Search

ABSTRACTMeasles (Me) and rubella (Ru) viral diseases are targeted for elimination by ensuring a high level of vaccination coverage worldwide. Less costly, more convenient MeRu vaccine delivery systems should improve global vaccine coverage, especially in low – and middle – income countries (LMICs). In this work, we examine formulating a live, attenuated Me and Ru combination viral vaccine with Nanopatch™, a solid polymer micro-projection array for intradermal delivery. First, high throughput, qPCR-based viral infectivity and genome assays were established to enable formulation development to stabilize Me and Ru in a scaled-down, custom-built evaporative drying system to mimic the Nanopatch™ vaccine coating process. Second, excipient screening and optimization studies identified virus stabilizers for use during the drying process and upon storage in the dried state. Finally, a series of real-time and accelerated stability studies identified eight candidate formulations that met a target thermal stability criterion for live vaccines (<1 log10loss after 1 week storage at 37°C). Compared to −80°C control samples, the top candidate formulations resulted in minimal viral infectivity titer losses after storage at 2–8°C for 6 months (i.e., <0.1 log10for Me, and ~0.4 log10for Ru). After storage at 25°C over 6 months, ~0.3–0.5 and ~1.0–1.4 log10titer losses were observed for Me and Ru, respectively, enabling the rank-ordering of the stability of candidate formulations. These results are discussed in the context of future formulation challenges for developing microneedle-based dosage forms containing stabilized live, attenuated viral vaccines for use in LMICs.

ABSTRACTFormulation development was performed with the live, attenuated, human neonatal rotavirus vaccine candidate (RV3-BB) with three main objectives to facilitate use in low- and middle- income countries including (1) a liquid, 2–8°C stable vaccine, (2) no necessity for pre-neutralization of gastric acid prior to oral administration of a small-volume dose, and (3) a low-cost vaccine dosage form. Implementation of a high-throughput RT-qPCR viral infectivity assay for RV3-BB, which correlated well with traditional FFA assays in terms of monitoring RV3-BB stability profiles, enabled more rapid and comprehensive formulation development studies. A wide variety of different classes and types of pharmaceutical excipients were screened for their ability to stabilize RV3-BB during exposure to elevated temperatures, freeze-thaw and agitation stresses. Sucrose (50–60% w/v), PEG-3350, and a solution pH of 7.8 were selected as promising stabilizers. Using a combination of an in vitrogastric digestion model (to mimic oral delivery conditions) and accelerated storage stability studies, several buffering agents (e.g., succinate, adipate and acetate at ~200 to 400 mM) were shown to protect RV3-BB under acidic conditions, and at the same time, minimize virus destabilization during storage. Several optimized RV3-BB candidate formulations were identified based on negligible viral infectivity losses during storage at 2–8°C and −20°C for up to 12 months, as well as by relative stability comparisons at 15°C and 25°C (up to 12 and 3 months, respectively). These RV3-BB stability results are discussed in the context of stability profiles of other rotavirus serotypes as well as future RV3-BB formulation development activities.

Protein therapeutics, monoclonal antibodies (mAbs) in particular, are large, structurally complex molecules that are prone to numerous modes of degradation during their production and long-term storage. Physical degradation via protein aggregation is a major concern when developing protein therapeutic candidates for clinical use. A dimer is perhaps the simplest element of protein aggregation, and thus, a better understanding of protein dimers in terms of their structures, intermolecular interactions, and chemical nature will help in the development of rational strategies for reducing aggregation propensity. In this study, two different mAb dimers were generated from an IgG2 monoclonal antibody solution, i.e., a native dimer generated under long-term storage and a thermal dimer from a thermal stress condition. Both IgG2 dimers were characterized in terms of their chemical and physical properties, bioactivity, and conformational dynamics. The native IgG2 dimer was formed mainly through noncovalent association. It displayed minimal differences in biophysical properties and higher-order structure compared to the monomer yet showed compromised in vitro potency, likely because of steric hindrance. In contrast, the thermal IgG2 dimer was mainly disulfide-linked, but even so, no new non-native disulfide bonds were detected by peptide mapping. Two regions within the Fc-CH2 domain of the thermal IgG2 dimer exhibited significantly increased flexibility as measured by hydrogen–deuterium exchange mass spectrometry, and notably, these regions are connected by an intrachain disulfide bond under natively folded conditions. These findings provide a better understanding of dimer formation under long-term storage and thermal stress conditions for this IgG2 mAb, and possible aggregation mechanisms are discussed.

