Suresh Babu C.V. 2 Agilent Technologies, Inc. Wilmington, DE...
1
Characterization of Intact Monoclonal Antibodies (mAb) and their fragments using Novel Reversed Phase Columns Suresh Babu C.V. 1 , Phu T Duong 2 , Andrew Coffey 3 , and Meenakshisundaram Palaniswamy 1 1 Agilent Technologies, Inc. Bangalore, India, 2 Agilent Technologies, Inc. Wilmington, DE 19808, 3 Agilent Technologies, Inc. Church Stretton, UK WCBP 2016 Poster # P203 W Introduction Experimental Therapeutic monoclonal antibodies were purchased from a local pharmacy and stored according to the manufacturers’ instructions. LC/UV: For intact mAb analysis, mAb samples were diluted to 2 mg/mL using PBS. For the separation of the light and heavy chains, an aliquot of 0.5 M TCEP stock was added to the mAb samples to obtain a final concentration of 10 mM. The mixture was held at 60 °C for 30 minutes. LC/MS: For intact mAb analysis, mAb samples were diluted to 1 μg/μL using 0.1% formic acid in 3% ACN, For the separation of the light and heavy chains, an aliquot of 1M DTT stock was added to the mAb samples and the mixture was held at 37 °C for 1 hr. Digestion with Papain and FabRICATOR was performed at 37 °C for 3 hr and 1 hr respectively. For LC/UV and LC/MS conditions, refer Agilent application notes: LC/UV: 5991-6274EN LC/MS : 5991-6296EN 2 * Fc F(ab’) 2 Fc 2 * Fab ScFc DTT reduction Papain digestion FabRICATOR LC HC 2 * Fab Intact or With the continued importance of monoclonal antibodies (mAbs) as biotherapeutics, comprehensive characterization is a prerequisite. It is critical at discovery, development and manufacturing stages to confirm that the antibody drug has the correct primary structure (number, location and order of amino acids) and to monitor variants and other potential post translational modifications that can impact safety and efficacy. Due to the complexity of the molecules, the characterization method with high resolution for mAb primary structure by reversed-phase liquid chromatography can be very lengthy and time consuming. In this presentation, the data analysis of intact mAb and fragments such as heavy/light chains and Fc/Fab regions with high speed and high resolution are presented. These data are performed by using the novel, unique bonding chemistries-C4, C8 and Diphenyl on a new particle design of 3.5 μm Poroshell technology. The data from the LC/UV and LC/MS methods will be included here in this presentation. LC-UV LC-MS Conclusions References • AdvanceBio RP-mAb columns: Fast and superior separation power for both intact and fragments of mAb • Demonstration of LC-UV-based approach to define the molecular similarity between a biosimilar and its innovator reference • Area and RT precision of intact and reduced analysis using AdvanceBio RP-mAb columns were excellent, and show the reliability of the method • The C4 and Diphenyl AdvanceBio RP-mAb columns, with an MS-compatible method, delivered fast and high-resolution analysis for intact and ADC mAbs • Gritti, F. Chromatog. Today, June 2012, 4-11. • Agilent Technologies, Inc. Publication number 5991-4266EN, 2014. • Navas, N; et al., Anal. Bioanal. Chem. 2013, 405, pp 9351-9363 • https://www.agilent.com/cs/library/brochures/5991- 5160EN_AdvanceBio%20mAb%20flyer_LR.pd • https://www.agilent.com/en-us/products/liquid-chromatography/lc- columns/biomolecule-separations/advancebio-rp-mab Intact mAb analysis on a AdvanceBio RP-mAb Diphenyl, 2.1 x 50 mm, 