DESC:MICROPARTICULATE DRY POWDER COMPOSITIONS AND A METHOD OF PREPARATION THEREOF

FIELD OF THE INVENTION
[0001] The present disclosure pertains to the field of microparticulate dry powder compositions, in particular, compositions for prevention of tuberculosis and more particularly to a composition formulated for pulmonary delivery.

BACKGROUND OF THE INVENTION
[0002] In the realm of microbiology and pharmaceuticals, the utilization of subunits of mycobacterium species in vaccine applications has garnered substantial attention. Mycobacteria, characterized by their unique biological properties, present promising avenues for the development of innovative antigenic compositions. Moreover, in the contemporary landscape of global health, the persistent threat posed by drug-resistant tuberculosis (TB) underscores the imperative for groundbreaking interventions. Traditional antibiotic regimens are increasingly challenged by the adaptability of Mycobacterium tuberculosis, necessitating a paradigm shift in prevention and therapeutic strategies. This complex milieu necessitates an exploration of unconventional approaches, and herein lies the genesis of a sophisticated microparticulate dry powder composition.
[0003] Tuberculosis (TB) is a chronic infectious disease caused by mycobacterium, mainly Mycobacterium tuberculosis. With an estimated one third of the world's population infected with tuberculosis, Mycobacterium tuberculosis is arguably one of the most successful pathologic micro-organisms worldwide and is the causative agent of the potentially infectious tuberculosis. It is a major disease in developing countries, as well as an increasing problem in developed areas of the world. Tuberculosis most commonly attacks the lungs, but can also affect the central nervous system, the lymphatic system, the circulatory system, the genitourinary system, bones, joints and even the skin. Symptoms of tuberculosis include a severe cough that lasts, weight loss, coughing up blood or mucus, weakness or fatigue, fever and chills, night sweats, loss of appetite.
[0004] Pulmonary TB is the most common clinical presentation of infection with Mycobacterium tuberculosis. Symptoms typically include chronic cough with or without haemoptysis, fever, night sweats, and weight loss. The pathogenesis of TB is complex, with the initial infection occurring as the result of the inhalation of aerosolized infectious Mycobacterium tuberculosis. Generally, person-to-person transmission occurs by aerosolized droplets generated by a person suffering from pulmonary TB (active disease). When a person breathes in TB bacteria, the bacteria can settle in the lungs and begin to grow. Only individuals with active pulmonary disease remain infectious as they can aerosolize the TB bacilli.
[0005] Furthermore, partial compliance with treatment may though lead to suboptimal therapeutic levels of the antimicrobials, and the micro-evolution of antibiotic resistance mutations. As a result of this, patients may remain infectious for longer durations, and the frequency of transmission is enhanced. The development of these resistance mutations, some of which are unresponsive to all known anti TB medications (multi-resistant), has recently caused heightened concern globally.
[0006] Despite the potential of mycobacterium-based therapies, conventional delivery methods face considerable challenges. Issues such as variable efficacy, limited stability, and concerns related to patient compliance have underscored the necessity for advancements in formulation design.
[0007] GB36270/67A and GB2920771A discloses pharmaceutical powders for the inhalatory use in which the micronised drug (0.01-10 µm) is mixed with carrier particles of sizes 30 to 80 µm and 80 to 150 µm, respectively; said mixtures can also contain a diluent of the same particle size as the micronised drug. GB9101551A discloses, for example, a controlled crystallization process for the preparation of carrier particles with smoother surfaces, and, in particular, characterized by a rugosity of less than 1.75 as measured by air permeametry; in practice their smoothness is readily apparent under electronic microscope examination. The use of said carrier particles allows to increase the respirable fraction of the drug.
[0008] EP92924673A claims the use of carriers for controlling and optimizing the amount of delivered drug during the aerosolisation phase, consisting of suitable mixtures of particles of size >20 µm and finer particles (<10 µm).
[0009] WO1995011666A1 combines both the aforementioned teachings (i.e. modification of the surface properties of the carrier and addition of a fine fraction) by exploiting the effects of a milling process, preferably carried out in a ball mill, referred to as corrasion (corrasion is a term used in geology and it describes either the effect of the wind on rocks and the filling of valley with stones during the ice age). Said process modifies the surface properties and it gets rid of the waviness of the carrier particles by dislodging any asperities in the form of small grains without substantially changing the size of the particles; the small grains, in turn, can be reattached to the surfaces of the particles either during the milling phase or after preventive separation followed by mixing, in order to saturate other high energy sites such as clefts. Said preliminary handling of the carrier causes the micronized drug particles to preferably link to the lower energy sites, thus being subjected to weaker interparticle adhesion forces.