ABSTRACTSecond-generation COVID-19 vaccines with improved immunogenicity (e.g., breadth, duration) and availability (e.g., lower costs, refrigerator stable) are needed to enhance global coverage. In this work, we formulated a clinical-stage SARS-CoV-2 receptor-binding domain (RBD) virus-like particle (VLP) vaccine candidate (IVX-411) with widely available adjuvants. Specifically, we assessed the in vitrostorage stability and in vivomouse immunogenicity of IVX-411 formulated with aluminum-salt adjuvants (Alhydrogel™, AH and Adjuphos™, AP), without or with the TLR-9 agonist CpG-1018™ (CpG), and compared these profiles to IVX-411 adjuvanted with an oil-in-water nano-emulsion (AddaVax™, AV). Although IVX-411 bound both AH and AP, lower binding strength of antigen to AP was observed by Langmuir binding isotherms. Interestingly, AH- and AP-adsorbed IVX-411 had similar storage stability profiles as measured by antigen-binding assays (competitive ELISAs), but the latter displayed higher pseudovirus neutralizing titers (pNT) in mice, at levels comparable to titers elicited by AV-adjuvanted IVX-411. CpG addition to alum (AP or AH) resulted in a marginal trend of improved pNTs in stressed samples only, yet did not impact the storage stability profiles of IVX-411. In contrast, previous work with AH-formulations of a monomeric RBD antigen showed greatly improved immunogenicity and decreased stability upon CpG addition to alum. At elevated temperatures (25, 37°C), IVX-411 formulated with AH or AP displayed decreased in vitrostability compared to AV-formulated IVX-411and this rank-ordering correlated with in vivoperformance (mouse pNT values). This case study highlights the importance of characterizing antigen-adjuvant interactions to develop low cost, aluminum-salt adjuvanted recombinant subunit vaccine candidates.

ABSTRACTImplementation of inactivated polio vaccines (IPV) containing Sabin strains (sIPV) will further enable global polio eradication efforts by improving vaccine safety during use and containment during manufacturing. Moreover, sIPV-containing vaccines will lower costs and expand production capacity to facilitate more widespread use in low- and middle-income countries (LMICs). This review focuses on the role of vaccine formulation in these efforts including traditional Salk IPV vaccines and new sIPV-containing dosage forms. The physicochemical properties and stability profiles of poliovirus antigens are described. Formulation approaches to lower costs include developing multidose and combination vaccine formats as well as improving storage stability. Formulation strategies for dose-sparing and enhanced mucosal immunity include employing adjuvants (e.g. aluminum-salt and newer adjuvants) and/or novel delivery systems (e.g. ID administration with microneedle patches). The potential for applying these low-cost formulation development strategies to other vaccines to further improve vaccine access and coverage in LMICs is also discussed.

ABSTRACTConcentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in CH3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.

The metal-catalyzed oxidation by [FeII(EDTA)]2–/H2O2of IgG-1 leads to the site-specific hydrolysis of peptide bonds in the Fc region. The major hydrolytic cleavage occurs between Met428 and His429, consistent with a mechanism reported for the site-specific hydrolysis of parathyroid hormone (1–34) between Met8 and His9 (Mozziconacci, O.; et al. Mol. Pharmaceutics2013, 10(2), 739–755). In IgG-1, to a lesser extent, we also observe hydrolysis reactions between Met252 and Ile253. After 2 h of oxidation (at pH 5.8, 37 °C) approximately 5% of the protein is cleaved between Met428 and His429. For comparison, after 2 h of oxidation, the amount of tryptic peptides containing a Met sulfoxide residue represents less than 0.1% of the protein. The effect of this site-specific hydrolysis on the conformational stability and aggregation propensity of the antibody was also examined. No noticeable differences in structural integrity and conformational stability were observed between control and oxidized IgG-1 samples as measured by circular dichroism (CD), fluorescence spectroscopy, and static light scattering (SLS). Small amounts of soluble and insoluble aggregates (3–6%) were, however, observed in the oxidized samples by UV–visible absorbance spectroscopy and size exclusion chromatography (SEC). Over the course of metal-catalyzed oxidation, increasing amounts of fragments were also observed by SEC. An increase in the concentration of subvisible particles was detected by microflow imaging (MFI).

ABSTRACTLow-cost, refrigerator-stable COVID-19 vaccines will facilitate global access and improve vaccine coverage in low- and middle-income countries. To this end, subunit-based approaches targeting the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein remain attractive. Antibodies against RBD neutralize SARS-CoV-2 by blocking viral attachment to the host cell receptor, ACE2. Here, a yeast-produced recombinant RBD antigen (RBD-L452K-F490W or RBD-J) was formulated with various combinations of aluminum-salt (Alhydrogel®, AH; AdjuPhos®, AP) and CpG 1018 adjuvants. We assessed the effect of antigen-adjuvant interactions on the stability and mouse immunogenicity of various RBD-J preparations. While RBD-J was 50% adsorbed to AH and <15% to AP, addition of CpG resulted in complete AH binding, yet no improvement in AP adsorption. ACE2 competition ELISA analyses of formulated RBD-J stored at varying temperatures (4, 25, 37°C) revealed that RBD-J was destabilized by AH, an effect exacerbated by CpG. DSC studies demonstrated that aluminum-salt and CpG adjuvants decrease the conformational stability of RBD-J and suggest a direct CpG-RBD-J interaction. Although AH+CpG-adjuvanted RBD-J was the least stable in vitro, the formulation was most potent at eliciting SARS-CoV-2 pseudovirus neutralizing antibodies in mice. In contrast, RBD-J formulated with AP+CpG showed minimal antigen-adjuvant interactions, a better stability profile, but suboptimal immune responses. Interestingly, the loss of in vivopotency associated with heat-stressed RBD-J formulated with AH+CpG after one dose was abrogated by a booster. Our findings highlight the importance of elucidating the key interrelationships between antigen-adjuvant interactions, storage stability, and in vivoperformance to enable successful formulation development of stable and efficacious subunit vaccines.

