ISA 720 has been shown to induce high antibody titers and specific cytotoxic T-lymphocyte (CTL) responses in a variety of animal species [10
]. It has been used as a vaccine adjuvant in malaria, HIV and cancer clinical trials [5
] and induced both humoral and cellular immune responses [5
]. However, a loss of potency with a pre-formulated AMA1/ISA 720 vaccine was reported due to the lack of vaccine stability [16
]. After an extensive screening of the effects of antioxidants and amino acids in the formulation, Miles et al reported that glycine or glycalglycine were able to stabilize the protein by eliminated the broadening of protein band and preventing multimer formation [25
]. The optimal concentration of glycine was later determined to be 50 mM after comparison of the glycine concentrations at 50, 134 and 267 mM in our laboratory (data not shown).
Due to difficulties in standardizing bedside formulation of ISA 720 emulsions, we chose to prepare and store AMA1-C1/ISA 720 with the addition of 50 mM glycine as an antigen stabilizer prior to initiating clinical trials [12
]. Therefore, monitoring the stability of the pre-formulated vaccine emulsions was critical, particularly given the previous example of loss of potency with a pre-formulated AMA1/ISA 720 vaccine [16
]. There are no generic methods available to evaluate the stability of ISA 720 based vaccines. In the present study, we report a set of analyses to assess the physical, chemical and biological stability of ISA720 emulsions. Appearance, sterility, integrity by particle size and SDS-PAGE with silver staining, identity by mAb recognition, and protein content by Modified Lowry Assay and ELISA were defined as stability indicating tests.
It is important to achieve a homogeneous distribution of small particles even if the smallest particles are not linked with stability [23
]. The manufacturer of ISA 720 (SEPPIC, Inc.) recommends an emulsion droplet size of approximately 1 μm diameter to achieve stability and immunogenicity. Larger droplets may cause the emulsion to prematurely collapse [25
]. In our study, although the droplet size of both the 10 μg/mL and 40 μg/mL emulsions still met the acceptable specification of VWM at 0.5-1.8 μm (based on the manufactures' recommendation and our experiences) after 18 month storage at 2-8°C, a significant increase, from 0.77 μm on the day of formulation to 1.26 μm at 18 months for 10 μg/mL and from 0.76 μm to 1.27 μm for 40 μg/mL was observed, suggesting that physical stability of emulsions changes over storage time at 2-8°C.
The AMA1-C1 proteins were also found to be unstable in the ISA720 emulsion after 18 months of storage. For the 10 μg/ml vaccine, the protein content at the 18 month time point was below the level of detection (5 μg/ml) in modified Lowry assay, and was significantly lower as measured by competition ELISA with anti-His mAb, and densitometry of Western blots. For the 40 μg/ml vaccine at the same time point, the protein content was 42 ± 2.6 μg/ml by modified Lowry assay, but was about 4 fold less by competition anti-His tag ELISA and densitometry of Western blots with mAb 1G4, 2C2 or 4G2. Because the modified Lowry assay indiscriminately measures intact as well as degraded protein fragments, data by competition ELISA using anti-His mAb and densitometry of Western blots using multiple mAbs recognizing AMA1 functional and conformational epitopes are better measurements of the AMA1 antigen integrity.
In contrast to biochemical measurement for antigen and formulation stability, in vivo
potency assay in mice, as measured by antibody ELISA, is a less sensitive method to evaluate formulation stability. Despite a considerable decrease in AMA1 antigen content after 12 months storage at 2-8°C, the vaccines induced antibody response levels comparable to a freshly formulated reference vaccine, thus considered to be fully potent. These could be due to the monoclonal antibodies used in present study recognize specific epitopes of AMA1, loss of these epitopes due to degradation or denaturation will result in loss of signal in ELISA or western blot. However, the in vivo potency assay measures polyclonal IgGs against multiple epitopes, it is thus less sensitive compared to biochemical tests. The vaccines remained within specifications during the course of the clinical trial, and both doses were also shown to be potent in humans [12
More degradation was found in the higher antigen dose (40 μg/mL) when compared to the lower antigen dose (10 μg/mL). This may be due to the higher amount of protein in the emulsion, the more polar and nonpolar groups present in the dispersed phase of emulsion, which will modify the global hydrophilic/lipophilic balance (HLB), and decrease the stability of the emulsion [23
]. This result suggests that different doses of vaccine may have different shelf lives although they were formulated with the same antigen.
In summary, we found that while the AMA1-C1/ISA720 emulsions remained within specifications during the clinical trial (within 12 months post vaccine manufacture), the emulsions were unstable at longer storage times, with partial degradation of AMA1-C1 after 12-18 months of storage at 2-8°C. Combinations of particle size analysis, SDS-PAGE, Western blot, a modified Lowry assay and competitive ELISA methods are likely to be more sensitive methods for determining protein degradation than animal potency assays.