[00010] While these patents collectively contribute to the understanding of mycobacterial composition and pulmonary delivery systems, none appear to anticipate or suggest the precise combination, manufacturing process, and therapeutic applications detailed in the current invention. Therefore, the proposed microparticulate dry powder composition, comprising a synergistic blend of mycobacterial antigenic subunits, stands as a novel and non-obvious advancement in the field.
[00011] Currently there are several new TB vaccines in clinical trials. However, they are primarily classical preventive vaccines based on a limited number of antigens expressed in the early stage of infection. As a direct consequence of the expression dynamic the epitope pattern that is presented to T cells changes radically over time - implicating how new vaccines should be designed. Presently, formulations predominantly centre around a single antigenic component of mycobacterium species, limiting the scope of applications. This one-dimensional approach leaves unaddressed the potential synergies and diverse biological activities that could arise from combining multiple antigenic subunits of mycobacterium species within a single prophylactic composition. Furthermore, challenges related to particle size distribution, aerosolization efficiency, and sustained release of the antigenic components necessitate a comprehensive solution. Existing formulations often fall short in providing a holistic and adaptable platform for delivering mycobacterium-based prophylactic compositions.
[00012] In response to the challenges and gaps identified in the prior art, the present invention introduces a novel microparticulate dry powder composition. This composition, meticulously designed, incorporates at least three distinct Mycobacterium tuberculosis species specific subunit antigens, each selected for its unique therapeutic attributes.
[00013] By combining these Mycobacterium tuberculosis species specific subunit antigens into a finely tuned microparticulate dry powder, the invention aims to address the limitations of current formulations. The synergistic interaction among the mycobacterial components enhances stability, improves aerosolization efficiency, and broadens the therapeutic spectrum and targeting to specific receptors in alveolar macrophages (AM), in particular the Dectin-1 receptor on AMs, making the composition a promising candidate for various applications in the fields of infectious diseases, immunotherapy, and beyond.
SUMMARY OF THE INVENTION
[00014] The present invention envisages a microparticulate dry powder composition, optimized for intrapulmonary delivery. The composition comprises a synergistically balanced blend of at least three Mycobacterium tuberculosis species specific subunit antigens selected from recombinant Escherichia coli strains, an adjuvant, sodium alginate and calcium chloride. In an embodiment of the present invention the concentration of each subunit antigens is finely tuned to elicit a cooperative immunomodulatory response and therapeutic effect against multidrug-resistant tuberculosis. In yet another embodiment of the present invention, the subunit antigens are selected from 38 kDa protein, Lpqh and ESAT-6.
[00015] The present invention also envisages a method for manufacturing the microparticulate dry powder composition. The method involves initially preparing an antigen blend of at least three Mycobacterium tuberculosis species specific subunit antigens selected from recombinant Escherichia coli strains. Once the blend is prepared, pre-determined amount of the carrier, ß-glucan is then mixed with the antigen blend to obtain glucan-antigen mixture; wherein the ratio ranges from 1:40 to 1: 80. ß-glucan facilitates the entrapment of the antigens within the pores of ß-glucan to obtain glucan-antigen mixture. The second step involves adding a pre-determined amount of sodium alginate to the glucan-antigen mixture such that a coat is formed over the glucan particles, entrapping the antigens within the pores of glucan to obtain coated glucan-antigen mixture. The concentration of sodium alginate is in the range of 0.1 to 0.5% w/v to the glucan-antigen mixture. The third step involves adding a pre-determined amount of calcium chloride to coated glucan-antigen mixture to cross-link the alginate layer surrounding the particles through ionic gelation technique, thereby rigidizing the coat to obtain the microparticulate dry powder composition. The concentration of calcium chloride is in the range of 2-6% w/v.
BRIEF DESCRIPTION OF THE DRAWINGS
[00016] The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate exemplary embodiments and, together with the description, serve to explain the disclosed principles:
FIG.1A, 1B and 1C illustrate the release profiles of entrapped antigens in the simulated lung fluid (pH 7.4) for glucan microparticles (GMP) prepared using varying concentrations of sodium alginate and calcium chloride and with varying gelation times.
