Review Potential of polymer microencapsulation technology for vaccine innovation

William Morris, Mark C. Steinhoff* and Philip K. Russell

Biodegradable polymer microspheres or microcapsules developed over the past decade for reliable, preprogrammed release o f contraceptive steroids have significant potential for adaptation to antigen release for immunization. In addition, polymeric encapsulation o f antigens could prevent the acid and enzymatic degradation that has been a barrier to the development of oral vaccines. This review summar- izes the published experience with microencapsulated hormones and antigens, describes the process o f microsphere production, discusses the strengths and weaknesses o f this approach to immunization, and outlines the gaps in knowledge. Microsphere technology has the potential benefits o f reducing the number o f inoculations, enhancing the immune response via both parenteral and oral vaccination routes, and in reducing the total antigen dose required to achieve immune protection.

Keywords: Microspheres; biodegradable; controlled release

IgG responses for mucosal as well as humoral defences 4. The polymeric microspheres that protect antigen from acidic and enzymatic degradation in the gastrointestinal tract also provide a stable vaccine vehicle with extended shelf life. In addition, the antigen sequestration in microcapsules may provide a mechanism to evade neutralization by maternal antibody.

Antigens can be microencapsulated in polymer matrices, or in microscopic phospholipid vesicles called liposomes 5. Liposomes show great potential for stable delivery of antigens parenterally as well as via mucous membranes in a manner that preserves or even enhances immunogenicity. Cholera toxin 6'~, malaria sporozoite protein s and tetanus toxoid 9 are a few of the antigens whose immunogenicity has been increased by liposomal delivery. This review, however, will focus on micro- encapsulation using polymers.

A heat-stable, single-dose, non-toxic vaccine that could be given by mouth to protect effectively against a number of common childhood diseases would be a welcome and important step towards increasing immunization coverage worldwide. The delivery of vaccines in biodegradable microspheres shows potential for achieving these ambitious goals.

Microencapsulation technology dates back to the 1950s, but its reliable application for drug delivery has been achieved only recently 1-3. Success in achieving consistent concentrations of reproductive hormones needed for contraception has encouraged the application of microsphere technology to the controlled release of other pharmaceuticals, including antigens for immuniz- ation. Fewer doses, pulsed release of antigen for up to 2 years, simultaneous administration of multiple antigens and enhanced immunogenicity are all theoretic- ally possible with encapsulated vaccines. Oral adminis- tration of antigen microspheres can induce both IgA and

Department of International Health, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD, USA. *To whom correspondence should be addressed at Center for Immunization Research, 624 N. Broadway (Rm 125), Baltimore, MD21205, USA. (Received 25 August 1992; revised 18 February 1993; accepted 1 March 1993)

P O L Y ( L A C T I D E - c o - G L Y C O L I D E ) M I C R O E N C A P S U L A T I O N

Spherical, sustained-release polymer particles can be either monolithic, with the antigen interspersed through- out the particle (microspheres), or consist of true core- wall capsule structures with a core reservoir of antigen surrounded by an outer polymer shell (reservoir micro- capsules). The size of the particles produced by the various manufacturing techniques generally ranges from 1 to 300/~m. A variety of inert and biodegradable substances have been investigated 2'3'7'1 o-1 a, but the most studied polymers for the controlled release of pharmaco- logical agents are made from lactic and glycolic acids, normal intermediates in mammalian energy metabolism (see Figure I ). The lack of toxicity of these poly(lactide- co-glycolide) polymers has been established by FDA licensure and years of use as absorbable suture materiaP 4'15. The FDA has recently approved a poly(lactide-co-glycolide) microsphere 30-day delivery system for leuprolide acetate (Lupran Depot ~ to be used in the treatment of prostate cancer 2.

This review will concentrate on poly(lactide-co- glycolide) polymers and their potential role in micro- encapsulation of vaccines. Other polymer controlled- release delivery systems have been reviewed elsewhere 2' 13.

