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The importance of the strength of antigen adsorption by aluminum-containing adjuvants on immunopotentiation was studied using HIV 1 SF162dV2gp140 (gp140), a potential HIV/AIDS antigen. The strengths of adsorption by aluminum hydroxide (AH) adjuvant and aluminum phosphate adjuvant, as measured by the Langmuir adsorptive coefficient, were 1900 and 400 mL/mg, respectively. The strength of adsorption by AH was modified by pretreatment of AH with two different concentrations of potassium dihydrogen phosphate to produce phosphate-treated aluminum hydroxide adjuvants having adsorptive coefficients of 1200 and 800 mL/mg. The four adjuvants were used to prepare vaccines containing either 1 or 10 μg of gp140 per dose. Antibody studies in mice revealed that the presence of an adjuvant increased the immune response in comparison with a solution of gp140 when the dose was 1 μg. Furthermore, the immune response was inversely related to the adsorptive coefficient. In contrast, no significant difference in immunopotentiation was observed between treatments in the presence or absence of an adjuvant when the dose of gp140 was 10 μg. Analysis of the binding of gp140 to CD4 and anti-gp140 monoclonal antibodies by surface plasmon resonance suggests that tight binding induced structural changes in the antigen.

Adsorption of the antigen to an aluminum-containing adjuvant is considered an important aspect of vaccine formulation. Adsorption is described by two parameters: the maximum amount that can be adsorbed as a monolayer, which is characterized by the adsorptive capacity and the strength of the adsorption force, which is described by the adsorptive coefficient. Research to date has focused on the adsorptive capacity with the goal of complete adsorption of the antigen. In this study, the relationship between the adsorptive coefficient and immunopotentiation was investigated. Four vaccines were prepared in which the adsorptive coefficient was varied by altering the number of phosphate groups on the antigen (alpha casein and dephosphorylated alpha casein) or the number of surface hydroxyls on the adjuvant (aluminum hydroxide adjuvant and phosphate-treated aluminum hydroxide adjuvant). In vitro elution upon exposure to interstitial fluid or normal human plasma was inversely related to the adsorptive coefficient. The geometric mean antibody titer in mice was also inversely related to the adsorptive coefficient. T-cell activation was not observed in mice that received the vaccine with the greatest adsorptive coefficient (alpha casein/aluminum hydroxide adjuvant). This suggests that antigen processing and presentation to T-cells is impaired when the antigen is adsorbed too strongly.

Aluminum-containing adjuvants are widely used in licensed human and veterinary vaccines. However, the mechanism by which these adjuvants enhance the immune response and predominantly stimulate a T(H)2 humoral immune response is not well understood. In this study, the effects of aluminum hydroxide and aluminum phosphate adjuvants on antigen presentation, expression of costimulatory molecules and cytokines by mouse dendritic cells (DCs) and the ability of DCs to induce T helper cell differentiation were investigated. Dendritic cells pulsed with ovalbumin (OVA) adsorbed to aluminum-containing adjuvants activated antigen-specific T cells more effectively than DCs pulsed with OVA alone. Aluminum hydroxide adjuvant had a significantly stronger effect than aluminum phosphate adjuvant. Both aluminum-containing adjuvants significantly increased the expression of CD86 on DCs but only aluminum hydroxide adjuvant also induced moderate expression of CD80. Aluminum-containing adjuvants stimulated the release of IL-1beta and IL-18 from DCs via caspase-1 activation. DCs incubated with LPS and OVA induced T(H)1 differentiation of naïve CD4(+) T cells. In contrast, DCs incubated with aluminum/OVA activated CD4(+) T cells to secrete IL-4 and IL-5 as well as IFN-gamma. Addition of neutralizing anti-IL-1beta antibodies decreased IL-5 production and addition of anti-IL-18 antibodies decreased both IL-4 and IL-5 production. Inhibition of IL-1beta and IL-18 secretion by DCs via inhibition of caspase-1 also led to a marked decrease of IL-4 and IL-5 by CD4(+) T cells. These results indicate that aluminum-containing adjuvants activate DCs and influence their ability to direct T(H)1 and T(H)2 responses through the secretion of IL-1beta and IL-18.

The degree of antigen adsorption by aluminum-containing adjuvants is considered an important characteristic of vaccines that is related to immunopotentiation by the adjuvant. This study examined immunopotentiation by aluminum phosphate adjuvant in three model vaccines in which the antigen was not adsorbed in the vaccine formulation nor when mixed in vitro with interstitial fluid. In the first model vaccine, aluminum phosphate adjuvant was pre-treated with 0.5 M KH2PO4 to minimize the adsorption of dephosphorylated alpha casein. The second model vaccine was composed of aluminum phosphate adjuvant and ovalbumin that was dephosphorylated by treatment with potato acid phosphatase. The third model vaccine consisted of aluminum phosphate adjuvant and lysozyme (LYS). In order to prevent adsorption of lysozyme, the aluminum phosphate adjuvant was pre-treated with fibrinogen, a protein present in interstitial fluid that binds strongly to aluminum phosphate adjuvant. Immunopotentiation was evaluated by measuring antibody production in mice. It was found that all three model vaccines induced antibody titers that were statistically higher than induced by a solution of antigen without adjuvant and similar to vaccines in which the antigens were adsorbed by aluminum phosphate adjuvant. Confocal microscopy experiments suggested that the antigens used in these experiments, even though not adsorbed to the aluminum phosphate adjuvant, were trapped in void spaces within the adjuvant aggregates, resulting in uptake of antigen by dendritic cells.

