Dr. Chisholm Presentation

Thermostable Lyophilized Ebola Vaccine Formulations

Carly Fleagle Chisholm, PhD

University of Colorado - Boulder

Center for Pharmaceutical Biotechnology

Department of Chemical and Biological Engineering

September 20th,2016 1

Cold chain requirements for vaccines

Current licensed vaccines require transport and storage under a tightly-controlled cold chain

Cold chain requirements are costly and particularly difficult to maintain in developing countries

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Ebola outbreaks occur in countries where maintaining the cold chain is challenging

2014 Ebola outbreak in West Africa

Total cases = 28,652 Total deaths= 11,325 3

No licensed vaccines against Ebola virus infections are currently available

We need a thermally stable vaccine!

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Lyophilization can be used to develop thermostable vaccines and could

eliminate the need for a cold chain

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Freezing-induced aggregation: A challenge for lyophilization of adjuvant-containing vaccines

Freezing typically causes aggregation of aluminum salt particles in vaccine formulations and can lead to losses in vaccine efficacy

Particle diameter (m)

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Post freeze-thaw

Clausi et al., J. Pharm. Sci., Vol. 97, (6), 2008 6

Aggregation of aluminum salt adjuvants during freezing can be avoided by using high concentrations of glass-forming excipients or faster cooling rates

Clausi et al., J. Pharm. Sci., Vol. 97, (6), 2008

2D Graph 2

Trehalose Concentration (w/v%)

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Shelf Cooled 0.5 C/min; Thawed

Shelf Cooled 3.5 C/min; ThawedImmersion in Liquid Nitrogen; ThawedSpray Frozen; ThawedShelf Cooled 3.5 C/min; Dried

Processing:

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Development of a thermostable Ebola vaccine

Formulations of the key antigen, Ebola glycoprotein, were prepared in liquid and lyophilized forms

Vaccine Formulation 0.1 mg/mL Ebola glycoprotein 10 mM ammonium acetate pH 7 9.5% (w/v) trehalose ± 0.5 mg/mL aluminum hydroxide (alum)

Lyophilized using fast cooling

Vaccine stability was tested after 12 weeks of incubation at 40°C

Vaccine potency was determined by measurement of antibody responses against Ebola glycoprotein in mouse models

Feldmann et al. 1999

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Ebola glycoprotein is thermally unstable in liquids

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Protein aggregation and degradation was minimized in lyophilized Ebola glycoprotein vaccine formulations

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Minimal aggregation of alum particles was observed after lyophilization of Ebola vaccine formulations

Formulation Mean particle diameter (ESD)

Liquid E-GP +alum 2.7 ± 0.1 µm

Lyo E-GP +alum 4.7 ± 0.1 µm

*Particle size was determined using microflow digital imaging with FlowCAM® 11

Near-native tertiary structure of Ebola glycoprotein was retained in all lyophilized formulations even after 12 weeks of incubation

Unfolded protein

native protein

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Ebola vaccines are equally potent in liquid and lyophilized forms

- ALUM + ALUM 13

More importantly, lyophilized Ebola glycoprotein vaccines are stable for 12 weeks at 40°C

- ALUM + ALUM 14

Conclusions

Using high trehalose concentrations and fast cooling, aggregation of aluminum salt particles can be avoided in lyophilized Ebola vaccines

Lyophilized Ebola vaccines are stable at 40°C eliminating the need for a cold chain

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Acknowledgments

University of Colorado- Boulder

Theodore Randolph

Taek Jin Kang, Dongguk University-Seoul

Milly Dong

Kasey Lewis

University of Hawaii- Manoa

Axel Lehrer

Oreola Donini

Funded by Soligenix, Inc.