and 3.5, almost throughout the reaction except the last 5-10% part of addition.

(6) The solutions are added to water under stirring at not less than 400 rpm, preferably between 500 and 600 rpm, and more suitably at the mid-to higher range.

(7) The solutions of aluminium chloride and trisodium phosphate are added to water at room temperature or at a temperature between 20 C. and 25 C.

(8) The solutions of aluminium chloride and trisodium phosphate are added and mixed with water in a vessel, which has baffles, minimum 2 and preferably between 2 and 4 in number. The stirring mechanism is top-mounted and the stirring rod has at least 3 impellers, each having not less than 4 blades.

(9) According to the process of the present invention, gel is formed at a pH range of 3.2 to 3.5 without addition of any other acid or alkali. This method may be used to provide a gel with a concentration not more than 3 mg/mL of aluminium, preferably, in the range up to 2.5 mg/mL of aluminium.

(10) The Aluminium phosphate gel prepared according to the present invention has a particle-size distribution wherein d(90) is in the range of <15 m, preferably 5 to 10 m.

(11) The efficiency of the method is improved significantly by adding the aluminium chloride and sodium phosphate tribasic to water at a constant ratio, and by maintaining pH & temperature. Such a process also makes the steps required in purification and concentration of final product, i.e. Aluminium Phosphate Gel, redundant.

(12) In yet another embodiment, the process involves, sterilizing the aluminium phosphate precipitate of step (i) by heating the aluminium phosphate precipitate to a temperature in excess of 120 C., preferably, 120-150 C. for a period of not less than 30 minutes, and preferably 30 to 90 minutes.

(13) Another improvement of the present invention is performing the process steps within a closed system, thereby increasing the sterility assurance of the final product and reducing the need for sterility testing. This makes the overall process more efficient since it reduces the number of sterility tests that need to be performed.

(14) In yet another embodiment, the present invention provides a process for the preparation of sterile Aluminium Phosphate gel aseptically within 60 minutes, more preferably within 45 minutes with the use of pre-sterilized WFI in a vessel, sterile-filtered chemical solutions and other process-related accessories, which can be immediately used for formulation of vaccines without any purification, sizing or other testing.

(15) In yet another embodiment, the Aluminium Phosphate gel produced according to present invention is more resistant to changes in particle sizing and other physicochemical parameters. Further, the pH of the gel, post-sterilization, is in the range more suitable for blending of antigens which have an iso-electric point (pI) above 5.0, and thereby requiring no pH adjustment.

(16) In yet another embodiment, the present invention provides a process for the preparation of sterile Aluminium Phosphate gel which, unexpectedly, aids in avoiding the steps of settling and thereby there is no formation of supernatant. Hence, the entire process is completed within a time period of 1 to 5 hours, towards the latter if a post-preparation sterilization mode is followed, which makes it simple, economical and operationally highly feasible.

(17) In a preferred embodiment, the present invention provides a process for the preparation of Aluminium Phosphate Gel which comprises the steps of:

(18) i) adding the solutions of aluminium chloride and trisodium phosphate to water for injection under stirring within 60 minutes, preferably between 35 to 55 minutes, by maintaining the pH of the precipitate formed between 3.0 and 4.0, preferably between 3.2 and 3.5;

(19) ii) sterilizing the aluminium phosphate precipitate of step (i) by heating the aluminium phosphate precipitate to a temperature in the range of 120-150 C. for a period of 30 to 90 minutes; and

(20) iii) obtaining the aluminium phosphate gel having particles with a size distribution having d(50) in the range of 3.0 m to 9.0 m and a mean particle size less than 7 m, wherein the process is devoid of the steps of settling the aluminium phosphate suspension and removal of supernatant.

(21) The Aluminium Phosphate adjuvant prepared according to the present invention remained stable during the shelf life when stored at or below room temperature.