The phosphate content of commercial ovalbumin was increased from 1.8 to 3.2 mol PO(4)/mol ovalbumin by conjugation of phosphoserine and reduced to 1.2 or 0.14 mol PO(4)/mol ovalbumin by treatment with potato acid phosphatase. The four ovalbumin samples were completely adsorbed by aluminum hydroxide adjuvant due to electrostatic attraction of the negatively charged ovalbumin and the positively charged aluminum hydroxide adjuvant as well as by ligand exchange of phosphate groups with surface hydroxyl groups. Elution from aluminum hydroxide adjuvant upon exposure to interstitial fluid was inversely related to the degree of phosphorylation of the ovalbumin. The ovalbumin sample containing 3.2 mol PO(4)/mol ovalbumin did not elute while the ovalbumin sample containing 0.14 mol PO(4)/mol ovalbumin eluted completely from aluminum hydroxide adjuvant during exposure to interstitial fluid for 30 min. Adsorption of the four ovalbumin samples by aluminum phosphate adjuvant was directly related to the degree of phosphorylation of ovalbumin. Adsorption was due to ligand exchange as an electrostatic repulsive force operated between the negatively charged ovalbumin samples and the negatively charged aluminum phosphate adjuvant. The potential for ligand exchange decreased as the phosphorylation of ovalbumin decreased. Elution upon exposure to interstitial fluid was inversely related to the degree of phosphorylation and was more extensive than observed for aluminum hydroxide adjuvant. Adsorption of ovalbumin by aluminum-containing adjuvants and elution upon exposure to interstitial fluid can be controlled by the degree of phosphorylation of both ovalbumin and the aluminum-containing adjuvant.

An important step in the induction of an immune response to vaccines is the internalization of antigens by antigen presenting cells, such as dendritic cells (DCs). Many current vaccines are formulated with antigens adsorbed to an aluminum-containing adjuvant. Following injection of the vaccine the antigens may either elute or stay adsorbed to the adjuvant surface. Antigens, which elute from the adjuvant surface, are internalized by dendritic cells through macropinocytosis while those that remain adsorbed are internalized with the adjuvant particle by phagocytosis. The relative efficiency of these two routes of internalization was studied. Alpha casein (AC) labeled with a green fluorescent dye was selected as the model antigen. In order to model vaccine antigens that elute from aluminum-containing adjuvants following administration, dendritic cells were incubated with a solution of fluorochrome-labeled alpha casein. To model vaccine antigens that do not elute from aluminum-containing adjuvants following administration, dendritic cells were exposed to fluorochrome-labeled alpha casein adsorbed to aluminum hydroxide adjuvant (AH). Alpha casein has eight phosphate groups and adsorbs to aluminum hydroxide adjuvant through ligand exchange. Alpha casein does not elute from aluminum hydroxide adjuvant upon exposure to cell culture media. The uptake of antigen by dendritic cells was determined at 0.5, 1, 2 and 3h by confocal microscopy and flow cytometry. Dendritic cells internalized both alpha casein in solution and alpha casein adsorbed to aluminum hydroxide adjuvant. However, the mean fluorescence intensity of dendritic cells incubated with adsorbed alpha casein was four times greater than dendritic cells incubated with alpha casein in solution. In addition, the internalization of alpha casein was enhanced when the mean aggregate diameter of the adjuvant in the cell culture media was reduced from 17 microm to 3 microm. It was concluded that antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed to the aluminum-containing adjuvant following administration and the aggregate size of the adjuvant was smaller than dendritic cells which are approximately 10 microm in diameter.

Calcium phosphate adjuvant is a commercially available vaccine adjuvant that potentiates the immune response to antigens. Although its name suggests that it is Ca 3 (PO 4 ) 2 , X-ray diffraction, FTIR spectroscopy, thermal analysis and the Ca/P molar ratio identify commercial calcium phosphate adjuvant as non-stoichiometric hydroxyapatite, Ca 10− x (HPO 4 ) x (PO 4 ) 6− x (OH) 2− x , where x varies from 0 to 2. The surface charge is pH-dependent (point of zero charge = 5.5). Consequently, commercial calcium phosphate adjuvant exhibits a negative surface charge at physiological pH and electrostatically adsorbs positively charged antigens. The presence of hydroxyls allows calcium phosphate adjuvant to adsorb phosphorylated antigens by ligand exchange with surface hydroxyls.

The rate of acid-catalyzed hydrolysis of glucose-1-phosphate (G1P) when adsorbed to aluminum hydroxide adjuvant was significantly slower than the rate of hydrolysis of a solution of G1P at the same pH. It was concluded that the positively charged aluminum hydroxide adjuvant (iep 11.4) electrostatically attracted anions including hydroxyls to form a double layer surrounding the adjuvant particles. Thus, the pH of the microenvironment surrounding the aluminum hydroxide adjuvant was higher than the bulk pH. Adsorbed G1P hydrolyzed at a rate associated with the pH of the microenvironment of the surface of the adjuvant rather than with the pH of the bulk solution. Comparison of the rate constant for the hydrolysis of adsorbed G1P to the pH-stability profile of G1P in solution revealed that adsorbed G1P hydrolyzed at a rate associated with a pH that was approximately two pH units higher than the bulk pH. The results suggest that the chemical stability of antigens that degrade by pH-dependent mechanisms can be optimized by modifying the surface charge of the aluminum-containing adjuvant to produce the pH of maximum stability in the microenvironment of the adjuvant.

The effect of the degree of adsorption after exposure to interstitial fluid on the immune response in mice to model vaccines containing ovalbumin, alpha casein or dephosphorylated alpha casein adsorbed to aluminum hydroxide adjuvant was studied. Ovalbumin and dephosphorylated alpha casein were adsorbed in the vaccine but were completely eluted when exposed to interstitial fluid for 4 h. The presence of aluminum hydroxide adjuvant in the vaccine produced immunopotentiation compared to a solution of the protein even though the protein desorbed rapidly upon subcutaneous administration. In contrast, alpha casein was completely adsorbed to aluminum hydroxide adjuvant in both the vaccine and upon exposure to interstitial fluid. Immunopotentiation by aluminum hydroxide adjuvant was also observed in this model vaccine compared to a solution of alpha casein. The results indicated that antigen presenting cells can take up desorbed antigen from interstitial fluid as well as antigen adsorbed to aluminum-containing adjuvants.