3.5μm column min 0.5 1 1.5 2 2.5 3 3.5 mAU 0 100 200 300 400 500 2.520 min 0.5 1 1.5 2 2.5 3 3.5 mAU 0 100 200 300 400 2.517 Innovator rituximab Biosimilar rituximab Intact mAb analysis on a AdvanceBio RP-mAb C4, 2.1 x 50 mm, 3.5μm column 0.5 1 1.5 2 2.5 3 3.5 0 50 100 150 200 250 300 1.963 mAU Innovator rituximab 0.5 1 1.5 2 2.5 3 3.5 0 50 100 150 200 250 300 1.961 mAU Biosimilar rituximab Biosimilar rituximab Innovator rituximab LC HC LC HC Reduced mAb analysis on a AdvanceBio RP-mAb column Diphenyl, 2.1 x 50 mm, 3.5μm C4.1 x 50 mm, 3.5μm Biosimilar rituximab Innovator rituximab LC HC LC HC Intact mAb mass analysis on a AdvanceBio RP-mAb C4, 2.1 x 100 mm, 3.5μm column 7 x10 0 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 mAb1 Counts 4 x10 0 0.4 0.8 1.2 1.6 2 2.4 2200 2400 2600 2800 3000 3200 3400 3600 3800 Counts 5 x10 0 0.4 0.8 1.2 1.6 2 2.4 148225.23 148063.92 148389.73 148545.04 147917.97 147800 148000 148200 148400 148600 G0/G0F G0F/G0F G0F/G1F G1F/G1F G1F/G2F Counts % 7 x10 0 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 mAb 2 Counts 4 x10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 2200 2400 2600 2800 3000 3200 3400 3600 3800 Counts 5 x10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 147244.07 147405.32 147079.63 147564.57 147000 147200 147400 147600 G0F/G0F G0F/G1F G0F/G2F G1F/G2F Counts % 7 x10 0 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 mAb3 (antibody drug conjugated ) Counts Acquisition Time (min) 3 x10 0 0.4 0.8 1.2 1.6 2 2.4 2.8 2200 2400 2600 2800 3000 3200 3400 3600 3800 Counts 3 x10 0 0.4 0.8 1.2 1.6 2 2.4 2.8 153018.05 151101.04 152058.74 149183.80 150143.59 153970.66 154928.31 156046.34 148225.52 148000 150000 152000 154000 156000 158000 DAR 0 DAR 1 DAR 2 DAR 3 DAR 4 DAR 5 DAR 6 DAR 7 DAR 8 Counts % Mass-to-Charge (m/z) Deconvoluted Mass (amu) mAb fragment analysis on AdvanceBio RP-mAb C4, 2.1 x 100 mm, 3.5μm column 8 x10 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 Acquisition Time (min) Counts LC HC 8 x10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2 3 4 5 6 7 8 9 10 11 Acquisition Time (min) Counts 2 * Fab 2 * Fc 9 x10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 2 3 4 5 6 7 8 9 10 11 12 Acquisition Time (min) Counts F(ab’) 2 ScFc 7 x10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 23036.20 23058.18 23074.94 23093.61 Deconvoluted Mass (amu) 22980 23020 23060 23100 23140 Counts % 5 x10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 50673.74 50511.62 50836.01 Counts vs. Deconvoluted Mass (amu) 50400 50600 50800 51000 Counts % 4 x10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 25646.24 25484.18 25808.33 Counts vs. Deconvoluted Mass (amu) 25350 25450 25550 25650 25750 25850 25950 Counts % Counts % Counts vs. Deconvoluted Mass (amu) 6 x10 0 0.4 0.8 1.2 1.6 2 2.4 2.8 3.2 25200.62 25362.81 24997.36 25524.86 24835.35 24631.98 25815.99 24469.80 25654.04 24400 24600 24800 25000 25200 25400 25600 25800 6 x10 0 1 2 3 4 5 6 7 8 9 96715.02 96808.97 96874.21 96400 96600 96800 97000 97200 Counts % Counts vs. Deconvoluted Mass (amu) mAb1 mAb2 mAb3 (antibody drug conjugate) LC HC 2 * Fc 6 x10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 47179.76 47342.31 Counts vs. Deconvoluted Mass (amu) 46900 47000 47100 47200 47300 47400 47500 47600 Counts % 2 * Fab ScFc F(ab’) 2 Publication Number 5991-6626EN
Transcript of Suresh Babu C.V. 2 Agilent Technologies, Inc. Wilmington, DE...
