Post on 14-Aug-2020
Development of an analytical reversed phase column for characterization
of 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.
IntroductionThe 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 with
silica particles.
Monolithic silica gel with an inner diameter of 2.1 mm was prepared via sol-gel processing of
tetraethoxysilane. 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 a
stainless steel tube.Intact protein (mAb) analysis were performed on a HPLC-UV and TripleTOF 6600 mass spectrometer
(Sciex).
Methods
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.
A mAb require buffering salt for stabilization in solution. However, to gain sensitivity in LC-MS analysis of
intact 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 developed
column provided excellent total ion chromatogram peak shapes compared with silica gel column.
The temperature effect for resolution was compared. The heat resistance of a monolithic column clad with
resin 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, peak
widths 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 acid
has also improved by cladded with a glass tube.
Results & Discussion
1 µm Through pore
60 nm Meso pore
Φ2.1mm Monolithic silica
Stainless steel tube
Glass tube
Assembly
Schematic of the developed monolithic silica column
Monolithic silica disk
SPE Spin columnAbsorbent material Capillary HPLC column
Monolithic silica application fields
0.0 1.0 2.0 3.0 4.0 5.0 6.0Time (min)
0.0
00.1
0V
olts
0.0 1.0 2.0 3.0 4.0 5.0 6.0Time (min)
0.0
00
.10
Vo
lts
0.0 1.0 2.0 3.0 4.0 5.0 6.0Time (min)
0.0
00.1
0V
olts
Developed column Silica gel
packed column
Polymer packed
column
4.5MPa 10.5MPaPressure drop 1.7MPa
Peak height 135451 25136 113250
ConditionsEluent: A:0.1 %TFA-H2O
B:0.1 %TFA-ACN
Gradient: A/B A/B=90/10-2.5min-10/90
Flow Rate: 0.3 mL/min
Oven Temp.: 80 ℃Injection Vol.: 5 μL R
ete
ntio
n tim
e[m
in]
Injection [times]
Developed monolithic
silica clumn
Conditions
Eluent: A) 0.075% HCOOH+0.025% TFA - H2O
B) 0.075% HCOOH+0.025% TFA - ACNA/B=95/5-5min-10/90
Flow Rate: 0.3 mL/min
Detection: UV 210 nm
Oven.Temp: 80 ℃Analyte: 1.4 mg/mL mAb
Column performance of the developed column with competition.
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
0 500 1000 1500 2000 2500 30002.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
0 500 1000 1500 2000 2500 3000
Re
ten
tio
n tim
e[m
in]
Injection [times]
0.0 1.0 2.0 3.0 4.0 5.0 6.0
Time (min)
0.0
0.2
0.4
Vo
lts
50 ℃
60 ℃
80 ℃
0.0 1.0 2.0 3.0 4.0 5.0 6.0
Time (min)
0.0
0.2
0.4
Vo
lts
0.0 1.0 2.0 3.0 4.0 5.0 6.0
Time (min)
0.0
0.2
0.4
Vo
lts
0.0 1.0 2.0 3.0 4.0 5.0 6.0
Time (min)
0.0
0.2
0.4
0.6
Vo
lts
40 ℃
Temperature effect
Peak tailing
Durability
The graph below shows the durability test. In response to the increase of injection times (x-axis), the
durability 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 of
phenyl groups modified column is much greater than that of the C4 modified columns. This assures stable
analysis over a long period of time.
LC/MS intact mAb analysis
>10 μm <1 μm
The pores can be sized according to application, resulting in a highly adaptive material for chromatography.
Developed columnSilica gel
packed column
0.0 1.0 2.0
Time (min)
0200
400
600
800
1000
mV
olt
0.0 1.0 2.0
Time (min)
0200
400
600
800
mV
olt
① ②
Conditions
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
Fab in CHO cell culture Purified sample
Flow rate: 0.3 mL/min
Col.Temp.: 60 ℃Detection: 210 nm
Injection Vol.: 1 μL
M
Electrophoresis
① ; Purified with MonoSpin ProL
② ; CHO cell culture
M ; Marker
Fab
CHO cell culture direct analysis
Purified by
ProteinL immobilized
Spin column
Conclusions
The developed monolith column has high porosity, it was possible to analyze the culture broth without clogging.
C4 group Phenyl group
Separation of Intact mAb using a developed monolithic column at different temperatures.
Analyte: 1.4mg/mL mAb
5 uL injection
CladdingConditionsEluent: A:0.1 %HCOOH-H2O
B:0.1 %HCOOH-ACN
Gradient: A/B=90/10-10min-10/90
Flow Rate: 0.3 mL/min
Detection: TripleTOF 6600
Oven Temp.: 80 ℃Injection Vol.: 5μL
Intact mAb mass analysis (total ion chromatogram) on (A) an developed column, (B) silica packed column.
(A) (B)
・ The developed column was shown to be very suitable for analysis of intact protein (mAb).
・ The Novel glass cladded monolithic silica column was developed.
Fab