There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C. A novel HX-MS experimental approach was then applied to directly monitor differences in local flexibility of mAb-C due to RSA at different protein concentrations in deuterated buffers. First, a stable formulation containing lyoprotectants that permitted freeze-drying of mAb-C at both 5 and 60 mg/mL was identified. Upon reconstitution with RSA-promoting deuterated solutions, the low vs. high protein concentration samples displayed different levels of solution viscosity (i.e., approx. 1 to 75 mPa.s). The reconstituted mAb-C samples were then analyzed by HX-MS. Two specific sequences covering complementarity-determining regions CDR2H and CDR2L (in the variable heavy and light chains, respectively) showed significant protection against deuterium uptake (i.e., decreased hydrogen exchange). These results define the major protein-protein interfaces associated with the concentration-dependent RSA of mAb-C. Surprisingly, certain peptide segments in the VHdomain, the constant domain (CH2), and the hinge region (CH1-CH2 interface) concomitantly showed significant increases in local flexibility at high vs. low protein concentrations. These results indicate the presence of longer-range, distant dynamic coupling effects within mAb-C occurring upon RSA.

Dominant negative inhibitor (DNI) is a translocation-deficient homolog of recombinant protective antigen of Bacillus anthracisthat is a candidate for a next generation anthrax vaccine. This study demonstrates that the biophysical characteristics of the DNI protein stored in lyophilized form at 4 °C for 8 y were similar to recombinant protective antigen (rPA). To provide information on the accelerated stability of DNI, samples in the lyophilized form were subjected to thermal stress (40 and 70 °C for up to 4 weeks) and thoroughly evaluated using various biophysical and chemical characterization techniques. Results demonstrate preserved structural stability of the DNI protein under extreme conditions, suggesting long-term stability can be achieved for a vaccine that employs DNI, as desired for a biodefense countermeasure. Furthermore, the biological activity of the stressed DNI bound to the adjuvant Alhydrogel®was evaluated in mice and it was found that the immunogenicity DNI was not affected by thermal stress.

A candidate vaccine to prevent human schistosomiasis is under development. The vaccine is comprised of a recombinant 9 kDa antigen protein corresponding to the large extracellular domain of a tetraspanin surface antigen protein of Schistosoma mansoni, Sm-TSP-2. Here, we describe the biophysical profile of the purified, recombinant Sm-TSP-2 produced in the yeast PichiaPink™, which in preclinical studies in mice was shown to be an effective vaccine against intestinal schistosomiasis. Biophysical techniques including circular dichroism, intrinsic and extrinsic fluorescence and light scattering were employed to generate an empirical phase diagram, a color based map of the physical stability of the vaccine antigen over a wide range of temperatures and pH. From these studies a pH range of 6.0–8.0 was determined to be optimal for maintaining the stability and conformation of the protein at temperatures up to 25 °C. Sorbitol, sucrose and trehalose were selected as excipients that prevented physical degradation during storage. The studies described here provide guidance for maximizing the stability of soluble recombinant Sm-TSP-2 in preparation of its further development as a vaccine.

There is great interest in the design and development of highly thermostable and immunogenic protein subunit vaccines for biodefense. In this study, we used two orthogonal and complementary computational protein design approaches to generate a series of single-point mutants of RiVax, an attenuated recombinant ricin A chain (RTA) protein subunit vaccine antigen. As assessed by differential scanning calorimetry, the conformational stabilities of the designed mutants ranged from 4°C less stable to 4.5°C more stable than RiVax, depending on solution pH. Two more thermostable (V18P, C171L) and two less thermostable (T13V, S89T) mutants that displayed native-like secondary and tertiary structures (as determined by circular dichroism and fluorescence spectral analysis, respectively) were tested for their capacity to elicit RTA-specific antibodies and toxin-neutralizing activity. Following a prime-boost regimen, we found qualitative differences with respect to specific antibody titers and toxin neutralizing antibody levels induced by the different mutants. Upon a second boost with the more thermostable mutant C171L, a statistically significant increase in RTA-specific antibody titers was observed when compared with RiVax-immunized mice. Notably, the results indicate that single residue changes can be made to the RiVax antigen that increase its thermal stability without adversely impacting the efficacy of the vaccine.