Figure 2 illustrates in-process antigen stability for optimization of gelation time; wherein A depicts 38 kDa; B depicts Lpqh; C depicts ESAT-6; D depicts Puregene PG-PMT-0782 Protein Ladder;
Figure 3 depicts SEM images of Antigen-loaded GMP; a double-sided carbon tape which was affixed to an aluminum stub and a small amount of GMP sample was mounted over the carbon tape. The aluminum stub holding the sample was placed in the SEM chamber and photographs of samples were taken to assess their surface appearance and shape. The images revealed distinct particles without any aggregation. The particles exhibited an irregular, hollow shape with an average size of 2-4 µm, which is suitable for inhalation.
Figure 4A and 4B illustrate in vitro release of BSA in simulated lung fluid (SLF, pH 7.4) and in vitro release of BSA in alveolar fluid (ALF, pH 4.5) respectively;
Figure 5 illustrates Andersen Cascade Impactor (ACI) and assembling of components;
Figure 6 depicts in vitro lung deposition of GMP-BSA using ACI;
Figures 7A depicts structure integrity of GMP-38 kDa during Accelerated Stability Testing; wherein 1 is pure 38 kDa; 2, 4, 6 and 8 are samples stored at 5°C±3°C analyzed at 1, 2, 4 and 6 months, respectively; 3, 5, 7 and 9 are samples stored at 25°C±2°C/60% RH±5% RH analyzed at 1, 2, 4 and 6 months, respectively;
Figure 7B depicts structure integrity of GMP-Lpqh during Accelerated Stability Testing; wherein 1 is pure Lpqh; 2, 4, 6 and 8 are samples stored at 5°C±3°C analyzed at 1, 2, 4 and 6 months, respectively; 3, 5, 7 and 9 are samples stored at 25°C±2°C/60% RH±5% RH analyzed at 1, 2, 4 and 6 months, respectively; and
Figure 7C depicts structure integrity of GMP-ESAT-6 during Accelerated Stability Testing; wherein 1 is pure ESAT-6; 2, 4, 6 and 8 are samples stored at 5°C±3°C analyzed at 1, 2, 4 and 6 months, respectively; 3, 5, 7 and 9 are samples stored at 25°C±2°C/60% RH±5% RH analyzed at 1, 2, 4 and 6 months, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[00017] The following is a detailed description of embodiments of the present disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[00018] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[00019] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[00020] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[00021] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
[00022] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[00023] All methods described herein can be performed in suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[00024] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[00025] Various terms are used herein. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[00026] At its core, this innovative composition transcends the singular focus on Mycobacterium tuberculosis, incorporating a nuanced blend of at least three Mycobacterium tuberculosis species specific subunit antigens. This blend is strategically interwoven for their unexplored immunomodulatory capacities. This amalgamation aims not only to directly combat the pathogen but also to orchestrate a dynamic interplay with the host immune milieu, creating a symbiotic strategy against the formidable backdrop of multidrug-resistant tuberculosis strains.
[00027] The carrier particles, a cornerstone of this composition, transcend mere encapsulation. Engineered with meticulous precision, they embody biocompatible matrices that transcend traditional delivery systems. The particle size distribution, maintained within the stringent confines of 2 to 4 micrometers, orchestrates an aerodynamic symphony that facilitates optimal deposition deep within the pulmonary recesses, thereby maximizing the bioavailability of the mycobacterial consortium.
[00028] In this avant-garde composition, stabilizing agents and pharmaceutical excipients play a dual role. Beyond ensuring prolonged shelf-life, they intricately interface with the mycobacterial consortium, harmonizing stability with the imperative for optimal pulmonary delivery. This convergence addresses not only the challenges of composition stability but also accentuates the precision required in drug development.
[00029] This nuanced invention illuminates the transformative potential of the microparticulate dry powder composition, positioned as an avant-garde response to the challenges of drug-resistant tuberculosis. As the global health community grapples with the imperative for innovative solutions, this composition emerges as a beacon of promise in the intricate tapestry of TB prevention. The present invention discloses a microparticulate dry powder compositions and a method of preparation thereof. In one aspect, the present disclosure discloses a microparticulate dry powder composition. The composition comprises at least three Mycobacterium species specific subunit antigens. In an embodiment of the present invention, the composition comprises:
- a synergistically balanced blend of at least three Mycobacterium tuberculosis species specific subunit antigens overexpressed and purified from recombinant Escherichia coli strains, wherein the concentration of each antigen is finely tuned to elicit a cooperative immunomodulatory and protective response effect against tuberculosis;
- an adjuvant;
- sodium alginate; and
- calcium chloride.
