Title of Invention

"A PROCESS FOR THE PREPARATION OF A SINGLE DOSE VACCINE"

Abstract

The present invention discloses a process for entrapping immunoreactive vaccine antigen in biodegradable polymer particles. Solvent evaporation methods are used to prepare polymer particles entrapping antigen. The denaturation of the antigen during the primary emulsification step of particle formulation is protected by use of suitable stabilizers in the internal aqueous phase. The particles are made of required size using both polylactide and polylactide-co-glycolide polymer. Improved immune responses are generated by immunizing the experimental animals with particles in combination with alum. The antibody titers generated in single injection of particles are comparable to that of two doses of vaccines.

Full Text

FIELD OP INVENTION The present investigation relates to a process for the preparation of single dose controlled released vaccines. In particular, the present invention relates a process for the preparation of single dose controlled released vaccines which comprises immunoreactive vaccines /antigen candidates entrapped in biodegradable polymer particles. A single dose, non-toxic, stable and easily administered vaccine to protect effectively against a number of common vaccine preventable infectious disease such as tetanus, malaria, hepatitis and many other would be an important considerations towards increasing the immunization coverage world wide. BACKGROUND During vaccination using multiple immunization schedule, high dropout rate for the next immunization results in lower coverage leading to low protective effect of many vaccines. Biodegradable polymers such as polylactide and polylactide-co-glycolide have been extensively used for the development of single dose controlled released vaccine formulations. The most important aspects for the development of single dose vaccine formulations using biodegradable polymer particles are the protection of the immunoreactivity of the entrapped antigen within the polymer particles and its release profiles from the particles. Most of the single dose vaccine formulations developed using biodegradable polymers give erratic release profile of the entrapped antigen leading to the generation of unwanted in vivo antibody titers. Protein denaturation during the formulation of particles by the organic solvent used for the solubilization of the polymer has been found to be the main cause for the lower immunogenecity of the entrapped antigen in the polymer particles. It is desirable that the formulation developed should not only release the immunoreactive antigen but also release in a manner that mimics the conventional immunization schedule of the vaccine. It is also essential that the polymer composition, size of the particle and the immunization protocol are optimally used for generation of long lasting sustained immune responses using single dose formulation. Preparation of polymer particles entrapping immunoreactive antigen and releasing them in a manner mimicking the conventional vaccination schedule is the essential requirement for the development of single dose vaccine formulation for many infectious diseases. Most of the polymer-based formulations elicit poor and/or erratic immune response. Development of single dose controlled release vaccine formulation using biodegradable polymers needs the protection of antigen denaturation from the organic solvent, preparation of required size particles using right polymer composition, and use of right immunization protocol. The present invention relates to the judicious combination of the above aspects resulting in the development of a single dose vaccine formulation. PRIOR ART Use of poly lactide-co-glycolide (PLGA) and polylactide (PLA) polymers for entrapping vaccine/antigens provide a viable alternative to the multidose vaccine injection schedule for immunization (Hanes et al. 1997, Cleland 1999). Stability of antigen particularly during particle formulation, and in vivo release is of paramount importance for the successful development of controlled release single dose vaccine formulations (Lu and Park 1995, Puteny and Bruke 1998, Lavelle et al. 1999, Zhu et al 2000). It has been suggested that the incomplete and erratic release profiles of the encapsulated antigens from polymer matrix as well as their lower in vivo responses could be due to the denaturation of the protein molecules during particle formulations (Crotts and Park 1995, Johansen et al. 2000). Multiple (W/O/W) emulsion technique used for the entrapment of proteins/antigens involves the emulsification of polymer solution in organic solvent