We have evaluated the stability profiles of adenovirus type-5 (Ad5)-based vaccine formulations to identify liquid formulations that are stable during long-term storage at 4°C. By identifying the major physiochemical inactivation pathway(s) during storage, formulations of Ad5 were designed with specific pharmaceutical excipients leading to greatly enhanced stability. For example, results indicate that Ad5 is stabilized by non-ionic surfactants and cryoprotectants as well as excipients known to inhibit free-radical oxidation. A non-ionic surfactant is necessary to prevent adsorption of adenovirus to glass surfaces during storage, and a cryoprotectant is needed to prevent freeze−thaw-induced virus inactivation. In a base formulation (A105) containing sucrose as the cryoprotectant and polysorbate-80 as the non-ionic surfactant, metal-ion catalyzed free-radical oxidation is an important mechanism of Ad5 inactivation. The free-radical oxidation inhibitors ethanol and histidine, combined with the metal-ion chelator ethylenediaminetetraacetic acid (EDTA), were determined to be effective stabilizers of Ad5. Arrhenius plots of stability data are consistent with a first-order inactivation mechanism with apparent activation energies for virus inactivation of 26.5±0.9 and 28.7±0.6 kcal/mol in the absence and presence of free-radical oxidation inhibitors, respectively. Optimization of formulation pH, as well as the EDTA and ethanol concentrations, allowed for the identification of formulations that further enhanced long-term storage stability. For example, Ad5 in an optimized liquid formulation (A195) lost <0.1 logs of infectivity after 24 months of storage at 4°C. The immunogenicity of a recombinant Ad5-based human immunodeficiency virus (HIV) vaccine candidate expressing HIV-1 gag (MRKAd5gag) formulated in A195, was shown to be equivalent to the same vaccine formulated in A105. Therefore, the use of EDTA, ethanol, and histidine did not significantly alter the immunogenicity of the vaccine in mice. The identification of 4°C stable liquid formulations should significantly enhance the utility of Ad5 as a vector for vaccines and gene therapy.

We have evaluated the stability profiles of adenovirus type‐5 (Ad5)‐based vaccine formulations to identify liquid formulations that are stable during long‐term storage at 4°C. By identifying the major physiochemical inactivation pathway(s) during storage, formulations of Ad5 were designed with specific pharmaceutical excipients leading to greatly enhanced stability. For example, results indicate that Ad5 is stabilized by non‐ionic surfactants and cryoprotectants as well as excipients known to inhibit free‐radical oxidation. A non‐ionic surfactant is necessary to prevent adsorption of adenovirus to glass surfaces during storage, and a cryoprotectant is needed to prevent freeze–thaw‐induced virus inactivation. In a base formulation (A105) containing sucrose as the cryoprotectant and polysorbate‐80 as the non‐ionic surfactant, metal‐ion catalyzed free‐radical oxidation is an important mechanism of Ad5 inactivation. The free‐radical oxidation inhibitors ethanol and histidine, combined with the metal‐ion chelator ethylenediaminetetraacetic acid (EDTA), were determined to be effective stabilizers of Ad5. Arrhenius plots of stability data are consistent with a first‐order inactivation mechanism with apparent activation energies for virus inactivation of 26.5 ± 0.9 and 28.7 ± 0.6 kcal/mol in the absence and presence of free‐radical oxidation inhibitors, respectively. Optimization of formulation pH, as well as the EDTA and ethanol concentrations, allowed for the identification of formulations that further enhanced long‐term storage stability. For example, Ad5 in an optimized liquid formulation (A195) lost <0.1 logs of infectivity after 24 months of storage at 4°C. The immunogenicity of a recombinant Ad5‐based human immunodeficiency virus (HIV) vaccine candidate expressing HIV‐1 gag (MRKAd5gag) formulated in A195, was shown to be equivalent to the same vaccine formulated in A105. Therefore, the use of EDTA, ethanol, and histidine did not significantly alter the immunogenicity of the vaccine in mice. The identification of 4°C stable liquid formulations should significantly enhance the utility of Ad5 as a vector for vaccines and gene therapy. © 2004 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:2458–2475, 2004

We describe the physiochemical characterization and immunological evaluation of plasmid DNA vaccine formulations containing a nonionic triblock copolymer adjuvant (CRL1005) in the presence and absence of a cationic surfactant, benzalkonium chloride (BAK). CRL1005 forms particles of 1–10 microns upon warming above its phase‐transition temperature (∼6–8°C) and the physical properties of the particles are altered by BAK. DNA/CRL1005 vaccines formulated with and without BAK were evaluated in rhesus macaques to determine the effect of CRL1005 and BAK on the ability of plasmid DNA to induce a cellular immune response. Immunogenicity results indicate that the addition of CRL1005 to human immunodeficiency virus‐1 gagplasmid DNA formulated in phosphate‐buffered saline leads to an enhancement in the gag‐specific cellular immune response. Moreover, the addition of BAK to human immunodeficiency virus‐1 gagplasmid DNA/CRL1005 formulations produces an additional enhancement in gag‐specific cellular immunity. In vitrocharacterization studies of DNA/CRL1005 formulations indicate no detectable binding of DNA to CRL1005 particles in the absence of BAK, suggesting that the enhancement of cellular immunity induced by DNA/CRL1005 formulations is not due to enhanced DNA delivery. In the presence of BAK, however, results indicate that BAK binds to CRL1005 particles, producing cationic microparticles that bind DNA through electrostatic interactions. If BAK is present at the phase‐transition temperature, it reduces the particle size from ∼2 microns to ∼300 nm, presumably by binding to hydrophobic surfaces during particle formation. Zeta potential measurements indicate that the surface charge of CRL1005‐BAK particles changes from positive to negative upon DNA binding, and DNA bound to the surface of CRL1005‐BAK particles was visualized by fluorescence microscopy. These results indicate that the addition of BAK to DNA/CRL1005 formulations leads to the formation of ∼300 nm CRL1005‐BAK‐DNA particles that enhance the cellular immune response in rhesus monkeys. © 2004 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1924–1939, 2004

