蛋白质稳定性及实验设计DOE去除蛋白聚合体.pptVIP

Title or job number 蛋白质稳定性与 实验设计（DOE）去除蛋白聚合体 Protein Stability and Aggregates Removal Based on DOE Protein Stability DOE for Protein Purification Optimization DOE Result Evaluation Aggregates Removal Based on DOE AKTAavant Introduction Protein Aggregation Proposed mechanisms Protein Aggregation Proposed mechanisms Native protein Protein aggregation can be minimized by stabilizing the native state reducing protein-protein interactions avoiding - mechanical stress- repeated freeze-thawing- high protein concentrations Additives proposed to stabilize the native state Additives proposed to reduce protein-protein interactions Commonly used additives to minimize protein aggregation Protein stability assessment - differential scanning calorimetry (DSC) Measures the temperature associated with a conformational change of a protein Tm is the thermal transition midpoint:50% native / 50% unfolded Tm is an indicator of stability Tm…an indicator of stability Protein A capture step development Low capacity of resin (2mg/ml). Increased loading caused precipitation in column eluate. Precipitation of the protein at low pH is a major obstacle. Protein must be stabilized in the elution buffer. Identification of optimal elution conditions DSC identified the most stabilizing elution conditions. This allowed increased loading capacity since mAb is stabilized. Liquid formulation stability evaluation Protein Stability and Aggregates Removal Based on DOE Protein Stability DOE for Protein Purification Optimization DOE Result Evaluation Aggregates Removal Based on DOE AKTAavant Introduction The Classical/Intuitive/”One-at-a-time” Approach Example: Improving Yield of a process (The higher the Better) From Past Experience: pH Cond. influence AIEX Yield. One factor changed at the time Example continued DoE benefits: Truly optimal conditions are found allowing better processes DoE benefits: reduces PD cost time DoE benefits: Validation  -Estab