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1、Method Development and Process Optimization Strategy for Protein-based Therapeutics 蛋白大分子纯化工艺优化策略蛋白大分子纯化工艺优化策略 Michael Li, Ph.D. 李国荣李国荣 博士博士 Sun Year Sept 11 上海申越公司上海申越公司 2011-09-11 Resume of Michael Li 个人简介 Ph.D. in Chemistry 化学系博士 Thirty years laboratory and process development of biopharmaceutica
2、ls 30年實驗和层析過程 Member of the Industrial Advisory Committee, Preparative 2010 and 2011, USA 美国工业层析会顧問之一 First author of a provisional patent on the development and applications of linear pH gradient to separate and to purify proteins 在美国巳申请用线牲pH梯度分开和纯化蛋白質 PAT-based In-Line Buffer Dilution in Downstrea
3、m Bioprocessing, PharmaTech Supplement, 10/2010 基于PAT的 缓冲液配制系统在下游的应用 PAT-based In Line Buffer Dilution: Serving the Paradigm of Quality by Design, Bioprocess International, 01/2010基于PAT 的缓冲液配制系统適合質量设计的要求 Table of Contents 内容内容 Protein Source and Potential Contaminants 蛋白质来源及其可能存在的杂质 Factors Affectin
4、g Protein Stability 影响蛋白质稳定性的因素 Purification and Process Optimization Strategy 纯化工艺优化策略 Scale-up Challenges 规模化生产的挑战 Chromatography and ultra filtration Protein Source and Potential Contaminants 蛋白质来源及其可能存在的杂质蛋白质来源及其可能存在的杂质 Common impurities常见的杂质常见的杂质 DNA脱氧核糖核酸 Host cell proteins宿主细胞蛋白 Proteases蛋白酶
5、Endotoxins (lipopolysaccharide)内毒素（脂多糖） Viruses病毒 Truncated proteins短链蛋白 Dimers二聚体 Misfolded proteins错误折叠的蛋白质 Degradation products降解产物 Factors Affecting Protein Stability 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 Physical and chemical property of the protein 蛋白质的理化性质蛋白质的理化性质 Solubility in water and in organic solvents
6、在水或有机溶剂中的溶解性 Hydrophilic or hydrophobic亲水性或疏水性 pI of the protein蛋白质的等电点 pH stability pH稳定性 Temperature stability 温度稳定性 Net charges 净电荷 Size and shape of the protein in relationship to other contaminants 相关 蛋白质杂质的大小和形状 Probability of unfolding and misfolding展开或错误折叠的可能性 De-naturation 变性 Factors Affect
7、ing Protein Stability 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 pI 等电点 pH Solubility behavior 溶解度行为 Light 光 照 Solvent 溶剂 Heat 热度 Presence of proteolytic enzymes蛋白分解酶的存在 Excipients 保护剂 Additives- PEGylation 聚乙二醇添加剂 Air 空气 Factors Affecting Protein Stability 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 Chemical- bond cleavage and formation a
8、ffecting primary structure 化学键断裂和构成 De-amidation脱酰胺作用 Hydrolysis 水解 Oxidation and reduction氧化还原作用 Disulfide exchange 二硫键交换 Physical- structural changes relating to secondary, tertiary and quarternary 有关蛋白质二级、三级、四级物理结构的变化 Factors Affecting Protein Stability 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 Physical- affects se
9、condary, tertiary and quarternary structure of the polypeptide 影响多肽的二级，三级和四级物理结构 Aggregation聚集体 Adsorption吸附 Unfolding展开 Misfolding错误折叠 Precipitation沉淀 Factors Affecting Protein Stability 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 Chemical- bond cleavage and formation affecting primary structure 化学键断裂和构成化学键断裂和构成 De-ami
10、dation- removal of amide bond 脱酰胺- 去掉酰胺键 R-CO-NHR”- R-COOH + RNHR” Asparagine to aspartic acid 天冬酰胺酸-天冬氨酸 Glutamine to glutamic acid 谷氨酰胺-谷氨酸 Facilitated by elevated temperature and pH 温度升高和pH的变化 Loss of biological activity 生物活性的丧失 Factors Affecting Protein Stability 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 R.M. Rigg
11、in et. al., Anal. Biochem. 167, Pg. 199-209 (1987) Factors Affecting Protein Stability 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 Hydrolysis and rearrangement 水解后重新组合 Factors Affecting Protein Stability 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 M.R. Schlittler, P.C. Toren, N.R. Siegel & B.N. Violand, Ninth ISPPP, 1989, Abst. 621 Factors
12、Affecting Protein Stability 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 Oxidation and reduction氧化和还原 Oxidation is the addition of oxygen(s) or the removal of hydrogen(s) from the molecule Methionine and cysteine 蛋氨酸和半胱氨酸 Air- oxygen 空气-氧气 Metal ions- copper and iron 金属离子-铜或铁离子 pH Light光照 Factors Affecting Protein Stabil
13、ity 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 M. Kunitani and D. Johnson, J. Chrom. 371, 313-333 (1986) Factors Affecting Protein Stability 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 Factors Affecting Protein Stability 影响蛋白质稳定性的因素影响蛋白质稳定性的因素 J.Miller et al. Biochem,32,5203-5213(1993) Purification and Process Optimization Strategy 纯化工艺优化策
14、略纯化工艺优化策略 Goal 目标目标 Purity 产品纯度 Recovery 产品回收率 Throughput 生产过程效率 Process economics 生产成本 Purification and Process Optimization Strategy 纯化工艺优化策略纯化工艺优化策略 Chromatography steps: 1. Capture- goal 捕获目标捕获目标 Isolation, rapid concentration, volume reduction and elimination of proteases 分离，快速浓缩，体积减少和去除蛋白酶 Com
15、mon techniques: IEX 常用技术：离子交换层析 Purification and Process Optimization Strategy 纯化工艺优化策略纯化工艺优化策略 Chromatography steps: 2. Intermediate goal中间目标中间目标 Removal of major impurities, 99+ purity 去除主要杂质，纯度达到99% Common techniques: IEX/HIC/AC 常用技术：离子交换层析/疏水作用层析/亲和层析 BioAssist Q 10x10 50mL Inject 阴阴离子交离子交换层换层析分离析分离 -5000500100015002000250030000510152025Retention time (min)mAU 280nm01020304050607080mS/cm-2003008001300180023000510152025Retention time (min)mAU 280nm020406080100120140160180200mS/cmBioAssist Phenyl 7.8mmIDx5cm 5mLInject 疏水作用疏水作用层层析分离析分离 Sepa