with antigens in aqueous medium (Couvreur et al 1997, Cleland 1998). Dichloromethane (DCM) generally used for the solubilization of PLGA / PLA polymers is a strong hydrophobic solvent and denatures protein during the primary emulsification step of particle formulation in the absence of any added stabilizers (Raghuvanshi et al 1998, Pean et al 1999, Sanchez et al. 1999). So, it becomes imperative to protect the protein from DCM induced denaturation for the preparation of immunoreactive/bioactive polymeric particles. Extensive studies with the use of PLGA microspheres have been carried out to explore the possibilities of developing a single shot vaccine for tetanus toxoid (TT) (Alonso et al 1993, Men et al 1995,). In most of the reported observations on controlled release formulation of TT from PLA or PLGA polymers, the antibody responses have always been lower than that obtained from two conventional doses of alum adsorbed TT (Xing, et al 1996, Kerstan et al. 1996, Audran et al 1998, Tobio et al 2000). Recently polymeric formulation encapsulating immunogenic TT by incorporation of additives have been reported (Schwendeman et al 1998, Audran et al 1998, Tobio et al 1999). Improved immunogenicity of polymer entrapped TT has also been reported (Johansen et al 200, Tobio et al 2000). However, very few studies have been reported on organic solvent induced denaturation of TT during particle formulation. Low antibody response generated from particle based immunization could have been due to organic solvent induced denaturation of the tetanus toxoid during particle formulation. The applicant has developed a process for single dose vaccine formulation for tetanus toxoid in biodegradable polymer matrix. Use of amphiphilic stabilizers in internal aqueous phase for protecting the immunoreactivity of the and promoting its uniform release from the polymer particles have been demonstrated. Particles formulation entrapping immunoreactive TT in biodegradable polymer matrix and evaluation of their performances both in vitro and in vivo have been investigated in detail. Immune response studies in Wistar rats using polymer particles have been carried out to demonstrate the feasibility of developing single dose vaccine using biodegradable polymer particle formulation. OBJECTS OF INVENTION Accordingly, it is the primary object of the present invention to provide a process for the preparation of a single dose vaccine formulation using biodegradable polymer particles, which mimic the conventional vaccination schedule i for many infectious diseases elicit high and uniform immune response. It is another object of the present invention to provide a process for entrapping antigens in the polymer particles while protecting its immunoreactivity or bioactivity. Another object of invention is to provide a process for preparing polymer particles of different size while entrapping immunoreactive antigen/vaccine inside it. Another object of invention is provide a process for preparing polymer particles which are capable of releasing immunoreactive antigens in vitro for a longer period of time. Another object of invention is to demonstrate the superiority of hydrophobic polymer particles for generating improved immune response. Another object of invention is to formulate immunization protocol for achieving long lasting sustained antibody responses using the polymer entrapped antigens. Yet another object of invention is to formulate immunization protocol using the polymer entrapped antigens along with alum as an adjuvant for potentiating the long lasting sustained antibody responses in experimental animal. SUMMARY OF THE INVENTION In accordance with the above and other objectives, the applicant has developed a process for entrapping immunoreactive vaccine antigen in biodegradable polymer particles. Solvent evaporation methods were used to prepare polymer particles entrapping antigen. The denaturation of the antigen during the primary emulsification step of particle formulation was protected by use of suitable stabilizers in the internal aqueous phase. The particles were made of required size using both polylactide and polylactide-co-glycolide polymer. Improved immune responses were generated by immunizing the experimental animals with particles in combination with alum. The antibody titers generated in single injection of particles were comparable to that of two doses of vaccines. In a most preferred embodiment, the preparation of the polymer particles were carried out as follows: Tetanus toxoid at a concentration 70 mg/ml protein concentration is used for the entrapment in polymer particles. Polylactide polymer