(W1330-01-01) Stabilization of Viruses for Vaccines and Gene Therapy by Spray Drying and Lyophilization

Purpose: Infectious viruses are capable of transfecting cells with genetic material for vaccines and gene therapy.^1,2 However, viral vectors are inherently unstable over long timer periods or exposed to moderate temperatures.^3,4 This instability contributes to high prices associated with these therapies and vaccines. Additionally, it prevents equitable global access to these products and can lead to vaccine wastage if storage requirements are not maintained.⁵ Thus, there is a need to enhance stability of viral vaccines and gene therapy vectors. In this work, we formulated a “live” virus in dry glassy solid for enhanced thermal stability.

Methods: Spray-drying:­ Green fluorescent protein (GF)-encoding Baculovirus (BV) was formulated in 9.5% trehalose, 2.5% hydroxyethyl starch, 10 mM l-histidine, and 40 mM ammonium acetate at pH 6.5. Formulated BV was spray-dried using a Buchi B-290 spray dry at an inlet drying gas temperature of 88°C or 50°C at 32 m3/h and an atomizing gas flowrate of 414 L/h.
Lyophilization: BV was formulated as described above then filled into 3-mL vials and loaded onto pre-cooled shelves at -1°C. The shelf temperature was decreased to -40°C before decreasing the pressure to 60 mTorr. Primary drying occurred at -20°C and 60 mTorr for 20 hours, before secondary drying at 30°C and 60 mTorr for 5 hours.
Powder characterization: Crystallinity of spray-dried powders, lyophilized cakes, and ground trehalose was determined by powder x-ray diffraction (XRD) with Cu K-alpha radiation. Glass transition temperature (T_g) was determined by differential scanning calorimetry (DSC). Size distributions of spray-dried particles were determined by flow imaging microscopy (FIM) using a FlowCam VS. Powder morphology was evaluated by scanning electron microscopy (SEM) using a Hitachi SU3500 Variable Pressure SEM.
Titer assays: GFP-encoding BV in insect cell media (Sf900 III SFM), liquid formulation, spray-dried powder, or lyophilized formulations were assessed for titer (activity) immediately after processing or after incubations between 4 – 97°C after 1 hour – 10 weeks. Titers were determined by resuspending BV preparation and transfecting insect cells. After 48 hours, cells were washed twice in fresh Sf900 III SFM and analyzed by flow cytometry. Titer was back-calculated based on the fraction of transfected cells, determined by gating for live single cells expressing GFP.

Results: Baculovirus (BV), an infectious enveloped virus, was formulated as described above. Spray-drying and lyophilizing BV in this formulation yielded amorphous glasses (by powder XRD) [Fig. 1] with T_gs between 70 – 80°C (by DSC). SEM and FIM reveal spray-dried BV particles are primarily less than 20 µm in diameter and have a dimpled morphology. Notably, BV retained all activity during spray-drying and lyophilization in this formulation [Fig. 2]. BV was stored between 4 – 40°C for up to 10 weeks. Liquid formulations of BV (as supplied in Sf900 III SFM [Fig. 3a], or in the formulation [Fig. 3b] listed above) showed significant titer loss when stored above 4°C [Fig. 3a-b]. At room temperature, BV lost over 3 log titer over 10 weeks, and at 40°C, BV lost all measurable activity in less than one week [Fig. 3a-b]. Spray-dried [Fig. 3c-3d] and lyophilized [Fig. 3e] BV preparations showed enhanced thermal stability, with less than one log loss titer occurring over 10 weeks. Additionally, spray-dried and lyophilized BV showed no titer loss after one hour incubation at up to 85°C, while liquid titer lost over 3 log loss titer at the same conditions.

Conclusion: Spray-drying and lyophilization are viable methods for embedding live viruses in a glassy matrix for enhanced thermal stability. The formulation and drying conditions used here yielded total activity retention of a live enveloped virus, and significantly enhanced thermal stability at room temperature and above. This has implications for developing vaccines and gene therapies which rely on viral vectors which are more stable, allowing for equitable global distribution and reduced product costs or wastage due to improper storage.

References: *Data presented in this poster is from a manuscript in preparation for submission and is expected to have a citation by the time of this conference*
1. Hansen, L. J. J., Daoussi, R., Vervaet, C., Remon, J. P. & De Beer, T. R. M. Freeze-drying of live virus vaccines: A review. Vaccine 33, 5507–5519 (2015).
2. Perry, C. & Rayat, A. C. M. E. Lentiviral vector bioprocessing. Viruses 13, 1–46 (2021).
3. Toniolo, S. P. et al. Excipient selection for thermally stable enveloped and non-enveloped viral vaccine platforms in dry powders. Int. J. Pharm. 561, 66–73 (2019).
4. Bee, J. S. et al. Impact of Time Out of Intended Storage and Freeze-thaw Rates on the Stability of Adeno-associated Virus 8 and 9: ‘Daisy Chain’ Stability of AAV8 and AAV9. J. Pharm. Sci. 000, 1–8 (2022).
5. Preston, K. B. & Randolph, T. W. Stability of lyophilized and spray dried vaccine formulations. Adv. Drug Deliv. Rev. 171, 50–61 (2021).

Acknowledgements: The authors have submitted a provisional patent related to this work. The patent application number is 63/456,720 (CU6185B).

XRD spectra of spray-dried and lyophilized placebo samples and unprocessed trehalose. Unprocessed trehalose (n = 1 scan) is highly crystalline and shows agreement with the literature for characteristic peaks of trehalose dihydrate. Spray-dried (SD) placebo powder, using heated air at an inlet temperature (Tin) of 88 or 50 °C, and lyophilized placebo cakes (n = 2 – 4 scans) were highly amorphous.

Titer of BV in liquid formulations prior to drying (dark bluebars) and in reconstituted lyophilized or spray-dried formulations (lightblue bars). No loss of titer occurred during spray-drying BV at drying gas inlet temperatures of 88 °C (n = 7) or 50 °C (n = 3) or lyophilization (n = 3).

Log loss titers for BV during storage in liquid, spray-dried powders, and lyophilized cakes. BV was incubated at 4 °C (blue squares), 25 °C (green circles), 30 °C (orange triangles) or 40 °C (red diamonds) as supplied (viral stock, a), after formulation (b), after spray-drying at a Tin of 88 °C (c) or 50 °C (d), and after lyophilization (e). Spray-dried and lyophilized BV show enhanced storage stability at 25 °C and above compared to liquid stock and formulated BV (n = 3). nd = not determined due to titer below detection limit of assay.