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1、蛋白质稳定性与 实验设计（DOE）去除蛋白聚 合体 Meets the Pace of Tomorrows Process Development Needs 孙文改 13911133967 Wen-gai.sun 2 / DOE and KTAavant * Protein Stability and Aggregates Removal Based on DOE Protein Stability DOE for Protein Purification Optimization DOE Result Evaluation Aggregates Removal Based on DOE A
2、KTAavant Introduction 3 / DOE and KTAavant * Solvated phaseLess solvated phase Adsorption Crystallisation Aggregation Change in: Temperature Solvent Ionic strength pH Protein Aggregation Proposed mechanisms 4 / DOE and KTAavant * Protein Aggregation Proposed mechanisms Native protein Larger aggregat
3、es Partial unfolding Oligomers of non-native protein Larger aggregates Oligomers of native protein 5 / DOE and KTAavant * Protein aggregation can be minimized by stabilizing the native state reducing protein-protein interactions avoiding - mechanical stress - repeated freeze-thawing - high protein c
4、oncentrations 6 / DOE and KTAavant * Additives proposed to stabilize the native state Hydration shell is strengthened by kosmotropes (”water structure makers”) Glycerol Sucrose Sorbitol Glycine 7 / DOE and KTAavant * Additives proposed to reduce protein- protein interactions Potential protein-protei
5、n interaction sites are shielded + - Hydration shell is weakened by chaotropes (”water structure breakers”) Polyethylene glycol Nonionic detergents Citrate Urea Arginine 8 / DOE and KTAavant * Commonly used additives to minimize protein aggregation AdditiveProposed mode of action Glycerol (5 - 40%)
6、Sucrose (10 - 40%) Glycine (0.02 0.5 M) Sorbitol (5 - 40%) Stabilizes native, intramolecular protein interactions Polyethylene glycol (1 - 15%) Nonionic detergents (below cmc) Shields surface exposed hydrophobic sites (reduces protein-protein interactions) Citrate (0.02 - 0.4 M)Shields surface expos
7、ed charged sites (reduces protein-protein interactions) Urea ( 2 M) Arginine ( 2 M) Reduces protein-protein interactions Dithiothreitol, DTT (0.1 1 mM)Prevents formation of intermolecular S-S bonds 9 / DOE and KTAavant * Protein stability assessment - differential scanning calorimetry (DSC) Measures
8、 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 MicroCal system 10 / DOE and KTAavant * 100% Unfolded protein Tm shift reflects increased stability Tman indicator of stability 11 /
9、DOE and KTAavant * 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. Further Processing Protein A Capture Elution Ho
10、ld Step UF/DF 12 / DOE and KTAavant * Identification of optimal elution conditions DSC identified the most stabilizing elution conditions. This allowed increased loading capacity since mAb is stabilized. 13 / DOE and KTAavant * Liquid formulation stability evaluation Excerpted from: Remmele, et. al.
11、, Pharmaceutical Res. 15, 200-208 (1998) Excepient typeExcepientTm (C) Control-48.1 SugarsManitol Glucose 46.7 49.6 Polymers / Polyols PEG Ethanol (low) Ethanol (high) 49.4 48.7 43.8 SaltsNaCl CaCl2 53.1 41.1 SurfactantsPluronic F6846.6 45.8 Glucose/NaCl-52.2 14 / DOE and KTAavant * Protein Stabilit
12、y and Aggregates Removal Based on DOE Protein Stability DOE for Protein Purification Optimization DOE Result Evaluation Aggregates Removal Based on DOE AKTAavant Introduction 15 / DOE and KTAavant * Introduction Process knowledge, development and outcome Traditional approach: Parameter 1 Fixed, Para
13、meter 2 changed or Parameter 2 Fixed, Parameter 1 changed DoE arranged approach: Vary all simultaneously - ”Design of Experience” Optimum Parameter 1 Parameter 2 Parameter 2 Parameter 1 16 / DOE and KTAavant * The Classical/Intuitive/”One-at-a-time” Approach Example: Improving Yield of a process (Th
14、e higher the Better) From Past Experience: pH MOR: Manufacturing Operating Range (BLA); PAR: Proven acceptable range (validation) pH7.0pH7.1pH6.9pH6.8pH6.7pH7.2pH7.3 23 / DOE and KTAavant * Totally benefits: Reduces Time to Market! Drug Discovery Pharmaceutical development Clinical trialsMarket One
15、year reduction of TTM a) $40m savings in R&D b) 20% increase in sales 20 years Ph1Ph2Ph3 Filed 24 / DOE and KTAavant * Introduction Responses: External data: Capacity DBC (Frontal analysis) Yield Purity/Selectivity Molecular weight Activity HCP DNA Aggregates Protein A Peak Data: Area Concentration
16、Amount Resolution Asymmetry Plates per meter Factors: Load pH Load conductivity Load concentration Mass load Wash volume Wash pH Wash conductivity Elution pH Gradient elution Step elution level Cut OD Elution Additive Media type Column size Bed Height Flow rate Residence time Sample conditions Wash conditions Elution conditions Entire process Compl