Poly(lactide-co-glycolide ) formation results in

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Microencapsulation: W. Morris et al.

random poly(DL-laetide-co-glycolide)

[ o ,°,l F ,o, , o , 1

CH 3 CH~ H20

oi[ o ] II II HO----CH--C---OH + HO---CHr--C OH

• Y CH3

Lactic acid Glycolic acid

Figure 1 Poly(DL-lactide-co-glycolide) structure and degradation

polymers of Mw ~< 40 000. In vivo, the polymer undergoes random, non-enzymatic hydrolysis of its backbone ester linkages (Fioure 1 ) at a rate that is influenced by the molecular weight, surface area, monomer stereoregularity and the ratio of lactide to glycolide. The incorporation of lactide into a glycolide polymer alters the crystallinity, solubility and water uptake of the final molecule, but most importantly it increases the copolymer's rate of biodegradation. In animals the half-life of 100% polyglycolide polymer microspheres is 5 months. The half-life decreases with increasing proportion of lactide to 1 week with a 50:50 copolymer. As the proportion of lactide increases further, the half-life increases, reaching 6 months for 100% polylactide 16. A variety of release patterns for encapsulated vaccines can be programmed by alteration of microcapsule sizes, the ratio of copolymer content, copolymer molecular weight or of antigen load. Additional flexibility is possible by combining reservoir microcapsules with monolithic microspheres of different characteristics.

Antigen is released from the microspheres by diffusion through matrix pores and by matrix degradation. Diffusion from polyester microparticles is relatively slow because of the copolymer's glassy rather than rubbery consistency. For example, the permeability of proges- terone to silicone rubber (used in Norplant ~ implants) is 2.6 x 1 0 - 9 g c m - l s -1 compared with 0.000033 x 10 - 9 g cm- 1 s- 1 for polylactide 17. Biodegradation of the polymer, therefore, is usually the rate-controlling step in antigen release from microspheres. This fact has been confirmed by scanning electron microscopy and gel- permeation chromatography 18. Monolithic microspheres release an initial surge of antigen from surface pores and then continue to release antigen at a nearly constant rate as the matrix of the microspheres degrades 19. Reservoir microcapsules will release steady low levels of antigen by diffusion until degradation disrupts the shell integrity and the core antigen is released. Pure polymers or thick- walled core-wall microspheres could have a slow enough rate of degradation to allow the majority of antigen to be released by diffusion 2°. In addition to spontaneous degradation of the copolymer matrix, ultrasound, light, magnetism and other external modalities have been investigated for their potential in influencing polymer breakdown and controlling the rate of antigen release 21-2 3

S U S T A I N E D - R E L E A S E SYSTEMS F O R H O R M O N E S

Early studies investigating sustained release of pharmaco- logical agents tested finely divided particles of poly(L-

lactide) containing 20wt% of norethisterone and demonstrated zero-order release kinetics in rats 24. Norethisterone microspheres demonstrated a rapid release of ~50% of the hormone load followed by a slower release over an extended period 25. Inhibition of the rat oestrous cycle was extended from the 2-day period seen with an intramuscular injection of soluble hormone to a full month with injection of the encapsulated formulation. No tissue reactions were noted over 6 months of histological follow-up, but the micro- spheres persisted beyond the 30-day hormone release. Subsequent studies with 100% lactide microspheres confirmed the pattern of an initial hormone burst, followed by a decline to a near steady-state discharge rate 26,27.

After evaluation in primates, norethisterone-containing monolithic microspheres made of poly(DL-lactide) polymer were evaluated in women. All but the lowest hormone doses retained the diffusion release kinetics demonstrated in the animal models, and the microspheres required approximately 12 months to fully degrade in situ 28. Different synthesis techniques, such as air- suspension coating, resulted in prolonged sustained release at a more steady rate 19. Addition of glycolide to the lactide polymer resulted in an increased degradation rate and a more consistent release of hormone over the life span of the microspheres 29-31. The investigators noted that the total dose of norethisterone required to inhibit ovulation was much lower when administered by injected microspheres than by daily oral doses.

Polyesters have been employed for the sustained release of other pharmacological agents, including antibiotics, antimalarials, antineoplastic agents and narcotic antagonists 32-3s.

