A new design of experiments—superlative box design (SBD), was adopted to optimize the adaptation of Chinese hamster ovary cells from adhesion culture to serum-free suspension culture. It is a general trend to use a serum-free medium instead of a serum-containing medium. The advantage of serum-free medium (chemically defended) is that it does not contain unknown components and avoids safety issues. SBD requires fewer experiments while ensuring a sufficient number of experiments and uniformity in the distribution of experiments amongst all the factors. Six factors were considered in this experimental design with 43 runs plus three more repeating center runs. The cell line was adapted to serum-free media by gradually reducing serum, and from adherent to suspension by rotating at various speeds in a shake flask. Response surface methodology was applied to find the optimum condition. The optimized cell density reached 7.02 × 10⁵ cells/mL, calculated by the quadratic model.

Experiments validated the predicted cell adaptation with the maximum cell density. Three suspension runs were selected randomly to perform in the bioreactor to validate cell stability and production homogeneity. This study provides an efficient method to transfer adherent cells to suspension cells and is the first to successfully use SBD and establish a parameter quadratic optimization model.