Five aluminum phosphate adjuvants having P/Al molar ratios ranging from 0.74 to 0.26 were prepared. The adjuvants were characterized by both protein adsorptive capacity and rate of acid neutralization at pH 2.25, 25 degrees C. The protein adsorptive capacity was not a useful parameter to compare the initial properties of the adjuvants, as differences in surface charge of the adjuvants required the use of different proteins. In contrast, the rate of acid neutralization allowed a comparison of the freshly precipitated adjuvants and revealed that the rate of acid neutralization was directly related to the P/Al molar ratio. The protein adsorptive capacity decreased slightly during 39 weeks of aging at room temperature. The changes in the rate of acid neutralization were much greater and indicated that a P/Al molar ratio of at least 0.5 was required to minimize the aging of the adjuvants. Thus, the rate of acid neutralization was found to be the most sensitive parameter to characterize aluminum phosphate adjuvants.

The effect of steam sterilization (dwell@121 degrees C) on the apparent viscosity of solutions of carbomer 940P, guar gum, hydroxyethylcellulose (HEC), and xanthan gum was studied. Guar gum and carbomer 940P could be steam sterilized for 30 min without any change in the apparent viscosity. Steam sterilization of HEC and xanthan gum resulted in a substantial decrease in the apparent viscosity and the desirable shear-thinning rheology. The addition of sodium chloride to either polymer prior to steam sterilization diminished the effect of sterilization on the rheology. The apparent viscosity of xanthan solutions was not significantly affected if the solution was made isotonic with sodium chloride (0.15 M). The addition of sodium chloride to xanthan gum after steam sterilization resulted in full recovery of the initial rheological properties. Carbomers, guar gum, and xanthan (in 0.15 M NaCI) are candidates for use in pharmaceutical solutions/suspensions that are subjected to steam sterilization.

Aluminum hydroxide adjuvant was pretreated with six concentrations of potassium dihydrogen phosphate to produce a series of adjuvants with various degrees of phosphate substitution for surface hydroxyl. The adsorption of three phosphorylated proteins (alpha casein, dephosphorylated alpha casein, and ovalbumin) by the phosphate-treated aluminum hydroxide adjuvants was studied. The phosphorylated proteins were adsorbed by ligand exchange of phosphate for hydroxyl even when an electrostatic repulsive force was present. However, the extent (adsorptive capacity) and strength (adsorptive coefficient) of adsorption was inversely related to the degree of phosphate substitution of the aluminum hydroxide adjuvant. Exposure of vaccines containing aluminum hydroxide adjuvant and phosphorylated antigens to phosphate ion in the formulation or during manufacture should be minimized to produce maximum adsorption of the antigen.

The traditional method of determining surface area, nitrogen gas sorption, requires complete drying of the sample prior to analysis. This technique is not suitable for aluminum hydroxide adjuvant because it is composed of submicron, fibrous particles that agglomerate irreversibly upon complete removal of water. In this study, the surface area of a commercial aluminum hydroxide adjuvant was determined by a gravimetric/FTIR method that measures the water adsorption capacity. This technique does not require complete drying of the adjuvant. Five replicate determinations gave a mean surface area of 514 m(2)/g and a 95% confidence interval of 36 m(2)/g for a commercial aluminum hydroxide adjuvant. The X-ray diffraction pattern and the Scherrer equation were used to calculate the dimensions of the primary crystallites. The average calculated dimensions were 4.5 x 2.2 x 10 nm. Based on these dimensions, the mean calculated surface area of the commercial aluminum hydroxide adjuvant was 509 m(2)/g, and the 95% confidential interval was 30 m(2)/g. The close agreement between the two surface area values indicates that either method may be used to determine the surface area of aluminum hydroxide adjuvant. The high surface area, which was determined by two methods, is an important property of aluminum hydroxide adjuvants, and is the basis for the intrinsically high protein adsorption capacity.

Six aluminum hydroxide adjuvants, poorly crystalline aluminum oxyhydroxide (AlOOH) were prepared using different thermal treatments of amorphous aluminum hydroxide (Al(OH)3) in an effort to increase the protein adsorption capacity. All of the adjuvants initially exhibited a higher protein adsorption capacity. However, the protein adsorption capacity decreased during aging at room temperature. X-ray and differential centrifugal sedimentation analysis revealed that complete dehydration of amorphous aluminum hydroxide to aluminum oxyhydroxide is required to produce a stable adjuvant. Any residual amorphous aluminum hydroxide will spontaneously transform to crystalline aluminum hydroxide during aging at room temperature. Since crystalline aluminum hydroxide has a small surface area, the protein adsorption capacity of adjuvants containing amorphous aluminum hydroxide decreased by 30-40% when stored for 6 months at room temperature.

The ability of interstitial fluid to change the degree of adsorption of ovalbumin to aluminum hydroxide adjuvant or lysozyme to aluminum phosphate adjuvant was studied. Ovalbumin and lysozyme were almost completely eluted after exposure at 37 degrees C to sheep lymph fluid for 4h or 15 min, respectively. The ability of sheep lymph fluid to elute lysozyme from aluminum phosphate adjuvant did not change as the model vaccine aged. However, only 60% of the ovalbumin adsorbed to aluminum hydroxide adjuvant was eluted during exposure to sheep lymph fluid for 24h after the model vaccine aged for 11 weeks at 4 degrees C.

The impact of the pH of precipitation on the physicochemical properties of aluminium phosphate adjuvants was investigated by precipitating aluminium phosphate adjuvants under constant pH conditions at pH values from 3.0 to 7.5 at intervals of 0.5. The pH of precipitation did not affect the morphology, but the point of zero charge (PZC) and rate of acid neutralization varied directly with pH of precipitation. Aggregation and protein adsorption capacity exhibited a parabolic relationship to the pH of precipitation. Minimum protein adsorption and maximum aggregation were observed at pH 5.5. In contrast to adjuvants precipitated from the same reactants but under uncontrolled pH conditions, the pH of all of the adjuvants precipitated under constant pH conditions remained constant for a 3-month aging period at room temperature.

