Background <p>High concentration protein formulation (HCPF) development needs to balance protein stability attributes such as conformational/colloidal stability, chemical stability, and solution properties such as viscosity and osmolality.</p> Methodology <p>A three-phase design is established in this work. In Phase 1, conformational and colloidal stability are measured by 384-well-based high-throughput (HT) biophysical screening while viscosity reduction screening is performed with HT viscosity screening. Collectively, the biophysical and viscosity screening data are leveraged to design the phase 2 of short-term stability study, executed using 96-well plates under thermal and freeze/thaw stresses. In phase 2, samples are analyzed by stability-indicating assays and processed with pair-wise Student’s t-test analyses to choose the final formulations. In phase 3, the final formulations are then confirmed through a one-month accelerated stability in glass vials.</p> Results <p>Using a model antibody A (mAb-A), the initial HT screening successfully established the 384-well based platform. A lead formulation was chosen from the second round based on statistical analyses and subsequently tested against the commercial formulation of mAb-A as a control. Compared to the control, the lead formulation reduced the viscosity of mAb-A by 30% and decreased subvisible particles after thermal stress by 80%.</p> Conclusions <p>HT biophysical screening in 384-well plates was demonstrated to effectively guide the rational design of a high-throughput stability screening study using 96-well plates. This platform enables the identification of a high concentration formulation within seven weeks within the first two phases of study that strategically balance stability with solution properties, thus achieving a rapid development of HCPF.</p>