having molecular weight of 45 Kd at a concentration of 50 mg/ml is used for the preparation of the particles. Aqueous phase containing tetanus toxoid and stabilizers such as rat serum albumin or polyvinyl alcohol at optimal concentration is used for the preparation of the primary emulsion. The resulting primary emulsion is again emulsified in presence of 2 % poly-vinyl alcohol to prepare the particles entrapping immunoreactive tetanus toxoid. Primary emulsion is carried out by using sonication where as secondary emulsion is preferably carried out using homogenization. Particle size of 3-4 µM are prepared using polylactide polymers. The particles are lyophilized and stored in powder form for immunization. The polymer particles when used for in vitro released experiments, showed continuous release of immunoreactive Tetanus toxoid for a longer period. The polymer particles along with alum are used for immunization. Single dose intramascular immunization of the particles in combination with alum resulted in a long lasting immune response. Serum antibody titers are comparable to that of two injection schedule of tetanus toxoid. Thus, according to the present invention, there is provided a process for the preparation of a single dose vaccine which comprises preparing an aqueous phase of a immunoreactive antigen of the kind such as herein described and subjecting said aqueous phase to a primary emulsification subjecting said pnmary emulsion so obtained to a secondary emulsification to obtain said single dose vaccine comprising of particles of said polymer entrapping said vaccine candidate/immunoreactive antigen. Preferably, said vaccine candidate/immunoreactive antigen is selected from Tetanus toxoid, diptheria toxoid, and B-subunit of human chorionic gonadotrpin hormone. In a preferred embodiment, said polymer comprises a biodegradable polymer. Preferably, said biodegradable polymer is selected from polylactide-co-glycolide and polylactide type polymers. In another preferred embodiment the aqueous phase is prepared in the presence of a stabilizer. Preferably, the stabilizer comprises one or more rat serum albumin or polyvinyl alcohol. Preferably, said primary and secondary emulsification are carried out by sonication. In another embodiment, said secondary emulsification is carried out by homogenization. Preferably, said secondary emulsification is carried out in the presence of 2% ply-vinyl alcohol. Preferably, said vaccine candidate/immunoreactive antigen and said polymer are present in a ratio of 1 : 20. In an embodiment the present invention uses solvent evaporation method for the preparation of polymer particles entrapping immunoreactive antigens/vaccine candidate. In another embodiment, the formulation comprises of different sizes of polymer particles entrapping immunoreactive antigen in it. In yet another embodiment, the invention relates to a process for preparing polymer particles entrapping immunoreactive antigen within it. In another embodiment, the process is carried out in the presence of ampiphilic stabilizer like serum albumin and polyvinyl alcohol in the internal aqueous phase during primary emulsion step of particle formulation for protecting the immunoreactivity of the antigen. In an embodiment, the polymer particles prepared by the process of the present invention releases immunoreactive antigen in vitro for along period of time, In another embodiment, particles prepared using high molecular weight polymer and hydrophobic polymer release the entrapped antigen slower in comparison to low molecular weight and hydrophilic polymers. In another embodiment, the polymer particles immunize and elicit immune response without the help of any adjuvants. In another embodiment, the immune response generated from these particles is in accordance with the in vitro release characteristics of antigen from the polymer particles. In another embodiment, intra-muscular immunization of the particles along with alum as an adjuvant potentiate the immune response in experimental animals. In another embodiment, the immune response generated by use of polymer particles entrapping antigen in combination with alum is similar to the two dose conventional vaccine immunization for tetanus. In another embodiment, immunization with polymer particles along with alum results in generation of long lasting immune response for more than 5 months. In yet another, the process describe a method of immunization without using multiple injection for achieving long lasting immune response. ^ Thus the present invention relates to a process for the preparation of single-dose vaccine using biodegradable