Aluminium phosphate adjuvant and aluminium hydroxide adjuvant became more ordered during aging at room temperature. The increased degree of order was accompanied by a decrease in protein adsorption capacity.

The present study explores the use of 27 Al magic‐angle‐spinning (MAS) NMR for the characterization of aluminum hydroxyphosphate adjuvants. Adjuvants were prepared by two different methods: batch‐precipitation and precipitation at constant pH, using a wide range of different conditions. The adjuvant compositions showed no evident stoichiometric restrictions and varied as a function of the precipitation conditions. All the aluminum hydroxyphosphate adjuvants were found by 27 Al MAS NMR to contain both tetrahedrally and octahedrally coordinated aluminum. The octahedral form was always predominant. The chemical shifts corresponding to octahedral aluminum were at values intermediate between that of aluminum hydroxide (9 ppm) and those of phosphate‐containing aluminum minerals such as variscite (−9 ppm) and varied with the phosphate content of the adjuvant. This was true even for adjuvants precipitated above pH 6 indicating that the phosphate is incorporated into the bulk solid phase contrary to predictions in the literature. Aside from the presence of tetrahedral and octahedral aluminum, there was no evidence in any of the adjuvants of distinct, structurally defined phases indicating that the adjuvants are not mixtures of distinct phases which differ significantly in the number of phosphorus atoms in the next‐nearest‐neighbor (NNN) position to aluminum. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 311–321, 2000

A small quantity of aluminum chlorohydrate (ACH), the active ingredient in antiperspirants, was labeled with the radioisotope 26Al. This labeled drug will be used in future studies to measure the absorption of aluminum from antiperspirant use. The purpose of this study was to demonstrate that the technique used to make the 26Al-labeled ACH resulted in a uniformly labeled complex, and therefore any measurement of 26Al would be indicative of total aluminum. The labeled ACH was fractionated using gel filtration chromatography into 72 evenly spaced samples. The fractions were then measured for 26Al and total aluminum content using accelerator mass spectrometry (AMS) and inductively coupled plasma atomic emission spectroscopy (ICP-AES). Results indicate that the ACH is uniformly labeled.

Aluminium phosphate adjuvant remained amorphous when autoclaved for 30 or 60 min at 121 degrees C. However, deprotonation and dehydration reactions occurred as evidenced by a decrease in the pH. The protein adsorption capacity, rate of acid neutralization at pH 2.5 and point of zero charge also decreased indicating that the deprotonation/dehydration reactions resulted in a decreased surface area. Autoclaving aluminium hydroxide adjuvant increased the degree of crystallinity as measured by the width at half height of the major band in the X-ray diffractogram. The pH decreased during autoclaving suggesting that the same deprotonation/dehydration reactions which reduced the surface area of aluminium phosphate adjuvant were responsible for the increased degree of crystallinity. These reactions also resulted in a reduced surface area as both the protein adsorption capacity and viscosity decreased following autoclaving.

Intravenous lipid emulsions are used for total parenteral nutrition and as carriers for lipophilic drugs. Exposure to the high temperature (121 degrees C) required for steam sterilization may cause coalescence and an increase in droplet size. The purpose of this study was to investigate whether an increase in the electrostatic repulsive force between oil droplets produced by formulation modification improves the thermal stability of lipid emulsions during autoclaving. The addition of a small amount, 0.66 or 1.32 mmol/kg (mm), of purified anionic phospholipid fractions (phosphatidic acid, phosphatidylglycerol, or phosphatidylinositol) to the standard formula increased the zeta potential from its normal value of -11 mV to -39 mV. Emulsions with the larger negative zeta potential did not exhibit any change in oil droplet size or distribution during steam sterilization at 121 degrees C for 15 min. The autoclaved emulsions having the larger negative zeta potential did not exhibit any evidence of coalescence when samples were stored for 1 month at 4 degrees C, room temperature, or 40 degrees C. Reduction of the negative surface charge of the oil droplets by the addition of stearylamine confirmed that the surface charge was an important factor, as emulsions having a reduced negative surface charge separated into two phases during autoclaving.

The effect of adjusting the pH of a model ovalbumin (iep = 4.7) and aluminum hydroxide adjuvant (iep = 11.5) vaccine or a model lysozyme (iep = 11.4) and aluminum phosphate adjuvant (iep = 5.0) vaccine to values ranging from 3 to 11 was studied. The model vaccine prepared at pH 7.4 served as the reference. Additional adsorption of ovalbumin occurred in the pH range 7.4 to 4.1. Elution of ovalbumin occurred when the pH was adjusted to values above 7.4 or below 4.1. Maximum elution occurred at pH 2.9 and 11.0. Elution of lysozyme from aluminum phosphate adjuvant occurred when the pH was adjusted to any value other than the reference pH of 7.4. More lysozyme was eluted at pH 2.9 than at pH 11.0. The adsorption/elution behavior could be explained by the effect of pH on: (1) the ionization state of the protein, 2) the solubility of the adjuvant, and (3) the electrostatic interaction between the protein and adjuvant. Copyright 1998 Academic Press.

