Development of a stable lyophilized adeno-associated virus gene therapy formulation

Abstract

Adeno-associated viruses (AAV) are among the most actively investigated vectors for gene therapy. Supply of early clinical studies with frozen drug product (DP) can accelerate timelines and minimize degradation risks. In the long-term, logistical challenges of frozen DP may limit patient access. In this work, we developed a lyophilized (freeze-dried) formulation of AAV. The mass concentration of AAV is typically low, and AAV also requires a minimum ionic strength to inhibit aggregation. These factors result in a low collapse temperature, which is limiting to lyophilization. Mannitol crystallization was found to cause extensive degradation and potency loss of AAV during the freezing step. With further development, we determined that AAV could be lyophilized in a sucrose and citrate formulation with a more desirable high glass transition temperature of the dried cake. An optimal residual moisture range (1–3%) was found to be critical to maintaining AAV8 stability. Glycerol was found to protect AAV8 from over-drying by preventing capsid damage and genome DNA release. A lyophilized formulation was identified that maintained potency for 24 months at 2–8 °C, indicating the feasibility of a dried formulation for AAV gene therapy.

Introduction

Gene therapy is designed to treat disease by delivering gene coding or editing material. In recent years, adeno-associated virus (AAV) vectors have attracted significant attention as a potentially safe and efficacious gene delivery vehicle. There are more than 190 AAV clinical trials at different stages that target many different indications, and the majority are for unmet medical needs (Auricchio et al., 2017, Boye et al., 2013, Rieser et al., 2020, Schön et al., 2015, Trapani et al., 2014). The fast-track/breakthrough designations and the criticality of those therapies to patient lives often result in greatly accelerated development timelines that reduce the time for formulation development (Kepplinger, 2015). The two FDA-approved AAV gene therapy products, LUXTURNA® (voretigene neparvovec-rzyl) and ZOLGENSMA® (onasemnogene abeparvovec-xioi), are stored at <−60 °C, indicating the challenges associated with stabilizing AAV for long-term storage under refrigerated conditions. The ZOLGENSMA package insert states that it is stable for 14 days from receipt when stored at 2–8 °C (ZOLGENSMA, 2019). Gruntman et al have reported a statistically significant decrease of in vivo potency after the storage of AAV1 at 4 °C for 4 weeks (p = 0.005) and 12 weeks (p = 0.0071), whereas there was minimal potency loss when samples were stored at −80 °C (Gruntman et al., 2015). The impetus for this work was based on our own similar findings that there was higher potency loss for AAV8 when stored at 2–8 °C than when stored at ≤−60 °C. Because gene therapy clinical trials for rare diseases can take several years to conduct, and because of large amount of resources needed to produce a batch, the drug product is currently stored frozen to minimize degradation that would occur in a liquid form at 2–8 °C. Lyophilization has been established as an approach to facilitate refrigerated storage of biologics that do not have adequate stability in a liquid form.

It is challenging to develop AAV formulations because of the complex nature of AAV particles, limitations of analytical methods, lack of prior knowledge about AAV physical and chemical stability, and limitations posed by routes of administration. AAV particles are complex macromolecular assemblies of 60 capsid proteins and up to a 4.7 kb single-stranded DNA (ssDNA) genome. AAVs are susceptible to a variety of chemical and physical degradation pathways (Wright, 2014, Wright, 2021). In addition to demonstrating safety and efficacy, an AAV product must remain stable and potent during manufacturing, shipping, storage, and administration. Viral particle degradation poses challenges for the fill/finish processes as well as product quality during long-term storage.

Lyophilized (freeze-dried) formulations offer the potential to improve stability by significantly reducing the degradation rate due to the presence of water. Wright et al have reported a minimum solution ionic strength is required to prevent particle aggregation (Wright et al., 2005). NaCl and buffer salts decrease the collapse temperature of the maximally freeze-concentrated solution unless they are completely crystallized (Her et al., 1995). The requirement for a minimum ionic strength is therefore a challenge to the development of a lyophilized formulation. The mass concentration of AAV is typically low, which does not help with increasing the collapse temperature as is often the case for other biologics such as monoclonal antibodies (Depaz et al., 2016).

In this study, we tested the possibility of inhibiting AAV aggregation with a low concentration of multivalent salt at a level that would still allow for a practical lyophilization cycle. In addition, the impact of buffer systems, crystalline and amorphous excipients, and moisture content on AAV stability during the lyophilization process were evaluated, and the critical factors for maintaining AAV stability were identified. More importantly, we demonstrated the potential of a lyophilization formulation for AAV long-term storage under refrigerated storage conditions.