PURPOSE: 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. METHODS: 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. RESULTS: 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-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin, as well as poloxamers 188 and 407, in half-filled shaken vials. CONCLUSIONS: A high-throughput screening approach can be followed for evaluating the performance of excipients against aggregation of a protein antigen at air-liquid interface.
PURPOSE: 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. METHODS: 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. RESULTS: 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-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin, as well as poloxamers 188 and 407, in half-filled shaken vials. CONCLUSIONS: A high-throughput screening approach can be followed for evaluating the performance of excipients against aggregation of a protein antigen at air-liquid interface.
Authors: John F Carpenter; Theodore W Randolph; Wim Jiskoot; Daan J A Crommelin; C Russell Middaugh; Gerhard Winter Journal: J Pharm Sci Date: 2010-05 Impact factor: 3.534
Authors: Bryan S Der; Christien Kluwe; Aleksandr E Miklos; Ron Jacak; Sergey Lyskov; Jeffrey J Gray; George Georgiou; Andrew D Ellington; Brian Kuhlman Journal: PLoS One Date: 2013-05-31 Impact factor: 3.240