Poly(lactide-co-glycolide) microparticles for the development of single-dose controlled-release vaccines

LAdvanced Drug Delivery Reviews 32 (1998) 225–246

Poly(lactide-co-glycolide) microparticles for the development of single-dosecontrolled-release vaccines

1*Derek T. O’Hagan , Manmohan Singh, Rajesh K. GuptaChiron Vaccines, 4560 Horton Street, Emeryville, CA 94608, USA

Received 5 August 1997; received in revised form 26 September 1997; accepted 18 October 1997

Abstract

With the exception of the provision of clean water supplies, vaccination remains the most successful public healthintervention strategy for the control of infectious diseases. However, the logistics of delivering at least two to three doses ofvaccines to achieve protective immunity are complex and compliance is frequently inadequate, particularly in developingcountries. In addition, newly developed purified subunit and synthetic vaccines are often poorly immunogenic and need to beadministered with potent vaccine adjuvants. Microparticles prepared from the biodegradable and biocompatible polymers,the poly(lactide-co-glycolides) or (PLG), have been shown to be effective adjuvants for a number of antigens. Moreover,PLG microparticles can control the rate of release of entrapped antigens and therefore, offer potential for the development ofsingle-dose vaccines. To prepare single-dose vaccines, microparticles with different antigen release rates may be combinedas a single formulation to mimic the timing of the administration of booster doses of vaccine. If necessary, adjuvants mayalso be entrapped within the microparticles or, alternatively, they may be co-administered. The major problems which mayrestrict the development of microparticles as single-dose vaccines include the instability of vaccine antigens duringmicroencapsulation, during storage of the microparticles and during hydration of the microparticles following in vivoadministration. In the present review, we discuss the adjuvant effect of PLG microparticles, and also their potential for thedevelopment of single-dose vaccines through the use of controlled-release technology. 1998 Elsevier Science B.V.

Keywords: Controlled release; Microparticles; Poly(lactide-co-glycolides); Biodegradable polymers; Vaccine adjuvants;Delivery systems; Single-dose vaccines

Contents

1. Introduction ............................................................................................................................................................................ 2262. The advantages of PLG polymers for vaccine development ........................................................................................................ 227

2.1. PLG controlled-release drug delivery systems .................................................................................................................... 2272.2. The biodegradability and biocompatibility of PLG.............................................................................................................. 2272.3. The preparation and characterization of PLG microparticles ................................................................................................ 229

3. The adjuvant effect of microparticles ........................................................................................................................................ 2293.1. Microparticles as adjuvants for antibody induction ............................................................................................................. 2303.2. The induction of cell-mediated immunity with microparticles.............................................................................................. 230

4. Microparticles as single-dose vaccines ...................................................................................................................................... 230

*Corresponding author. Tel.: 1 1 510 9237662; fax: 1 1 510 9232586; e-mail: derek o’[email protected]]1Present address: Wyeth-Lederle Vaccines and Pediatrics, Pearl River, NY 10965, USA.

0169-409X/98/$32.00 1998 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 98 )00012-X

226 D.T. O’Hagan et al. / Advanced Drug Delivery Reviews 32 (1998) 225 –246

4.1. Tetanus toxoid ................................................................................................................................................................. 2314.2. Diphtheria toxoid ............................................................................................................................................................. 2344.3. Hepatitis B surface antigen (hepatitis B vaccine) ................................................................................................................ 2344.4. Envelope glycoprotein gp120 (HIV vaccine) ...................................................................................................................... 2354.5. Human chorionic gonadotropin (hCG)-based birth control vaccines ..................................................................................... 236

5. Recent progress in the development of PLG controlled-release protein delivery systems............................................................... 2375.1. Protein stability during microparticle preparation................................................................................................................ 2375.2. Protein stability during freeze drying ................................................................................................................................. 2385.3. Protein stability during storage .......................................................................................................................................... 2395.4. Protein stability following in vivo administration................................................................................................................ 239

6. Conclusions ............................................................................................................................................................................ 240Acknowledgements ...................................................................................................................................................................... 241References .................................................................................................................................................................................. 241

1. Introduction in licensed human vaccines are based on insolublealuminum compounds. However, aluminum adju-

As a consequence of the wide-spread use of vants have several limitations; they are not effectivevaccines during the last several decades, the inci- for all antigens, induce local reactions, induce IgEdence of many infectious diseases has declined antibody responses and generally fail to induce cell-considerably, particularly in developed countries. mediated immunity, particularly cytotoxic T cellThe eradication of smallpox in 1977 represents the responses [1,3,4]. Even with aluminium adjuvants,clearest success story for vaccination policy. Never- two to three doses of vaccine are normally requiredtheless, there are still significant challenges for to achieve protective immunity.vaccine development, including: (1) the need to To increase immunization coverage and to reduceimprove existing vaccines by making them more safe the cost of immunization, efforts have been made toand potent; (2) the need for the development of reduce the number of doses. Early studies concen-vaccines against diseases for which no vaccines trated mainly on increasing antigen and adjuvantcurrently exist; and (3) the need for improved doses [5–12]. However, despite promising results,vaccines to extend coverage in certain populations, these high dose formulations were never used on aparticularly in the developing world. large scale for routine immunization. More recently,

One of the most important current issues in the Children’s Vaccine Initiative (CVI) proposed thevaccinology is the need for new adjuvants and development of a single-dose, heat stable vaccinedelivery systems. Many of the vaccines currently in that could be delivered at birth and would protectdevelopment are based on purified subunits, recom- against multiple diseases [13]. Although at first sightbinant proteins, or synthetic peptides. It is clear that this daunting goal appears difficult to achieve, it maythis new generation of vaccines will be less immuno- be approached through improvements in existinggenic than traditional vaccines and will require better technologies, starting with a reduction in the numberadjuvants and delivery systems to induce optimal of doses required for immunization. Several novelimmune responses [1,2]. Even potent immunogens, approaches are being pursued to achieve these aims,such as tetanus toxoid (TT), require at least two or including the use of live attenuated vectors [14],three doses to achieve protective immunity. In many DNA vaccines [15], potent adjuvants [16] and thedeveloping countries, compliance with multi-dose controlled release of antigens from microparticlesvaccine regimens is difficult to achieve, resulting in prepared from biodegradable polymers [17–19]. Inhundreds of thousands of deaths per annum from this review, the use of biodegradable microparticlesneonatal tetanus. Consequently, there is an urgent prepared from poly(lactide-co-glycolide) (PLG)need for the development of a TT vaccine which polymers as systemically administered adjuvants andwould be effective with fewer doses, preferably controlled-release vaccines will be discussed. Thefollowing a single dose. principal focus will be the development of single-

The only adjuvants which have been widely used dose vaccines.

