新一代层析介质Capto(丁晓萍)

2/ GE Title or job number /
7/20/2008
Capto 系列层析介质的基架
高流速琼脂糖 High flow agarose • 亲水性 Æ 蛋白和基架间的非特异
吸附非常小 “protein friendly” • 化学稳定性 • 开放的孔结构 • 公认的生物分离介质
现在增加两大特点： 刚性更强、传质速度更快
Generic elution conditions
25 / GE MabSelect SuRe /
Ghose et al., (2005) Biotech Bioeng 92, 665-673 Amgen data
洗脱条件更温和, 避免抗体聚集
26 / GE Title or job number /
MabSelect SuRe的优势
Engineered Protein A
Stability in CHO cell lysate
E D A BC Sure ligand
rProtein A SuRe ligand
123
1) Time 0 2) 18 hour 3) Control
Low leakage (1~10ppm)
2000: rmp Protein A Sepharose 4FF (multi-point attached)
2001: Mabselect
(single-point attached thioether bond)
2005: Mabselect SuRe, Xtra (single-point attached thioether bond)
7/20/2008
HiTrap™ Capto™ IEX Selection Kit
5 x 1 ml HiTrap Capto Q HiTrap Capto S HiTrap Capto DEAE HiTrap Capto MMC HiTrap Capto adhere
Code no: 28-9343-88
MabSelect SuRe: 清洗效果更好！
40 minute in GuHCl 3 Hour in GuHCl
Resin after 300 cycles using phosphoric acid CIP protocol
Treatment with 0.1 M NaOH
40 min
3 hours
Capto adhere
Ion exchange media
Capto Q
Traditional
Capto S
ligands
Capto DEAE
12 / GE Title or job number /
7/20/2008
Capto 阴离子交换层析介质
Capto Q, and Capto DEAE
+
7/20/2008
重组蛋白A介质的发展
Argarose: from sea weed to separation meida
1991: Protein A Sepharose 4FF
(multi-point attached CNBr)
1996: rProtein A Sepharose 4FF (single-point attached thioether bond)
SuRe Ligand for Mabselect SuRe Mr 26748
OH
O
O
OH
O O + HS– Prot A
O
O
O
S – ligand
OH
Single-point attached thioether bond
24 / GE Title or job number /
7/20/2008
10 / GE Title or job number /
7/20/2008
新一代的Capto 系列介质
使日产百公斤级蛋白成为可能
目标：24小时内纯化100KG目标蛋白
目标蛋白: recombinant Green Fluorescent Protein (GFP) expressed in E. coli
抗体纯化
adhere - Ligand
Multimodal strong anion
Multiple types of interactions
ionic interaction
hydrophobic interaction
hydrogen bonding
17 / GE Title or job number /
~30mg hIgG/ml medium 1
~30mg hIgG/ml medium 1
~41mg hIgG/ml medium 2
18 / GE Title or job number /
7/20/2008
那一种凝胶是最佳选择呢?
纵使7根不同层析柱也很容易处理
19 / GE /
20 / GE /
Capto系列亲和介质
Mab select ™系列抗体亲和介质
Capto Blue 染料亲和介质
VIII select 重组VIII因子亲和介质
6/ GE Title or job number /
7/20/2008
传质速度更快
7/ GE Title or job number /
7/20/2008
动态结合载量更高
QB10% BSA (mg/mL)
160 140 120 100
80 60 40 20
0 0
Capto Q Q Sepharose FF
Ig Select ™专用于人IgG纯化的亲和介质
21 / GE Title or job number /
7/20/2008
抗体的结构
heavy chain
VH
VH
可变区
VH
VH
VL
CH
CL -S-S-
CH
VL
-S-S- CL
VL
CH CH
VL
Fab-fragments
-S-S-
light CL -S-S-
新一代层析介质
Capto & Mabselect
丁晓萍
迎接现代下游工艺的挑战
• 培养条件的优化，使得产品的表达量提高，由 以前的mg/l到现在的 g/L
• 培养体积的增大，由升级－－吨级 • 产品产量的提高，由克级－－公斤级
要求下游： • 单位时间的处理量增大－－高流速 • 单位时间的产量提高－－高动态载量
Surface extender Animal free dextran
Ligand Sulphoethyl (Strong cation exchange)
A high flow agarose base matrix modified with dextran and strong cation exchange groups for optimal rigidity, capacity and mass transfer
-S-S- CL (papain digest)
chain
-S-S-
(pepsin digest)
VH
F(ab’)2
VL
-S-S-
Fc fragment
CH -S-S- CL
Fab
恒定区
Protein A结合部位 IgG
VL
亲和层析是抗体纯化的通用方法！
VH ScFv
22 / GE Title or job number /
3/ GE Title or job number /
7/20/2008
刚性更强：高流速/低反压
Pressure/flow specification: >700 cm/h in 1 m diameter column, 20 cm bed height, below 3 bar
Capto™ Q and Sepharose™ 6 Fast Flow in BPG™ 300, 20 cm bed height
ED
N21 N23
N28
N52 N43 N11
A
ቤተ መጻሕፍቲ ባይዱ
B
CX
rProtein A IEGRNC Mr 34 300
B domain Mutant (Asp replaced with alkali-stable amino acid )
Zstab
N6
N3
Cys Z Z Z Z stab stab stab stab
O
N
OH
+
Base matrix
High flow agarose 90 µm (average)
+
Surface extender
Animal-free dextran
Capto Q ligand
Quaternary ammonium
CH3 CH3
N H+
CH3 H
N+
CH3
CH3
CH3 +
N
N CH3
7/20/2008
MabSelect 家族
Mabselect
Mabselect SuRe Mabselect Xtra
Dynamic Binding Capacity
Flow Rate 3 PrA Ligand Particle Size Matrix Ligand density Coupling
pI of target protein = 6.2
表达水平 ~2.5 mg GFP/ml 需要上样体积：40000L