[00030] In another embodiment of the present invention, the composition comprises Mycobacterium tuberculosis specific antigens selected from TB10.4, Ag85 complex, culture filtrate proteins and pharmaceutically acceptable carriers; wherein the carriers are selected from chitosan, tripolyphosphate, pectin, phospholipids and delivery systems selected from liposomes and polymeric or lipidic microparticle-based nano systems.
[00031] In an exemplary embodiment of the present invention, at least three Mycobacterium tuberculosis specific subunit antigens are obtained from recombinant Escherichia coli strains. The subunit antigens are selected from 38 kDa protein, Lpqh and ESAT-6.
[00032] In still another embodiment of the present invention, the composition is formulated for intrapulmonary delivery to a mammalian host, and wherein said composition comprises an immunologically protective dose when delivered to said host.
[00033] In another embodiment of the present invention, the antigens are produced by recombinant DNA technology. Typically, the 38 kDa, Lpqh and ESAT-6 antigens are overexpressed and purified from recombinant Escherichia coli strains. The said antigens can also be procured commercially.
[00034] In another embodiment of the present invention, the composition comprises an adjuvant to enhance an immune response in said host. Typically, beta-glucan is used as an adjuvant having a particle size in the range of 2 to 4µm.
[00035] In yet another embodiment of the present invention, the composition comprises a pharmaceutically acceptable carrier. Typically, the pharmaceutically acceptable carriers are selected from the group consisting of, but not limited to, chitosan, tripolyphosphate, pectin and phospholipids. Additionally, delivery systems like liposomes, polymeric or lipidic microparticle-based nano systems can also be used as pharmaceutically acceptable carriers.
[00036] In still another embodiment of the present invention, the composition is formulated for oral inhalation intrapulmonary delivery to said host using any conventional available techniques. The composition of the present invention is administered directly to the lungs and targeted to the alveolar macrophages. In an exemplary embodiment of the present invention, the composition is a microparticulate composition which facilitates the deposition in the alveolar region.
[00037] In an exemplary embodiment of the present invention, the composition is lyophilized.
[00038] In another embodiment of the present invention, the subunit antigens are intricately encapsulated within carrier particles and the particle size distribution is precisely controlled within the range of 2 to 5 µm, ensuring optimal aerodynamic properties for deep lung deposition and enhanced bioavailability.
[00039] Typically, the composition is formulated for pulmonary and mucosal delivery to a subject. The composition when delivered to the lung or mucosal/nasal mucosa of a subject is postulated to elicit a much stronger immune response.
[00040] In an embodiment of the present invention, the microparticulate dry powder composition comprises Mycobacterium tuberculosis specific subunit antigens selected from the group consisting of, but not limited to, 38 kDa protein, Lpqh and ESAT-6. The microparticles are composed of ß-glucan, which are porous particles. Typically, these particles have size in the range of 2 to 4µm in diameter.
[00041] In another embodiment of the present invention, the antigens are entrapped in the pores of these particles and sealed inside by ionic gelation method using sodium alginate and calcium chloride. Typically, the sodium alginate sealant sustains the release of entrapped antigens, with only 25% antigens releasing in 24 hrs; thereby boosting the effect of the antigens upon in vivo administration.
[00042] In still another embodiment of the present invention, the particle size of the composition is in the range of 2 to 5 µm. The composition in said particle size aids in the delivery to deep lungs which is the portal of entry of tuberculosis bacilli. The composition can be delivered into the lungs by any of the conventional methods known in the art.
[00043] In an exemplary embodiment of the present invention the composition targets the dectin-1 receptors on alveolar macrophages and dendritic cells. The composition is advantageous as it is effective for prophylaxis of active as well as latent tuberculosis.
[00044] In another aspect of the present invention, disclosed is a method of preparation of the microparticulate dry powder composition. The method comprises the following steps:
- preparing an antigen blend of at least three Mycobacterium tuberculosis species specific subunit antigens selected from recombinant Escherichia coli strains;
- mixing the antigen blend with pre-determined amount of ß-glucan,
wherein the ratio ranges from 1:40 to 1: 80 to facilitate entrapment of the antigens within the pores of ß-glucan to obtain glucan-antigen mixture;
- adding pre-determined amount of sodium alginate to the glucan-antigen mixture such that a coat is formed over the glucan particles, entrapping the antigens within the pores of glucan to obtain coated glucan-antigen mixture;
wherein the concentration of sodium alginate is in the range of 0.1 to 0.5% w/v to the glucan-antigen mixture; and
- adding pre-determined amount of calcium chloride to coated glucan-antigen mixture to cross-link the alginate layer surrounding the particles through ionic gelation technique, thereby rigidizing the coat to obtain the microparticulate dry powder composition;
wherein the concentration of calcium chloride in the range of 2-6% w/v.

