(W1030-07-37) Formulation with Hydroxypropyl ß-Cyclodextrin Preserves the Potency of Adenovirus Vectors Under Heat and Light Stress

Purpose: Adenoviral vectors have emerged as pivotal tools in the fields of vaccine development and gene therapy for their remarkable adaptability and efficiency. These vectors offer the unique advantage of easy genome manipulation, allowing researchers to precisely modify their genetic content for diverse applications such as vaccines/gene therapy. However, their in-process and long-term storage stability, especially in non-cold-chain situations, is a challenge to overcome for biopharmaceuticals.¹ The selection of appropriate excipients to protect these biologics during development, biomanufacturing, and final formulation is critical in ensuring product stability against various physical stresses throughout their shelf life.² In this study, the objective is to explore the use of hydroxypropyl ß-cyclodextrin to provide improved protection of adenoviral vector-based biotherapeutics against heat and light stress. Adenovirus serotype 5 was identified as the model virus vector due to its well-known pharmacology and wide application. The purpose is to identify alternate stabilizers that can mitigate the impact of heat and/or light stress in destabilizing the virus vectors that harbor and protect the genomic content of the biological drug. This approach aims to offer an added option to biomanufacturers of adenovirus-based biologics to improve the in-process stability of their products, as well as provide their drug products with better efficacy and/or shelf life.

Methods: Adenovirus serotype 5-Cre-GFP (Cre recombinase fusion with GFP) virus vector was purchased from SignaGen Laboratories (Maryland, USA), while the HEK293T cell line was acquired from ATCC (Virginia, USA). Biopharma grade hydroxypropyl ß-cyclodextrin (KLEPTOSE® HPB) was obtained from Roquette Frères (Lestrem, France). Ad5-Cre-GFP virus vector was quantified with the Adeno-X™ qPCR Titration Kit from Takara Bio USA (California, USA), according to the manufacturer’s instructions, using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, California, USA). In brief, Ad5-Cre-GFP virus vectors were buffer-exchanged into a citrate buffer with/without 0.02% (v/v) polysorbate 80 or 5% (w/v) KLEPTOSE® HPB (see Table 1). Formulated samples were subjected to heat stress at 25 °C/65% relative humidity (RH), 40 °C/75% RH or light and UV stress (following ICH Q1B guidelines; 1.2 million Lux hours and UV light for 200 watt-hours per square meter) for one cycle. Samples were collected and frozen at their respective time points before quantification using a 50% tissue culture infectious dose (TCID₅₀) assay. HEK293T cells were seeded in 96-well plates and allowed to reach 70-80% confluency. Virus vector samples were then diluted 10-fold (i.e., 10⁰ to 10^-7) in MEM (Life Technologies, California, USA) and used to infect a row (i.e., 10 wells) of HEK293T cells for 4 hours. Diluted virus vector samples were then removed, and wells were replaced with complete growth media (DMEM with L-glutamine and 10% FBS, Life Technologies, California, USA). Infected cells were incubated for 72 hours at 37 °C, 8% CO₂, before GFP fluorescence was measured using the TECAN Infinite® 200 Pro (PerkinElmer, Massachusetts, USA).

Results: The formulation of our model Adv5-CMV-Cre-GFP virus vector containing 5% KLEPTOSE® HPB was able to maintain a significantly higher TCID₅₀ under heat stress at 25 °C over 21 weeks (see Figure 1A). In the presence of 0.02% polysorbate 80 (i.e., F2), allowed for a 3–5-fold improvement in potency versus the control buffer F1 up to week 12. However, beyond that, the infectibility of Adv5-CMV-Cre-GFP in F2 buffer fell to levels that of the control buffer, eventually illustrating a 2-log reduction of TCID₅₀ at week 21. In contrast, F3, containing KLEPTOSE® HPB, conferred superior protection, showing 10-fold higher retention in potency vs. those without after 21 weeks. Similarly, at 40 °C, although all formulated Adv5-CMV-Cre-GFP virus vectors fell in potency, samples in F3 remained above detection levels up to week 12 (see Figure 1B). When subjected to white light-only or UV-only stresses, formulation F3 containing 5% KLEPTOSE® HPB demonstrated 100% potency while virus vectors in F1 and F2 fell nearly a log in TCID₅₀ value (see Figure 2). When treated with both white light and UV stress, F1-formulated samples plummeted 2-logs in infectability, while Adv5-CMV-Cre-GFP in F2 and F3 showed less reduction in potency (i.e., 1-log reduction).

Conclusion: Adenoviral vectors are known to have stability issues during their manufacturing processes as well as in long-term storage without a robust cold chain. The study demonstrated the effective use of KLEPTOSE® HPB as an alternative excipient to protect adenovirus vectors against heat and light/UV stresses. In addition, compendia excipients that are safe and nonhazardous and can thus be easily incorporated into the product stream without the requisition of removal validation. Our findings also suggest the function of cyclodextrins in extending the stability and shelf life of the adenoviral delivery vehicle, and potentially, other similar systems.

References: 1. Rexroad, Evans, et al. “Effect of pH and ionic strength on the physical stability of adenovirus type 5.” J Pharm Sci. 2006 Feb;95(2):237-47. doi: 10.1002/jps.20496.
2. Ma, Su, et al. “The Effect of Residual Triton X-100 on Structural Stability and Infection Activity of Adenovirus Particles.” Mol Ther Methods Clin Dev. 2020 Dec 11; 19: 35–46. doi: 10.1016/j.omtm.2020.08.013.

Figure 1. TCID50 values of Ad5-Cre-GFP in the presence of different excipients after being subjected to heat stress at (A) 25 °C/65% RH and (B) 40 °C/75% RH.

Figure 2. TCID50 values of Ad5-Cre-GFP in the presence of different excipients after being subjected to UV stress of 1.2 million Lux hours and UV light for 200 watt-hours/m2.

Table 1. Formulation buffers for Adv5-CMV-Cre-GFP virus vector.