polymer particles. The immunoreactivity of the entrapped antigen is maintained by incorporating optimal amount of stabilizers in the internal aqueous phase during the particle formulation. The polymer particles release immunoreactive antigen for a longer period of time. Intramuscular injection of particles generate antibody response in rats without the help of aduvants. Use of adjuvants along with polymer particles potentiated the immune response. DETAILED DESCRIPTION The invention is described in detail with the aid of following examples and the accompanying drawing. Various modification that may be apparent to one in the art are intended to fall within the scope of invention. BRIEF DESCRIPTION OF THE ACCOMPANING DRAWINGS Figure 1. Flow chart of water in oil double emulsion (W/O/W) method for the preparation of TT loaded particles. Secondary emulsification with sonication leads to formation of nanoparticles where as use of homogenization results in formation of microparticles. Figure 2. In vitro release profiles of entrapped antigen from different PLGA nanoparticles (-A -, 45 KD PLGA without RSA : -■-, 45 KD PLGA particles with RSA : -● - , 14 KD PLGA particles with RSA ). Figure 3. In vitro release of entrapped antigen from stabilized nanoparticles made from PLA 45 KD (-■-) and PLA 14 KD (-● -) polymer (size 0.69 and 0.73 µm respectively). Figure 4. In vitro release profiles of entrapped antigen from stabilized nanoparticles (-● -) and microparticles (-■-) made from PLA 45 KD polymer. Figure 5. Anti-TT antibody titer from group of rats immunized with (a) 15 Lf TT in saline (- -), (b) 15 Lf TT in PLGA nanoparticles (-■-) single dose and (c) two doses of alum adsorbed TT (5 Lf each dose) on day 1 and 28 (-● -). Figure 6. Anti-TT antibody titer from group of rats immunized with (a) single dose of 15 Lf TT in PLA (45KD) nanoparticles (-■-) and microparticles (-● -) and (b) two doses of alum adsorbed TT (5 Lf each dose) on day 1 and 28 (-A -). Figure 7. Anti-TT antibody titer from group of rats immunized with (a) single dose of 15 Lf TT in PLA (45KD) microparticles (-●-) along with alum and (b) two doses of alum adsorbed TT (5 Lf each dose) on day 1 and 28 (--). Example 1 Use of the process for the development of single dose vaccine for tetanus toxoid Poly lactide-co-glycolide and polylactide polymer particles entrapping immunoreactive tetanus toxoid (TT) were prepared with a view to develop a single shot controlled release vaccine formulation. Denaturation of TT by dichloromethane (DCM) during primary emulsification stage of particle formulation was minimized by incorporation of optimal amount of rat serum albumin (RSA) in the internal aqueous phase. Incorporation of RSA as a stabilizer during the primary emulsification stage of polymer particle formulation protected the immunoreactivity of TT, enhanced its encapsulation efficiency and also led to uniform polymer particle formation. Use of sonication, both during primary and secondary emulsification processes, resulted in formation of nanoparticles whereas microparticles were formed when the secondary emulsion was carried out by homogenization. Immunoreactive TT particles made from different polymers incorporating stabilizers released antigen continuously for more than 4 months in vitro. Single injection of both type of particles encapsulating stabilized TT elicited anti-TT antibody titers in rats for more than five months which was higher than that obtained with TT injected in saline. Anti-TT antibody titers in vivo were in accordance with the in vitro release characteristics of immunoreactive TT from the particles. Immune responses with hydrophobic polymer particles were better than those made using hydrophilic polymer. Polymer particles entrapping immuoreactive TT when immunized along with alum as an adjuvant improved the immune response in rats considerably. Single dose Immunization with polylactide particles along with alum resulted in anti-TT antibody response very close to that observed with two dose of TT vaccine adsorbed on alum. Example 2 Preparation of polymer particles Polylactide and ploy (lactide-co-glycolide) polymers were prepared using solvent evaporation method (figure 1). For the preparation of particles from PLGA (50:50, 45 KD) polymer, the ratio of protein to polymer was 1:20 and the ratio of internal aqueous phase to polymer was always at 1. For the preparation of particles from PLA and other PLGA copolymers, the aqueous to polymer ratio was varied depending on the molecular weight of the polymer to have consistency in polymer solution viscosity during particle formulation. Primary emulsion between internal