Enhancing lyophilized cake morphology in biopharmaceutical products through stepped annealing at freezing phase

Lyophilization is widely used to stabilise biopharmaceuticals, yet split‑cake formation remains a challenge. Cake elegance, a critical quality attribute (CQA), is influenced by both formulation and process parameters. As biosimilar manufacturers are constrained from altering formulation composition, optimisation of process parameters becomes the primary strategy. We hypothesised that precooling combined with two‑step annealing during freezing would facilitate freeze‑concentrate migration into microchannels, yielding homogeneous ice crystallisation and elegant cakes. rt‑PA protein was produced using CHO cells and the protein formulation contained L-Arginine and Polysorbate 20. Critical temperatures were determined using Freeze‑Drying Microscopy and Integrated Impedance‑DTA. The formulation was filled into 20 mL Lyo glass vials and lyophilization parameters were optimized to obtain an elegant cake of the rt-PA protein. Protein aggregation and potency were assessed by size‑exclusion chromatography and biological assays respectively. Replacing traditional one‑step annealing at -10 °C with precooling at -8 °C combined with two‑step annealing at -15 °C and -10 °C promoted uniform ice crystallisation, and decreased split‑cake formation. As expected, the residual moisture decreased, with a slight increase in reconstitution time for elegant cake. Robustness of the optimized method was confirmed by varying annealing conditions (±2 °C). Optimisation of freezing‑phase parameters significantly influenced cake morphology, producing elegant lyophilized matrices with desirable quality attributes. Such physical appearance is considered a CQA and is highly valued by regulatory agencies, as it reflects appropriate process control and product quality in biopharmaceutical products. This study thus establishes a process development framework that can be extended to other molecules to confirm broader applicability.