(22) In a more preferred embodiment, the present invention provides a method of producing aluminium phosphate gel having a particle size distribution wherein d(90) is in the range of <15 m, which comprises the steps of:

(23) i) adding the solutions of aluminium chloride and trisodium phosphate at a concentration between 400 and 500 milliMolar (mM) to Water For Injection, wherein initial amount of aluminium chloride solution is added 2 to 10 seconds, prior to the addition of sodium phosphate solution, under stirring within 60 minutes, preferably between 35 to 55 minutes, by maintaining the pH of the precipitate formed between 3.0 and 4.0, preferably between 3.2 and 3.5;

(24) ii) sterilizing the aluminium phosphate precipitate of step (i) by heating the aluminium phosphate precipitate to a temperature in the range of 120-150 C. for a period of 30 to 90 minutes and,

(25) iii) obtaining the aluminium phosphate gel having particles with a size distribution having d(50) in the range of 3.0 m to 9.0 m and a mean particle size less than 7 m, wherein the process is devoid of the steps of settling the aluminium phosphate suspension and removal of supernatant.

(26) The mechanism of adjuvanticity of aluminium compounds includes formation of a depot, efficient uptake of aluminium adsorbed antigen particles by antigen presenting cells due to their particulate nature and optimal size (<10 m); and stimulation of immune competent cells of the body through activation of complement.

(27) In yet another embodiment, the present invention provides a formulation process of vaccine which comprises the steps of:

(28) i. adding the solutions of sterile-filtered aluminium chloride and trisodium phosphate to pre-sterilized water for injection under stirring within 60 minutes, preferably between 35 to 55 minutes, by maintaining the pH of the precipitate formed between 3.0 and 4.0, preferably between 3.2 and 3.5;

(29) ii. adding the antigens to the aluminium phosphate gel obtained in step (i) immediately without any purification, sterilization, sizing.

(30) In yet another embodiment, the present invention provides use of Aluminium Phosphate Gel prepared according to the process for adsorption of antigens in vaccine preparations.

Advantages of the Invention

(31) 1. The process of the present invention does not involve any addition of acid/alkali for pH adjustment.

(32) 2. The process does not involve any step for particle size reduction of aluminium phosphate gel.

(33) 3. The process does not involve the steps of settling and thereby removal of supernatant.

(34) 4. The process does not involve any washing step.

(35) 5. The process avoids high temperature conditions and longer durations for maturation.

(36) The present invention will be more specifically illustrated with reference to the following examples. However, it should be understood that the present invention is not limited by these examples in any manner, but includes variations thereof within the parameters described herein, as can be known to those well-versed in the art.

(37) Example-1. Preparation of Aluminium Phosphate Gel.

(38) Solutions of aluminium chloride and trisodium phosphate at a concentration of 4851 mM were used as raw materials and Water For Injection (WFI) as the solvent for producing the aluminium phosphate gel. Both solutions were added as shown in the Table given below at a constant rate, within 4510 minutes, to WFI (at a volume 3.1-3.15X of a solution's volume) under stirring at 55050 rpm in a vessel having 2-4 baffles, a top-mounted stirrer with minimum 3 impellers at different heights and each having 4-blades. Addition of aluminium chloride solution was started 2-5 seconds prior to start of addition of Sodium phosphate solution to maintain the pH of precipitation below 3.5 throughout the process, except for the last 5-10% of addition time, during which the pH was seen to rise to around 5.0. Post-completion of addition, the gel was sterilized in situ using steam at 121.1 C. for 30-45 minutes. pH of the gel, post-sterilization, reduced by 1 unit to reach around 4.00.3.

(39) TABLE-US-00001 TABLE 1 Aluminium Phosphate Gel Preparation Solution volumes required for gel volume of . . . Stock Solutions Qty./L 1.8 L 18 L 40 L Aluminium Chloride 117.1 g/L 350 ml 3.5 L 7.7 L (Solution 1) Tri-Sodium Phosphate 184.3 g/L 350 ml 3.5 L 7.7 L (Solution 2) WFI required 1.1 L 11 L 24.6 L Final Concentration of both salts in the gel is 94.3 mM; 1:1 ratio.

(40) Aluminium phosphate gel, using the above process, was produced from 1L to 40L scales, giving results reproducibly at each scale provided the parameters are within the given range. There is no other settling, purificationi.e. washing of the gel using any buffers for e.g. saline, etc. in this process and hence it is very simple, straight-forward and results in a gel which is ready-to-use for blending. The process, thereby, proved its versatility, scalability and its cost-effectiveness, thereby confirming its suitability for commercial production.