The elutability of proteins from adjuvants in model vaccines composed of ovalbumin adsorbed by aluminum hydroxide adjuvant or lysozyme adsorbed by aluminum phosphate adjuvant following treatment with surfactant solutions was studied. Nonionic (Triton X-100, lauryl maltoside), zwitterionic (lauryl sulfobetaine), anionic (sodium dodecyl sulfate), and cationic (cetylpyridinium chloride, dodecyltrimethylammonium chloride) surfactants were investigated. Cetylpyridinium chloride produced the greatest degree of elution (60%) of ovalbumin from aluminum hydroxide adjuvant. Sodium dodecyl sulfate completely eluted lysozyme from aluminum phosphate adjuvant. The effectiveness of surfactants in removing preadsorbed proteins was directly related to their ability to denature the protein. Micellar solubilization and electrostatic repulsion may also contribute to desorption. Copyright 1998 Academic Press. Copyright 1998Academic Press

The effect of adding phosphate, sulfate, or nitrate anions to model aluminum-adjuvant-containing vaccines at pH 7.4 was studied. Three different aluminum phosphate adjuvants were used that had phosphate to aluminum molar ratios of 1.31, 1.18, and 0.58. One aluminum hydroxide adjuvant was used. The isoelectric points of the adjuvants were inversely related to the degree of phosphate substitution for hydroxyl. The addition of phosphate anion and to a lesser degree sulfate anion caused additional adsorption of positively charged lysozyme by a model vaccine composed of lysozyme adsorbed by aluminum phosphate adjuvant. The addition of phosphate anions and to a lesser degree sulfate anions caused desorption of negatively charged ovalbumin from a model vaccine composed of ovalbumin adsorbed by aluminum hydroxide adjuvant. Nitrate anions had no effect on either model vaccine. The effect of phosphate or sulfate anions did not change as the model lysozyme-aluminum phosphate vaccine aged, but it decreased with time in the model ovalbumin-aluminum hydroxide vaccine. The effect of phosphate or sulfate anions in producing desorption was also influenced by the amount of adsorbed protein. More desorption occurred when the ovalbumin concentration was at the plateau region. The adsorption of phosphate anions was found to lower the isoelectric point of both adjuvants. Thus, one mechanism acting to produce adsorption or desorption may be an increase in the negative surface charge of the aluminum-containing adjuvants. This in turn decreased the electrostatic interaction with negatively charged proteins and increased the electrostatic interaction with positively charged proteins.

The optimum formulation of vaccines containing multivalent antigens may require that more than a single type of aluminum-containing adjuvant be used. In some cases, in order to maximize the binding of the negatively charged antigen(s), a positively charged adjuvant such as aluminum hydroxide could be used. In other cases, if the antigen(s) were positively charged, a negatively charged adjuvant such as aluminum phosphate might be preferred. The multivalent vaccine would therefore be prepared by combining the individual monovalent bulks resulting in a suspension consisting of mixed aluminum-containing adjuvants. Studies of such mixed suspensions revealed that some phosphate ions from the aluminum phosphate adjuvant desorbed upon the dilution which occurred when the monovalent bulks were combined. The desorption of phosphate reduced the negative surface charge of the aluminum phosphate adjuvant. The desorbed phosphate anions were subsequently readsorbed by the aluminum hydroxide adjuvant resulting in a decrease of its positive surface charge. Desorption of the adsorbed antigens may also occur when the monovalent suspensions are mixed. In the model system studied, a significant fraction (25%) of adsorbed lysozyme desorbed from the aluminum phosphate adjuvant upon dilution (1:2). In contrast, almost no bovine serum albumin was desorbed from an aluminum hydroxide adjuvant upon similar dilution. A method based on measuring the electrophoretic mobility of the adjuvants was developed to assess the interactions that take place between the different adjuvants. Rapid aggregation was observed for the system consisting of oppositely charged adjuvants. The rate of aggregation of the positively charged aluminum hydroxide adjuvant with the negatively charged aluminum phosphate adjuvant was reduced by the adsorption of proteins. Colloidal stability was enhanced by increased surface coverage of the proteins on the adjuvants. It was concluded that protein adsorption reduces the rate of aggregation of the mixed adjuvant system by minimizing the difference in surface charge between the aluminum-containing adjuvants and by providing steric repulsion.

Heterocoagulation in suspensions containing both aluminum hydroxycarbonate and magnesium hydroxide leads to the development of an aluminum hydroxycarbonate coating on the magnesium hydroxide particles. For this reason, mixed suspensions exhibit isoelectric points similar to those or aluminum hydroxycarbonate, but markedly different from magnesium hydroxide. The finding is significant in relation to physical properties of the systems as mixed suspensions demonstrated theological behavior which responded to changes in media pH in a manner analogous to aluminum hydroxycarbonate suspensions. Thus, a maximum yield value of the mixed suspension was observed at a pH corresponding to the isoelectric point of the aluminum hydroxycarbonate coating.

An increase in the rate of bleeding of aluminum lake dyes was observed in mixed suspensions containing both titanium dioxide and an aluminum lake dye when pH conditions caused the titanium dioxide to have a net negative surface charge. In mixed suspensions in which titanium dioxide had a high positive surface charge, no difference in bleeding was observed in comparison to lake dye suspensions at the same pH. The degree of bleeding was directly related to the magnitude of the negative surface charge of the titanium dioxide. It is hypothesized that negatively charged titanium dioxide accelerates bleeding of aluminum lake dyes because of surface acidity. The concentrations of aluminum cation and dye in solution in mixed suspensions were initially higher than in control suspensions containing only the aluminum lake dye. The concentration of dye in solution increased with time in both the mixed and control suspensions. The concentration of aluminum cation in solution increased with time in the control suspension but decreased in the mixed suspension. It is believed that aluminum cations which are solubilized by the surface acidity of the titanium dioxide are adsorbed by the negatively charged titanium dioxide particles. Evidence in support of this hypothesis is the change in the electrophoretic mobility of the mixed suspension from negative to positive with time.

The formation of hydrotalcite in mixed suspensions of magnesium hydroxide and aluminum hydroxycarbonate was found to be influenced by factors affecting heterocoagulation of the separate particles. A model of the system employing large particles of magnesium hydroxide exposed to an aluminum hydroxycarbonate suspension demonstrated rapid hydrotalcite formation when pH conditions were adjusted to prohibit heterocoagulation. Scanning electron micrographs of the particles indicated that nucleation and growth of the mixed crystal was occurring directly on the magnesium hydroxide surface. The appearance of hydrotalcite was delayed in mixed suspensions wherein heterocoagulation resulted in an occlusive coating of aluminum hydroxycarbonate on the magnesium hydroxide. Formation of hydrotalcite occurred sooner in systems having higher initial pH values presumably due to earlier exposure of magnesium hydroxide surfaces resulting from more rapid aging of the aluminum hydroxycarbonate coating. Mixed suspensions of magnesium hydroxide and other aluminum-containing hydroxides, wherein an occlusive coating did not form on the magnesium hydroxide particles, demonstrated rapid hydrotalcite formation.