We describe the physiochemical characterization and immunological evaluation of plasmid DNA vaccine formulations containing a nonionic triblock copolymer adjuvant (CRL1005) in the presence and absence of a cationic surfactant, benzalkonium chloride (BAK). CRL1005 forms particles of 1–10 microns upon warming above its phase‐transition temperature (∼6–8°C) and the physical properties of the particles are altered by BAK. DNA/CRL1005 vaccines formulated with and without BAK were evaluated in rhesus macaques to determine the effect of CRL1005 and BAK on the ability of plasmid DNA to induce a cellular immune response. Immunogenicity results indicate that the addition of CRL1005 to human immunodeficiency virus‐1 gag plasmid DNA formulated in phosphate‐buffered saline leads to an enhancement in the gag‐specific cellular immune response. Moreover, the addition of BAK to human immunodeficiency virus‐1 gag plasmid DNA/CRL1005 formulations produces an additional enhancement in gag‐specific cellular immunity. In vitro characterization studies of DNA/CRL1005 formulations indicate no detectable binding of DNA to CRL1005 particles in the absence of BAK, suggesting that the enhancement of cellular immunity induced by DNA/CRL1005 formulations is not due to enhanced DNA delivery. In the presence of BAK, however, results indicate that BAK binds to CRL1005 particles, producing cationic microparticles that bind DNA through electrostatic interactions. If BAK is present at the phase‐transition temperature, it reduces the particle size from ∼2 microns to ∼300 nm, presumably by binding to hydrophobic surfaces during particle formation. Zeta potential measurements indicate that the surface charge of CRL1005‐BAK particles changes from positive to negative upon DNA binding, and DNA bound to the surface of CRL1005‐BAK particles was visualized by fluorescence microscopy. These results indicate that the addition of BAK to DNA/CRL1005 formulations leads to the formation of ∼300 nm CRL1005‐BAK‐DNA particles that enhance the cellular immune response in rhesus monkeys. © 2004 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1924–1939, 2004

A refrigerator-stable rotavirus (RV) vaccine that withstands gastric acid is anticipated to permit more widespread use of RV vaccine.

A single-shot Hepatitis B vaccine formulation using poly(d, l)-lactide-co-glycolide acid (PLGA) microspheres as a delivery system was examined using a variety of biophysical and biochemical techniques as well as immunological evaluation in C3H mice. PLGA microsphere encapsulation of the Hepatitis B surface antigen (HBsAg), a lipoprotein particle, resulted in good recoveries of protein mass, protein particle conformational integrity, and in vitroantigenicity. Some partial delipidation of the HBsAg, however, was observed. The loading and encapsulation efficiency of HBsAg into the PLGA microspheres were measured along with the morphology and size distribution of the vaccine-loaded PLGA microspheres. The in vitrorelease kinetics of HBsAg from the PLGA microspheres was evaluated and found to be affected by experimental conditions such as stirring rate. HBsAg showed enhanced storage stability at 37°C in the slightly acidic pH range reported to be found inside PLGA microspheres; thus, the antigen is relatively stable under conditions of temperature and pH that may mimic in vivoconditions. The immunogenicity of the microsphere formulations of HBsAg was compared with conventional aluminum adjuvant formulated HBsAg vaccine in C3H mice. Comparisons were made between aluminum formulations (one and two injections), PLGA microsphere formulations (single injection), and a mixture of aluminum and PLGA microsphere formulations (single injection). The nine-month serum antibody titers indicate that a single injection of a mixture of aluminum and PLGA-formulated HBsAg results in equal or better immune responses than two injections of aluminum-formulated HBsAg vaccine. Based on these in vitroand in vivostudies, it is concluded that HBsAg can be successfully encapsulated and recovered from the PLGA microspheres and a mixture of aluminum-adjuvanted and PLGA-formulated HBsAg can auto-boost an immune response in manner comparable to multiple injections of an aluminum-formulated vaccine.

