Maturation of Aluminium Adsorbed Antigens Contributes to the Creation of Homogeneous Vaccine Formulations

Abstract

Although aluminium-based vaccines have been used for almost over a century, their mechanism of action remains unclear. It is established that antigen adsorption to the adjuvant facilitates delivery of the antigen to immune cells at the injection site. To further increase our understanding of aluminium-based vaccines, it is important to gain additional insights on the interactions between the aluminium and antigens, including antigen distribution over the adjuvant particles. Immuno-assays can further help in this regard. In this paper, we evaluated how established formulation strategies (i.e., sequential, competitive, and separate antigen addition) applied to four different antigens and aluminium oxyhydroxide, lead to formulation changes over time. Results showed that all formulation samples were stable, and that no significant changes were observed in terms of physical-chemical properties. Antigen distribution across the bulk aluminium population, however, did show a maturation effect, with some initial dependence on the formulation approach and the antigen adsorption strength. Sequential and competitive approaches displayed similar results in terms of the homogeneity of antigen distribution across aluminium particles, while separately adsorbed antigens were initially more highly poly-dispersed. Nevertheless, the formulation sample prepared via separate adsorption also reached homogeneity according to each antigen adsorption strength. This study indicated that antigen distribution across aluminium particles is a dynamic feature that evolves over time, which is initially influenced by the formulation approach and the specific adsorption strength, but ultimately leads to homogeneous formulations.

Keywords: adsorption; ageing; aluminium hydroxide; compounding strategy; distribution; homogeneity; maturation; populations.

Figure 1

Figure describing three different formulation approaches for aluminium-based vaccines. The values expressed as 2X in competitive adsorption refers to antigens and AlumOH content of intermediate drug product, while those 4X in separate adsorption refers only to antigens content of monovalent bulks. In all formulation samples, the final drug products have exactly same content of both AlumOH and antigens.

Figure 2

pH value of each single formulation sample at different time points.

Figure 3

PSD results at different time points for each sample. PSD results are expressed as the mean value of three independent measurements with standard deviation.

Figure 4

SDS-PAGE results at time points 0 and 100 days which show antigens identity in respect to standard controls and degree of antigens adsorption after steps of centrifugation and separation of supernatants from AlumOH pellet. STD: Standard antigens solution of known amount; Ag: Antigen; TCA: Supernatant precipitated with tri-chloro acetic acid after steps of centrifugation and AlumOH separation; Ads: Adsorption; DES: AlumOH desorbed with desorption buffer following steps of centrifugation and supernatant separation.

Figure 5

Mean and standard deviation of ZP value for three different replicates of each single formulation sample at different time points.

Figure 6

Fluorescence histogram at T0 and after 100/130 days of different formulation approach samples are reported. The IEP of each antigen is reported in brackets. On the X axis Fluorescence Intensity is reported, while on the Y axis number of events is reported. All numbers refer to Mean Fluorescence Intensity (MFI) values.

Figure 7

Fluorescence histogram at T0 of separate formulation approach sample (red) in comparison with negative control (light blue) and monovalent formulation (light green) are reported. On the X axis Fluorescence Intensity is reported, while on the Y axis number of events is reported. All numbers refer to Mean Fluorescence Intensity (MFI) values.

Figure 8

Separate adsorption sample, histogram overlay of time points 0-30-130 days for antigen A, B and D, while of 0-50-130 days for antigen C. On the X axis Fluorescence Intensity is reported, while on the Y axis number of events is reported. All numbers refer to Mean Fluorescence Intensity (MFI) values.

Figure 9

Western blot results of sorted AlumOH populations P1 and P2 after Flow Cytometry staining for antigen A in separate adsorption sample at T0, for orthogonal evaluation of delivered antigen content. Std ag: Standard antigen A solution of known amount used as references; P1 and P2: AlumOH populations sorted from separate adsorption sample at T0 after antigen A staining (see Figure 7).