Investigation of the Impact of a Protein Source on the Purification of l‑Asparaginase Type II from.

l-asparaginase type II (EcA2) is essential for treating childhood acute lymphoblastic leukemia (ALL), improving survival rates since its introduction. After the expiration of its original patents, interest in producing biosimilars has increased, particularly to reduce treatment-related side effects. In this study, we compared two production methods for EcA2, purifying the enzyme from broth and from the soluble fraction of the cell pellet lysate. Using a converging purification workflow, we obtained 66.3 (±2.3) mg/L of l-asparaginase from broth and 29.2 (±4.6) mg/L from the cell pellet lysate, with specific activities of 136.3 (±13.3) and 123.5 (±10.3) IU/mg, respectively. Both versions showed similar three-dimensional structures, thermal stability, and specific activity, with no significant differences in performance. Proteomic analysis revealed that purification from broth resulted in fewer host cell proteins than purification from the cell pellet lysate. Our results further suggest that the purification process from cell lysate is more susceptible to variability than purification from the broth. These findings demonstrate that while both production methods yield comparable enzymes in terms of structure and activity, purifying from broth may offer advantages in terms of lower contamination and better reproducibility.