Spray drying of virosomes to produce stable vaccines Laura Masona, Jack Sorrella, Andrew Naylora, Mario Amackerb, Toon Stegmannc, Sylvain Fleuryb and Richard Johnsona

a Upperton Ltd, Biocity, Pennyfoot Street, Nottingham, NG1 1GF, UK b Mymetics SA, 1066 Epalinges, Switzerland c Mymetics BV, 2333 CH Leiden, The Netherlands

Introduction Vaccines are poorly accessible in developing countries Vaccines require cold-chain storage and are often delivered by injection, which is undesirable, less safe and more expensive to administer.

Developing thermostable solid form vaccines through non-invasive routes may represent a long-term global solution to the vaccination

challenge (Amorij, 2008).

Virosomes are an efficient vaccine delivery system Virosomes are spherical, unilamellar lipid-based carriers, intercalated with functional glycoproteins to reflect the natural virus, however the lack

of viral RNA means there is no risk of infection (Figure 1). Virosomes can be tagged with different antigens and adjuvants, meaning they can

be tailored to target different viruses, and offer increased immunogenicity over inactivated viruses.

Currently, virosomal influenza vaccines are only available in liquid form (Amorij, 2008).

Spray drying can produce dry powders for a range of dosage forms, including inhaled or nasal drug delivery. A dry powder is formed when a liquid feed solution or suspension is atomised using a spray nozzle, and rapidly dried using hot air. However,

while the drying process is gentle due to evaporative cooling, there is still the potential to stress and inactivate vaccine components. It has

been found that subunit and live-attenuated vaccines (and other delicate molecules such as proteins) can be protected during processing b

by incorporating them in an amorphous sugar matrix, which also offers longer term stability during storage (Kanojia, 2016).

A method has been developed to produce a powder form of virosome based influenza vaccine using spray-drying. Formulations have been optimised for oral and nasal delivery.

Figure 1: Representation of virosome structure

Materials and Methods Influenza virosomes were manufactured with intercalated HA (A/H1N1),

a recombinant protein fragment (rgp41) and a synthetic lipopeptide (P1)

derived from the HIV rgp41 protein.

Three formulations were developed to target oral and nasal drug

delivery. Solutions were sprayed from a 10% w/v feed solution

Results The effect of spray-drying on virosome structure and antigen integrity

Figure 3 shows the hydrodynamic diameter of the virosomes, measured by DLS before and after spray drying.

• The diameter was between 140 – 152 nm (PdI 0.197 – 0.334) virosomes intact after spray drying.

SDS page electrophoresis, using a 4-12% Bis Tris gel plate and non-reducing conditions, showed;

• The functional proteins, marked in the stock virosomes, remained intact after spray drying.

Spot blot analysis of serial dilutions and antibody probing showed no significant difference in antibody binding

• No significant differences found for P1, rgp41 and HA antigens in liquid and spray dried powder

Conclusions Virosomes were successfully spray dried into three dry powder formulations, designed for oral and nasal drug

delivery.

The integrity of the virosome structure and antigen reactivity was maintained after spray drying and processing

parameters could influence the physical powder.

An enteric-coated oral capsule to deliver the formulation to the Peyer’s patch-rich ileac region of the small intestine,

and a single dose nasal spray have been developed.

Studies are currently being conducted to assess the long-term stability of the powder. The efficacy and safety is

currently being evaluated in preclinical studies.

References and Acknowledgements Amorij, J-P., Huckriede, A., Wilschut, J., Frijlink, H.W., Hinrichs, W.L.J., 2008. PharmRes, 25 (6), 1256-1273. Kanojia, G Willems, G-J., Frijlink, H.W., Kersten, G.F.A., Soema, P.C., Amorij, JP., 2016. IntJPharm, 511, 1098-1111.

Project funded by MACIVIVA, a Horizon 2020 European Union funded project (grant no 646122). Mymetics SA is supported by the State Secretariat for Education, Research and Innovation (SERI) of Switzerland.

The impact of spray-drying conditions on powder properties The particle size of powders manufactured at an outlet temperature of 60 °C, flow rate of 2 mL/min and

pressure of 4 bar, was typically between 2.5 – 4 µm. Whilst this particle size is suitable for oral drug

delivery from a filled capsule, larger particles (> 10 µm) are recommended for the nasal route to limit

pulmonary deposition.

Particle size could be changes by adjusting flow rate and air pressure.

• At an air pressure of 1 bar and a flow rate of 0.5 ml/min, the mean particle size increased to > 8 µm

• Particle size further increased to > 10 µm when the spray feed stock contained 30% w/v solids.

Figure 2: Spray drying process Powders were produced using a ProcepT spray dryer

using a range of drying temperatures

Testing of spray dried product Virosomes, pre and post spray drying were

characterised for particle size (DLS,

NanoSight, Malvern) and the presence of

intact protein structure (SDS-page

electrophoresis).

Antigen integrity was assessed by spotblot

analysis

Powder particle size was measured using

laser diffraction (HELOS, Sympatec).

Oral Virosomes Trehalose

Water

Nasal 1 Virosomes Trehalose Leucine Water

Nasal 2 Virosomes Trehalose Alginate Water

Feed rate 0.5 – 2.0 mL/min

Outlet temperature 60 & 80 °C

Air pressure 1 – 4 bar

Figure 3: Hydrodynamic diameter of virosomes pre-post spray drying

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Figure 4: Spot blot analysis of specific antigens on virosomes