Background Streptococcus pneumoniae causes widespread morbidity and mortality. Current vaccines contain free polysaccharides or protein-polysaccharide conjugates, and do not induce protection against serotypes that are not included in the vaccines. An affordable and broadly protective vaccine is very desirable. The goal of this study was to determine the optimal formulation of a killed whole cell pneumococcal vaccine with aluminum-containing adjuvants for intramuscular injection. Methods Four aluminium-containing adjuvants were prepared with different levels of surface phosphate groups resulting in different adsorptive capacities and affinities for the vaccine antigens. Mice were immunized three times and the antigen-specific antibody titers and IL-17 responses in blood were analyzed. Results Although all adjuvants induced significantly higher antibody titers than antigen without adjuvant, the vaccine containing aluminum phosphate adjuvant (AP) produced the highest antibody response when low doses of antigen were used. Aluminum hydroxide adjuvant (AH) induced an equal or better antibody response at high doses compared with AP. Vaccines formulated with AH, but not with AP, induced an IL-17 response. The vaccine formulated with AH was stable and retained full immunogenicity when stored at 4°C for 4 months. Conclusions Antibodies are important for protection against systemic streptococcal disease and IL-17 is critical in the prevention of nasopharyngeal colonization by S. pneumoniae in the mouse model. The formulation of the whole killed bacterial cells with AH resulted in a stable vaccine that induced both antibodies and an IL-17 response. These experiments underscore the importance of formulation studies with aluminium containing adjuvants for the development of stable and effective vaccines.

A dissolution and reprecipitation process contributes significantly to the formation of an aluminum hydroxycarbonate coating on magnesium hydroxide particles in aqueous suspensions containing the two types of particles. Shortly after the two suspensions are mixed, heterogeneous particles form consisting of magnesium hydroxide crystallites coated with aluminum hydroxycarbonate. The alkaline nature of the magnesium hydroxide surface enhances dissolution of the aluminum hydroxycarbonate particles and promotes desorption of carbonate anion from potential-determining sites on these particles. Loss of carbonate anion results in an increased isoelectric point of the aluminum hydroxycarbonate phase of the mixtures and thus of the heterogeneous particles as well. For this reason the isoelectric points of mixtures are increased compared to those of aluminum hydroxycarbonate suspensions. Both the particle size of magnesium hydroxide and media pH influence the magnitude of this increase.

Magnesium hydroxide and aluminum hydroxycarbonate interact in mixed suspensions resulting in the formation of agglomerates containing both types of particles. Direct visual evidence of the phenomenon was gained from scanning electron microscopy studies carried out on a model of the system employing a highly crystalline magnesium hydroxide of large particle size and from transmission electron microscopy of mixed colloidal suspensions. Doppler electrophoretic light scattering measurements on mixed colloid systems supported the findings as two distinct electrokinetic populations of particles were initially present which corresponded to the zeta potentials of magnesium hydroxide and aluminum hydroxycarbonate. The two populations were observed to transform over time into a single population having an intermediate zeta potential. Solubilization of both types of particles apparently takes place, leading to changes in bulk pH and ionic strength of the mixtures.

1. Rat gut perfusion studies in vivo at pH 4, 6 or 8 using aluminium chloride or equimolar aluminium chloride and sodium citrate showed that elevated plasma aluminium concentrations were associated with aluminium solubility in the perfusion. Elevated plasma aluminium levels and soluble aluminium in the perfusate occurred with perfusion of equimolar aluminium chloride and sodium citrate at all three pH values. 2. Partitioning studies in vitro, utilizing water and ethyl acetate, revealed that uncomplexed aluminium exhibited maximum partitioning into the ethyl acetate phase at pH 2.5. When complexed with citrate, aluminium exhibited partitioning over a much broader pH range, pH 2.5–8.0. 3. A direct linear relationship was observed between the soluble aluminium concentration of the perfusate and the increase in the plasma aluminium level, suggesting that soluble aluminium is absorbed by a passive diffusion mechanism.

The isoelectric point of aluminum hydroxycarbonate suspension increases upon dilution with deionized water. The increase in the isoelectric point is directly related to the degree of dilution. Dilution with bicarbonate ion-containing solutions reduced the change in isoelectric point in comparison to dilution with deionized water. The change in isoelectric point was inversely related to the concentration of bicarbonate ion in the diluting solution. The increase in isoelectric point observed following dilution with deionized water was reversed by the addition of bicarbonate ion. The observations suggest that carbonate ions occupying potential determining sites in the primary particles are in equilibrium with carbonate/bicarbonate ions in solution. Formulation or processing factors which affect the equilibrium will affect the isoelectric point of aluminum hydroxycarbonate suspensions and will influence their physical properties.

The relationship between depot formation and immunopotentiation was studied by comparing the retention of antigen at the inoculation site with antibody production in rats. A model (111)In-labeled alpha casein (IDCAS) antigen was formulated into four vaccines: IDCAS adsorbed onto either aluminum hydroxide adjuvant (AH) or aluminum phosphate adjuvant (AP); non-adsorbed IDCAS with phosphate-treated AP (PTAP); and IDCAS solution. Gamma scintigraphy showed the order of retention following subcutaneous administration to be: AH adsorbed>AP adsorbed>non-adsorbed with PTAP=solution. The antibody titers followed the order: non-adsorbed with PTAP=AP adsorbed>AH adsorbed>>solution. The presence of an aluminum-containing adjuvant was essential for immunopotentiation, but retention of the antigen at the inoculation site was not required.