I M M U N O P O T E N T I A T I O N

In animals, sustained release of antigen from a non-biodegradable polymer showed an enhanced immune response for over 6 months 7. The possible mechanisms of immunopotentiation by sustained release of antigen include a depot effect analogous to that of aluminium salt adjuvants, the delivery of antigen directly to antigen-presenting cells such as macrophages, or continuous exposure to antigen as occurs in chronic infections. Direct uptake of intact microspheres of < 10/~m diameter into macrophages may provide a route to evade maternal antibody. Biodegradable particle systems have an advantage over implants because they obviate the need for implant removal, and can be engineered to release antigen with up to four peaks over 1 year.

Iminocarbonate polymers with added tyrosine, a known adjuvant, were evaluated for an adjuvant effect independent of the sustained-release effect. An investi- gation comparing the antibody titres of mice in response to bovine serum albumin (BSA) encapsulated with an iminocarbonate with and without tyrosine showed significantly enhanced antibody titres with the tyrosine- containing polymers 36.

V A C C I N E A P P L I C A T I O N S

The first applications of microencapsulation technology to immunization in 1979 demonstrated that polymeric delivery systems were indeed capable of continuous

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Table 1 Summary of polymer microencapsulation immunization studies

Reference Polymer/part ic le (date) Antigen size (wt% antigen) Animal Route Comments

7 (1979) BSA, bovine Ethylene-vinyl acetate; Mice s.c. implant gammaglobul in 0.3 mm (10)

36 (1986) BSA Tyrosine-based Mice s.c. implant polyiminocarbonate; 0.5 mg (10)

44 (1992) Tetanus toxoid PLG 55-45; (0.45) Mice s.c.

42 (1990) Human chorionic PLG Rabbits i.rn. gonadotropin

40 (1989) Ovalbumin Polyacrylamide; Rats i.p., p.o. 2.557/zm (10)

50 (1993) Ovalbumin PLG; 1.2-3/Jm Mice i.p., p.o.

4 (1989) SEB PLG; 50:50/1-10/zm Mice i.p., p.o. (0.23)

47 (1993) E. Coil CFA/I PLG; 50:50; (0.1) Rabbits i.d.

48 (1993) E. coli CFA/II PLG; 5-10 #m Rabbits i.d.

38, 39 (1989, 1990) SEB PLG; 85:15; 50:50; Mice p.o. 1-10/Jm (0.23)

49 (1993) E. coil adhesin PLG; 1-12/zm (0.62) Rabbits i.d.

41, 51 (1989, 1991) Inactivated influenza PLG; 2,10/zm ( ~ 1.0) Mice i.p., p.o. A virus

Enhancement of immune response comparable to CFA; no tolerance

Intrinsic adjuvant activity with tyrosine

Antigenicity preserved, alum did not increase response

Mix of microsphere sizes for sustained response

Low-level enhancement of IgA response

CT lymphocyte response

60 x greater specific serum IgG than p.o. soluble antigen

Serum and copro-antibody response

Effective oral route, safety tests

Microspheres uptake by Peyer's patches documented. Combined sizes provided a booster response Protected against challenge

Immunogenic, good priming and good boosting

Abbreviations: SEB, staphylococcal enterotoxoid B; PLG, poly(oL-lactide-co-glycolide); CFA, complete Freund's adjuvant;, s.c., subcutaneous; i.m., intramuscular; i.p., intraperitoneal; p,o., peroral; i.d., intraduodenal

antigen release and stimulation of sustained antibody formation. A single injection of albumin coated with ethylene-vinyl acetate (EVAc) polymer produced an antibody response in mice over a 6-month period that was comparable to a primary-booster series of two intramuscular injections with the same amount of antigen 7. Similar responses were obtained with other antigens of widely varying molecular weights 7'12. The sustained immune response was of the IgG class and did not exhibit tolerance 12.

Although this early work using polymer delivery systems demonstrated clear potential, reports in this area remain sparse and repetitive '~'7'12'37--44. Table 1 summarizes the published polymer microencapsulation research in animals. No trials of human immunization were found in an extensive literature search.