aqueous phase (TT in PBS, pH 7.4) and organic phase (polymer solution in DCM) was prepared by sonication. Secondary emulsification was carried out either by sonication or homogenization in order to produce particles of different size. Sonication (20 watts, 80% duty cycle, 20 cycles) (Brason, Sonifier 450, USA) was used both for primary and secondary emulsification to obtain nanoparticles. Briefly, 7mg of the TT in 100 µL of aqueous buffer and was sonicated in presence of RSA (2.5 % of the internal aqueous phase) with the polymer (100 mg) dissolved in 900 µL of dichloromethane. The resulting primary emulsion was again emulsified by sonication (20 watt, 80 % duty cycle, 20 cycles) with 10 ml of 1 % PVA solution to obtain a secondary emulsion. For preparation of microparticles, the secondary emulsification step was carried out by homogenization (10,000 rpm for 10 minutes) (Virtis, Cyclone I.Q., USA). The secondary emulsion was then stirred overnight at room temperature for evaporation of DCM. The resulting polymeric particles were washed twice with cold PBS and lyophilized to get free flowing powder. The size of the particles was measured using GALAI-CIS-1 particle size analyzer. Example 3 Estimation of protein content of polymer particles To measure the protein content of particles, known weight of particles were dissolved in acetonitrile to solubilize the polymer while precipitating the encapsulated protein. The precipitated protein was pelleted by spinning at 5,000 rpm for ten minutes and was dissolved in 1% SDS solution. The protein content of the solution was determined by micro BCA assay. The protein loading was calculated as the percent weight of protein per unit weight of polymer. Entrapment efficiency was calculated as the percentage of protein entrapped with respect to the known starting amount. Example 4 In vitro release experiments In vitro release experiments of the entrapped TT from nano and microparticles were carried out in eppendorf tubes by taking known amount of particles in 1 ml PBS (pH, 7.4) containing sodium azide (0.05%). The tubes were kept in orbital shaker at 37°C. At regular time intervals, the supernatant was removed from the tubes by centrifugation (5000 rpm, 10 min) and was replaced with fresh buffer. The amount of protein released into the medium at different time point was determined by analyzing its protein content using micro BCA assay. Immunoreactivity of the released TT was determined by ELISA (Raghuvanshi et al 1998). Example 5 In vivo animal studies Immunogenicity of the encapsulated TT in particles was checked in animal model using Wistar rats. Rats were injected intramuscularly with 30 jag of either free, alum adsorbed or particle encapsulated TT. Both PLGA and PLA polymer particles entrapping TT were used for animal experiments (six animals in each group). Animals were bled at different time interval through retro-orbital plexus and the serum was analyzed for anti-TT antibody titers by ELISA. Two injections of TT (5 Lf each, total 30 n g) adsorbed on alum was given at four weeks interval. Antibody titers were estimated in duplicates with reference to a standard antibody and their concentration (ng/ml) determined as geometric mean (Raghuvanshi et al. 1998). Results and Discussion Characterization of particles containing stabilized TT The most important requirement for the successful development of single-dose tetanus vaccine is that the protein encapsulated and released remains competent as an immunogen to invoke immune response. Particles from PLA and PLGA polymer were prepared using multiple emulsion method with incorporation of 2.5% RSA in the internal aqueous phase along with TT. Nanoparticles entrapping TT were formulated from a variety of polymers using sonication during secondary emulsion. When homogenization was used for secondary emulsification, particles in the size range of 2 to 6 nm (microparticle) were obtained. The encapsulation efficiency was around 65% in all the nano and microparticle batches and the average TT loading was around 4 % of the polymer weight (Table 1). Size of both micro and nanoparticles fall within a range and the spread was a typical Gaussian distribution (Table 1). The mean size of microparticles and nanoparticles were 3 (am and 630 nm respectively. Incorporation of serum albumin in the internal aqueous phase during particle formation enhanced the encapsulation efficiency of TT. It has been reported earlier that addition of small amount of albumin, forms an interfacial film between the aqueous phase containing protein and organic phase containing polymers and thereby stabilize the inner emulsion leading