[00045] In an embodiment of the present invention, the step of rigidization is carried out for a time duration in the range of 2 to 15 hours for retarding the release of the entrapped antigen.
[00046] In another embodiment of the present invention, the ß-glucan has a concentration in the range of 2 to 10 mg.
[00047] In yet another embodiment of the present invention, the antigen has a concentration in the range of 25 to 50 µg.
[00048] The scope of the present invention is not only limited to as mentioned microbial species and products derived therefrom but also extends to microbially closely related microbes specially belonging to same family, preferably belonging to same genus, still preferably belonging to same species having substantially similar characteristics.
[00049] In still another embodiment of the present invention, the microparticulate dry powder composition is utilised for the immunization against tuberculosis, wherein the concerted action of the mycobacterium-specific antigens not only establishes an enhanced host immune response but generates mucosal immunity directly at the portal of entry of the pathogen, thereby mitigating the emergence of infection and promoting more effective disease management.
[00050] In yet another embodiment of the present invention the microparticulate dry powder composition is utilised for targeted delivery of mycobacterial subunit antigens to alveolar macrophages, exploiting the unique characteristics of the composition to enhance intracellular localization and maximize antimycobacterial immunity.
[00051] The present invention also envisages a comprehensive pharmaceutical kit designed for the optimal administration of the microparticulate dry powder composition, comprising the composition, a sophisticated inhalation device equipped with precise dosing capabilities, and a comprehensive user guide delineating intricate details of administration techniques, ensuring maximal therapeutic efficacy and patient compliance.
[00052] EXAMPLES:
[00053] EXAMPLE 1: PREPARATION OF GMP-BSA/GMP-ANTIGEN
Batch size: 10 mg
Amount of Glucan microparticles (GMP): 10 mg
Vol and amount of Bovine Serum Albumin (BSA)/antigen: 50µl containing 25-50µg of protein.
Volume and concentration of sodium alginate solution: 50 µl of 0.25% w/v solution
Volume and concentration of calcium chloride solution: 50 µl of 2% w/v solution

Preparation steps
GMP-BSA was prepared based on ionic gelation of sodium alginate (Alg) in the presence of calcium chloride (CaCl2) as reported previously, with slight modification. Briefly, the procedure followed, in general, was as follows:
1. BSA solution (2.5 mg/ml) was prepared in Phosphate Buffered Saline (PBS), pH 7.4.
2. GMPs were mixed with BSA solution (5 µl/mg of GMP) in a microcentrifuge tube (MCT) to obtain a uniform paste and incubated at 4ºC for 2 hrs.
3. The protein was sealed inside the GMPs by addition of Alg solution. For this, Alg solution (5 µl/mg of GMP) was mixed with the GMP-BSA mixture and incubated at 4ºC for 1 hr.
4. Then, CaCl2 solution (5 µl/mg of GMP) was added and mixed uniformly to crosslink with Alg. This mixture was allowed to gel overnight at 4ºC.
5. The samples were centrifuged (Biofuge Stratos, Heraeus) at 5,000 rpm for 5 mins to remove unentrapped BSA and unreacted Alg and CaCl2. The supernatant was collected for determination of % entrapment efficiency (%EE) and the pellets were lyophilized (Labconco Triad) for 24 hrs.
6. The lyophilized pellets were passed through 200# sieve to obtain a dry powder, which was filled in MCT and stored refrigerated in a desiccator until further use.