The non-biodegradable EVAc polymer has been replaced by the biodegradable lactide-co-glycolide polyesters in most current development work 2 . Despite data that suggest improved control of drug release in polyanhydride microparticles 2"1z and enhanced adjuvant effect with poly(CTTH-iminocarbonate) polymers 36, polyesters remain the only FDA-approved biodegradable polymers in common use for encapsulation.

Subcutaneous administration of microencapsulated staphylococcal enterotoxoid B (SEB) strongly potentiated serum IgG responses in comparison to soluble antigen 3a. The presentation of antigen in microspheres also resulted in an enhanced early antibody response. Parenteral immunization with one I00/tg dose of mieroencapsulated SEB resulted in an antibody level equal to that produced by three doses of soluble SEB. No evidence of tolerance was seen at higher antigen doses, and systemic immunization effectively primed for a subsequent mucosal IgA response to an orally administered booster dose.

1000000

I-"

.~ 800000 -

• 1-10 um 600000. • 20-50 um

" • 1-10 + 20-50 um <

a. 0 10 20 30 40 so s0 Day Post SC Immunization

Figure 2 Effect of a mixture of microsphere sizes on anti-SEB antibody response in mice. Microspheres consisting of 50:50 glycolide-lactide polymer sized 1-10 pm produced a peak antibody response at 30 days, those sized 20-50/zm had a peak at 50-60 days. A mixture of microspheres of both sizes caused an antibody response tenfold higher than either size alone. (Reprinted from: Biodegradable microspheres as a vaccine delivery system. Molecular Immunology 1991, 28, 287-294, with kind permission from Pergamon Press, Ltd, Oxford, UK)

Mixtures of microspheres with varied antigen release characteristics based on size and composition have been shown to provide an enhanced antibody response. The injection of SEB in a mixture of poly(lactide-co-glycolide) microspheres of size 1-10/~m and 20-50/~m resulted in a synergistic, prolonged immune response 38 which was greater than that seen with particles of single size given alone (Figure2). A single injection of a mixture of particles with varied polymer ratios has a similar synergistic effect (Eldridge, personal communication). An evaluation in mice of tetanus toxoid encapsulated in microspheres which were sterilized with gamma irradi- ation showed that a good primary immune response was produced, that gamma irradiation modified some epitopes and that encapsulation of alum-absorbed toxoid

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did not enhance the primary response 44. In addition to proteins and peptides, whole viruses have been encapsu- lated. Formalin-inactivated whole influenza A virus retained immunogenicity when encapsulated in lactide- co-glycolide microspheres 41. Although encapsulation did not enhance immune response, oral administration of an encapsulated antigen has potential advantages over parenteral immunization.

O R A L I M M U N I Z A T I O N

Observations that microparticles were taken up by the Peyer's patches of the gastrointestinal tract 45'46 suggested that oral administration of microencapsulated vaccines was feasible, and might overcome the problems of the low pH and lytic enzymes in the gastric environment and the high antigen doses needed for mucosal immunization. For both systemic and parenteral immunization, the size of the microspheres is an important determinant of the initial uptake and cellular trafficking of encapsulated antigen. Fluorescence microscopy analysis of pathology sections revealed that orally administered microspheres > 10 #m in diameter were not absorbed 4'3~. Microspheres >5/~m were taken up by the Peyer's patch lymphoid tissue of the gut and remained detectable there for up to 35 days. Particles < 5 # m , however, were found in draining mesenteric lymph nodes and the spleen 4'37'38.

Hydrophobicity appears to be a determinant of the Peyer's patch uptake of microspheres. Similar sized biodegradable microspheres made of ethyl cellulose triacetate or cellulose acetate hydrogen phthalate were not taken up by Peyer's patches, whereas the more hydrophobic poly(hydroxybutyrate) and lactide/glyco- lide copolymer microspheres were absorbed more efficiently 39.

Adsorption of antigen on to biodegradable particles was demonstrated to enhance secretory immune response in comparison with soluble antigen in gastrically inoculated rats 4°'~3. Oral immunization with micro- spheres containing SEB not only induced circulating lgM, IgG and IgA anti-toxin antibody, but also a disseminated mucosal IgA response in mice 4"37'39. In contrast, oral immunization with an equivalent amount of soluble SEB resulted in minimal to absent antibody titres of all immunoglobulin isotypes.