(41) Example-2. Particle size of Aluminium Phosphate Gel Preparation.

(42) The particle size of the aluminium phosphate gel prepared as described in Example 1 was determined and the mean particle size was found to be in the range of not more than 7 m, with the d(10), d(50) & d(90) ranges being >1 m, 3-8 m and <14m, respectively, without any additional processing step, e.g. homogenization, fines selection/removal, etc. Representative data for pH and particle sizes, along with PZC and Zeta-potential values, estimated during preparation of gel lots made at 18L and 40L scales, both before and after sterilization, is given in Table-2. The data indicates that without any of the additional process requirements, the process has proven its simplicity and reproducibility.

(43) TABLE-US-00002 TABLE 2 pH, Particle Size Ranges & Other Parameters Tested on 3 Lots Each of Aluminium Phosphate Gel (AlPhos Gel) prepared at 18 L and 40 L Scales Batch No. 18 L 40 L APG 18/01 APG 18/02 APG 18/03 APG 40/01 APG 40/02 APG 40/03 BS AS BS AS BS AS BS AS BS AS BS AS pH 5.2 4.31 5.23 4.1 4.88 3.91 4.9 4.06 5.1 4.2 4.9 3.82 Particle Size Mean 3.64 4.75 3.59 5.64 3.68 4.18 4.26 4.85 3.42 4.63 3.64 4.33 (m) d.sub.10 2.07 3.15 2.07 2.68 2.01 2.71 2.73 3.26 2.03 3.19 2.28 2.66 d.sub.50 4.77 6.7 4.51 7.84 4.35 5.62 4.74 6.13 3.92 6.54 4.07 5.28 d.sub.90 8.26 10.9 7.7 12.7 8.89 9.88 7.33 10.0 6.75 10.2 6.54 8.83 PZC 5.19 5.4 5.23 5.39 5.48 5.44 5.1 5.18 5.27 5.35 5.27 5.34 Zeta Potential 31.1 35.7 31.8 32.0 26.5 28.8 33.5 35.0 31.9 32.4 30.8 32.3 @ pH 7.5 BS = Before Sterilization; AS = After Sterilization.

(44) Example-3. Formulation of Aluminium Phosphate Gel Adjuvant in Immunogenic Compositions.

(45) The aluminium phosphate gel prepared as described in the Example-1, was used in formulation of liquid pentavalent vaccine (LPV) comprising Diphtheria toxoid (DT), Tetanus Toxoid (TT), Whole-cell pertussis (wP), Hepatitis-B surface antigen (HBsAg) and Haemophilus influenzae type-b Polyribosyl Ribitol phosphate-TT conjugate (Hib) as the antigenic components, in saline as the final diluent. In this formulation, to the gel obtained as in Example-1 under stirring at 200-300 rpm the HBsAg, DT and TT antigens were added one after the other without requirement of any pH adjustment, as it is the ideal pH for their adsorption to the gel (adjuvant) as per literature and proved in our studies. This blend was then added with up to 80% of saline required for final volume make-up followed by addition of wP; then the whole blend was chilled to below 10 C., and Hib component was added with stirring. The final volume was then made-up to the required level with saline. The pH of the blend, if required, was then adjusted to be between 6.2 and 6.5, but was not found to be necessary in most cases.

(46) The process, as described above, was optimized for the LPV blending process using the aluminium phosphate gel prepared as described in Example 1 at different scales. LPV blends of volumes ranging from 100 mL to 60L was made using this process, and all were tested and proved to meet the specifications for the vaccine, including the safety and potency parameters of each antigen used in the formulation.

(47) The aluminium phosphate gel was also shown to be suitable in the preparation of Liquid Quadravalent vaccine (LQV) comprising Diphtheria toxoid (DT), Tetanus Toxoid (TT), Whole-cell pertussis (wP), and Haemophilus influenzae type-b Polyribosyl Ribitol phosphate-TT conjugate (Hib) as the antigenic components, in saline as the final diluent. The formulation of this vaccine follows the same antigens and sequences of their addition, except non-use of HBsAg which is added first in the LPV preparation.

