Improvement of Separation of Monoclonal
Antibodies Using Core-Shell Column
Norikazu Nagae, Tomoyasu Tsukamoto, Makoto Sato
ChromaNik Technologies Inc. Namiyoke, Minato-ku, Osaka 552-0001 Japanwww.chromanik.co.jp

Following low-molecular medicine, the antibody medicine group is attracting attention and recognized as the most important medicine
due to its effectiveness. It is well known that size exclusion chromatography (SEC), hydrophobic Interaction chromatography (HIC) and reversed
phase chromatography are used as a typical separation analysis method for monoclonal antibodies.
In this study, silica base material, pore diameter and stationary phase were compared in reversed phase chromatography. Although it is known that thecore-shell silica column has a higher number of plate and higher efficiency than the totally porous silica column, the same result was obtained for theseparation of the monoclonal antibodies. Although a packing material with 30 nm of pores has been frequently and satisfactorily used for a separation ofproteins whose molecular weight was 100,000 or less, 100 nm of pore leaded a better separation of monoclonal antibodies whose molecular weight wasaround 150,000 to compare with 30 nm of pore. As a result, core-shell silica with 100 nm pore bonded with butyl group (C4) showed the best separation formonoclonal antibodies (IgG) which was obtained by cell culture and purified on a protein G affinity column.

Core shell particle for separation of peptides and proteins
Porous layer

1.6 μm

5 μm for routineCore

0.5 μmCore

2.6 μmParticle size: 2.6 μm
Pore diameter: 100 nm

0.6 μmPore diameter
9 nm,16 nm,
30 nm,
100 nm

3.4 μm

0.9Desorption Dv (log d) (cc/g)

2.6 μmCore4.6 μm

3.4 μm for protein

Particle size: 2.6 μm
Pore diameter: 16 nm

Pore diameter 9 nm

0.2 μmParticle size: 3.4 μm
Pore diameter: 30 nm

2 μm
0.4 μm
3.0 μmCore

1.2 μm

3.4 μmCore2 μm

SunShell C4-30
2.1 x 100 mm

2.6 μm
2.6 μm

1.6 μm
1.6 μm

0.5 μm
0.5 μm

100 nmSurfaceareaCarbon
loading

22 m2/g

30 nm

40 m2/gCore shell 2.6um, 100nm

0.4
0.3
0.2
0.1

0.6%

101001000

Figure 3. Pore size distribution of core shell silica

Figure 2. Electron micrograph of core shell silica

21Surfacecoverage
3µmol/m2

43Table 1. Specification of used core shell columns

SunShell C4-100
2.1 x 100 mm

Core shell 3.4um, 30nm

0.5

Pore Diameter (nm)Separation of standard proteins
PorediameterCore shell 2.6um, 30nm

0.6

1Figure 1. Schematic diagrams of core shell silica

Thickness ofporous
layerCore shell 2.6um, 16nm

0.70Pore diameter 9 nm

Pore diameter 30 nm

ParticleCoresize
diameter

0.85Endcapping
Yes

0.9%

3µmol/m20.9%

2.5µmol/m2

Yes

Yes

SunShell C8-100
2.1 x 100 mm

2.6 μm

1.6 μm

0.5 μm

100 nm

22 m2/g

SunShell C8-30
2.1 x 100 mm

2.6 μm

1.6 μm

0.5 μm

30 nm

40 m2/g

1.2%2.5µmol/m2

Yes

SunShell C8-30HT
3.4 μm2.1 x 100 mm

3.0 μm

0.2 μm30 nm

15 m2/g

0.5%2.5µmol/m2

Yes

Retention time / minFigure 4. Comparison of C4 and C8 phases

Separation of purified antibody obtained using cell culture technique(Purified with IgG using protein G affinity column)

Core shellC8, 300 Å

SunSec Diol30, 300 x 4.6 mm

Core shell C4, 300 Å
SunShell C4-100, 1000 ÅC4-100 C4-30 C8-100 C8-30 C8-30HTC8-30HT
0.5uL

Sampleconcentration

0.167

0.177

0.160

0.155

0.212

0.144

0.050%

Lysozyme

0.164

0.180

0.153

0.166

0.196

0.145

0.050%

BSA

0.308

0.410

0.276

0.514

0.422

0.330

0.100%

Myoglobin

0.197

0.221

0.180

0.199

0.238

0.176

0.050%

Ovalbumin

0.391

0.889

0.247

0.428

0.184

0.176

0.050%

Size exclusion mode (SEC)
Column: SunSec Diol30, 4 μm 300 x 4.6 mm i.d. X 2Mobile phase:
0.1M Phosphate buffer + 0.2 M NaCl (pH6.8)
Flow rate: 0.10 mL/min
Temperature: 25 ºC
Detection: UV at 220 nm
Injection: 2 µL
Sample: Purified antibody obtained by cell culture(Purified with IgG using protein G affinity column)

Not eluted

Totally porous
C4, 300 Å

Table 2. comparison of peak width (W0.5, min)
Cytochrome C

Calibration curve

Molecular weight

Reversed- phase mode (RP)Column: listed on the table 1
Mobile phase: A) 0.1% TFA in waterB) 0.1 % TFA in acetonitrile
Gradient program: Time 0 min 60 min%B 20% 65%
Flow rate: 0.5 mL/min
Temperature: 80 oC
Detection: UV@215 nm
Injection volume: 1.0 μL or 0.5 μL
Sample:1 = Cytochrome C, 2 = Lysozyme, 3 = BSA,
4 = Myoglobin, 5 = Ovalbumin
UHPLC instrument: HITACIHI Chromaster

Column dimension: 100 x 2.1 mm i.d.
Mobile phase: A) 0.1% TFA in waterB) 0.085% TFA in acetonitrile
30-45%B (0-30 min)Flow rate: 0.4 mL/min
Temperature: 80 ºC
Detection: UV at 215 nm
Injection: 5 µL
Sample: Purified antibody obtained by cell culture(Purified with IgG using protein G affinity column)

Retention time/min

Elution volume/mL

300 x 4.6 mm i.d. X 1
0.35 mL/min

300 x 4.6 mm i.d. X 2
0.10 mL/min

4567
Elution volume/mL8Figure 4. Separation of purified antibody obtained by cell culture using RP column and SEC column

Conclusion

SunShell C4-100, 1000 Å 100 x 0.15 mm i.d.

Retention time/min

Column dimension: 100 x 0.15 mm i.d.
Mobile phase: A) 0.1% TFA in waterB) 0.085% TFA in acetonitrile
30-45%B (0-15min）
Flow rate: 0.004 mL/min
Temperature: 80 ºC
Detection: UV at 215 nm
Injection: 0.1 µL
Sample: Purified antibody obtained by cell culture(Purified with IgG using protein G affinity column)

Figure 5. Separation of purified antibody obtained by cell culture using nano RP column

A core shell silica with from 9 nm to 100 nm pore is available.
C4 and C8 phase with 100 nm pore showed a better separation ofstandard proteins than those with 30 nm pore.
In case of small sample injection, C8 with 0.2 μm thickness ofporous layer and 30 nm pore showed good separation.
Regarding reversed phase separation of monoclonal antibody (IgG),
not only core shell C4 with 30 nm pore showed the better
separation than totally porous C4, but also 100 nm of pore leaded
the best separation.
Nano column showed almost the same separation of IgG as semimicro column.