Microencapsulated oral vaccines against enterotoxi- genic Escherichia coli pilus proteins are in the early stages of development. Encapsulated whole pilus protein is immunogenic in vivo, but is destroyed by gastric acidity 47. Retention of immunogenicity with encapsulation has been demonstrated along with protection from subsequent pathogen challenge in a rabbit model 48'49. Systemic IgG, as well as local IgA response, are evident in animals immunized intragastrically and intraduodenally 4~-~9. Similar results have been seen with microencapsulated cholera toxin B subunit 5°.

Oral administration of microencapsulated influenza A vaccine virus induced levels of serum antibody in mice comparable to parenterally administered microspheres 4~. Mice vaccinated orally or systemically with the encapsulated antigen had higher salivary IgA antibody titres than mice given free virus. A second series of studies showed that microencapsulated influenza virus provided protection against virus challenge, and that the oral route boosted antibody titres resulting from primary oral or systemic immunization 51.

Oral boosting of mice systemically primed with SEB in 1-10 #m microspheres was as effective at inducing mucosal IgA antitoxin antibodies as three oral doses. Intratracheal boosting with microspheres proved even more efficient at stimulating both circulating IgG and mucosal IgA specific antibody 38.

P R O D U C T I O N O F M I C R O S P H E R E S

Microsphere preparation can be achieved by a number of different methods. The most common techniques are described below.

Solvent evaporation or emulsion polymerization tech- nique. This involves adding an aqueous solution to a copolymer/antigen/solvent suspension to form an 'oil-in-water' emulsion of copolymer/antigen micro- particles 2°'52. The microparticles are then dried and characterized after the removal of the solvents by evaporation and/or solvent extraction. The specific organic solvent, temperature of solvent evaporation, volume of organic phase per unit volume of aqueous phase, nature and amount of emulsifer dissolved in the aqueous phase, polymer structure and molecular weight, and antigen solubility all influence the final microsphere product obtained. Very small microspheres can be produced with this technique, but smaller sizes may result in low carrying capacity of the microsphere and absorption of antigen on to the microsphere surface rather than inside. Microspheres with less than 30% of their weight consisting of antigen are produced with this technique 53. A potential problem is that the removal of solvent contaminants from the final microspheres becomes more difficult as microsphere size decreases.

Phase-separation technique. This utilizes solubilized polyesters and antigen which are then mixed with salts, non-solvents or, more commonly, with an incompatible second polymer resulting in a separation of the polyester phase that engulfs the antigen particles 54. The liquid- polyester/antigen phase is desolvated and solidified by the addition of excess non-solvent at low temperatures. This approach is limited to antigens which remain insoluble in the organic solvent used to dissolve the polyester. Biodegradable polyesters like poly(L-lactide) and lactide-co-glycolide polymers are solubilized only under relatively strong solvent conditions that favour the solubilization of many drugs, making this technique difficult to use, especially when retention of antigenicity is important. A higher degree of antigen loading can be achieved using this method in comparison with the solvent evaporation technique, but the low antigen content required for vaccines (less than 1% core loading by weight) is easily achievable by either technique. In contrast to the solvent evaporation technique, which produces microspheres with antigen dispersed throughout the polymer sphere, phase-separation techniques create reservoir microcapsules.

Spray-drying. These techniques aerosolize a suspen- sion of polyester and antigen, to create microdroplets that solidify in a brief blast of heated air. Knowledge of copolymer/antigen suspension rheology and nozzle design is used to control the microparticle size and shape.

8 Vaccine 1994 Volume 12 Number 1

Film-casting technique. Copolymer/antigen suspen- sions are cast into thin films and then ground into microparticles by shredding, pulverization or cryogenic grinding.

Air-suspension coating technique. Reservoir micro- capsules are formed by coating particles or droplets of antigen in copolymer while they are suspended in a stream of air s4. Tfiis technique is limited to production of microcapsules >50/A in size, as smaller particles adhere together in the air flow.