to the formation of uniform matrix type microparticle encapsulating the protein (Schugens et al. 1994). Thus, apart from reducing DCM induced denaturation the incorporation of serum albumin will be helpful in multiple ways for better formulation of antigen entrapped particles. Another feature of albumin incorporation was that the nanoparticle or microparticle formulated showed a uniform loading of the TT (60-70 % encapsulation efficiency) irrespective of the size of the particles. This was also an important criterion for the controlled release of proteins from PLGA/PLA polymer because most of them degrade by bulk degradation in homogeneous way (Gopferich, 1996). The more uniform is the distribution of TT in the polymer matrix a better continuous release pattern will be expected during polymer degradation. Continuous release of immunoreactive TT from stabilized particles The particles containing stabilized TT were analyzed for their in vitro release performance (Figure 2). Continuous release of TT from particles made from PLGA (50:50, 45 Kd and 14 Kd) was observed after an initial burst phase (20%). Particles made without the incorporation of stabilizers during primary emulsification step did not show any continuous release, only 20% of the encapsulated TT were released during burst phase. This is in accordance with the recently published observation that very little immunoreactive TT was released from particle prepared without the use of stabilizers (Johansen et al. 2000). TT released from the stabilized particles was found to be immunoreactive. More than 70% of the encapsulated TT was released in a continuous manner over an extended period of 140 days. The in vitro release profiles of immunopotent TT from polymer particles obtained in this study was more uniform in comparison to the previously reported information (Alonso et al. 1993, Xing et al 1996, Johansen et al. 2000). Continuous release of entrapped antigen was also observed from particles made from polylactide polymer (Figure 3 and 4). Nanoparticles made of high molecular weight PLA showed a slower release profile as compared to those using low molecular weight PLA in vitro. It was also observed that the in vitro release profile of TT from PLA polymers were slower than that observed with PLGA polymers. These observations are concomitant with the nature and degradation of polymer as they degrade by bulk erosion through hydrolysis (Vert et al. 1991). Higher the molecular weight and hydrophobicity of the polymer, lower will be the degradation rate of the polymer due to low water uptake. This might be attributed for the slow release pattern of TT observed with PLA particles (Park 1994) The other interesting aspect of the in vitro release experiments is that TT released from polymer particles showed immunoreactivity in in vitro ELISA tests. These results from the in vitro experiments showed improved immunoreactivity of the present TT formulation, which is the most important requirement for the development of controlled release vaccine formulation. Interestingly in spite of extensive work carried out for development of microencapsulated TT such incomplete release profiles has been reported very recently (Johansen et al. 2000). However in the present formulation, incorporation of serum albumin has resulted in uniform microparticle formation along with continuous release of the protein from the polymer matrix for more than five months. Probably, in the present formulation, use of optimal amount of stabilizer helped in maximum protection of the antigen against DCM induced denaturation at the interface during primary emulsification step, thereby releasing immunoreactive TT continuously for a longer time. These in vitro results correlated well with the in vivo response of the stabilized particles, which showed much improved immune response in comparison to other formulations reported in literature. In vivo response of TT from biodegradable particles Results of anti-TT-antibody titres from two groups of rat, immunized with 15 Lf TT in saline and 15 Lf TT encapsulated in polymer particles (PLGA 50:50, 14 KD) showed that the antibody titres in group immunized with encapsulated TT was significantly higher than that obtained with soluble TT (Figure 5). These observations suggested that polymer particles encapsulating TT have adjuvant property due to their particulate nature and the small size, which helps in better uptake by the antigen presenting cells. The most important observation in the in vivo response was the similarities in the trend of anti-TT antibody with that of TT release in vitro. An early peak of anti-TT antibody titers