(48) LQV blends of volumes ranging from 1L to 60L have been made using this process, and all have been tested and proved to meet the specifications for the vaccine, including the safety and potency parameters of each antigen used in the formulation.

(49) Example-4. Antigen Adsorption Capacity Procedure and Results.

(50) Among the antigens used for formulating LPV HBsAg, DT & TT were the three which bind to aluminium phosphate gel. The in-house requirement for binding in LPV for HBsAg was 90%, for that of DT it was 28% and for TT it was 30%. The aluminium phosphate gel produced at different scales has consistently met these requirements, when tested as produced up to 40L gel lot and LPV blend volume of up to 60L. The variations in the % adsorption values between LPV blends produced using the Aluminium Phosphate gel prepared as per the procedure described in Example-1, both made at different scales, is insignificant. Also, exposure of the vaccine to stress conditions (37 C. for 14 days) did not change the % adsorption values significantly. A representative example of this property of the gel, made at different scales (1L and 18L) and used for LPV blending at 2L and 60L scales, along with a 2L and 60L blends of LPV made using Adju-Phos of Brenntag Biosector (commercial supplier) is summarised in Table-3.

(51) As an example of the proof of stability of the gel/LPV blend made using the Aluminium Phosphate gel prepared as per Example-1, the % adsorption results for the three antigens of a 4L LPV blend made and tested on days 0 (release), after incubation at 37 C. on days 5, 7 & 14 are summarised in Table-4.

(52) TABLE-US-00003 TABLE 3 % Adsorption Results, in LPV Blends with Different Gel Sources & Blend Sizes Prepared as per the Adju-Phos procedure described S. (Brenntag) in Example-1 No. Antigen 2 L LPV 60 L LPV 2 L LPV 60 L LPV 1 HBsAg 94.8 99.7 98.8 98.9 2 Diphtheria 47.6 59.6 50.0 58.3 Toxoid 3 Tetanus Toxoid 41.7 46.7 44.8 46.7

(53) The data presented in Table-3 confirm that the % adsorption of the aluminium phosphate gel prepared as per the procedure described in Example-1 matches with that of Adju-Phos of Brenntag.

(54) TABLE-US-00004 TABLE 4 % Adsorption Results - 4 L LPV Blend at Release (Day-0) & After Incubation at 37 C. for 14 days Sampling Day Diphtheria Tetanus HBsAg Day-0 50.0 40.72 96.5 Day-5 77.1 53.8 98.7 Day-7 41.7 48.6 99.2 Day-14 56.8 47.5 98.9

(55) Example-5. Other Procedures for Characterizing the Gel and Their Results.

(56) Several lots of aluminium phosphate gel produced as described in Example 1 were analysed for various physico-chemical properties so that they are fully characterized. Other than pH, particle size and aluminium content, few more parameters were also tested which, along with the reasons for their analysis, are summarised in Table-5.

(57) TABLE-US-00005 TABLE 5 Physico-chemical Properties of AlPhos Gel to be Tested & Rationale Impact/Reason for Parameter Limit Analysis Appearance White turbid Describes the general suspension in which physical appearance the mineral carrier tends to settle down slowly upon Aluminium content Not Less Than 2 Determines the binding mg/mL capacity Particle Mean Nor More Than Determines surface area, size (NMT) 7 aggregation potential, (m) D.sub.10 1 binding capacity D.sub.50 3-8 D.sub.90 14 PZC (pI) 5.1 0.5 Zeta Potential 28 to 40 mV Determines adsorption (@ pH 7.5) ratio/rates; main factor determining aggregation: also determines the stability of suspension Antigen Adsorption rHBsAg: 90% Determines relative capacity DT: 28% distribution of antigens in TT:: 30% sol/gel phases Impurity Free NMT 50 ppm Gives indication of gel Profile aluminium purity Soluble NMT 0.5% as PO.sub.4 Phosphates Specific Gravity/ 1.00-1.03 Determines weight/unit Sedimentation Rate NMT 35% Determines the settleability of the gel Osmolality 550 50 mOsm/Kg Reflects the concentration of solutes in the gel

(58) Representative quantitative data from the six batches of Aluminium Phosphate Gel produced at 18L scale prepared as per the procedure described in Example-1 for process validation batches of LPV and LQV batches are summarised in Tables 6 & 7. All these batches complied with requirements for all the parameters tested at both post- & pre-sterilization stages; while all batches complied with the specifications for appearance, sterility and impurities levels, the quantitative parameters among those were analysed in these tables.