The adsorptive behavior of the recombinant malarial antigens R32tet32, R32NS181 and NS181V20 to aluminum hydroxide and aluminum phosphate gels was studied as a function of pH and buffer ions. The Plasmodium falciparum antigen, R32NS181, and the P. vivax antigen, NS181V20, with isoelectric points (pI) of 5.9 and 5.5, respectively, adsorbed readily to the positively charged boehmite form of aluminum hydroxide gel. These two antigens displayed reversible, linear adsorption behavior in the pH range 5-9, with maximal adsorption observed at the lowest pH studied. The addition of acetate buffer ions had little effect on adsorption, while the presence of phosphate decreased adsorption for R32NS181 and NS181V20 by 25 and 40% respectively. The adsorptive behavior of these two antigens with the negatively charged adjuvant, aluminum phosphate, was markedly decreased. The converse situation was observed with the R32tet32 antigen, whose pI is estimated to be 12.8. There was minimal interaction of this antigen with aluminum hydroxide gel except in the presence of phosphate counter ions and significant, nonreversible adsorption with aluminum phosphate gel. Enhanced adsorption of R32tet32 to aluminum hydroxide gel in the presence of phosphate is suggested to be the result of a covalent bond between a surface aluminum and a phosphate anion that modifies the surface charge of the aluminum hydroxide gel. These results indicate that the role of complementary surface charges, both for the ionization state of the protein and for the aluminum adjuvants, is the key in optimizing conditions for significant antigen-adjuvant interactions.

The pure dye content of FD&C yellow lake #5 and FD&C red lake #40 was found to be inversely related to the point of zero charge. The effect of the adsorbed anionic dye on the zeta potential of aqueous suspensions of FD&C yellow lake #5 at pH 5 was demonstrated.

The in-vitro adsorptive capacities of aluminum hydroxide gel, dried aluminum hydroxide gel, and boehmite for cholate anion were determined at pH 7.5, 37°C and compared to cholestyramine. The adsorptive capacity of aluminum hydroxide gel was similar to cholestyramine. However, spray drying reduced the adsorptive capacity by approximately 50%. Boehmite, a crystalline aluminum oxyhydroxide, had an adsorptive capacity similar to dried aluminum hydroxide gel. The results support the hypothesis that adsorption of bile salts contributes to the constipation which is sometimes associated with aluminum hydroxide therapy.

The binding of bile salts by cholestyramine may be influenced by other anions, as the Langmuir adsorption coefficients for three bile salts tested were similar to the model anion, citrate. However, the selectivity coefficient indicated preferential binding of cholate anion in comparison to citrate anion. Binding experiments confirmed cholestyramine's preference for bile salts as the presence of other anions reduced but did not prevent the binding of cholate anion. Binding of cholate anion was reduced in direct relationship to the citrate anion concentration. Prior exposure of cholestyramine to citrate anion caused the binding of cholate anion to decrease slightly. Sequential exposure of cholestyramine to simulated gastric fluid and simulated intestinal fluid containing cholate anion resulted in a decrease in cholate binding which was attributed to competition with anions present in the gastrointestinal fluids. Components of tomato juice and orange juice, fluids commonly used to enhance ingestion of cholestyramine, also reduced the binding of cholate anion.

The structure of nine commercially manufactured aluminum-containing adjuvants was investigated by X-ray diffraction, infrared spectroscopy, transmission electron micrography, and energy dispersive spectrometry. Seven samples which were labeled as aluminum hydroxide were identified as boehmite, a crystalline aluminum oxyhydroxide [AlO(OH)]. However, the degree of crystallinity varied between the samples. Two samples which were labeled as aluminum phosphate were found to be amorphous aluminum hydroxyphosphate. Buffer anions and sulfate anions substitute for hydroxyls in the amorphous aluminum hydroxide formed by the in situ alum precipitation method. Finally, the aluminum-containing adjuvant in diphtheria and tetanus toxoid, U.S.P., produced by three manufacturers was characterized.

Aluminum-containing adjuvants are an important component of many vaccines because they safely potentiate the immune response. The structure and properties of aluminum hydroxide adjuvant, aluminum phosphate adjuvant and alum-precipitated adjuvants are presented in this review. The major antigen adsorption mechanisms, electrostatic attraction and ligand exchange, are related to the adjuvant structure. The manner by which aluminum-containing adjuvants potentiate the immune response is related to the structure, properties of the adjuvant and adsorption mechanism. Immunopotentiation occurs through the following sequential steps: inflammation and recruitment of antigen-presenting cells, retention of antigen at the injection site, uptake of antigen, dendritic cell maturation, T-cell activation and T-cell differentiation.

Steam sterilization of xanthan gum solutions at 121 degrees C caused a decrease in the helix conformation as well as the molecular weight distribution with a corresponding increase in the coil structure. The effect was directly related to the exposure time and inversely to the xanthan gum concentration, thus suggesting a two-step mechanism of disentanglement followed by degradation with the first step being predominant at higher concentrations. Mark-Houwink exponent of 0.9002 for the intrinsic viscosity of xanthan gum compared favorably with reported values in the literature. The model for intrinsic viscosity of a free draining coil yielded an expansion coefficient of 1.2 (independent of molecular weight) and a root mean square radius of unperturbed chain in the range of 189.5-368 nm. The root mean square unperturbed chain length increased with molecular weight without reaching an asymptotic value, thus indicating that the xanthan molecule behaved as a stiff chain.

The objective of this research was to determine how the mechanism by which antigens adsorb to aluminum-containing adjuvants affects the elution upon exposure to interstitial fluid. Antigens (alpha lactalbumin, bovine serum albumin, lysozyme and myoglobin) that adsorb to aluminum-containing adjuvants principally by electrostatic attraction were found to elute readily in vitro when exposed to interstitial fluid. Phosphorylated antigens (alpha casein, hepatitis B surface antigen and phosphorylated bovine serum albumin) that adsorb to aluminum-containing adjuvants principally by ligand exchange exhibit little if any elution during 12-24 h in vitro exposure to interstitial fluid. Dephosphorylated alpha casein, which contains less than two phosphate groups, was less strongly adsorbed by ligand exchange in comparison to alpha casein, which contains eight phosphate groups. Dephosphorylated alpha casein was completely eluted when exposed to interstitial fluid. The results of this study lead to the generalization that antigens that adsorb to aluminum-containing adjuvants by electrostatic attraction are more likely to elute upon intramuscular or subcutaneous administration than antigens that adsorb by ligand exchange.

