Development of an analytical reversed phase column for characterizationof intact antibodies using wide pore monolithic silica.
Shigenori Ota, Yuko Yui, Shota Miyazaki, Chiaki Aoyama, Ken Miyashita, Manami Takeda, Shunta Futagami, Tsutomu Sato
GL Sciences Inc.
Results & Discussion
Column performance of the developed column with competition.
The recoveries of intact mAbs was compared on the two particle packed columns (silica gel type and polymer
gel type) and developed columns, it appeared that adsorption was less pronounced on the silica gel particle
packed, probably due to the size of the small pore size. Developed monolithic column was shown the excellent
recoveries to intact mAb. The pressure drop of the developed column is typically low that of a column packed
with polymer column.
Polymer packed
column

Silica gel
packed column

Developed column

Monolithic silica application fields

Phenyl group

C4 group
3.8

4

3.6

3.8

3.4

3.6

Retention time [min]The production and purification of monoclonal antibodies (mAb), comprehensive characterization can be
performed at the intact protein on a mass spectrometry. Reversed phase columns with a wide pore(>30nm) particle are used for intact protein chromatography analysis. However, analytical column packed
wide pore show low pressure resistance due to the physicochemical durability of support material.
Monolithic silica have been widely studied due to their unique structures that have many advantages
as separation media(Figure). Monolith structure and pore size were easily controlled by starting material.
In this study, we have developed the monolithic silica column with wide pore size suitable for the intact
protein analysis. The performance of the developed column was compared with columns packed withsilica particles.

The graph below shows the durability test. In response to the increase of injection times (x-axis), thedurability of each column is plotted at y-axis with the retention time of the mAb. The developed column
modified phenyl groups shows great durability under low pH and high temperature conditions. The lifetime ofphenyl groups modified column is much greater than that of the C4 modified columns. This assures stable
analysis over a long period of time.

Retention time [min]Introduction

Durability

3.232.8
2.6
2.40SPE Spin column3.23Gradient:
Flow Rate:Oven Temp.:
Injection Vol.:

2.8
2.65001000

Capillary HPLC column

1500

2000

2500

3000

0500

1000

2000

2500

3000

CHO cell culture direct analysis

Volts

Volts

0.10

0.100.10
Volts

1500

Injection [times]

Injection [times]

Monolithic silica disk

A：0.1 %TFA-H2O
B：0.1 %TFA-ACN
A/B A/B=90/10-2.5min-10/90
0.3 mL/min
80 ℃
5 μL

2.4

2.2

Absorbent material

Conditions
Eluent:

3.4

4.0

5.0

6.0

0.0

1.0

2.0

3.0
Time (min)

4.0

5.0

6.0

800

3.0
Time (min)25136

113250Pressure drop

1.7MPa4.5MPa

10.5MPa

FabPurified byProteinL immobilized
Spin column

400135451

600

2.0

Flow Rate:
Detection:
Oven.Temp:
Analyte:MethodsA) 0.075% HCOOH+0.025% TFA - H2OB) 0.075% HCOOH+0.025% TFA - ACN
A/B=95/5-5min-10/90
0.3 mL/min
UV 210 nm
80 ℃
1.4 mg/mL mAb

0.0

1.0Conditions
Eluent:A)0.08%HCOOH-0.02%TFA-H2O
B)0.08%HCOOH-0.02%TFA-ACN
A/B=80/20-2.5 min-10/90

Temperature effect

Schematic of the developed monolithic silica column

0.0

1.0

2.0
Time (min)Flow rate：
Col.Temp.：
Detection：
Injection Vol.：

0.3 mL/min
60 ℃
210 nm
1 μL

Electrophoresis① ; Purified with MonoSpin ProL
② ; CHO cell culture
M ; Marker

LC/MS intact mAb analysis
A mAb require buffering salt for stabilization in solution. However, to gain sensitivity in LC-MS analysis ofintact mAbs, it is critically important to remove the salt before LC-MS analysis. Figures show the LC/MS
data for the intact mAbs with developed column and silica gel columns, respectively. The developedcolumn provided excellent total ion chromatogram peak shapes compared with silica gel column.
Silica gel
packed column

Developed column

Analyte: 1.4mg/mL mAb
5 uL injection
0.6

Φ2.1mm Monolithic silica

2.0Time (min)The temperature effect for resolution was compared. The heat resistance of a monolithic column clad withresin was typically up to 50 °C, but clad the monolithic material with glass improved the column‘s heat
resistance to 80℃. Recovery of mAb were significantly improved with elevated temperature, and adsorption
was generally acceptable in the temperature range between 60 and 80℃. As temperature increases, peakwidths of the protein peaks has decreased and absolute retention of each peak was reduced with increasing
temperature. The Stability of the developed column at higher temperature and presence of trifluoroacetic acidhas also improved by cladded with a glass tube.

Fab0Conditions
Eluent:

mVolt

1.0

400

0.0

200

6.0

1000

5.0

800

4.0

Peak height

The pores can be sized according to application, resulting in a highly adaptive material for chromatography.

Monolithic silica gel with an inner diameter of 2.1 mm was prepared via sol-gel processing oftetraethoxysilane. Then, monolithic silica was treated with alkali solution to prepare wide pore size
(60nm). The resulting monolithic silica was cladded with a glass tube. After cutting it to an appropriate
length, monolithic silica surface was modified with the phenyl group or the C4 group. Reversed phase
modified monolithic silica column in this study was fabricated by sealing the cladded silica in astainless steel tube.
Intact protein (mAb) analysis were performed on a HPLC-UV and TripleTOF 6600 mass spectrometer(Sciex).

3.0
Time (min)

200

<1 μm

2.00>10 μm

1.0

Purified sample

Fab in CHO cell culture

mVolt
600

0.0

① ② M

0.00

0.00

0.00

The developed monolith column has high porosity, it was possible to analyze the culture broth without clogging.

(B)

Volts

60 ℃

Peak tailing

0.2

40 ℃

0.2

Glass tube

Volts
0.4

0.4

(A)

Conditions
Eluent:0.0

Cladding

0.0

Gradient:
Flow Rate:
Detection:
Oven Temp.:
Injection Vol.:

0.0

1.0

2.0

Assembly

3.0Time (min)

4.0

5.0

6.0

0.0

1.0

2.03.0Time (min)

4.0

5.0

6.0

A：0.1 %HCOOH-H2O
B：0.1 %HCOOH-ACN
A/B=90/10-10min-10/90
0.3 mL/min
TripleTOF 6600
80 ℃
5μL

Volts

Volts

Conclusions

0.2

0.2

80 ℃

0.0

60 nm Meso pore

50 ℃

0.0

Stainless steel tube

Developed monolithic
silica clumn

Intact mAb mass analysis (total ion chromatogram) on (A) an developed column, (B) silica packed column.

0.4

0.4

1 µm Through pore

0.0

1.0

2.0

3.0
Time (min)

4.0

5.0

6.0

0.0

1.0

2.0

3.0
Time (min)4.0

5.0Separation of Intact mAb using a developed monolithic column at different temperatures.

6.0

・The Novel glass cladded monolithic silica column was developed.

・The developed column was shown to be very suitable for analysis of intact protein (mAb).