Streamlining process development and scale-up: Risk assessment to reduce workload in primary protein recovery

Risk assessment is an integral aspect of the Quality-by-Design strategy to identify potential obstacles at every stage of biopharmaceutical production, from process development to tech transfer. We explored Flow Process Charts, root cause analysis, and failure mode and effects analysis, to assess the scale-up of bacterial cell disruption and its influence on centrifugation and filtration steps. The Ishikawa diagram suggests that data on the impact of homogenizer valve design on product release, impurity profile, particle size distribution, viscosity, and dsDNA fragment size are missing which were collected experimentally for this study. Cell lysates from micro-, lab- and pilot scales cell disruption were analyzed for the above-mentioned parameters. Process parameters affecting these output parameters were identified on each individual scale. Cell disruption on the micro scale was performed in a bead mill. High pressure homogenization was used on lab- and pilot scales. Cell disintegration by bead milling delivers homogenates of product and impurity content comparable to those on bench scale but with 3-fold higher viscosity and significantly larger dsDNA fragments, 8.0 instead of 1.0 kbp, respectively. Miniaturized pressure flow curves identified dsDNA fragment sizes as critical for filter performance during clarification. Combining risk assessment, micro scale cell disintegration and bench scale pressure flow curves allows for selective and efficient process development, and scale up for primary recovery steps. • Risk assessment for primary recovery of biopharmaceuticals recombinantly produced in Escherichia coli • Comparative analysis between microscale bead milling and high-pressure homogenization conducted. • Importance of risk assessment throughout the biopharmaceutical production process, emphasizing its utility from development to scale-up and tech transfer. • Combination of risk assessment, microscale experiments and miniaturized pressure flow curves allows an efficient process development and scale up of primary recovery steps