Integrated Lab-Scale Process Combining Purification and PEGylation of l-Asparaginase from Zymomonas mobilis

Abstract

During the last quarter of a century, PEGylation has been used to enhance the clinical effectiveness of various therapeutic proteins. However, the conjugation process significantly increases the costs of the process. This parameter is critical, especially for biosimilar products that do not have patent protection. The integration of protein purification to its conjugation could potentially reduce total costs. Following this hypothesis, in this work, the purification of L-asparaginase from Zymomonas mobilis, produced recombinantly in Escherichia coli, linked to its conjugation with a PEG of 12 kDa, was studied. The effect of operating parameters on cell rupture by high-pressure homogenization was analyzed, and the optimal values for the conditions in this study were determined. The chromatographic capture and final purification steps were also studied. It was possible to elute the protein from ion-exchange chromatography, used as final purification, in the optimal buffer for the PEGylation step. This result allowed us to eliminate the buffer change step that is usually carried out at the beginning of the PEGylation process. The conjugate obtained by this continuous process showed higher thermal stability and slower protein degradation compared to the native protein. This continuous process not only has great potential to reduce costs but could also be useful for studying the effect of different parameters on the overall performance of the process. This knowledge is especially important to establish a quality by design approach at the industrial scale.