title: Towards development of stable formulations of a live attenuated bacterial vaccine: a preformulation study facilitated by a biophysical approach.
abstract: Development of optimal formulation conditions stabilizing live attenuated bacterial vaccines is impeded by traditional methods used for viability measurement. To facilitate preformulation studies of such vaccines, spectroscopic techniques capable of providing real-time and high throughput information have been employed to obtain a global stability profile for a live attenuated Ty21a bacterial typhoid vaccine over a wide range of pH (4 to 8) and temperature (10 to 85 degrees C). Using the data obtained from fluorescence and circular dichroism techniques, an empirical phase diagram (EPD) has been subsequently constructed, which suggests that Ty21a cells exist in at least four apparent physical phases related to different viability states, with the most stable phase at pH 6 and 7 at temperatures below 30 degrees C. A slightly basic pH (pH 8) appears to decrease the fluidity of the cell membrane, whereas acidic pH conditions are detrimental to membrane integrity over the entire temperature range. Based on the above stability profile, a fluorescence-based high throughput screening assay has been developed to test the stabilizing effects of various compounds at different concentrations. Amongst other promising stabilizers, 10% sucrose and 0.15 M glutamic acid display the greatest protective effects, with an increase of about 10 degrees C in the transition temperature of Ty21a cells. Preliminary studies have also been performed on foam dried formulations as an alternative approach to further stabilize Ty21a cells. The data show that 10% sucrose and trehalose both increase the in-process and storage stabilities of the cells.
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Laboratory, University of Kansas, Lawrence, Kansas 66047, USA.
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Zeng
authors.forename: Haihong
authors.initials: H
authors.lastname: Fan
authors.forename: Gary
authors.initials: G
authors.lastname: Chiueh
authors.forename: Binh
authors.initials: B
authors.lastname: Pham
authors.forename: Russ
authors.initials: R
authors.lastname: Martin
authors.forename: David
authors.initials: D
authors.lastname: Lechuga-Ballesteros
authors.forename: Vu L
authors.initials: VL
authors.lastname: Truong
authors.forename: Sangeeta B
authors.initials: SB
authors.lastname: Joshi
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
mesh_terms: D002942:Circular Dichroism; D004355:Drug Stability; D005079:Excipients / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D050296:Microbial Viability; D011135:Polysaccharides, Bacterial / Q000235:genetics / Q000276:immunology*; D013057:Spectrum Analysis / Q000379:methods; D013395:Sucrose / Q000494:pharmacology; D013696:Temperature; D014436:Typhoid-Paratyphoid Vaccines / Q000235:genetics / Q000276:immunology*; D014613:Vaccines, Attenuated / Q000235:genetics / Q000276:immunology
chemical_list: D005079:Excipients; D011135:Polysaccharides, Bacterial; C072772:Ty21a typhoid vaccine; D014436:Typhoid-Paratyphoid Vaccines; D014613:Vaccines, Attenuated; D018698:Glutamic Acid; D013395:Sucrose