(T1330-04-19) Developability Assessment as an Early De-risking Tool for Biopharmaceuticals: Case Study with Fusion-Proteins

Purpose: Developability assessments of large molecule drug candidates can help to identify and address issues related to instability, manufacturability, delivery, safety and efficacy during early stage development, reducing development costs and time. Such developability assessments have been widely applied to monoclonal antibody (mAb) candidates. In this study, we assessed the developability of fusion proteins, which may pose development challenges due to their increased propensity for aggregation.¹ With the limited material (300 mg) and time (3 months) available in the early stages of development, we established a developability assessment workflow (Figure 1) for assessing biological drug candidates.

Methods: We used three fusion-protein candidates (X, Y, Z) as a model system to establish a developability assessment workflow. All three fusion-protein candidates were first dialyzed into 10 mM acetate pH 5.0, 10 mM histidine pH 6.0, and 10 mM phosphate pH 7.0 buffers, respectively. Due to incompatibility (precipitation during dialysis and/or concentration steps) of all three candidates with 10 mM phosphate pH 7.0, no further testing was performed in this buffer. The structural stability assessment was performed using DSC (Differential Scanning Calorimetry) and fluorescence spectroscopy where scans were recorded from 10 to 90°C at 60°C/hr scanning rate. For colloidal stability assessment, hydrodynamic radius was measured in the concentration range from 1 to 20 mg/mL using DLS (Dynamic Light Scattering). For solubility and viscosity assessments, three candidates were dialyzed and then concentrated to ~100 mg/ml in 10 mM acetate pH 5.0 and 10 mM histidine pH 6.0 buffers, respectively. For solubility assessment at 100 mg/mL proteins, appearance and turbidity at 400 nm was recorded at T0 and after storage at 2-8°C for 2 days. Viscosity assessment was performed at various temperatures (ranging from 5°C to 30°C). The storage stability of all three candidates was studied at 10 mg/mL in 10 mM acetate pH 5.0 and 10 mM histidine pH 6.0 at 37°C for up to four weeks. The storage stability of all three candidates was also studied at 100 mg/mL in 10 mM acetate, pH 5.0 at 25°C for up to four weeks. For storage stability assessment, the samples were analyzed for size and charge variants using SE-UPLC (Size Exclusion-Ultra performance Liquid Chromatography) and iCE (Imaged Capillary Electrophoresis) respectively.

Results: The structural stability assessment by DSC and fluorescence suggested that all molecules had a T_onset  value of > 40°C and a T_m value of > 50°C in both buffers (10 mM acetate pH 5.0 and 10 mM histidine pH 6.0). The colloidal stability assessment by DLS revealed that all molecules had positive k_D values in 10 mM acetate pH 5 and negative k_D values in 10 mM histidine pH 6, implying that all molecules have better colloidal stability in acetate buffer pH 5.0 than in histidine buffer pH 6.0. The viscosity assessment at 100 mg/mL showed a steeper increase in viscosity values with decrease in temperature for all molecules except for molecule Y. However, the viscosity of all molecules ranged from 3 to 7 cP at 20°C, indicating that all molecules are developable in a pre-filling syringe (PFS) presentation. Solubility assessment at 100 mg/mL showed no measurable change in turbidity and appearance in 10 mM acetate buffer while in 10 mM histidine buffer showed notable change in turbidity and phase separation after storage at 2-8°C. The storage stability assessment at 10 mg/mL showed measurable change in size variants (%HMW species) in 10 mM histidine pH 6.0 in comparison to 10 mM acetate pH 5.0 for two of the candidates (X and Y) after storage at 37°C for up to four weeks. All three candidates showed notable increase in charge variants (% acidic species) in 10 mM histidine pH 6.0 in comparison to 10 mM acetate pH 5.0. The storage stability assessment at 100 mg/mL in 10 mM acetate, pH 5.0 revealed some differences (within method variability) in the rate of HMW species and acidic species formation after four weeks of storage at 25°C.

Conclusion: Based on the developability assessment results (Figure 2), none of the molecules pose significant developability risk. This developability assessment workflow required only 300 mg for each candidate and took approximately 3 months to complete which is useful not only for selecting a candidate for further formulation optimization but facilitating the selection of initial pH/buffer conditions for Phase 1 formulation, thereby shortening development timelines.

References: 1. Fast, J. L., Cordes, A. A., Carpenter, J. F., & Randolph, T. W. (2009). Physical instability of a therapeutic Fc fusion protein: domain contributions to conformational and colloidal stability. Biochemistry, 48(49), 11724-11736.

Acknowledgments: I would like to thank my manager shalini minocha and our department at Alexion-AstraZeneca Rare Disease for giving me the opportunity to present our work. This work is funded by Alexion-AstraZeneca Rare Disease.

Figure 1. Developability assessment workflow



Figure 2. Summary of results from developability assessment in 10 mM Acetate buffer, pH 5.0