D.T. O’Hagan et al. / Advanced Drug Delivery Reviews 32 (1998) 225 –246 227

2. The advantages of PLG polymers for vaccine bulk erosion, the polymer chains are cleaved bydevelopment hydrolysis until water-soluble fragments are pro-

duced, followed by the monomeric acids, which are2.1. PLG controlled-release drug delivery systems eventually eliminated from the body as CO . The2

potential role of enzymes in the biodegradation ofThere are a number of examples of the successful PLG has been somewhat controversial. Brady et al.

commercial use of PLG controlled-release technolo- [70] concluded that bioerosion of these materialsgy for improved delivery of low-molecular weight occurred strictly through hydrolysis with no en-drugs, hormones and peptides. However, there are zymatic involvement. However, several investigatorsmany significant challenges remaining for the suc- [83–85] have reported that enzymes do play acessful delivery of high-molecular weight proteins, significant role in the breakdown of the polymer andincluding vaccine antigens [20]. The major problems that the rate of degradation is dependent on cellwith PLG microencapsulation include instability of uptake. Holland et al. [86] concluded that littleproteins during microparticle preparation, during enzyme involvement is expected in the early stages,storage of the microparticles, during hydration of the when the polymer is in the glassy state, but thatmicroparticles in vivo and during extended periods in enzymes can play a significant role in degradation ofthe body at 378C [21,22]. Nevertheless, in recent polymer in the rubbery state. Recently, the rate ofyears, a number of studies in a range of animal degradation of PLG has been confirmed to be fastermodels [24–66] have described the induction of in vivo than in vitro [87]. Occasionally, an unusualpotent immune responses following systemic ad- combination of polymeric excipients and a bioactiveministration of biodegradable polymers with entrap- agent can lead to an unexpected biodegradationped antigens (Table 1). PLG microparticles have also profile. Maulding et al. [88] showed that the tertiarybeen used as mucosal vaccine delivery systems and amine thioridazine (free base) accelerated the rate ofthis area has recently been reviewed in detail [67,68]. degradation of PLG in vitro and in vivo.

Pitt et al. [89] determined that the first stage in the2.2. The biodegradability and biocompatibility of biodegradation process was a decrease in polymerPLG molecular weight, caused by random hydrolytic

cleavage of the ester linkages. The second stage wasPLG polymers were initially developed by the onset of polymer mass loss, accompanied by a

pharmaceutical industry for use as degradable sur- change in the rate of chain scission. It has beengical sutures [69,70]. Subsequently, characteristics established that 50:50 lactide /glycolide co-polymerssuch as biocompatibility, controllable degradation have the fastest degradation rate, with these poly-kinetics, ease of fabrication and established regula- mers degrading in about 50–90 days in vivo. Thetory approval for human applications have attracted 65:35, 75:25 and the 85:15 PLG polymers haveinterest in these polymers for alternative biomedical progressively longer lifetimes, with 85:15 lastingapplications. Among the first reports of PLG for about 150 days, while PLG 100:0 (Poly DL-lactide)controlled-release applications were those of requires about 12–14 months to completely degrade.Bosewell and Scribner [71], Yolles et al. [72], Microparticles of poly(L-lactide) are more crystallineSinclair [73], Wise et al. [74] and Beck et al. [75]. than the DL-lactide and less hydrophilic. Consequent-These polymers have been developed as controlled- ly, they resist water uptake more effectively andrelease delivery systems for narcotic antagonists, have been found to persist in vivo for 1.5–2 yearscontraceptive hormones, conventional drugs and [90]. In addition to the polymer characteristics, inantibiotics [74,75]. PLG polymers have also attracted vivo biodegradation times also vary depending onattention for a variety of additional biomedical the surface area and porosity of the device. Poly-applications, including tracheal replacement [76], glycolide and a 90:10 polymer used for suturesligament reconstruction [77,78], fracture repairs [79– (polyglactin) in the form of microparticles (1.6 mm)81] and surgical dressings [82]. were found to interact with cultured monocytes and

Biodegradation of PLG occurs by homogeneous macrophages and biodegradation was evident within


Table 1Model proteins and vaccine antigens encapsulated in controlled-release polymers and evaluated in animals for immune response byparenteral routes

Antigen Polymer Use Immunogenicity (References)

Bovine serum albumin (BSA) Ethylene-vinyl acetate sc, implant, mice, rabbits Antibody response similar to CFA [24–26]

tyrosine-based sc, implant, mice Antibody response similar to CFA; adjuvant effect of tyrosine [27]

Poly(iminocarbonate)

Poly(lactide-co-glycolide) sc, microspheres, mice High-titered and long-lasting immune response [45]

Human g-globulin Gelatin microspheres sc, mice Enhanced antibody response and delayed hypersensitivity [49]

Human serum albumin Polyacryl starch im, ip, iv, microspheres, mice Humoral response similar to CFA, DTH [48]

Ovalbumin Poly(lactide-co-glycolide) sc, oral, microsplieres, mice Antibody response similar to CFA, high IgG antibody

levels for 1 year, higher IgA than soluble [32,39]

Poly(lactide-co-glycolide) sc, ip, oral, microspheres, mice High IgG; DTH similar to ISCOMs, intestinal IgA; CTL [42]

Poly(lactide-co-glycpolide) ip, sc, microspheres, mice IgG antibodies higher than soluble antigen [55,60]

Poly(lactide-co-glycolide) sc, microspheres, mice High serum IgG antibodies for 6 months to 1 year [57]

Influenza Polymethylmethacrylate ip, microsphere, mice, guinea pigs Enhanced antibody response than fluid or alum [23]

Poly(lactide-co-glycolide) ip, microsperes, mice Good priming and boosting [28]

Human parainfluenza virus Poly(lactide-co-glycolide) ip, microsplieres, mice High serum antibodies [40]

Staph. enterotoxin B Poly(lactide-co-glycolide) sc, ip, oral, microspheres, mice Circulating and local antibodies, , 10 m more immunogenic [30,31]

Human chorionic gonadotrophin Poly(lactide-co-glycolide) im, microspheres, rabbits Sustained antibody response [29]

HSD-DT Poly(lactide-co-glyoclide) im, microspheres, rats, monkeys Antibody response after one dose similar to three doses of alum [50]

Hib-TT Poly(lactide-co-glyoclide) sc, microspheres, mice Antibody response similar to two doses of soluble vaccine [19]

Tetanus toxoid PLGA-glucose sc, im, microspheres, Immunogenicity preserved [35]; antibodies similar to soluble [56,66]

Poly(lactide-co-glycolide) mice, guinea pigs, rats or two to three doses of alum [37,46,64]; antitoxin higher than soluble [2,19]

[21,38,41,65] and similar to AlpO [2,19,41,56,65]4

Diphtheria toxoid Poly(lactide-co-glycolide) sc, microspheres, mice IgG similar to three doses of CaPO [33,34,117]4

Plague antigen Poly(lactide-co-glycolide) ip, microspheres, mice Mucosal and systemic immunity better than whole cell vaccine [54]

Hepatitis B surface antigen Poly(lactide-co-glycolide) ip, microspheres, guinea pig Antibody levels higher than alum [36]

Poly(lactide-co-glycolide) im, microspheres, mice Antibody levels similar to three doses of Al(OH) [63]3

Rotavirus Spermine, alginate im, microspheres, mice Enhanced humoral response [61]

HIV-1(gp120) and QS21 Poly(lactide-co-glycolide) im, microspheres, guinea pig Enhanced antibody response than alum [44,99,128,129]

FIW-env protem Poly(lactide-co-glycolide) Sc, ip, microspheres, mice Induction of CTL and CMI [51]

Vibrio cholerae antigens Poly(lactide-co-glycolide) sc, iv, microspheres, rabbits Better carrier of antigens by sc route [43]

Ricin toxoid Poly(lactide-co-glycolide) im or sc, microspheres, mice Early, long-lasting IgG response [47]

Tuberculosis antigen Poly(lactide-co-glycolide) sc, microspheres, mice Similar antibodies to IFA, superior T cell responses [52]

Venezuelan equine encephalitis Poly(lactide-co-glycolide) sc, microspheres, mice Enhanced antibody responses than free virus [53]

Peptides (malaria, influenza A) Poly(lactide-co-glycolide) im, microspheres, mice Induction of CTL [58]

Peptides (malaria) Poly(lactide-co-glycolide) im, microspheres, mice Antibody response similar to IFA [59]

Peptides (rabies) Poly(lactide-co-glycpolide) sc, microspheres, mice Immune response similar or superior to CFA [62]

sc, subcutaneous; ip, intraperitoneal; im, intramuscular; iv, intravenous; CFA, complete Freund’s adjuvant; DTH, delayed typehypersensitivity; ISCOMs, immunostimulating complexes; CTL, cytotoxic T lymphocytes; HSD-DT, conjugate of diphtheria toxoid withhetero species dimer a-ovine lutenizing hormone and b-human chrionic gonadotropin hormone; Hib-TT, conjugate of tetanus toxoid withcapsular polysaccharide of Hemophilus influenzae type b.

