Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening.

The adsorption mechanism of antigen on aluminum adjuvant can affect antigen elution at the injection site and hence the immune response. Our aim was to evaluate adsorption onto aluminum hydroxide (AH) by ligand exchange and electrostatic interactions of model proteins and antigens, bovine serum albumin (BSA), ß-casein, ovalbumin (OVA), hepatitis B surface antigen, and tetanus toxin (TT). A high-throughput screening platform was developed to measure adsorption isotherms in the presence of electrolytes and ligand exchange by a fluorescence-spectroscopy method that detects the catalysis of 6,8-difluoro-4-methylumbelliferyl phosphate by free hydroxyl groups on AH. BSA adsorption depended on predominant electrostatic interactions. Ligand exchange contributes to the adsorption of ß-casein, OVA, hepatitis B surface antigen, and TT onto AH. Based on relative surface phosphophilicity and adsorption isotherms in the presence of phosphate and fluoride, the capacities of the proteins to interact with AH by ligand exchange followed the trend: OVA < ß-casein < BSA < TT. This could be explained by both the content of ligands available in the protein structure for ligand exchange and the antigen's molecular weight. The high-throughput screening platform can be used to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant.

Automated Gravimetric Calibration to Optimize the Accuracy and Precision of TECAN Freedom EVO Liquid Handler.

High-throughput screening technologies are increasingly integrated into the formulation development process of biopharmaceuticals. The performance of liquid handling systems is dependent on the ability to deliver accurate and precise volumes of specific reagents to ensure process quality. We have developed an automated gravimetric calibration procedure to adjust the accuracy and evaluate the precision of the TECAN Freedom EVO liquid handling system. Volumes from 3 to 900 µL using calibrated syringes and fixed tips were evaluated with various solutions, including aluminum hydroxide and phosphate adjuvants, ß-casein, sucrose, sodium chloride, and phosphate-buffered saline. The methodology to set up liquid class pipetting parameters for each solution was to split the process in three steps: (1) screening of predefined liquid class, including different pipetting parameters; (2) adjustment of accuracy parameters based on a calibration curve; and (3) confirmation of the adjustment. The run of appropriate pipetting scripts, data acquisition, and reports until the creation of a new liquid class in EVOware was fully automated. The calibration and confirmation of the robotic system was simple, efficient, and precise and could accelerate data acquisition for a wide range of biopharmaceutical applications.

Development of a high-throughput screening platform to study the adsorption of antigens onto aluminum-containing adjuvants.

Aluminum-containing salts are important adjuvants in the formulations of many licensed human vaccines. However, in the early stage of the design of a new vaccine, a thorough understanding of the adsorption mechanisms of an antigen onto an aluminum salt is required. Therefore, we have developed a robust, rapid, and reproducible high-throughput screening (HTS) platform to study the adsorption capacity of aluminum-containing vaccines. The adsorption isotherms on aluminum hydroxide and aluminum phosphate of two model proteins, ß-casein, and bovine serum albumin, were evaluated using a liquid handling system, which permitted rapid sample preparation in a small volume without nonspecific adsorption. Highly reproducible adsorption capacities and adsorptive coefficients were estimated based on the Langmuir model. To demonstrate the potential of this HTS platform, we evaluated the adsorption isotherms for two antigens, hepatitis B surface antigen and a pneumococcal serotype polysaccharide conjugated to a protein-D carrier, onto aluminum-containing vaccines at either a constant protein or a constant aluminum concentration. The automated assay enabled the rapid quantification of antigen adsorption with a significant reduction in operator workload and reagent use. This platform should accelerate data acquisition during the development of a new vaccine.