A single-shot Hepatitis B vaccine formulation using poly(d,l)-lactide-co-glycolide acid (PLGA) microspheres as a delivery system was examined using a variety of biophysical and biochemical techniques as well as immunological evaluation in C3H mice. PLGA microsphere encapsulation of the Hepatitis B surface antigen (HBsAg), a lipoprotein particle, resulted in good recoveries of protein mass, protein particle conformational integrity, and in vitro antigenicity. Some partial delipidation of the HBsAg, however, was observed. The loading and encapsulation efficiency of HBsAg into the PLGA microspheres were measured along with the morphology and size distribution of the vaccine-loaded PLGA microspheres. The in vitro release kinetics of HBsAg from the PLGA microspheres was evaluated and found to be affected by experimental conditions such as stirring rate. HBsAg showed enhanced storage stability at 37°C in the slightly acidic pH range reported to be found inside PLGA microspheres; thus, the antigen is relatively stable under conditions of temperature and pH that may mimic in vivo conditions. The immunogenicity of the microsphere formulations of HBsAg was compared with conventional aluminum adjuvant formulated HBsAg vaccine in C3H mice. Comparisons were made between aluminum formulations (one and two injections), PLGA microsphere formulations (single injection), and a mixture of aluminum and PLGA microsphere formulations (single injection). The nine-month serum antibody titers indicate that a single injection of a mixture of aluminum and PLGA-formulated HBsAg results in equal or better immune responses than two injections of aluminum-formulated HBsAg vaccine. Based on these in vitro and in vivo studies, it is concluded that HBsAg can be successfully encapsulated and recovered from the PLGA microspheres and a mixture of aluminum-adjuvanted and PLGA-formulated HBsAg can auto-boost an immune response in manner comparable to multiple injections of an aluminum-formulated vaccine. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1019–1035, 2002

The stability of highly purified supercoiled plasmid DNA formulated in simple phosphate or Tris-buffered saline solutions has been characterized to establish the overall degradation processes that occur during storage in aqueous solution. Plasmid DNA stability was monitored during accelerated stability studies (at 50°C) by measurements of supercoiled, open-circle, and linear DNA content, as well as the accumulation of apurinic sites and 8-hydroxydeoxyguanosine residues over time. The effects of formulation pH, demetalation, metal ion chelators, and ethanol (hydroxyl radical scavenger) on the supercoiled content of plasmid DNA during storage at 50°C were also determined. The results indicate that free radical oxidation may be a major degradative process for plasmid DNA in pharmaceutical formulations unless specific measures are taken to control it by the addition of free radical scavengers, specific metal ion chelators, or both. The generation of hydroxyl radicals in phosphate-buffered saline was confirmed by examining the hydroxylation of phenylalanine over time by reverse phase high-performance liquid chromatography. Ethanol was found to enhance plasmid DNA stability and to inhibit the hydroxylation of phenylalanine; both observations are consistent with the known ability of ethanol to serve as a hydroxyl radical scavenger. Moreover, the combination of ethylenediamine tetraacetic acid (EDTA) and ethanol had a synergistic enhancing effect on DNA stability. However, the metal ion chelator diethylenetriaminepentaacetic acid (DTPA) was as potent as the combination of EDTA and ethanol for enhancing the stability of plasmid DNA. By controlling free radical oxidation with EDTA and ethanol, the rate constants of plasmid DNA degradation by means of depurination and β-elimination were then determined, allowing accurate predictions of DNA storage stability as a function of formulation pH and temperature. The ability to predict plasmid DNA storage stability in the absence of free radical oxidation should prove to be a valuable tool for the design of stable pharmaceutical formulations of plasmid DNA. © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 76–87, 2000

The stability of highly purified supercoiled plasmid DNA formulated in simple phosphate or Tris‐buffered saline solutions has been characterized to establish the overall degradation processes that occur during storage in aqueous solution. Plasmid DNA stability was monitored during accelerated stability studies (at 50°C) by measurements of supercoiled, open‐circle, and linear DNA content, as well as the accumulation of apurinic sites and 8‐hydroxydeoxyguanosine residues over time. The effects of formulation pH, demetalation, metal ion chelators, and ethanol (hydroxyl radical scavenger) on the supercoiled content of plasmid DNA during storage at 50°C were also determined. The results indicate that free radical oxidation may be a major degradative process for plasmid DNA in pharmaceutical formulations unless specific measures are taken to control it by the addition of free radical scavengers, specific metal ion chelators, or both. The generation of hydroxyl radicals in phosphate‐buffered saline was confirmed by examining the hydroxylation of phenylalanine over time by reverse phase high‐performance liquid chromatography. Ethanol was found to enhance plasmid DNA stability and to inhibit the hydroxylation of phenylalanine; both observations are consistent with the known ability of ethanol to serve as a hydroxyl radical scavenger. Moreover, the combination of ethylenediamine tetraacetic acid (EDTA) and ethanol had a synergistic enhancing effect on DNA stability. However, the metal ion chelator diethylenetriaminepentaacetic acid (DTPA) was as potent as the combination of EDTA and ethanol for enhancing the stability of plasmid DNA. By controlling free radical oxidation with EDTA and ethanol, the rate constants of plasmid DNA degradation by means of depurination and β‐elimination were then determined, allowing accurate predictions of DNA storage stability as a function of formulation pH and temperature. The ability to predict plasmid DNA storage stability in the absence of free radical oxidation should prove to be a valuable tool for the design of stable pharmaceutical formulations of plasmid DNA. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 76–87, 2000