(59) TABLE-US-00006 TABLE 6 Analysis of Results of Physico-chemical Parameters Testing of Aluminium Phosphate Gel Prepared as per the procedure described in Example-1 - Pre-Sterilization Stage In LPV Batches In LQV Batches Analyses S. No. Test LPV-1 LPV-2 LPV-3 LQV A LQV-B LQV-C Avg. Std. Dev. % CV 1 Particle size Mean 3.50 3.19 3.24 3.50 3.62 3.73 3.46 0.21 6.10 (m) D.sub.10 2.03 1.86 1.93 1.97 2.12 2.26 2.03 0.14 7.09 D.sub.50 4.2 3.67 3.68 4.4 4.19 4.29 4.07 0.32 7.77 D.sub.90 7.34 6.43 6.28 7.82 7.26 7.19 7.05 0.59 8.31 2 PZC (pI) 5.33 5.44 5.41 5.49 5.50 5.36 5.38 5.43 0.06 32.8 32.6 31.8 33.4 30.7 33.7 32.3 32.3 1.5 3 Zeta Potential 32.8 32.6 31.8 33.4 30.7 33.7 32.3 32.3 1.5 (@ pH 7.5) 4 Sedimentation 27 25 25 25 25 25 25 25.0 0.0 Rate @ 48 hours (in %)

(60) The CV (Co-efficient of Variation) values for all parameters tested, without considering the individual distribution ranges of particle sizes, were well within 10% and more so with the post-sterilization stage; this indicated good consistency of the gel preparation procedure over six batches analysed, produced over a period of 6 months with different lots of input materials. The data also confirmed that only minor, insignificant changes occur post-sterilization in physico-chemical parameters of the gel.

(61) TABLE-US-00007 TABLE 7 Analysis of Results of Physico-chemical Parameters Testing of Aluminium Phosphate Gel Prepared as per the procedure described in Example-1 - Post-Sterilization Stage In LPV Batches In LQV Batches Analyses S. No. Test LPV-1 LPV-2 LPV-3 LQV A LQV-B LQV-C Avg. Std. Dev. % CV 1 Aluminium 2.42 2.42 2.395 2.05 2.15 2.10 2.26 0.17 7.71 content 2 pH 3.77 4.28 4.31 3.91 4.04 4.21 4.09 0.22 5.32 3 Particle size Mean 4.13 3.91 4.00 4.24 3.90 4.00 4.03 0.13 3.28 (m) D.sub.10 2.49 2.57 2.39 2.52 2.46 2.38 2.47 0.07 3.00 D.sub.50 5.48 4.07 4.97 5.73 5.15 4.68 5.01 0.59 11.81 D.sub.90 9.29 7.63 8.45 9.63 8.51 7.85 8.56 0.78 9.14 4 PZC (pI) 5.33 5.48 5.54 5.47 5.44 5.4 5.44 0.07 1.33 5 Zeta Potential 32.8 31.27 33.43 33.9 33.03 34.17 33.7 0.60 1.77 (@ pH 7.5) 6 Specific 1.020 1.020 1.019 Not Checked Not Applicable Gravity 7 Osmolality 528 542 509 Not Checked Not Applicable 8 Sedimentation 27 28 28 28 27 28 27.7 0.52 1.87 Rate @ 48 hours (in %)

(62) Some of these parameters, for a few batches, have been tested over the gel's storage period (up to 7 days) at different stages viz. after preparation, after in-situ sterilization, and after autoclaving of in-situ sterilized gel. Representative data from one batch tested for storage effect on different parameters is summarized in Table-8. The data confirm that the parameters do not change much up to 7 days post-sterilization/autoclaving. These characterization data and their analyses confirm the consistency of the gels made at different scales and also reconfirm the robustness of the gel preparation process.