78 h [91]. This observation supported the approach greater than 1 year. In addition, variation of themicroparticle size can control the ability of theof targeting drugs or antigens to macrophagesparticles to be taken up by antigen-presenting cells.through microencapsulation in PLG polymers.

PLG polymers have been used to prepare severalThe rate of release of peptides and proteins fromcommercially available controlled-release drug deliv-PLG microparticles can be controlled by manipulat-

TMery systems, including Zoladex (Zeneca),ing parameters such as the co-polymer composition,TM TMthe molecular weight and crystallinity [89]. There- Decapeptyl (Ipsen Biotech) and Prostap SR

fore, the duration of release of an entrapped antigen (Lederle) which are licensed for use in humans incan be varied from a few weeks, to months, to Europe and in the USA [92,93]. Hence, the safety of


this polymer for biomedical use has been demon-strated through the development of several productsfor the controlled release of drugs.

2.3. The preparation and characterization of PLGmicroparticles

The most commonly used method for the prepara-tion of microparticles with entrapped antigens in-volves the use of aqueous solutions of antigen, whichare dispersed in an organic solvent containing thedissolved polymer. Various modifications of thisprocess have been reported, most commonly involv-ing the preparation of microparticles by solventevaporation [94–97] or extraction [98,99] fromwater-in-oil-in-water emulsions. Various alternativeapproaches have also been described, including spraydrying [100] and phase separation [101]. The processfor the preparation of microparticles from a water-in-oil-in-water emulsion is shown in Fig. 1. Briefly, anaqueous solution of the antigen is homogenized orsonicated with PLG in an organic solvent, which isusually methylene chloride or ethyl acetate. The ratiobetween the aqueous and organic phases is variableand is an important parameter controlling the charac-teristics of the resulting microparticles. The water-in-oil emulsion prepared from the organic and aqueous

Fig. 1. A diagrammatic representation of the solvent evaporationphases is subsequently dispersed into a larger volumemethod for the preparation of PLG microparticles with entrappedof an aqueous solution of a particle stabilizer (e.g.antigens.polyvinyl alcohol) by homogenization, to form a

water-in-oil-in-water double emulsion. The organicsolvent is then removed by extraction or evaporation, croparticle size and morphology, antigen loading,allowing the polymer to collapse around the internal antigen integrity, in vitro release rates and immuno-aqueous droplets and to entrap the antigen in poly- genicity in animals.mer. The microparticles are normally collected bycentrifugation and are then washed and dried. De-tailed methods for the preparation of both small and 3. The adjuvant effect of microparticleslarge microparticles with entrapped antigens havebeen published previously [95–97]. As an alternative The adjuvant effect achieved as a consequence ofapproach to the process described above, the antigen the entrapment of antigens within microparticles hascan also be dispersed in the organic solvent as a dry been known for many years [23]. Studies evaluatingsolid, with or without stabilizers [98]. the ability of microparticles to induce enhanced

Following the preparation of microparticles, it is immune responses are summarized in Table 1. Theimportant to undertake detailed physicochemical enhanced immunogenicity of particulate antigens ischaracterizations of the product, to ensure consis- unsurprising, since pathogens are particulates oftency and to minimize lot to lot variability. Various similar dimensions and the immune system hasroutine parameters have been found to be useful in evolved to deal with these [67]. Particulate deliverycontrolling product consistency, including mi- systems present multiple copies of antigens to the


immune system and promote trapping and retention systems. The adjuvant effect of microparticles can beof antigens in local lymph nodes. Moreover, particles further enhanced by their administration in vehiclesare taken up by macrophages and dendritic cells, with additional adjuvant activity [104], or by theleading to enhanced antigen presentation and the microencapsulation of adjuvants. Only very limitedrelease of cytokines, to promote the induction of an studies have so far been performed to evaluate theimmune response. Many alternative antigen delivery antibody isotypes induced by antigens in microparti-systems are available, which are also particulates, cles. Immunization of mice with a microencapsulatedincluding liposomes, ISCOMs, micelles and emul- recombinant antigen (38 kDa) from Mycobacteriumsions [18,67]. In recent years, developments in tuberculosis induced antibodies of predominantly thebiotechnology have also allowed the expression of IgG2a isotype, while Freunds’ adjuvant mainly in-protein-based particulate antigen delivery systems, duced the IgG1 isotype [52]. However, the totale.g. virus-like particles. levels of antibodies induced by 38-kDa antigen in

microparticles was comparable to the responses3.1. Microparticles as adjuvants for antibody induced by Freund’s adjuvant [52].induction

3.2. The induction of cell-mediated immunity withIn studies from 1976 onwards, Kreuter and Speiser microparticles

[23] described the use of polymeric nanoparticles asadjuvants for adsorbed and entrapped vaccines. Recent studies have shown that microparticles alsoHowever, the polymethyl methacrylates used in these exert an adjuvant effect for the induction of cell-studies are degraded in vivo only very slowly. More mediated immunity [51,52,58,105,106]. Microparti-recently, the adjuvant effect achieved by the entrap- cles induced cytotoxic T lymphocyte (CTL) re-ment of antigens in biodegradable PLG microparti- sponses in mice following systemic [51] and mucosalcles has been described [31,32,39]. O’Hagan et al. immunization [106], with protein [51,106] and pep-[31] showed that microparticles with entrapped tide [58] antigens. Microparticles also induced aovalbumin (OVA) had comparable immunogenicity delayed-type hypersensitivity (DTH) response [106],to OVA dispersed in Freund’s adjuvant, the most which is thought to be mediated by Th1 cells, andpotent adjuvant available. Eldridge et al. [32] con- potent T cell proliferative responses. The limitedfirmed that antigens entrapped in microparticles had data available on the induction of cytokine responsescomparable immunogenicity to Freund’s adjuvant, in cells from animals immunized with microencapsu-using staphylococcal B enterotoxoid as an antigen. lated antigens indicates that microparticles preferen-Particle size was shown to be an important parameter tially induce a Th1-type response [51,52]. Macro-affecting the immunogenicity of microparticles, since phages are reportedly responsible for phagocytosissmaller particles ( , 10 mm) were significantly more and presentation of particulate antigens through theimmunogenic than larger ones [32,39]. The effect of cytosolic MHC class I-restricted pathway [107].particle size on immunogenicity is likely to be a However, dendritic cells may also play a role in theconsequence of enhanced uptake into lymphatics and presentation of particulate antigens and the release ofgreater access to antigen-presenting cells for the cytokines to promote a Th1-type response [108].smaller sized particles [102]. Enhanced immuno- Preliminary studies have indicated that particle sizegenicity for smaller sized particles has also been may be an important parameter influencing theobserved with emulsion droplets [103]. It appears efficacy of microparticles as adjuvants for CTLthat antigen entrapment in microparticles is not induction [58].necessary for an adjuvant effect, since the immuno-genicity of antigens adsorbed to the surface of themicroparticles was also enhanced [39]. However, the 4. Microparticles as single-dose vaccinesdelivery of antigens on the particle surface ratherthan entrapped restricts the potential of the mi- In recent years, a number of studies have beencroparticles to perform as controlled-release delivery undertaken to evaluate the potential of microparticles


for the induction of potent long-term immune re- nificantly higher anamnestic antibody responses thansponses following a single dose. The majority of this those primed with TT adsorbed to aluminum phos-work has concentrated on TT, with the objective of phate. Men et al. [46] prepared microparticles withdeveloping a single-dose vaccine to prevent neonatal entrapped TT and induced high TT antibody and Ttetanus. However, additional work has also been cell responses in mice following a single immuniza-performed with other antigens, including DT, gp120, tion. The antibody responses in mice immunizedhepatitis B surface antigen and human chorionic with a single injection of a mixture of three PLGgonadotrophin. This work will now be reviewed in microparticle formulations induced IgG antibodydetail. levels similar to three injections of alum adsorbed to