was observed (Figure 5) which presumably could be due to the burst release of TT from nanoparticles as observed in the in vitro release experiments (Figure 2). There after the antibody titers were higher than that of saline for more than 140 days. The immune response from PLGA microparticles was similar to that obtained from nano particles (data not shown). The immune response generated from PLA particles (both nano and microp article) was sustained and was in accordance to the in vitro release profile of the antigen (Figure 6). The most interesting observation from PLA particles was that the anti-TT antibody titers were higher than those obtained using PLGA nanoparticles (Figure 5). This is probably due to the hydrophobic nature of the PLA in comparison to PLGA. Improved immune response from hydrophobic polymer particles is expected, as they are preferably taken up by macrophage for antigen presentation. The experimental matching of the in vivo titers with that of in vitro release profile of TT is an important consideration for the development of a single shot vaccine formulation. It not only compliments with the degradation characteristics of the polymers but also will help as quality control parameters for the microparticle formulation for mass immunization (Johansen et al 2000). Results of antibody titres obtained from groups immunized with two injections of 5 Lf TT each on alum and encapsulated TT showed that the conventional immunization schedule gives significantly higher antibody titre than immunization with encapsulated TT (Figure 5 and 6). This indicated that even though these polymeric particles have adjuvant property, alum proved to be a better adjuvant under these conditions of immunization. The anti-TT-antibody titres from alum adsorbed TT was almost twice as compared to that obtained from TT encapsulated in polymer matrix. However, the antibody titers obtained from this present particle formulation was better than the earlier reported values (Alonso et al. 1993, Kersten et al. 1996, Singh et al. 1997). Higher antibody titres in the present formulation could be due to the prevention of DCM induced denaturation of TT during primary emulsification step of particle formulation. Improvement in immune response from the polymer particles was achieved by incorporating alum along with the particles during immunization (Figure 7 A). Single dose imunization of polymer particles entrapping TT along with alum elicited anti-TT antibody response similar to that observed by two doses of alum adsorbed TT injection (Figure 7B). Thus, by use of hydrophobic polymers, preparing polymer particles of diameter POLYMER % ENCAPSULATION MEAN DIAMETER Nanoparticles Microparticles Nanoparticles Microparticles PLGA 50:50 72 640.67 3?75 (45 Kd) PLGA 50:50 69 76 0.52 3.42 (14 Kd) PLGA 65:35 64 69 0.71 4.9 (22 Kd } PLA(70 Kd) 59 81 0.61 3.34 PLA(45Kd) 76 69 0.69 4.0 PLA(14Kd) 64 74 0.73 2.8 REFERENCES Alonso, M. J., Gupta, R. K., Min, C., Siber, G. R. and Langer, R., 1993, Biodegradable microspheres as controlled-release tetanus toxoid delivery systems. Vaccine, 12, 299-306. Audran, R., Men, Y., Johansen P., Gander, B., Corradin, G., 1998, Enhanced immunogenicity of microencapsulated tetanus toxoid using stabilizing agents. Pharmaceutical Research, 15, 1111-1116. Cleland, J. L., 1998, Solvent evaporation processes for the production of controlled release biodegradable microsphere formulations for therapeutics and vaccines. Biotechnology Progress, 14, 102-107. Cleland, J. L., 1999, Single immunization vaccines: controlled release technology to mimic repeated immunization. Trends in Biotechnology, 17, 25-29. Couvreur, P., Blanco-Prieto, M. J., Puisieux, F., Roques, B. and Fattal, E., 1997. Multiple emulsion technology for the design of microspheres containing peptide and oligopeptides. Advanced Drug Delivery Reviews, 28, 85-96. Crotts G. and Park T. G., 1997. Stability and release of bovine serum albumin encapsulated within poly (D,L-lactide-co-glycolide) microparticles, Journal of Controlled Release, 44, 123-134. Gopferich, A., 1996, Mechanism of polymer degradation and erosion. Biomaterials, 17, 103-114. Griebenow, K. and Klibanov, A. M., 1996, On protein denaturation in aqueous organic mixture but not in pure organic solvent. Journal of American Chemical Society, 118,11695-11700. Hanes, J., Cleland, J. L. and Langer, R., 1997, New advances in microspheres based single dose vaccine. Advanced Drug Delivery Reviews, 28, 97-119. Johansen, P., Gander, B., Merkele, H. P., Sesardic, D., 2000, Ambiguities in the preclinical quality assessment of microp articulate vaccines. Trends in Biotechnology, 