[00054] EXAMPLE 2:
In accordance with the preparation method described above, nineteen different trials were carried out using BSA as the model protein by changing the different formulation parameters as given below in Table 1:
Table 1- Optimization trials carried out with BSA as a model protein
Trial No. Batch Code GMP:BSA Ratio by Weight Alg Conc
(% w/v) CaCl2 Conc (% w/v) Gelation Time (hrs) Particle size (µm) %EE

1. G1 80:1 0.25 2 15 3.57±1.25 99.33
2. G2 200:1 0.25 2 15 3.82±1.28 96.81
3. G1A1 80:1 0.1 2 15 4.28±1.88 99.14
4. G1A2 80:1 0.25 2 15 3.57±1.25 91.43
5. G1A3 80:1 0.5 2 15 4.76±1.94 95.99
6. G1A1C1 80:1 0.1 2 15 4.52±2.08 98.29
7. G1A1C2 80:1 0.1 4 15 3.33±1.66 96.49
8. G1A1C3 80:1 0.1 6 15 3.57±1.68 99.23
9. G1A2C1 80:1 0.25 2 15 3.81±1.66 96.88
10. G1A2C2 80:1 0.25 4 15 3.81±2.01 97.54
11. G1A2C3 80:1 0.25 6 15 4.05±1.61 92.52
12. G1A1C3-2 80:1 0.1 6 2 3.57±1.68 93.37
13. G1A1C3-5 80:1 0.1 6 5 3.81±1.66 95.11
14. G1A1C3-10 80:1 0.1 6 10 3.81±2.01 97.49
15. G1A1C3-15 80:1 0.1 6 15 3.57±1.68 99.42
16. G1A2C1-2 80:1 0.25 2 2 3.57±1.68 96.01
17. G1A2C1-5 80:1 0.25 2 5 4.05±1.61 93.19
18. G1A2C1-10 80:1 0.25 2 10 3.57±1.68 96.44
19. G1A2C1-15 80:1 0.25 2 15 3.81±1.66 97.66
As shown in Table 1 and Figures 1A, 1B and 1C the particles sizes were less than 5µm, entrapment efficiencies were more than 90% and satisfactory sustained release of protein was observed for trials 12 to 19 and were suitable for further optimization.
[00055] EXAMPLE 3:
Trials 12-19 were again carried out using the antigens instead of BSA to assess the in-process stability of the antigens during the preparation of the batches and to optimize the gelation time. In all, 12 trials were carried out as follows:
Table 2: Trials for Optimization of Gelation Times using Antigens
Trial Antigen GMP:Antigen Ratio by Weight Alg Conc
(% w/v) CaCl2 Conc (% w/v) Gelation Time (hrs)
Trial 1 38 kDa 80:1 0.25 2 2
Trial 2 38 kDa 80:1 0.25 2 5
Trial 3 38 kDa 80:1 0.25 2 10
Trial 4 38 kDa 80:1 0.25 2 15
Trial 5 Lpqh 80:1 0.25 2 2
Trial 6 Lpqh 80:1 0.25 2 5
Trial 7 Lpqh 80:1 0.25 2 10
Trial 8 Lpqh 80:1 0.25 2 15
Trial 9 ESAT-6 80:1 0.25 2 2
Trial 10 ESAT-6 80:1 0.25 2 5
Trial 11 ESAT-6 80:1 0.25 2 10
Trial 12 ESAT-6 80:1 0.25 2 15
As illustrated in Figure 2, the single sharp bands of desired antigens in the SDS-PAGE of trials 1 to 12 of example 3 implied that the integrity of all the three antigens was maintained irrespective of the gelation times of 2, 5, 10 and 15 hrs. From the trials 12-19 of example 2, gelation time of 15 hrs was found to provide more sustained BSA release compared to gelation times of 2, 5 and 10 hrs. Hence, gelation time of 15 hrs was considered as optimum for preparation of GMP-Antigens.