TT [46]. Recently, Singh et al. [64] confirmed the4.1. Tetanus toxoid earlier observations of Raghuvanshi et al. [37] in rats

with TT microparticles. It was further shown that aNeonatal tetanus remains a significant problem in combination of TT entrapped in microparticles and

most of the developing world and results in an adsorbed to alum induced greater antibody responsesunacceptable level of mortality, despite the fact that than microparticles alone [64].the disease can easily be prevented by immunization Although in most of the studies described, potentof pregnant women with TT. However, the major antibody responses were induced which persisted forproblem is full compliance with the two- to three- periods of up to 1 year, none of the studies showed adose schedule of vaccine needed to induce protective typical booster response in animals following aimmunity [15]. Therefore, the development of a single injection of a mixture of two to three differentsingle-dose TT vaccine would lead to tremendous microparticle formulations. It had previously beenprogress in the control of neonatal tetanus and would claimed with an alternative antigen, staphylococcalresult in the prevention of many deaths. Various enterotoxin B, that in vivo booster responses couldattempts have already been made to develop a single- be achieved with combinations of small and largerdose TT vaccine, including increasing the antigen microparticles as a single injection [30]. However,and adjuvant doses [6,9–12]. However, develop- studies with TT entrapped in small and large mi-ments in controlled-release technology have allowed croparticles did not provide evidence of in vivoa number of research groups to apply their expertise boosting, although the antibody responses wereto the problem of neonatal tetanus. Several publi- maintained at high levels for 1 year [64].cations have described initial observations in relation The major caveat with most of the TT studies isto the potential development of a single-dose TT that a relatively large dose of TT was used in smallvaccine based on PLG microparticles. Esparza and animals, | 15 mg or higher. It has been shownKissel [35] and Raghuvanshi et al. [37] induced recently that a similiar dose of TT adsorbed ontolong-lasting immune responses in mice and rats, aluminum adjuvants is capable of inducing potentrespectively, by immunization with PLG microparti- and long-lasting immune responses in mice andcles. As previously reported [104], the antibody guinea pigs following a single immunization [65].responses were enhanced when the microparticles Early studies had suggested that a model antigenwere administered in an oil-based vehicle [35]. The adsorbed onto aluminium can induce long-lastingantibody responses obtained were similar to those immune responses in rodents following a singleinduced by two injections of TT adsorbed to alum. immunization [39]. Additional studies also showedAlonso et al. [38] and Gupta et al. [21] reported high that lower doses of TT entrapped in microparticles,anti-TT IgG antibodies and tetanus antitoxin levels in | 1.5 mg, were not potently immunogenic. Lowthe sera of mice and guinea pigs immunized with doses of TT entrapped in microparticles were poorlyPLG microparticles. The antibody levels were sig- immunogenic mainly as a consequence of the in-nificantly greater than those induced by soluble TT stability of TT during microencapsulation and alsoand were similar to those elicited by a single following in vivo administration [109]. Most of theinjection of TT adsorbed to aluminum adjuvant. The studies involving TT indicated that there were sig-animals primed with TT in microparticles had significant instability problems with this antigen in


microparticles. A number of parameters during the such as ABA-triblock copolymers of PLG and PEGmicroencapsulation process contributed to the in- [66]. The inclusion of gelatin or human serumstability problems, which mainly manifested as ag- albumin in microparticles with TT significantlygregation of entrapped TT [110]. The aggregation of increased the release of antigenic TT in vitro [111].the antigen prevented complete release of TT from The stabilization effect of these excipients wasthe microparticles and, therefore, affected its im- attributed to a reduction in the interactions betweenmunogenicity. TT and polymers and to better control of the pH

Schwendeman et al. [110] investigated the causes inside the microparticles. Kissel et al. [66] attributedof TT instability, in order to enhance the solubility the reduction of antigenicity of TT in microparticlesand antigenicity of TT released from microparticles. to the hydrophobicity of the PLG polymers. TheIt was shown that lyophilized TT aggregated on immunogenicity of TT was improved followingexposure to moisture, even in the absence of mi- encapsulation in more hydrophilic PLG/PEG co-croparticles. The aggregation of TT was due to three polymers, in comparison to encapsulation in PLGmechanisms, which depended on the water content polymers [66]. Overall, significant progress has beenof the lyophilized protein: (1) noncovalent interac- made during the last 2–3 years in stabilizing TT.tions, (2) disulfide interchange, and (3) form- However, the effects of these stabilization ap-aldehyde-mediated conversion of labile intramolecu- proaches on the immunogenicity of microencapsu-lar bonds to stable intermolecular cross-links [110]. lated TT still needs to be evaluated in vivo.It was further shown that formaldehyde-mediated An additional problem contributing to the poorcross-linking was the principal cause of moisture- immunogenicity of low doses of microencapsulatedinduced aggregation of TT. The presence of residual TT is the relatively low immunogenicity of solubleformaldehyde in the protein was due to incomplete TT. At low doses of microencapsulated TT, it isreaction of tetanus toxin with formaldehyde during likely that the initial ‘burst’ release of TT from thethe de-toxification of TT. A similar mechanism of microparticles would not be enough to initiate aaggregation was found for an additional vaccine, potent immune response. For example, for a 1.5-mgdiphtheria toxoid (DT) which is also prepared by dose of encapsulated TT, a 10% burst from theformalinization of a toxin. Hence, these studies microparticles would only release 150 ng. However,indicated that the principal cause of antigen instabili- when the microparticles were mixed at low dosesty in microparticles for TT and DT was the presence with aluminum adjuvants, potent antibody responsesof residual formalin in the toxoided vaccines. Based were induced in rodents (unpublished observations).on these observations, rational approaches to the Based on these observations, a mixed formulationstabilization of TT at the molecular level were consisting of microencapsulated TT and TT adsorbedproposed, which included: (1) reaction of TT with onto an aluminum adjuvant at a ratio of 10:1 wassuccinic anhydride, which reacts with lysine and developed [19]. Combination formulations of mi-tyrosine residues, to prevent intermolecular aggrega- croparticles and alum had previously been evaluatedtion; (2) reduction of TT by reaction with cyano- at higher doses by Singh et al [64] in rats and hadborohydride, which reduces reactive imines and been shown to be better than microparticles alone. Instabilizes the bound formaldehyde; and (3) addition the combined vaccine formulations, each mouseof low-molecular weight excipients, particularly received 1.5 mg of TT entrapped in PLG microparti-sorbitol. However, the effects of these chemical cles in addition to 0.15 mg of TT adsorbed tomodifications on the immunogenicity of TT have yet aluminum phosphate. Control groups of animalsto be evaluated. received two doses of 0.15 mg TT adsorbed to

