(T1030-10-58) Rapid Development of High Concentration Protein Formulation Driven by High-Throughput Technologies

Purpose: High concentration protein formulation (HCPF) is of considerable interest to developers. To develop HCPF, protein stability attributes such as conformational stability, colloidal stability, chemical stability often need to be balanced with solution properties such as viscosity and osmolality. It is well-known that some of these attributes reach their optimum at different pH and with different types of excipients. To successfully balance the need to optimize these multiple attributes, high-throughput experimentation often needs to be employed to cover more design space and increase statistical power.

Methods: We present a two-round design for HCPF development. The first round of high-throughput biophysical screening uses 384-well plates to maximize the throughput of Tm by DSF, Tagg by SLS, solubility by PEG-challenge, and ΔG by ICD. We further present high-throughput viscosity screening by strictly controlling formulation accuracy and viscosity measurements using chip-based micro-TFF, variable pathlength UV measurements, and capillary-based viscosity measurements. Taken together, these high-throughput biophysical screening studies allow for rationally designed stability study, which is executed in the second round using 96-well plates for thermal and freeze/thaw stress. In the short-term stability using 96-well plates, samples are analyzed by SEC, R/NR-CGE, and charge variants. the results are passed through statistical analyses using pair-wise Student's t-test. The formulation chosen from the high-throughput stability study are confirmed in drug product stability study with one-month accelerated stability in glass vials.

Results: Using a guselkumab biosimilar, the initial high-throughput screening successfully established the 384-well based platform, allowing for a streamlined automated operation using commercial 384-well plates. The results show high degrees of correlation between Tagg, PEG assay, and A2. Moderately good correlation was found between Tm and ΔG. Viscosity screening data identified three viscosity-reducer to be used in the stability study. These results were summarized in contour plot and used to narrow down the design space for the second round. A lead formulation was chosen from the second round based on statistical analyses. this formulation was tested against the commercial formulation of guselkumab as control. Compared to control, the lead formulation reduced viscosity of guselkumab biosimilar by 30% and reduced subvisible particle after thermal stress by 80%.

Conclusion: We successfully demonstrate the effectiveness of using high-throughput biophysical screening in 384-well plates to guide the rational design of a high-throughput stability screening study using 96-well plates. The resultant formulation chosen based on high-throughput analytics and statistical analyses is shown to be better-performing in both viscosity and stability compared to the commercial formulation control.

Acknowledgements: This work is fully funded by Wuxi Biologics and all authors are current employees of Wuxi Biologics USA.

Correlational analyses shows the good correlation among conformational stability indicators such as Tonset, Tm1, Tm2, and Cm. Excellent correlation was also found between Tagg, C-PEG, and A2.

A contour plot was used to find the optimum conditions using Tm, Cm and C-PEG. This result was checked against viscosity profiling data to rationally design the stability study plate.

Statistical analyses based on SEC, NR-CGE, and iCE on the thermal stress compared to T0. Pair-wise Student's t test shows the best conditions among the 96 formulations. The lead formulation was tested against the commercial formulation in drug product development study.