提高蛋白载量 改善质量传递
Conventional Surface
Protein
Porous particle
Grafted Layer of a Hydrophilic Polymer
高动态载量和更快的质量传递
Capto Q/SP ImpRes --快流速高分辨率 填料
40 μm粒径 Capto™ ImpRes
同样适用于使传统IMAC填料金属离子脱落的样品
Sample kindly provided by Drs.Wolfgang Knecht (CVGI iMed Bioscience) and Paul Wan (Discovery Sciences), AstraZeneca R&D, Mölndal, Sweden
蛋白质层析新填料应用与选择
Imagination at work.
做好分离纯化需要什么？
层析设备
控制软件
技术方案
填料
生物分子特性
Ion exchange chromatography (IEX) 离子交换层析
净电荷 生物学亲和性
Tag, PTM
Affinity chromatography (AC) 亲和层析
Capto Core 700 优点:
• 高纯度和高回收率 • 高载样量和更短的接触时间 • 操作灵活适合工艺放大 高生产能力的同时提高工艺 的经济性
Capto Core 700 vs Gel Filtration
LAIV virus purification
HCP and fragmented Virus in void DNA in total volume Virus in FT HCP and fragmented DNA in CIP
+ 分子质量小，免疫原性低 + 易于进入实体瘤 + 容易表达，可以使用酵母或者大肠杆菌
- 抗原结合活性相对较弱 - 半衰期短
Capto Core 700 凝胶过滤和吸附层析复合模式
天然琼脂糖基架外壳
-没有表面基团 -排阻大分子>700kDa
激活内核-带辛氨基 (多通道配体)
-目标物穿透
-进入孔内的小分子被捕捉 -通过CIP再生
Zstab
Zstab Zstab Zstab Zstab
N6 N3
Cys
SuRe Ligand for Mabselect SuRe Mr 26748
OH O O O OH
OH O O O O + HS–
Prot A
S – ligand
Single-point attached thioether bond
for reduction to below detection limit of < 3 organisms /mL
0.5 M NaOH effectively inactivates bacteria, mold and yeast
endotoxin (ng/mL)
Inactivation of endotoxin
Media
Sepharose™ 4 FF
Virus Recovery (HA*) [%]
100
Host Cell Protein Removal [%]
95
Amount of loaded feed material [CV]
Organism C. albicans A. niger P. aeruginosa S. aureus
1
Type yeast mold bacteria gram bacteria gram +
Time1 1h 1h 1h 1h
On average, 0.5 M NaOH results in 0.5 LRV higher inactivation versus 0.1 M NaOH
SDS-PAGE (reduced conditions 1. LMW-SDS Marker Kit 2. 0 mM EDTA 3. 2 mM EDTA 4. 10 mM EDTA
Sample kindly provided by Drs.Wolfgang Knecht (CVGI iMed Bioscience) and Paul Wan (Discovery Sciences), AstraZeneca R&D, Mölndal, Sweden
纯化CHO细胞培养基中分泌表达的蛋白 Ni Sepharose™ excel vs. Ni Sepharose 6 FF
Sample: 250 ml mPAI-1-(his)8 secreted into GIBCO™ CD CHO medium, pH 7.0 RESULTS Blue – Ni Sepharose excel packed in Tricorn ™ 5/50: Purity >98% Green - Ni Sepharose 6 FF packed in Tricorn 5/50: No recovery of target protein
Increase 小粒径，大改变
Superdex 200 Increase 更高的分辨率，更短的纯化时间
ed resolution
0.5 ml/min -> 48 min
Decreased runtime
0.5 ml/min -> 48 min
Superdex 200 10/300 GL:
R
O O CH2OH O O O O HO O O
6.0 5.0 4.0 3.0 2.0 1.0 0.0
Capto Family (Medium porosity)
R
Increasing flow rate
Capto高流速琼脂糖
表面修饰
减少非特异性吸附 控制内表面积 通过提高内表面积提高载量
表面衍生物
Final concentration and clarification IMAC with conventional Ni resin
Final concentration and clarification
Time savings IMAC with conventional up to 50% Ni resin
3 小时
0.5 M NaOH 有效灭活病毒，降低生物负荷及内 毒素
Increased viral inactivation
10.0 LRV 5.0 0.0 HIV BVDV CPV BHV POL 0.5 M SV-40 MLV ADV 0.1 M
Increased bioburden control
SDS-PAGE (reducing conditions) 1. LMW-SDS Marker Kit 2. Start material 3. Ni Sepharose excel, flowthrough 4. Ni Sepharose excel, wash 5. Ni Sepharose excel, eluate 6. Ni Sepharose 6 FF, flowthrough 7. Ni Sepharose 6 FF, wash 8. Ni Sepharose 6 FF, eluate
0.5 ml/min -> 48 min
1.5 ml/min -> 16 min
Superdex 200 Increase 10/300 GL:
磁感应蛋白MagR
Superose 6 Increase
专为大蛋白和蛋白复合体纯化设计
Protein A抗体亲和填料
MabSelect SuRe (2005) MabSelect (2001) MabSelect Xtra (2005) MabSelect SuRe LX (2012) rmProtA Sepharose FF
Ni Sepharose excel简化工作流程
Conventional workflow NEW workflow
Cell culturing
Time
Cell culturing
Removal of cells
Removal of cells
Diafiltration
Ni Sepharose™ excel/ His Mag Sepharose excel
Ni Sepharose™ excel在位清洗
• 即使在0.01HCl中孵育 1个星期镍离子都不会 脱落
•可进行 1 M NaOH在 位清洗
MabSelect SuRe™ LX 动态载量较 MabSelect SuRe提高45%
human IgG monoclonal antibodies
抗体和抗体片段 全抗 Fab ScFv Dabs
E E
N21 N23 N52 N43 N28 N11
cell wall binding domain
C C X X
IEGRNC
Native protein A Mr ~ 42 000
D D
A A
B B
rProtein A Mr 34 300
B domain Mutant (Asp replaced with alkali-stable amino acid )
时间更短，分辨率更高，用途更广
Superdex 200 Matrix Fractionation range Flow/pressure properties Average bead size pH stability Regular use Short term (CIP) Superdex 200 Increase Cross-linked agarose and dextran Molecular weights from 10 000 to 600 000 Regular 13 µm pH 3 – 12 pH 1-14 Rigid beads with high flow/pressure resistance 8.6 µm