Alternative strategies to stabilize TT and to en- aluminum phosphate or soluble TT 4 weeks apart.hance the release of antigenically intact protein High antitoxin levels were induced in mice immun-include the use of stabilizers, such as gelatin and ized with a mixture of microencapsulated TT andhuman serum albumin. In addition, TT can be aluminium adsorbed TT and the antibody levelsentrapped in microparticles prepared by modified increased over a period of 14 weeks (Fig. 2). Inpolymers, which are less hydrophobic than PLG, contrast, two doses of aluminium-adsorbed TT in-


Fig. 2. The immunogenicity in mice (female, CD-1, 4–6 weeks old, six per group) of two doses of tetanus toxoid adsorbed to AlPO at three4

different dose levels and protocols as follows: (i) two doses of 0.15 mg each at 4-week intervals; (ii) first dose of 0.15 mg and second dose1.5 mg after 4 weeks; (iii) one single dose of 1.65 mg. These responses are compared to additional groups as follows: two doses of solubletetanus toxoid (first dose of 0.15 mg, second dose 1.5 mg after 4 weeks) and a single dose of a combined controlled release formulation,comprising a mixture of 1.5 mg of toxoid in PLG microspheres and 0.15 mg of AlPO -adsorbed tetanus toxoid. The graph shows tetanus4

antitoxin titers of the pooled sera determined by a toxin neutralization assay in mice [41].

duced higher antitoxin levels after the second in- (Hib) conjugated to TT [19]. In this formulation, thejection but, at 14 weeks, the levels were similar to microencapsulated TT was stabilized by the capsularthose induced by a single dose of the combined polysaccharide from Hib. The microencapsulatedformulation (Fig. 2). At 14 weeks, the TT antitoxin Hib/TT conjugate induced TT antitoxin responses inlevels in mice immunized with the combined formu- mice and high levels of anti-Hib IgG antibodies,lation were 9.6 AU/ml, which are approximately following a single immunization.1000 times higher than the minimum protective level Based on these various observations, we feel thatfor tetanus (0.01 AU/ml). From earlier studies in TT microparticle formulations should be evaluated inmice and guinea pigs, it is clear that the decay of humans. However, many studies have shown thathigh-affinity TT antibodies is very slow. Therefore, it small animal models are often poor predictors of thewould be expected that the mice in this study would immunogenicity of vaccines in humans. For exam-have at least 10 times higher levels than the mini- ple, unlike humans, rodents produce a very highmum protective levels of TT antibodies for at least 1 antibody response after a single injection of TTyear. These results in mice confirmed that an optimal adsorbed to aluminum phosphate. In addition, TTsingle-dose TT vaccine formulation should consist of adsorbed to calcium phosphate, which is significantlya mixture of TT entrapped in PLG microparticles and less potent in mice and guinea pigs than TT adsorbedTT adsorbed to an aluminium adjuvant [64]. The onto aluminum adjuvants, elicited comparable anti-aluminum-adsorbed TT is likely to optimally initiate body levels in humans to those induced by TTthe immune response, while the continuous release adsorbed onto aluminum adjuvants [112–114].of TT from PLG microparticles should maintain Therefore, studies are needed in large animal modelsantibodies at high levels for extended periods. to confirm the observations made in rodent studies.

An alternative approach to the development of a Recent work has confirmed that the mechanism ofsingle-dose TT vaccine is represented by the en- adjuvant activity for TT microparticles is differentcapsulation in PLG microparticles of the capsular from that of TT adsorbed to aluminum. Microparti-polysaccharide of Haemophilus influenzae type b cles were shown to form a depot at the injection site


from which antigen is released over a prolongedperiod of time, but this was not the case foraluminum adjuvants [87].

4.2. Diphtheria toxoid

The incidence of diphtheria declined rapidly indeveloped countries after the introduction of avaccine in the 1940s and 1950s. During the early1980s, when its incidence was at the lowest levelever, there was optimism that indigenous respiratorydiphtheria might be eliminated from Europe. How-ever, the recent diphtheria epidemics in easternEurope, particularly in Russia and Ukraine[115,116], have caused concern that diphtheria out-breaks could occur in developed countries. These Fig. 3. The antibody responses in rats following a single immuni-epidemics were attributed to inadequate immuniza- zation with a controlled release formulation of diphtheria toxoid

(DT) comprising microparticles prepared by combining threetion coverage for DT, due to the requirement forpolymer types, PLG 50/50, 85/15 and100/0 and mixed withmultiple injections.alum. One group received a combination of alum and microparti-DT like TT, requires three injections during itscles as a single injection, the other was injected three times at 0, 4

immunization schedule and, therefore, represents a and 8 weeks with equivalent doses of DT adsorbed to alum [117].suitable target for conversion into a single-dosecontrolled-release vaccine. However, less work hasbeen published involving DT controlled-release vac- doses. Moreover, Schwendeman et al. [110] alsocine delivery systems. Singh et al. [33] were the first reported moisture-induced aggregation of DT, anto describe the use of PLG polymers for the mi- observation similiar to that reported for TT.croencapsulation of DT. They reported that a singledose of DT entrapped in microparticles induced 4.3. Hepatitis B surface antigen (hepatitis Bcomparable serum IgG titers in mice to three divided vaccine)dose of DT adsorbed to calcium phosphate adjuvant[33]. Similarly, we have recently reported that a Hepatitis B infection is a major worldwide healthsingle injection of DT in PLG microparticles induces problem, causing progressive liver cirrhosis andcomparable serum IgG antibody responses to three hepato-cellular carcinoma [118,119]. Immunizationdivided doses of DT on alum in rats (Fig. 3). In represents the optimal approach to prevent the spreadaddition, we have also shown that optimal immune of the virus. The hepatitis B vaccine, comprisingresponses are obtained with combinations of PLG hepatitis B surface antigen (HBsAg), is now used asmicroparticles and alum [117]. A similiar observa- a component of the EPI schedule, along withtion was previously reported using microparticle and diphtheria, pertussis and tetanus (DPT) in manyalum combinations for TT [64]. These studies also geographical regions. Efforts are also underway toshowed that combining two antigens, DT and TT, include HBsAg in the worldwide EPI schedule towithin the same microparticles reduced responses, in control the disease on a global scale. Despite thecomparison to administration of individual antigens availability of an effective recombinant vaccine, thein microparticles [64,117]. The studies performed to relatively high cost of the vaccine, the geographicaldate indicate that PLG microparticles have signifi- location of the population at risk and the multiplecant potential for the development of single-dose injection schedules used have all contributed to thevaccines for TT and DT. However, these studies under-utilization of HBsAg vaccines. Hence, thewere performed at human dose levels of DT in development of an improved delivery system forrodents and they need to be repeated with lower DT hepatitis B vaccine which could induce the desired


antibody response from a single injection would be velopment of a controlled-release hepatitis B vac-of enormous benefit. cine, it is clear that the HBsAg needs to be protected