The optimized formula GMP-BSA/GMP-Antigens is as follows:
Table 3. Parameters for Optimized Batch of GMP-BSA/GMP-Antigens
Batch Parameter Optimized Value
GMP:BSA/GMP:Antigen ratio 80:1 (2 mg:25 µg)
Sodium alginate concentration 0.25% w/v
Calcium chloride concentration 2% w/v
Gelation time 15 hrs

[00056] Characterization of GMP-BSA and GMP-Antigen
The GMP-BSA and GMP-Antigen (GMP-38 kDa, GMP-Lpqh, GMP-ESAT-6) had the physicochemical properties as summarized in Table 4 below:
Table 4. Physicochemical Properties of GMP-BSA and GMP-Antigens

Sr. No. Physicochemical Property GMP-BSA GMP-38 kDa GMP-Lpqh GMP-ESAT-6
1. Color Buff colored
2. Odor Odorless
3. Shape Near spherical
4. Entrapment Efficiency (%) 97.66 ± 1.50 98.42 ± 0.89 97.25 ± 0.73 99.54 ± 0.88
5. Particle Size (µm) by Imaging Microscopy (under 10X) D10: 2.828
D50: 5.200
D90: 7.271 D10: 2.046
D50: 5.277 D90: 7.121 D10: 2.683
D50: 4.682 D90: 8.809 D10: 2.530
D50: 4.682 D90: 8.209
6. Surface Morphology by Scanning Electron Microscopy (SEM) Irregular, hollow shaped distinct particles with an average size 2-4 µm (Figure 5)
7. Zeta Potential (mV) -1.53 -6.53 -5.57 -4.27
8. Moisture Content (% w/w) 19.36 -- -- --
9. Flow properties Bulk density (g/ml) 0.13 -- -- --
Tapped density (g/ml) 0.15 -- -- --
Hausner’s ratio and Flow Characteristics 1.15
Good -- -- --
Carr’s Compressibility Index and Flow Characteristics 13.04
Good -- -- --
10. Angle of Repose [degrees (°)] and Flow Characteristics 23.58
Excellent -- -- --
11. % Protein Content 98.73 ± 1.21 99.86 ± 2.57 100.00 ± 0.59 99.66 ± 0.84

[00057] In Vitro Release:
In vitro release studies were carried out in triplicate for plain BSA and GMP-BSA as reported earlier, with some modifications. The protocol for the release study is described in Table 5 below:
Table 5. Protocol for In Vitro Release Study

Parameters Specifications
Method Sample dispersed in release medium in a polypropylene MCT and incubated in a shaker incubator at 170 ± 30 rpm (Meta-Labs Scientific Industries)
Sample quantity Equivalent to 125 µg of BSA
Release media SLF (pH 7.4)
ALF (pH 4.5)
Volume of release medium 1 ml
Temperature 37 ± 2ºC
Aliquot withdrawal time points 1, 3, 5, 24, 48 and 72 hrs
Aliquot volume 200 µl
Method of aliquot withdrawal Centrifugation at 12,000 rpm and 4°C for 2 mins and withdrawing the supernatant
Method of analysis BCA assay
The release profiles of plain BSA and of BSA from GMP-BSA in SLF and ALF are depicted in Figures 4A and 4B, respectively. Plain BSA immediately dissolved rapidly in both the release media, SLF and ALF; almost 100% release could be seen at the first time-point of analysis. The release of entrapped BSA was more sustained in ALF than in SLF. Only 25% of the entrapped BSA was released in ALF at the end of 24 hrs whereas, GMP-BSA exhibited complete release in SLF, achieving a 100% release profile within a 48-hour timeframe.
The observed 25% release of the GMP-BSA over 72 hours implies a potential susceptibility of the protein to degradation within acidic conditions.
This suggests that the release of BSA (model protein) will be more sustained after the uptake of microspheres by the macrophages. This is essential for booster effect of the administered vaccine formulation.
• In Vitro Lung Deposition by Andersen Cascade Impactor:
In vitro lung deposition of GMP-BSA was evaluated using Andersen Cascade Impactor (ACI). All the component parts of the ACI were assembled as depicted in Figure 5. Aerosolization and deposition parameters of the BSA microparticles are given in Figure 6 and Table 6 (hereinbelow).
Table 6: Aerosolization Properties of GMP-BSA
Parameter Observation
Emitted dose 1.094 ± 0.19 mg
% Emitted dose 88.41 ± 0.93 %
% Recovered dose 82.54 ± 2.9 %
% Fine particulate fraction (FPF) (<5.8 µm) 45.33 ± 5.8 %
% Fine particulate fraction (FPF) (<4.74 µm) 35.0 ± 5.9 %
Mean mass aerodynamic diameter (MMAD) 3.4 ± 0.58 µm
Geometric standard deviation (GSD) 2.9 ± 0.48
• Stability Studies of Selected Vaccine Delivery Systems
Accelerated stability studies were carried out for the following batches:
1. GMP-BSA, equivalent to 25 µg BSA, filled in size 3 HPMC capsules
2. GMP-38 kDa, equivalent to 25 µg 38 kDa, filled in size 3 HPMC capsules
3. GMP-Lpqh, equivalent to 25 µg Lpqh, filled in size 3 HPMC capsules
4. GMP-ESAT-6, equivalent to 25 µg ESAT-6, filled in size 3 HPMC capsules
The capsules were packaged in blister packs with Alu-PVC backing membrane.
