Rapid and effective purification of IgG by using Ion Exchange Media BioPro IEX SmartSep S30
R150521AE

BioPro IEX SmartSep Q/S are ion exchange media dedicated to high-throughput intermediate purification stepand polishing step of biopharmaceuticals. BioPro IEX SmartSep media are available in strong ion exchangers ofhydrophilic porous polymer beads with low nonspecific adsorption and high binding capacity over a wide range offlow rate. BioPro IEX SmartSep media show both high resolution and recovery even at a high flow rate and highloading condition. DBC is influenced by such as pH, linear velocity and salt concentration. BioPro IEX SmartSep
Q/S shows the high DBC in any conditions. BioPro IEX SmartSep Q/S make a big improvement in productivity ofbiologics, especially, antibody therapeutics.

High Dynamic Binding Capacity (DBC) for IgG
Comparison of DBC of various proteins
BioPro IEXSmartSep
S30 S30
SmartSep
TSKgel SP-3PW
Brand
T (Porous S type)

100GE
SOURCE
30S
Brand
G (PorousS type)

DBC (mg/mL-resin)

1208060

DBC (mg/mL-resin, 10% breakthrough)InsulinLysozyme
Human Polyclonal IgG(MW : 5,800) (MW : 14,300)(MW : 150,000)
BioPro IEX SmartSep S30

73111

93

Brand T (Porous S type, 30 mm)

67

72

42

Brand G (Porous S type, 30 mm)

64

85

41

More than 2 times
higher thancompetitors

40
20

Conditions of DBC measurement *Column size
SampleFlow rate

0Insulin(MW : 5,800)

Lysozyme(MW : 14,300)

HumanPolyclonal
Polyclonal IgG
HumanIgG(MW :: 150,000)
150,000)(MW: 50 X 5.0 mmI.D.
: 1.5 mg/mL in equilibration buffer: 400 cm/hr (1.32 mL/min)*Please inquire us for deails.

DBC of human polyclonal IgG at various flow rates
Conditions of DBC measurement

120110

BioPro IEXSmartSep
S30S30
BioProSmartSep

90
80

Even at 800 cm/hr,
More than 1.5 times higher
DBC than competitors
at 200 cm/hr

70
60
50Column: 50 X 5.0 mmI.D.
Equilibration buffer : 20 mM citric acid-NaOH(pH 5.3)
Elution buffer: Equilibration buffer
containing 0.5 M NaClFlow rate
: 200-800 cm/hr (0.66-2.62 mL/m in)
Temperature: ambient (25℃)
Detection: UV at 280 nm
Sample: 1.5 mg/mL human polyclonal
IgG in equilibration buffer

Brand T (Porous S type)

40
30

Change of Pressure at various flow rate

Brand G (Porous S type)200.3

100200

400

600

800

1000

Linear velocity (cm/hr)
DBC (mg/mL-resin, 10% breakthrough)Linear velocity
200 cm/hr

400 cm/hr

800 cm/hr

BioPro IEX SmartSep S30

1109384

Brand T (Porous S type, 30 mm)

61

4226

Brand G (Porous S type, 30 mm)

5841230.25

Pressure (MPa)

DBC (mg/mL-resin)

1000.20.150.1
0.050020040060080010001200

Linear velocity (cm/hr)

・ BioPro IEX SmartSep S30 provides higher DBC on various proteins.
For IgG, BioPro IEX SmartSep has more than twice as high DBC as competitors'
Enabling column downsizing (gel cost reduction) during antibody purification
・Larger difference of DBC at high flow rate
→Expect improvement of productivity by increasing flow rate.
BioPro IEX SmartSep S30 makes purification productivity per unit time double or more.
http://www.ymc.co.jp1/2

High DBC under various conditions
Effect of sample buffers/salts concentration

Effect of equilibration buffer pHSample : 1.5 mg/mL human polyclonal IgG
in equilibration buffer containing 0, 25, 50 mM NaCl
＊Equilibration buffer pH : 5.3

Conductivity : 1.30 mS/cm (pH 5.3)
1.40 mS/cm (pH 6.0)
120

110

110

100

BioPro IEX SmartSep S30

DBC (mg/mL-resin)

8070

Brand T (Porous S type)

60
50
40

100

DBC (mg/mL-resin)

90

Brand G (Porous S type)30
20

BioPro IEX SmartSep S30

9080

Brand T (Porous S type)

7060Brand G (Porous S type)50403020

1000.0

101.0

2.0

3.0

4.0

5.0

6.0

7.00
5Conductivity (mS/cm)

65.3

7pHColumn: 50 X 5.0 mmI.D.
Equilibration buffer : 20 mM citric acid-NaOH (pH 5.3 or 6.0)
Elution buffer: Equilibration buffer
containing 0.5 M NaCl
Flow rate: 200 cm/hr (0.66 mL/min)
Temperature: ambient (25℃)
Detection: UV at 280 nm
Sample: 1.5 mg/mL human polyclonal
IgG in equilibration buffer

DBC (mg/mL-resin, 10% breakthrough)
5.35.30 mM
25 mM
50 mM
1.36 mS/cm 4.14 mS/cm 6.8 mS/cm
10797501106455276158491958

pHNaCl concentration
Conductivity
BioPro IEX SmartSep S30
Brand T (Porous S type, 30 mm)
Brand G (Porous S type, 30 mm)

6.01036458

・High DBC than competitors even in the presence of 50 mM NaCl.
Eluate from Protein A column chromatography could be directly subjected
・High DBC at buffer pH range commonly used in antibody purification by cation exchange chromatography

Purification of IgG1（Anti-hTNFalpha IgG）
This is an example that an IgG1 monoclonal antibody was purified from cell culture medium. After clarified, it wassubjected to initial purification (capture step) by affinity chromatography (rProtein A), followed by ion- exchange
chromatography. In the capture step rProtein A derived from affinity media contaminated the eluate, then they wereseparated and removed by following ion exchange chromatography.
Intermediate purification (cation exchange chromatography)

Purification Scheme of IgG1mAU

Sample： Anti-h TNF alpha IgG1

BioPro IEX SmartSep S30Raw material

Culture 1000 mL

0.032 mg/mL

rProtein A
Clarification

Dialysis

20 mM Sodium Phosphate pH 7.0

Brand T (Porous S type)

CaptureAffinity chromatography(rProtein A)
Brand G (Porous S type)Intermediate
purification

Ion exchange chromatography(BioPro IEX SmartSep S30)

048Column
EluentPolishing
Flow rate
Temperature
Detection
SampleInjection

1216202428

32

36

40

4448min: 50 X 5.0 mmI.D.
: A) 20 mM citric acid-NaOH (pH 5.3)
B) 20 mM citric acid-NaOH (pH 5.3) containing 0.5 M NaCl0-100 %B, 30 column volumes: 180 cm/hr (0.59 mL/min): ambient: UV at 280 nm: Anti-hTNFalpha IgG1(Purified by Affinity chromatography): 0.25 mL (0.1 mg IgG1)

・ BioPro IEX SmartSep S30 ion exchange media is effective to remove desorbed rProtein Aligand in the capture step.
http://www.ymc.co.jp