Nellore et al. [36] were the first to report on the from organic solvents during the microencapsulationimmunogenicity of HBsAg in PLG microparticles. process.Subsequently, Singh et al. [63] compared the anti-body responses induced by a single injection of the 4.4. Envelope glycoprotein gp120 (HIV vaccine)HBsAg entrapped in PLG microparticles to thatobserved with three injections of HBsAg adsorbed to According to the World Health Organizationalum (Fig. 4). Although the long-term antibody (WHO), the number of cases of human immuno-responses from a single injection of the microparti- deficiency virus (HIV-1) infection by the turn of thecles were encouraging, it seemed likely that the century is expected to total around 40 million [121].immunogenicity of the HBsAg had been damaged by Hence, the development of an effective HIV vaccineexposure to organic solvents, since the microen- is an important objective on a global scale. This is acapsulated HBsAg was less immunogenic than the formidable task, due to the variability of HIV, thealum-adsorbed antigen [36,63]. Because of this various transmission routes of the virus and a lack ofproblem, Lee et al. [120] adopted an alternative understanding of the possible mechanisms of im-approach and entrapped HBsAg into double-walled mune protection [122,123]. Nevertheless, severalmicroparticles, with an initial coating to protect the studies performed with the HIV-1 envelope glycopro-antigen from the organic solvent. The HBsAg en- tein (gp120) have demonstrated that neutralizingtrapped in the double walled microparticles retained antibodies against the principal neutralizing deter-immunogenicity to a greater extent than antigen minant (PND) in the V3 region of gp120 can protectentrapped in standard PLG microparticles. It seems chimpanzees from virus challenge [124–127]. How-likely that exposure to organic solvents removes ever, although neutralizing antibodies may be re-lipids from the HBsAg, which are important for the sponsible for elimination of cell-free virus particles,induction of functional antibody responses. Although cell-mediated immune responses, particularly cyto-PLG microencapsulation holds promise for the de- toxic T lymphocytes (CTL) are responsible for

clearance of virus-infected cells and are likely to bean important component of protective immunity.

Cleland et al. [44,99,128,129] evaluated the effectof the adjuvant QS21 in combination with PLGmicroparticles containing an entrapped recombinantgp120 vaccine in guinea pigs. The addition ofsoluble QS21 increased the initial antibody titersfive-fold when compared to gp120 alone in PLGmicroparticles [44]. However, the addition of QS21to the ‘boost’ release of gp120 did not increase theantibody titers over the release of antigen alone[128]. Therefore, the use of additional adjuvants, inassociation with microparticles, appears to be usefulonly for the initial immunization [128].

It should be noted that the microparticles used byCleland et al. were relatively large ( . 20 mm) and,consequently, were not designed to have optimal

Fig. 4. Anti-HBsAg antibody response in two groups of CD1 mice ‘adjuvant’ effect, but were designed for controlledimmunized either with a single injection of a combined mixture of release. The work of Singh et al. [64] also demon-alum and microparticles containing 30 mg of HBsAg divided

strated that the immune response to controlled-re-equally into four components or three 10-mg HBsAg injections onlease microparticles is enhanced if the microparticlesalum at 0-, 1- and 6-month intervals [63]. Data shown are

geometric mean6S.E. IU/L in each group. are combined with an additional adjuvant that is not


entrapped. Cleland et al. confirmed that, unlike shown to induce neutralizing antibodies and T cellalternative approaches involving more standard ad- responses [127]. In preliminary studies, a singlejuvants, controlled-release microparticles were able injection of the p200M microparticle formulation into induce high titer long-lasting antibody responses mice induced a comparable serum IgG response toin guinea pigs [129]. The gp120 microparticle that obtained with three divided doses of the peptideformulations were optimized to release stable, un- adsorbed to alum [130]altered antigen and to provide an in vivo auto boostresponse, as a consequence of the delayed release of 4.5. Human chorionic gonadotropin (hCG)-basedthe antigen entrapped in slower degrading mi- birth control vaccinescroparticles [99]. Fig. 5 shows the serum-neutralizingtiters obtained in guinea pigs with a single injection The rationale of birth control vaccines is to induceof the gp120 vaccine in microparticles. humoral and/or cell-mediated immune response to

We have recently described a controlled-release selectively intervene with one or more critical stepsPLG microparticle formulation for a HIV-1 synthetic in the reproductive process. Mammalian reproduc-peptide vaccine. The peptide (p200M) consisted of a tion is regulated by a cascade of hormones to30-amino acid sequence from the V3 loop of gp120, generate gametes. Some of the hormone and antigenrepresenting the PND of HIV-1, which was syn- targets which have been evaluated as potential birththesized as a branched octameric peptide [127]. No control vaccines include; gonadotropin-releasing hor-additional adjuvants were included with the mi- mone, human chorionic gonadotropin (hCG), follic-croparticle formulations. The octameric peptide had le-stimulating hormone, zona pellucida and spermpreviously been shown to be highly immunogenic antigens. hCG is a hormone which has an importantand induced significant titers of neutralizing anti- role in sustaining the corpus luteum and in thebodies in a small animal model [96,127]. Moreover, establishment of pregnancy. Therefore, immunologi-this immunogen has subsequently undergone Phase I cal intervention with hCG can result in effectiveclinical evaluation in human volunteers and has been contraception [131,132]. The effects of antibody-

mediated contraception are reversible in humans anddo not appear to have residual side effects followingreversal.

Two birth control vaccines based on hCG havebeen evaluated in humans [29,132]. The two ap-proaches involved a synthetic peptide-based vaccine[29] and the b-subunit of hCG [132], which bothrequired repeated immunizations to achieve contra-ception in humans. Stevens et al. [29] demonstratedthe feasibility of using PLG-based microparticles togenerate long-lasting anti-hCG titers with a singleinjection of the peptide vaccine. Singh et al. [50]evaluated the alternative approach, involving the b-subunit of hCG conjugated with the a-subunit ofovine luteinizing hormone to form a hetero speciesdimer (HSD). Following preparation, the dimer wasconjugated to the protein carrier DT to enhance theimmunogenicity of the construct. Finally, the HSD-

Fig. 5. HIV-1 envelope gp120 antibody titers and MN neutraliza- DT vaccine was entrapped in PLG microparticlestion titers in guinea pigs immunized with a single injection of 50 and evaluated in rats and bonnet monkeys. Highmg QS-21 and 30 mg soluble gp120, combined with 30 mg gp120

neutralizing titers were induced in bonnet monkeysentrapped in PLG microparticles at day 0 [129]. The graph showsfollowing a single injection of the hCG vaccine ina gradual increase in antibody titers over time (data provided by

Jeff Cleland of Genentech). PLG microparticles made from two different poly-


depend, to some extent, on the chosen microencapsu-lation process and partly on the desired duration ofprotein release from the microparticles. The potentialproblems encountered with protein instability arelikely to be greater with vaccine antigens than withtherapeutic proteins, since the duration of releaserequired for an effective controlled-release vaccine islikely to be much longer. Protein instability problemsmay also be encountered during storage of themicroparticle formulations. For example, significantpotential exists for interactions to occur between thepolymer and the protein, including adsorption pro-cesses and ionic interactions [133,134]. These inter-actions have the potential to modify, or even tocompletely prevent protein release from the mi-Fig. 6. hCG binding capacity in ng/ml for two groups of fourcroparticles [133–135]. Moreover, the potential forbonnet monkeys (Macaca radiata) immunized with 300 mg of

hCG vaccine (HSD-DT) in microparticles as a single injection at interactions between the polymer and microencapsu-day 0 or as three divided doses on alum at days 0, 28 and 56. Both lated proteins may be enhanced following adminis-groups were boosted at day 225 with 100 mg of HSD-DT in tration, when both are wetted. Protein instability inmicroparticles or alum, respectively [50].

microparticles is a critical issue, the importance ofwhich cannot be over emphasized. Only recently, has

mers. The titers induced were comparable to those some success has been achieved in terms of theinduced by three doses of HSD-DT on alum given at effective stabilization of proteins during microen-monthly intervals. Fig. 6. shows the anti-hCG anti- capsulation [136,137]. However, much greater pro-body titers induced in bonnet monkeys following a gress needs to be made in appreciating the basicsingle dose of the HSD-DT vaccine in PLG mi- mechanisms of protein instability during microen-croparticles [50]. capsulation and their underlying causes.