18,203-211. Kersten, G. F. A., Donders, D., Akkermans, A. and Beuvery, E. C., 1996, Single shot with tetanus toxoid in biodegradable microsphere protects mice despite acid-induced denaturation of the antigen. Vaccine, 14, 1627-1632. Lu, W. and Park, T. G., 1995, Protein release from poly (lactic-co-glycolic acid) microspheres: protein stability problems. Pharmaceutical Science and Technology, 1, 231-241. Men, Y., Thomasin, C., Merkle, H. P., Gander, B. and Corradin G., 1995, A single administration of tetanus toxoid in biodegradable microspheres elicit T cell and antibody responses similar or superior to those obtained with aluminum hydroxide. Vaccine, 13, 683-689. Park, T. G., 1994, Degradation of poly (D,L-lactic acid) microspheres: effect of molecular weight. Journal of Controlled Release, 30, 161-173. Pean, J-M., Boury, F., Julienne, M. C. V., Menei, P., Proust, J. E. and Benoit, J.P., 1999, Why does PEG 400 co-encapsulated improve NGF stability and release from PLGA biodegradable microspheres. Pharmaceutical Research, 16, 1294-1299. Putney, S. C and Bruke, P. A., 1998, Improving protein therapeutics with sustained release formulation. Nature Biotechnology, 16, 153-157. Raghuvanshi, R. S., Goyal, S., Singh, O. and Panda A. K., 1998, Stabilization of dichloromethane induced protein denaturation during microencapsulation. Pharmaceutical Development Technology, 3,269-276. Sanchez, A., Villamayor, B., Guo, Y., Mclver, J., and Alonso, M. J., 1999, Formulation strategy for the destabilization of tetanus toxoid in poly (lactide-co-glycolide) microsphere. International Journal of Pharmaceutics, 185, 255-266. Schugens, C., Laruelle, N., Nihant N., Grandfils C., Jerome R. and Teyssie P., 1994, Effect of the emulsion stability on the morphology and porosity of semicrystalline poly L-lactide microparticle prepared by w/o/w double emulsion-evaporation, Journal of Controlled Release, 32, 161-176. Schwendeman, S. P., Tobio, M., Joworowicz, M., Alonso, M. J. and Langer, R., 1998, New strategies for the microencapsulation of tetanus vaccine. Journal of Microencapsulation, 15, 299-318. Tobio, M., Nolley, J., Guy, Y., Mclver, J., and Alonso, M.J., 1999, A novel system based on a polaxomer/PLGA blend as tetanus toxoid delivery vehicle. Pharmaceutical Research, 16, 682-688. Tobio, M., Sachwendeman, S. P., Guy, Y., Mclver, J., and Alonso, M. J., 2000, Improved immunogenicity of a core coated tetanus toxoid delivery system. Vaccine, 18, 618-622. Vert, M., Li, S. and Garreau, H., 1991, More about the degradation of LA/GA derived matrices in aqueous media. Journal of Controlled Release, 16, 15-26 Xing, D. K. L., Crane, D. T., Bolgiano, B., Corbel, M. J., Jones, C. and Sesardic D., 1996, Physicochemical and immunological studies on the stability of free and microsphere-encapsulated tetanus toxoid in vitro. Vaccine, 14, 1205-1213. Zhu, G., Mallery, S. R. and Schwendenman, S. P., 2000, Stabilization of proteins encapsulated in injectable poly (lactide-co-glycolide). Nature Biotechnology, 18, 52-57. We claim: 1. A process for preparation of a single dose vaccine which comprises preparing an aqueous phase of a immunoreactive antigen of the kind such as herein described and a biodegradable polymer selected from polylactide-co-glycolide and polylactide type polymer in a ratio of 1:20, in the presence of a conventional stabilizer, subjecting in a conventional manner said aqueous phase to a primary emulsification and subjecting in a conventional manner said primary emulsion so obtained to a secondary emulsification to obtain said vaccine comprising of particles of said polymer entrapping said immunoreactive antigen. 2. A process as claimed in claim 1 wherein said immunoreactive antigen is selected from Tetanus toxoid, diptheria toxoid, and ß-subunit of human chorionic gonadotrpin hormone. 3. A process as claimed in claim 1 wherein said stabilizer comprises of rat serum albumin or polyvinyl alcohol or a mixture thereof. 4. A process as claimed in claim 1 or 2 wherein said primary emulsification is carried out by sonication. 5. A process as claimed in any preceding claim wherein said secondary emulsification is carried out by sonication. 6. A process as claimed in any one of claims 1 to 4 wherein said secondary emulsification is carried out by homegenization. 7. A process as claimed in any preceding claim wherein said secondary emulsification is carried out in the presence of 2% poly-vinyl alcohol. 8. A process as claimed in claim 1 for the preparation of a single dose vaccine substantially as herein described with reference to the foregoing examples.

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