The solvent evaporation technique with proprietary variations is used most frequently by current product developers. Improvements in phase-separation techniques have resulted in microspheres of similar quality, but the harsh organic solvents required in this process make it difficult to use in antigen encapsulation. Changes in antigenic epitopes due to temperature fluxes during the manufacturing process are another potential problem area. More data are required on the effect of each technique on the immunogenicity of the encapsulated antigens. The success in preserving the biological activity of labile peptides such as human interferon in poly(lactide-co-glycolide) implants s5 is encouraging.

M I C R O E N C A P S U L A T I O N P R O C E S S : I M P L I C A T I O N S F O R V A C C I N E D E V E L O P M E N T

Vaccine manufacture, regulatory issues and vaccine evaluation are crucial areas of linkage between basic development and implementation of improved vaccines. A review of the manufacturing process is important in highlighting the areas in which problems may develop with application to vaccines. There are five sequential stages in the microencapsulation process:

1. Monomer synthesis: The process of lactide and glycolide generation was developed for the production of absorbable sutures and is well established 2°. Another potential polymer, polyanhydride, may have a more controllable degradation profile than the polyesters, and it appears to be safe in numerous animal models and in humans 2'56'57.

2. Polymerization: A variety of catalysts can be used, but organometallic catalysts are required to produce high-molecular-weight polymers. Trialkyl aluminium compounds are preferred for biomedical purposes 5s. The possibility of toxicity from residual catalysts in the microspheres is a potential hindrance to licensure for human use.

3. Polymer-antigen blending: Each antigen will require individual solvent testing to determine dissolution characteristics and solvent effect on antigenicity and stability. Residual solvent toxicity is a concern, and effort towards minimizing the use of solvents is needed. Functional peptide side groups may be reactive with the polymer matrix 36'59, indicating that antigen-polymer interaction may be a problem.

4. Microsphere formation: The microspheres produced by each method discussed previously should be evaluated by scanning electron microscopy or light scattering, in vitro antigen release studies and antigen content analysis. Classification of each technique by

.

Microencapsulation: W. Morris e t a l .

cost and capabilities, i.e. microsphere size, uniformity, quality, and technique flexibility, would be helpful when considering which method or combination of methods to employ for large-scale production. The microencapsulation literature emphasizes that in vitro release studies are based on diffusion, not biodegrad- ation, since the antigen concentration gradient between the microspheres and a solvent bath is extreme and solvents act to increase the permeability of microspheres. Comparison of degradation in vitro with in vivo 14C release studies is required. Once studies are performed in vivo, the profiles can provide information on microsphere quality and batch-to- batch reproducibility. Advances in magnetic resonance imaging (MRI) technology will probably gain importance in characterizing antigen release and copolymer degradation as well 59.

Discussion regarding the relative merits of mono- lithic microsphere versus core-wall microcapsule antigen release kinetics is absent in the scientific literature. The majority of current microsphere developers employ solvent evaporation techniques that produce monolithic microspheres. It has been suggested that the ideal release patterns for encapsul- ated vaccines should mimic the antigen concentrations seen over time in natural infections 6°. A rapid increase in antigen load within a few days influences memory T-cell formation favourably, and a subsequent steady decrease in antigen load may be important in antibody affinity maturation. Exposure to a pulse of antigen after an interval of weeks or months would provide a booster effect. The reservoir microcapsules produced by phase-separation or air-suspension coating tech- niques are more likely to achieve this antigen release pattern. Investigations into the immune response to different combinations of antigen in monolithic microspheres, reservoir microcapsules and soluble forms are needed. Packaging and sterilization: The optimum dose and volume for microsphere vaccines have not been established. Hormonal preparations have been administered by intramuscular injection by 18 gauge needle of microspheres sized 25-90 #m suspended in 3 ml of sterile saline solution with 2% carboxymethyl cellulose and 1% Tween 20.