Aluminium hydroxide adjuvant has an isoelectric point (i.e.p.) of ca 11 and is a good adsorbent for acidic proteins due to the contribution of electrostatic attractive forces. However, electrostatic repulsive forces reduce its ability to adsorb basic proteins. Pretreatment of aluminium hydroxide adjuvant with carefully selected concentrations of phosphate anion reduces the positive surface charge which exists at pH 7.4. Treatment with higher concentrations of phosphate anion produces a negative surface charge. The adsorption of lysozyme (i.e.p = 11.1) was found to be directly related to the concentration of phosphate anion used to pretreat the aluminium hydroxide adjuvant.

The distribution of alpha-casein, bovine serum albumin (BSA), myoglobin and recombinant protective antigen (rPA) in mono-valent and combination vaccines containing aluminum hydroxide adjuvant was studied by fluorescence microscopy and flow cytometry. Green and red fluorescent probes were conjugated to the antigens. Adsorption isotherms of the fluorescently labeled proteins to aluminum hydroxide adjuvant demonstrated that incorporation of the fluorescent probe did not significantly affect the adsorption. In mono-valent vaccine systems, antigen adsorption occurred within one minute and uniform surface coverage of the adjuvant aggregates was observed within 1 h. Content uniformity was achieved through a cycle of de-aggregation and re-aggregation of the aluminum hydroxide adjuvant aggregates caused by mixing. For combination vaccines, two antigens were adsorbed separately to the aluminum hydroxide adjuvant prior to combination. Following combination, cycles of de-aggregation and re-aggregation occurred due to mixing, which led to uniform distribution of both antigens. The results of this study indicate that content uniformity should not be an issue during the production of mono-valent or combination vaccines as long as adequate mixing procedures are followed. © 2003 Elsevier Ltd. All rights reserved.

Recombinant protective antigen (rPA) is the active pharmaceutical ingredient in a second generation anthrax vaccine undergoing pre-clinical evaluation. This rPA vaccine differs from the currently licensed vaccine, anthrax vaccine adsorbed (AVA), in that the sole component is a recombinant form of protective antigen (PA). Unlike AVA the rPA vaccine contains no lethal factor (LF) or edema factor (EF), components of the two bipartite toxins, nor many other Bacillus anthracis-related contaminating proteins that are present in AVA. The proposed clinical protocol involves adsorption of the rPA to an aluminum-based adjuvant. The adsorptive characteristics of rPA and two aluminum-containing adjuvants were examined in a physiological buffer with and without EDTA. Based on the pI of rPA (pI=5.6) and the zero charge point of aluminum hydroxide adjuvant (11.5) and aluminum phosphate adjuvant (4.5), it was predicted and demonstrated that rPA bound in a more efficient manner to aluminum hydroxide adjuvant than to aluminum phosphate adjuvant in the physiological buffer. Binding of the rPA to the aluminum hydroxide adjuvant was decreased by increasing amounts of phosphate in the buffer. The adsorptive capacity for rPA onto aluminum hydroxide adjuvant in the physiological buffer and in water were calculated to be 0.46 mg rPA/mg aluminum in DPBS and 0.73 mg rPA/mg aluminum in water. This study also demonstrated that upon desorption from the aluminum hydroxide adjuvant the rPA was physically intact and free of detectable aggregates. Further, the eluted material was biologically active in an in vitro cytotoxicity assay. Desorption was only possible after an overnight incubation of 2-8 degrees C and not after a room temperature incubation reflecting increased contact with the aluminum hydroxide adjuvant over time. These data suggest that the interaction between rPA and aluminum hydroxide adjuvant is predominantly electrostatic in character.

Hepatitis B surface antigen (HBsAg) differs from many antigens because of its associated lipid bilayer that is largely composed of phospholipids. In general, phosphate groups adsorb strongly to hydroxylated mineral surfaces by ligand exchange. The purpose of this study was to investigate the mechanism of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant with emphasis on the role of phospholipids in this adsorption. The adsorption of HBsAg by aluminum hydroxide adjuvant exhibits a high affinity adsorption isotherm. The Langmuir equation was used to calculate the adsorptive capacity (1.7 microg/microg Al), which is the amount of HBsAg adsorbed at monolayer coverage and the adsorptive coefficient (6.0 ml/microg), which is a measure of the strength of the adsorption force. The relatively high value of the adsorptive coefficient indicates that adsorption is due to a strong attractive force. Ligand exchange between a phosphate of the antigen and a surface hydroxyl of the adjuvant provides the strongest adsorption mechanism. The adsorption capacity of HBsAg was not affected by increased ionic strength indicating that electrostatic attraction is not the predominant adsorption force. Adsorption was also not affected by the addition of ethylene glycol indicating that hydrophobic interactions were not the predominant adsorption force. The strength of the adsorption force was indicated by the resistance of HBsAg to elution when exposed to interstitial fluid. Less than 5% of the HBsAg adsorbed to aluminum hydroxide adjuvant in a model vaccine was eluted during a 12 h in vitro exposure to interstitial fluid at 37 degrees C. Less than 1% of the adsorbed HBsAg in two commercial vaccines was eluted by in vitro exposure to interstitial fluid for 48 h at 37 degrees C. Thus, it was concluded that adsorption of HBsAg by aluminum hydroxide adjuvant is predominantly due to ligand exchange between the phospholipids in HBsAg and surface hydroxyls in aluminum hydroxide adjuvant.