(63) TABLE-US-00008 TABLE 8 Physico-Chemical Parameters of an Aluminium Phosphate Gel Batch at Different Stages of Production and Storage Sedimentation Particle Size (m) ZP (@ Osmolality Rate (%), Stage D.sub.10 D.sub.50 D.sub.90 Mean pH PZC pH 7.5) (mOsm/Kg) @ 48 hrs. After Preparation 2.21 4.73 8.38 3.79 5.51 5.2 32.87 518 25 Post in situ sterilization 2.90 5.60 9.30 4.44 4.20 5.0 31.93 28 Post autoclaving of 2.99 5.79 9.73 4.52 4.20 5.0 32.10 Not 28 in-situ sterilized gel Checked (Day-0) Post autoclaving of 2.94 5.79 10.04 4.54 4.65 5.0 34.67 28 in-situ sterilized gel (Day-7) Post- Day-0 2.19 4.74 8.45 3.78 4.30 5.1 32.00 523 26 autoclaving Day-7 2.33 4.92 9.19 3.94 4.68 5.2 33.27 524 25 only

(64) Example-6. Efficacy & Stability of AlPhos gel as Adjuvant in Vaccine Preparations.

(65) Three batches of 60L LPV were formulated with three different batches of aluminium phosphate gel prepared as described in Example-1, and one 60L LPV batch was formulated with Brenntag's Adju-Phos gel as a controlfor comparison of the product produced using the procedure as described in Example-1 and Brenntag's AlPhos gels. The aluminium phosphate gel was prepared at 18L scale using sterilized solutions & in sterile vessel and was transferred in sterile glass bottles for sterilization by autoclaving; this sterile gel was transferred to blending vessel for LPV blending.

(66) Three Final Bulks of LPV were produced using in-house aluminium phosphate gel, with the following batch numbers (as mentioned in tables 6 & 7, under Example-5): LPV-1, LPV-2 and LPV-3. The reference batch of LPV Final Bulk produced with Brenntag's Adju-Phos was given the number LPV-4.

(67) The lots of antigens used for formulation of LPV-1 and LPV-4 were the same, to enable comparison of impact of gel made prepared according to the present invention and that of Adju-Phos available commercially, made by Brenntag Biosector. The results of all qualitative & quantitative tests complied with the acceptance criteria for respective parameter, and thus confirm that the AlPhos Gel prepared according to the present invention produced the LPV lots which were highly comparable with that produced using the Adju-Phos of Brenntag, thus confirming the suitability of in-house AlPhos gel in making the vaccine preparations.

(68) Similar to the LPV, three Final Bulks of LQV (D-T-P-Hib vaccine) were also produced using aluminium phosphate gel prepared according to the present invention, with the following batch numbers (as mentioned in tables 6 & 7, under Example-5): LQV-A, LQV-B and LQV-C. The results of all qualitative & quantitative tests complied with the acceptance criteria (same as that for LPV) for respective parameter, thus reconfirming that the gel prepared according to the present invention for blending of LQV lots were also highly consistent. These data reaffirm the suitability of AlPhos gel of the present invention in making the vaccine preparations.

(69) All the lots of LPV and LQV produced as above were filled into glass vials in single (0.5 mL) dose 10-dose (5.0 mL) presentations and subjected to stability studies at real-time (2-8 C.) and accelerated (252 C.) temperature storage conditions. The test parameters were evaluated to check the stability were all complying with the specifications up to 3 months storage, when last tested, at both conditions for both vaccines; and the results of stability parameters tested on the Final Lots filled from LPV-1 and LPV-4 up to 9 months' time-point when stored at Real-time storage conditions are again comparable, indicating no change in any of the parameters tested due to change in the AlPhos gel source.

(70) Hence the process of the present invention to prepare Aluminium-Phosphate gel using the unique process has proven to be easily reproducible, scalable and highly suitable for use in vaccine preparations as an adjuvant.