Running on the verge of collapse in lyophilization: What is the impact of the edge vial effect on colloidal protein stability and particle morphology?

Lyophilization is frequently employed to stabilize sensitive biopharmaceuticals. The potential importance of the edge vial effect, defined as the discrepancy in temperature and drying behavior between vials located in different positions of the batch, attracts considerable attention from the lyophilization community. Here, we lyophilized two model fusion-protein formulations by applying a design space representation to select conservative as well as aggressive lyophilization protocols on the verge of structural collapse of the cake. The effect of vial position and number of neighbor vials on cake appearance, cake volume, and residual moisture content was quantified. Moreover, the stability of the fusion-protein was assayed in relation to monomer content, size distribution of submicron and subvisible particles, as well as the presence of visible particles. Additionally, the images of micron-sized particles were analyzed by using a machine-learning based "fingerprinting" method to identify potential morphological differences induced by the edge vial effect. We concluded that the investigated quality attributes, including cake appearance, residual moisture, particle burden, and particle morphology, were not significantly affected by the edge vial effect. In contrast, the aggressive lyophilization cycle on the verge of collapse showed a minor, formulation-dependent impact on cake volume and particle count.