High-Throughput Screening of Excipients Intended to Prevent Antigen Aggregation at Air-Liquid Interface

The aim was to develop a high-throughput screening method compatible with low protein concentrations, as present in vaccines, in order to evaluate the performance of various excipients in preventing the aggregation at air-liquid interface of an experimental recombinant antigen called Antigen 18A. Aggregation of Antigen 18A was triggered by shaking in a half-filled vial or by air bubbling in a microplate. Size-exclusion chromatography, turbidimetry, Nile Red fluorescence spectroscopy, and attenuated total reflection Fourier-transform infrared spectroscopy were used to assess Antigen 18A aggregation. A high-throughput method, based on tryptophan fluorescence spectroscopy, was set up to screen excipients for their capability to prevent Antigen 18A aggregation at air-liquid interface. While a similar aggregation profile was obtained with both stress tests when using size-exclusion chromatography, spectroscopic and turbidimetric methods showed an influence of the stress protocol on the nature of the aggregates. The high-throughput screening revealed that 7 out of 44 excipients significantly prevented Antigen 18A from aggregating. We confirmed the performance of hydroxypropyl-beta-cyclodextrin and hydroxypropyl-gamma-cyclodextrin, as well as poloxamers 188 and 407, in half-filled shaken vials. A high-throughput screening approach can be followed for evaluating the performance of excipients against aggregation of a protein antigen at air-liquid interface.