5.1. Protein stability during microparticle5. Recent progress in the development of PLG preparationcontrolled-release protein delivery systems

PLG polymers are soluble in only a limited rangeThe most significant limitation which might re- of organic solvents and are insoluble in water. The

strict the potential development of single-dose vac- most commonly used solvent for PLG is dichlorome-cines and controlled-release delivery systems for thane (DCM), although ethyl acetate and others havetherapeutic proteins using PLG polymers is the also been used. The techniques most commonly usedinstability of proteins in microparticles. During the for the preparation of microencapsulated vaccinesmicroparticle preparation process, during storage and involves emulsion-based solvent evaporationfollowing in vivo administration, proteins are ex- [31,33,97,136] or solvent extraction [38]. Duringposed to a range of conditions which might result in microparticle preparation, the vaccine is normallytheir denaturation. The conditions include; dispersion dissolved in an aqueous solution, which is dispersedin organic solvents, high-speed shear, exposure to as an emulsion in a solution of the polymer in anaqueous /organic solvent interfaces and freeze dry- organic solvent. Hence, the preparation of mi-ing. Microencapsulated proteins are also exposed to croparticles using PLG polymer necessarily involvespotentially adverse conditions following in vivo some exposure of antigens to organic solvent. How-administration, including elevated temperature and ever, exposure to solvent can be minimized by thelow pH in the presence of moisture. dispersion of antigen into the solvent as a dried solid

The extent of exposure to these conditions will [136,137]. Although dissolving the antigen in an


aqueous phase and the preparation of an emulsion antigen denaturation may not be a significant prob-normally limits the direct exposure of the antigen to lem if the objective is to induce cell-mediatedthe organic solvent, this also creates an organic / immunity. In this context, microparticles have beenaqueous interface, which may promote the denatura- shown to induce specific cytotoxic T cell responsestion of some antigens. against small linear peptides [58]. In contrast, B cell

In addition to solvent exposure, antigen instability receptors recognize specific amino acid sequences,problems may also arise during microparticle prepa- often in a three-dimensional conformation. There-ration as a consequence of the high shear forces used fore, the induction of antibody responses againstduring preparation. The extent of shear applied discontinuous epitopes will be more dependent onduring microparticle preparation is generally greater the maintenance of the native conformation ofas the desired microparticle size decreases [97]. antigens. On a number of occasions, the nativeThere are several well-documented reports of the structure of microencapsulated antigens has beeninstability of peptides and proteins in microparticles maintained as determined by the binding of anti-[141–143]. In recent studies, the instability problems bodies and the induction of neutralizing antibodyencountered with TT, which is prone to moisture- responses [19,21,41,65,144]. Following microparticleinduced aggregation, have been identified [110]. preparation, the presence of residual solvents in theAggregation problems were also encountered with formulation might conceivably affect antigen stabili-BSA entrapped in PLG microparticles, which were ty. However, the level of DCM in microparticlesminimized by the elimination of a free thiol group in prepared by solvent evaporation, was shown to bethe protein [133]. Nevertheless, despite the potential below the lowest limit of detection using gas chro-problems, it should be noted that a range of proteins matography, i.e. , 10 ppm [95]. Furthermore, Lup-have been successfully entrapped in PLG microparti- ron Depot, a marketed product which is manufac-cles prepared by solvent evaporation, without evi- tured by solvent evaporation, contains , 50 ppmdence of significant changes in their native structure DCM. Consequently, the level of residual DCM in[97,136–138]. During microencapsulation, the po- microparticles is low enough to not represent antential for protein instability problems may be mini- issue for protein stability, although it is an importantmized by limiting the direct exposure of the antigens regulatory concern.to organic solvents and to interfaces. This can beachieved using novel techniques such as the encapsu- 5.2. Protein stability during freeze dryinglation of antigen in an oily reservoir within themicroparticles [145]. Alternatively, the protein may The preparation of microencapsulated vaccines hasbe dispersed in solvent and encapsulated as a dry often involved a freeze-drying step [31,97], andpowder [136,137]. Several microencapsulated vac- proteins need to be stabilized during this process. Forcines have induced potent immune responses and some microparticle preparation processes, the pro-protection against challenge in animal models of teins are dried with excipients prior to microencapsu-infectious diseases [40,53,64,139,140,144] (Table 1). lation [136,137]. In order to obtain functional nativeOverall, the extent to which protein instability prob- proteins following lyophilization, it is necessary tolems can be overcome through modifications in the stabilize the proteins during both the freezing and themicroparticle preparation process needs to be better drying stages. Each process applies very differentappreciated and the process of microencapsulation stresses to the protein and each stage requiresneeds to be modified to be more compatible with the different stabilizing agents. The stabilization ofspecific antigen of interest. proteins during freeze drying is a complex area, but

The extent to which antigen instability in mi- the mechanisms by which proteins are damagedcroparticles is a real problem may depend on during lyophilization are gradually becoming betterwhether the objective is to induce T or B cell- understood [146,147,153]. For the future develop-mediated immunity. Since T cell receptors recognize ment of microencapsulated vaccines, it is importantshort linear sequences of amino acids in association that recent advances in the stabilization of therapeu-with the major histocompatibility (MHC) antigens, tic proteins during lyophilization should also be


applied to the stabilization of antigens in microparti- that large PLG devices degraded more quickly thancles. smaller devices, as a consequence of autocatalysis

due to the entrapment of acidic oligomers within thedevice during degradation. It was suggested that

5.3. Protein stability during storagedevices with diameters of less than 200–300 mmwould not be prone to autocatalysis, since oligomers

The stability of microencapsulated vaccines duringwould be free to diffuse out from these smaller

storage is likely to be only a minor issue in com-structures [148,149]. In all of the studies described in

parison to other more serious stability issues. Inthis review, the microparticles used for vaccine

general terms, the stability of proteins in the dry statedelivery have been smaller than 200 mm, to allow for

is often greater than solution formulations. There-easy injection.

fore, microencapsulated vaccines might be expectedBecause of the generation of acidity during degra-

to be more stable than traditional vaccine formula-dation, it was suggested that PLG polymers may be

tions, which are often suspensions of antigen ad-suitable only for the long-term delivery of acid-stable

sorbed to alum. Since microencapsulated vaccinesagents [150]. However, this conclusion was based on

are dried formulations, they may be amenable towork involving the use of polymeric implants of

storage without refrigeration, which would be aseveral milimeters in diameter. Studies with cholera

tremendous advantage for use in the developingtoxin B subunit (CTB), which is unstable at low pH

world. The storage stability requirements for vac-and separates into monomeric fragments, have indi-

cines to be included in the Expanded Programme forcated that molecules which are acid unstable might

Immunization of the WHO have been defined andstill be successfully entrapped in PLG microparticles.

these requirements have already been met by aCTB was entrapped in microparticles and was re-

candidate microparticle vaccine [96]. Moreover,leased intact both in vitro and in vivo [151]. Never-

several microparticle products are already marketedtheless, studies with a model protein, BSA, indicated

worldwide, with acceptable shelf-lives, althoughthat significant acid-catalyzed degradation can occur

these products contain peptides rather than proteins.during in vitro release from microparticles at 378C[143]. This work served to highlight the importance