[00058] Storage Conditions and Time Period
The batches to be evaluated for accelerated stability studies were stored at the following temperature conditions, as per ICH guidelines Q1A (R2) and Q5C for products to be stored in refrigerator:
1. 5°C±3°C
2. 25°C±2°C/60% RH±5% RH
The stability studies were carried out for a period of six months. The samples were evaluated at 1-, 2-, 4- and 6- months’ intervals for various physicochemical parameters.
The GMP-BSA and GMP-Antigen formulations exhibited no marked change in visual appearance upon storage up to 6 months.
The results of the particle size, protein/antigen content analyses and in vitro lung deposition of the formulations are presented in Tables 7-9.
Table 7. Particle Size Data for GMP-BSA and GMP-Antigens during Accelerated Stability Testing (Optical Microscopy under 45X Magnification)


Table 8. Protein/Antigen Content for GMP-BSA and GMP-Antigens during Accelerated Stability Testing

Table 9. In vitro Lung Deposition (%FPF) for GMP-BSA during Accelerated Stability Testing by Twin Stage Impinger
There were no marked changes in the particle size or in %FPF of the microparticles at both the storage conditions during the testing period. There was no significant change in the protein/antigen content at the conditions of stability assessment.
The results of the protein structure integrity analysis of GMP-Antigens by SDS-PAGE are presented in Figures 7A, 7B and 7C.
The antigens were found to remain stable at both the conditions of stability assessment.
Hence, it can be concluded that the developed microparticulate vaccine delivery systems are stable under the conditions of accelerated stability testing. The stability study data indicates that it may be possible to avoid cold-chain storage and transport, which may be advantageous for tropical regions.
[00059] While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure.
,CLAIMS:1. A microparticulate dry powder composition, optimized for intrapulmonary delivery, comprises;
- a synergistically balanced blend of at least three Mycobacterium tuberculosis species specific subunit antigens selected from recombinant Escherichia coli strains, wherein the concentration of each subunit antigens is finely tuned to elicit a cooperative immunomodulatory response against tuberculosis;
- an adjuvant;
- sodium alginate; and
- calcium chloride.
2. The composition as claimed in claim 1, wherein said subunit antigens are selected from 38 kDa protein, Lpqh and ESAT-6.
3. The composition as claimed in claim 1, wherein said composition further comprises Mycobacterium tuberculosis specific antigens selected from TB10.4, Ag85 complex, culture filtrate proteins and pharmaceutically acceptable carriers;
wherein the carriers are selected from chitosan, tripolyphosphate, pectin, phospholipids and delivery systems selected from liposomes and polymeric or lipidic microparticle-based nano systems.
4. The composition as claimed in claim 1, wherein adjuvant is beta glucan having a particle size in the range of 2 to 4µm.
5. The composition as claimed in claim 1, wherein said composition is lyophilized.
6. The composition as claimed in claim 1, wherein said subunit antigens are intricately encapsulated within carrier particles and the particle size distribution is precisely controlled within the range of 2 to 5 µm, ensuring optimal aerodynamic properties for deep lung deposition and enhanced bioavailability.
7. A method of manufacturing the microparticulate dry powder composition comprising the following steps:
- preparing an antigen blend of at least three Mycobacterium tuberculosis species specific subunit antigens selected from recombinant Escherichia coli strains;
- mixing said antigen blend with pre-determined amount of ß-glucan,
wherein the ratio ranges from 1:40 to 1: 80 to facilitate entrapment of the antigens within the pores of ß-glucan to obtain glucan-antigen mixture;
- adding pre-determined amount of sodium alginate to said glucan-antigen mixture such that a coat is formed over the glucan particles, entrapping the antigens within the pores of glucan to obtain coated glucan-antigen mixture;
wherein the concentration of sodium alginate is in the range of 0.1 to 0.5% w/v to the glucan-antigen mixture; and
- adding pre-determined amount of calcium chloride to coated glucan-antigen mixture to cross-link the alginate layer surrounding the particles through ionic gelation technique, thereby rigidizing the coat to obtain said microparticulate dry powder composition;
wherein the concentration of calcium chloride in the range of 2-6% w/v.
8. The method as claimed in claim 7, wherein said step of rigidization is carried out for a time duration in the range of 2 to 15 hours for retarding the release of the entrapped antigen.
9. The method as claimed in claim 7, wherein said ß-glucan has a concentration in the range of 2 to 10 mg.
10. The method as claimed in claim 7, wherein said antigen has a concentration in the range of 25 to 50 µg.