Orally administered pharmaceuticals are not required to be sterile, but the unique uptake of microspheres into Peyer's patches may necessitate sterilization of microspheres intended for oral vaccines. The gamma radiation used for sterilization has been shown to decrease microsphere molecular weight and thus increase the biodegradation rate 17, but hormonal activity appears to be unaffected 61. Although some producers have successfully used gamma radiation, others have reported a loss of tetanus toxoid antigenicity from an unspecified dose of gamma radiation in the sterilization process 44. Documentation of sterility of microspheres capable of holding one or two bacteria and designed to degrade over weeks to months presents a challenge. The use of organic solvents during the microsphere production results in a sterile processing environment and may establish internal microsphere sterility, but residual traces of these harsh solvents create additional barriers to FDA approval. Methylene chloride, the most widely used solvent for the solvent evaporation

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Mic roencapsu la t ion : W. Mor r i s et al.

technique, is toxic and has been shown in microspheres 6z.

Quality control of the final packaged product requires sterilization testing in addition to evaluation of antigen content, polymer molecular weight and determination of residual solvent. One study of microspheres prepared under GMP conditions showed acceptable levels of non-pathogenic bacteria and solvents 48. Characterization of shelf life and requirements for storage has not been performed, but the possibility of spontaneous hydrolysis of the polymer and/or aggregation of encapsulated proteins in the presence of ambient moisture is a concern 56.

C O N C L U S I O N S

Experience with sustained-release preparations of hor- mones and animal immunization data suggest that lactide-co-glycolide polymer microsphere technology is feasible and holds great promise for improving human vaccines.

The release of microencapsulated antigen can be programmed by altering the size of the microparticles, the ratio of monomers in the matrix, the copolymer molecular weight, the chemical characteristics of the antigen and the antigen load.

Although there is extensive information on the antigen-release kinetics of differing microspheres, more data are needed on the effect of these kinetics on the human immune response. Similarly, the effect of cellular targeting through microsphere size should be defined in humans. As the correlations between antigen delivery and the immune response are better defined, microparticle technology can be focused on those characteristics that are major determinants of the antibody response.

The process of microencapsulation and selection of an effective microsphere size distribution, polymer molecular weights, and ratios that maximize immunogenicity remains complex, however. The degree of complexity is illustrated by the time required for development of a microencapsulated human chorionic gonadotropin vaccine. An intensive 5-year effort was needed to establish the formulation that would induce an effective contraceptive immune response lasting 1 year in experimental animals.

Despite the potential of microencapsulated vaccines, a number of unsolved questions persist. The degree to which different antigens can be combined in the same microsphere or different microspheres in the same injection has not been determined. T-cell independent antigens, such as bacterial capsular polysaccharides, may not benefit from encapsulation without first being conjugated to a protein carrier.

The additional concern of safety must also be addressed. Residual solvents in the microspheres remain a problem. Adverse reactions may occur with slowly released antigen that persists for long periods in the tissue. Indeed, allergic reactions may not be easily controlled in the presence of continued antigen release.

The use of microspheres as oral vaccines creates additional challenges. Microencapsulation can protect antigens from the gastrointestinal environment. The application of microencapsulation for enhancement of antigen uptake and prolonged release of vaccine will be more difficult. The requirement for oral poly(lactide-co- glycolide ) microspheres to be less than 10/~m in size for

efficient gastrointestinal uptake may limit the patterns of antigen release and booster abilities. Altering the copolymer ratios and molecular weight would restore some flexibility in controlling antigen-release kinetics.

Review of the microencapsulation process reveals that the initial steps of monomer preparation, polymer synthesis and polymer-antigen blending are well developed, with good manufacturing protocols already established. Indeed, there are industrial plants already in existence that could easily be converted to large-scale lactide-glycolide microencapsulation of vaccines under appropriate GM P conditions for human pharmaceuticals.

In summary, poly(lactide-co-glycolide) microsphere technology has the potential benefits of reducing the number of inoculations, enhancing the immune response, and in reducing the total antigen dose required for effective immunization. In addition, poly(lactide-co- glycolide) microspheres have the potential to protect antigen from the harsh gastric environment and in delivering antigen to lymphatic tissues, permitting effective oral primary or booster immunization.

A C K N O W L E D G E M E N T

This work was supported by the Children's Vaccine Initiative.

R E F E R E N C E S

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Medicine (Ed. Gregoriadis, G.) Academic Press, London, 1979, Chapter 12

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