5.4. Protein stability following in vivo of the experimental conditions employed during inadministration vitro release studies with PLG microparticles. It was

shown that during in vitro release, low pH wasThe area which is likely to cause the greatest generated in the release medium, which catalyzed

stability problems for microencapsulated vaccines is both polymer and protein degradation. Protein degra-the area in which least is currently known, protein dation was significantly reduced when the in vitrostability in microparticles following in vivo adminis- release study was performed in a dialysis bag, whichtration. PLG polymers degrade by random hydrolytic allowed the low-molecular weight polymer frag-scission of the polyester and in the absence of ments to be removed from the release media. It wassufficient buffering capacity, polymer degradation suggested that release in the dialysis bag was a moremay generate low pH due to the increasing pro- accurate representation of protein release from mi-duction of carboxylic acidic end groups. croparticles in vivo, since polymer degradation frag-

The generation of low pH in the external medium ments would not be expected to remain at thein which microparticles are suspended has been injection site once they had diffused from thedemonstrated [143], but the generation of low pH microparticles [143]. An alternative approach towithin microparticles during degradation has not. avoid the possible generation of low pH due to buildIndeed, it is thought likely that low-molecular weight up of polymer degradation fragments is the use of aoligomers of PLG would diffuse out from the continuous flow-through system, rather than an en-degrading microparticles and that this would prevent closed system for in vitro release [151]. Alternative-the development of low pH within the microparticle ly, the amount of particles suspended in buffer cancore. Grizzi et al. [148] and Li et al. [149] showed be adjusted so that the buffering capacity of the


release media is not exhausted, even after significant ated with developing successful delivery systems forpolymer degradation. the controlled release of proteins is much greater.

Various approaches may be adopted to minimize Nevertheless, recent progress in the stabilization ofthe possible effects of low pH generated within proteins during microencapsulation has indicated thatmicroparticles during polymer degradation. For ex- the development of controlled-release formulationsample, the internal environment of the microparticles for therapeutic proteins is only a matter of time. Themay be buffered with poorly soluble excipients, problems associated with the development of con-which will not be released from the particles during trolled-release vaccines may be greater than those fordegradation. Alternatively, the microparticles may be therapeutic proteins, since the required duration ofdesigned so that the majority of the entrapped protein release is likely to be much longer.is released in advance of the occurrence of signifi- Despite encouraging observations in early studiescant polymer degradation. Rapid antigen release with microencapsulated TT, it soon became clearmight be acceptable and even advantageous if the that this antigen had serious instability problems inmicroparticles are being used simply as adjuvants, or microparticles. It had been hoped that this might notfor the mucosal delivery of antigen. However, early be the case, since TT is toxoided and, therefore, wasantigen release would not be an acceptable option if not thought to contain sensitive conformationalthe overall objective was to develop a single-dose epitopes. Paradoxically, the fact that this antigen wascontrolled-release vaccine. Although high-molecular a toxoid proved to be its greatest problem. Detailedweight polymers are available which would not be analysis of the stability of lyophilized TT demon-expected to undergo significant degradation until strated that the protein aggregated, mainly as aperhaps 1 year after immunization and antigen consequence of the presence of residual formalde-release could be designed to be completed within this hyde. The protein did not need to be in the environ-time frame. ment of microparticles for this aggregation to occur,

Protein instability problems in microparticles may the presence of moisture was all that was required.also occur as a consequence of polymer /protein Subsequent work demonstrated that these instabilityinteractions; based on adsorption, charge or ionic problems might be overcome through the chemicalstrength, or even through protein /protein interac- modification of TT. However, the modified TT needstions. The extent to which these problems can be to be evaluated for immunogenicity following inovercome for each individual protein by formulation vivo administration. Despite the TT stability prob-modifications needs to be determined. In vitro re- lems, a number of studies in small animal modelslease studies should be performed at 378C under showed the induction of toxin neutralizing antibodiesconditions in which acidic degradation products do which persisted at high levels for more than 1 yearnot accumulate and the stability of both ‘early’ and after a single injection with microencapsulated TT.‘late’ released antigen should be determined. More- Overall, more work on the development of control-over, alternative stabilizers and additives need to be led-release vaccines has been performed with TTevaluated for their ability to stabilize each individual than with any other antigen. This work has served toantigen of interest. Overall, the observation that indicate the approaches that need to be undertaken ifmicroencapsulated vaccines can induce protective controlled-release vaccines are to be developed.immunity of lasting duration following a single Antigen stability problems, if present, should beimmunization is an encouraging finding [64]. defined as thoroughly as possible, as early as pos-

sible. Furthermore, pre-formulation studies, e.g. sol-vent compatibility, excipient selection, buffer choice,

6. Conclusions etc., should be undertaken to overcome, or minimizethe antigen instability problems. Unfortunately, each

Controlled-release formulations based on PLG individual antigen may require specific formulationmicroparticles have been proven to be successful for modifications to overcome its problems and thethe release of low-molecular weight drugs and choice of excipients is currently empirical. However,peptides. However, the degree of difficulty associ- as more becomes known about the causes and


Novel Strategies in Design and Production of Vaccines,mechanisms of antigen instability in microparticles,Plenum Press, New York, 1996, pp. 105–113.the easier it will be to overcome these problems.

[3] R.K. Gupta, E.H. Relyveld, E.B. Lindblad, B. Bizzini, S.Recent work with a well-defined recombinantBen-Efraim, C.K. Gupta, Adjuvants—a balance between

glycoprotein antigen, gp120 from HIV-1, has further toxicity and adjuvanticity, Vaccine 11 (1993) 293–306.demonstrated the exciting potential of PLG mi- [4] R.K. Gupta, B.E. Rost, E. Relyveld, G.R. Siber, Adjuvant

properties of aluminum and calcium compounds, in: M.F.croparticles for the development of single-dose vac-Powell, M.J. Newman (Eds.), Vaccine Design: The Subunitcines. Recombinant gp120 has been entrapped inand Adjuvant Approach Plenum Press, New York, 1995, pp.microparticles with full maintenance of antigen229–248.

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[6] J.P. Stanfield, D. Gall, P.M. Brasken, Single dose antenatalfor at least 1 year [152]. This work, along with tetanus immunization, Lancet i (1973) 215–219.similiar work by others, serves to highlight the [7] R. MacLennan, L. Levine, K.W. Newell, G. Edsall, The earlycrucial importance of the stability of antigens in primary immune response to adsorbed tetanus toxoid in man.

A study of the influence of antigen concentration, carriermicroparticles. For antigens which are inherentlyconcentration, and sequence of dosage on the rate, extent,stable in the microparticle environment, such asand persistence of the immune response to one and twogp120, there is great optimism that controlled-releasedoses of toxoid, Bull. WHO 49 (1973) 615–626.

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Cooverji, M.P. Anand, Response to single dose of tetanusrange of antigens which are suitable for encapsula-vaccine in subjects with naturally acquired tetanus antitoxin,tion in controlled-release microparticles is yet to beLancet ii (1981) 219–222.

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[13] P. Freeman, A. Robbins, Introduction: Vaccine technologiesof our various colleagues and collaborators to muchand public health: Why a critical review now?, Int. J.of the data presented in this review.Technol. Assess. Health Care 10 (1994) 1–6.

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Poly(lactide-co-glycolide) microparticles for the development of single-dose controlled-release vaccines