The design of an aqueous formulation for acidic fibroblast growth factor (aFGF) requires an understanding of the type of compounds that can either directly or indirectly stabilize the protein. To this end, spectrophotometric turbidity measurements were initially employed to screen the ability of polyanionic ligands, less specific compounds, and variations in solution conditions (temperature and pH) to stabilize aFGF against heat-induced aggregation. It was found that in addition to the well-known protection of aFGF by heparin, a surprisingly wide variety of polyanions (including small sulfated and phosphorylated compounds) also stabilizes aFGF. These polyanionic ligands are capable of raising the temperature at which the protein unfolds by 15–30°C. Many commonly used excipients were also observed to stabilize aFGF in both the presence and the absence of heparin. High concentrations of some of these less specific agents are also able to increase the temperature of aFGF thermal unfolding by as much as 6–12°C as shown by circular dichroism and differential scanning calorimetry. Other compounds were found which protect the chemically labile cysteine residues of aFGF from oxidation. Aqueous formulations of aFGF were thus designed to contain both a polyanionic ligand that enhances structural integrity by binding to the protein and chelating agents (e.g., EDTA) to prevent metal ion-catalyzed oxidation of cysteine residues. While room-temperature storage (30°C) leads to rapid inactivation of aFGF in physiological buffer alone, several of these aFGF formulations are stable in vitro for at least 3 months at 30°C. Three aFGF topical formulations were examined in an impaired diabetic mouse model and were found to be equally capable of accelerating wound healing.

A variety of biophysical techniques have been employed to examine the size and conformational integrity of highly purified hepatitis A virus (HAV) in solution (purified HAV particles are subsequently formalin-inactivated and adsorbed to aluminum salts for use as the vaccine VAQTA). The size of HAV particles was assessed by a combination of electron microscopy, sedimentation velocity, and dynamic light scattering. The effect of ionic strength and temperature on the overall conformational stability of HAV was determined by a combination of intrinsic HAV protein fluorescence, fluorescent probes of both RNA and protein, and UV–visible spectroscopy. A major structural change in HAV occurs near 60 °C with the addition of 0.2 M magnesium chloride enhancing the thermal stability of HAV by ~10 °C. Salt concentrations above 0.2 M, however, decrease the solubility of HAV. The effect of pH on the physical properties of HAV particles was monitored by dynamic light scattering, analytical size exclusion HPLC, and interaction with fluorescent dyes. HAV particles undergo a substantially reversible association/aggregation at pH values below 6 with the concomitant exposure of previously buried hydrophobic surfaces below pH 4. These results are in good agreement with previous studies of HAV thermal stability under extreme conditions in which the irreversible inactivation of the viral particles was measured primarily by the loss of viral infectivity. The wide variety of biophysical measurements described in this work, however, directly monitor structural changes as they occur, thus providing a molecular basis with which to monitor HAV stability during purification and storage.

The deamidation of polyanion‐stabilized acidic fibroblast growth factor (aFGF; FGF‐1) can be induced by prolonged storage under accelerated conditions of elevated pH and temperature. A urea–isoelectric focusing (urea‐IEF) method has been developed to monitor aFGF deamidation in the presence of highly negatively charged polyanions which are required to maintain the conformational stability of the protein. The kinetics of aFGF deamidation have been established by a combination of urea‐IEF and an enzymatic ammonia assay. Native, non‐deamidated aFGF (complexed with heparin) has a half‐life of 16 weeks at pH 7, 30 °C, and 4 weeks at pH 8, 40°C. The mitogenic activity and biophysical properties of deamidated aFGF were compared to the non‐deamidated protein. These initial deamidation events have no significant effect on the protein's overall conformation, thermal stability, interaction with heparin, or bioactivity. At longer times, however, limited aggregation of the protein was observed after prolonged storage under some conditions. N‐terminal protein sequencing of the protein's first 21 amino acid residues have identified one of the deamidation sites in a flexible, peptide‐like region of the protein (Asn8‐Tyr9).

It has been demonstrated that the amino acids Asp537, Asp812, Lys631, His811 and Tyr639 are involved in bacteriophage T7 RNA polymerase catalysis. In the present paper, we report kinetic, spectroscopic and calorimetric characterization of the wild-type and mutant T7 RNA polymerases generated at these five loci (D537N, E; K631M, R; Y639F, S, A, W; H811Q, A; D812N, E). The wild-type enzyme has a substantial amount of secondary structure as determined by CD analysis (α-helix, 43%; β-sheet, 14%; β-turn, 25%; unordered, 18%). The CD spectra of 12 mutants at five loci are very similar to that of the wild-type, except for the mutant Y639W. Within experimental error, the thermal transition temperatures measured by CD and DSC as well as the λ_max values of the fluorescence spectra were the same for the wild-type and all of the mutants. Therefore, the overall folding and stability of the mutant enzymes are very similar to those of the wild-type enzyme, although small local conformational changes cannot be excluded. For the synthesis of the pentamer pppGGACU, the mutants D537E and D812E showed an approximately two-to threefold decrease in (k_cat)_app and an approximately two- to threefold increase in (K_m)_app, relative to the wild-type, in contrast to the mutants D537N and D812N which exhibited no detectable activity. The mutant K631R showed a sevenfold reduction in (k_cat)_app and a two- to threefold increase in (K_m)_app supporting our earlier observation with the mutant K631M that Lys631 may be involved in phosphodiester bond formation. The mutant Y639S can systhesize the trimer GGA with an approximately 50-fold decrease in (k_cat)_app and a tenfold increase in (K_m)_app relative to the wild-type, underlining the importance of the phenyl ring of Tyr639. The mutant H811A, in which the side-chain at position 811 is incapable of forming a hydrogen bond, can synthesize the trimer GGA with an approximately tenfold decrease in (k_cat)_app and an approximately 35-fold increase in (K_m)_app. Thus, either the hydrogen-bonding capacity of this residue is non-essential or some other group can functionally substitute for the His811 side-chain. The wild-type enzyme showed significant effects of the base position in the sequence on the apparent binding constants for the NTPs. The kinetics of GpG-primed trimer, tetramer and pentamer synthesis on three 22 bp templates were investigated for the wild-type and mutant enzymes with measurable activity. The rate of oligonucleotide synthesis depends strongly on the message sequence, and abortive termination occurs more frequently following UMP incorporation. For a given template, the rate of synthesis by the wild-type and all the mutants is trimer>pentamer (run-off transcript)>tetramer. The effect of the 5'-triphosphate in the initiating nucleotide is minimal for the wild-type and mutant enzymes. The apparent pK values obtained from the high-pH side of the pH activity profiles of the wild type and the mutants K631R and Y639F are the same within experimental error, implying that an amino acid(s) other than Lys631 or Tyr639 is responsible for the high-pH side of the pH-activity profile. Copyright 1994, 1999 Academic Press

The origin of the microheterogeneity of a highly purified antiinflammatory humanized monoclonal antibody prepared in mammalian cell culture has been investigated. This antibody is an IgG directed toward human CD 18 (a subunit of leukocyte integrins). When the IgG preparation is subjected to isoelectric focusing, it is found to contain four major species with pI values ranging from 6 to 7. Although the relative amounts of each form differ and some species are present only in small quantities, each has been isolated by a combination of high-resolution anion-exchange chromatography and isoelectric focusing. Comparative studies reveal no detectable differences in overall secondary (far UV circular dichroism) or tertiary (intrinsic fluorescence) structure, molecular weight (laser-desorption mass spectroscopy), or antigen binding activity. When each of the isolated species is incubated under conditions which favor deamidation, it is converted to forms of lower pI which appear to correspond to naturally observed species. While the isolated light chain is relatively homogeneous, the heavy chain exhibits a pattern of isoelectric focusing bands similar to that of the intact immunoglobulin. These results suggest that in this case, charge microheterogeneity is due to the sequential deamidation of the immunoglobulin heavy chain.

A variety of biophysical techniques have been employed to examine the size and conformational integrity of highly purified hepatitis A virus (HAV) in solution (purified HAV particles are subsequently formalin-inactivated and adsorbed to aluminum salts for use as the vaccine VAQTA). The size of HAV particles was assessed by a combination of electron microscopy, sedimentation velocity, and dynamic light scattering. The effect of ionic strength and temperature on the overall conformational stability of HAV was determined by a combination of intrinsic HAV protein fluorescence, fluorescent probes of both RNA and protein, and UV–visible spectroscopy. A major structural change in HAV occurs near 60°C with the addition of 0.2M magnesium chloride enhancing the thermal stability of HAV by ∼10°C. Salt concentrations above 0.2 M, however, decrease the solubility of HAV. The effect of pH on the physical properties of HAV particles was monitored by dynamic light scattering, analytical size exclusion HPLC, and interaction with fluorescent dyes. HAV particles undergo a substantially reversible association/aggregation at pH values below 6 with the concomitant exposure of previously buried hydrophobic surfaces below pH 4. These results are in good agreement with previous studies of HAV thermal stability under extreme conditions in which the irreversible inactivation of the viral particles was measured primarily by the loss of viral infectivity. The wide variety of biophysical measurements described in this work, however, directly monitor structural changes as they occur, thus providing a molecular basis with which to monitor HAV stability during purification and storage.

ABSTRACTPassive immunization with antibodies is a promising approach against enterotoxigenic Escherichia colidiarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamianaplants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68–81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitromodel. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivochallenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68–61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitrofunctional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.