Characterization of Intact Monoclonal Antibodies (mAb) and their fragments
using Novel Reversed Phase ColumnsSuresh Babu C.V.1, Phu T Duong2, Andrew Coffey3, and Meenakshisundaram Palaniswamy1
1Agilent Technologies, Inc. Bangalore, India, 2Agilent Technologies, Inc. Wilmington, DE 19808,
3Agilent Technologies, Inc. Church Stretton, UKWCBP 2016 Poster # P203 W
Introduction Experimental
Therapeutic monoclonal antibodies were purchased from a local pharmacy and stored according to the
manufacturers’ instructions.
LC/UV: For intact mAb analysis, mAb samples were diluted to 2 mg/mL using PBS. For the separation of the light
and heavy chains, an aliquot of 0.5 M TCEP stock was added to the mAb samples to obtain a final concentration of
10 mM. The mixture was held at 60 °C for 30 minutes.
LC/MS: For intact mAb analysis, mAb samples were diluted to 1 μg/μL using 0.1% formic acid in 3% ACN, For the
separation of the light and heavy chains, an aliquot of 1M DTT stock was added to the mAb samples and the
mixture was held at 37 °C for 1 hr. Digestion with Papain and FabRICATOR was performed at 37 °C for 3 hr and 1
hr respectively.
For LC/UV and LC/MS conditions,
refer Agilent application notes:
LC/UV: 5991-6274EN
LC/MS : 5991-6296EN
2 * Fc
F(ab’)2
Fc
2 * Fab
ScFc
DTT reduction Papain digestion
FabRICATOR
LC HC
2 * Fab
Intact
or
With the continued importance of monoclonal antibodies
(mAbs) as biotherapeutics, comprehensive
characterization is a prerequisite. It is critical at discovery,
development and manufacturing stages to confirm that the
antibody drug has the correct primary structure (number,
location and order of amino acids) and to monitor variants
and other potential post translational modifications that
can impact safety and efficacy. Due to the complexity of
the molecules, the characterization method with high
resolution for mAb primary structure by reversed-phase
liquid chromatography can be very lengthy and time
consuming. In this presentation, the data analysis of intact
mAb and fragments such as heavy/light chains and
Fc/Fab regions with high speed and high resolution are
presented. These data are performed by using the novel,
unique bonding chemistries-C4, C8 and Diphenyl on a new
particle design of 3.5 µm Poroshell technology. The data
from the LC/UV and LC/MS methods will be included here
in this presentation.
LC-UV
LC-MS
Conclusions References
• AdvanceBio RP-mAb columns: Fast and superior separation power for both intactand fragments of mAb
• Demonstration of LC-UV-based approach to define the molecular similaritybetween a biosimilar and its innovator reference
• Area and RT precision of intact and reduced analysis using AdvanceBio RP-mAbcolumns were excellent, and show the reliability of the method
• The C4 and Diphenyl AdvanceBio RP-mAb columns, with an MS-compatiblemethod, delivered fast and high-resolution analysis for intact and ADC mAbs
• Gritti, F. Chromatog. Today, June 2012, 4-11.
• Agilent Technologies, Inc. Publication number 5991-4266EN, 2014.
• Navas, N; et al., Anal. Bioanal. Chem. 2013, 405, pp 9351-9363
• https://www.agilent.com/cs/library/brochures/5991-
5160EN_AdvanceBio%20mAb%20flyer_LR.pd
• https://www.agilent.com/en-us/products/liquid-chromatography/lc-
columns/biomolecule-separations/advancebio-rp-mab
Intact mAb analysis on a AdvanceBio RP-mAb Diphenyl, 2.1 x 50 mm, 3.5µm column
min
0.5 1 1.5 2 2.5 3 3.5
mA
U
0
100
200
300
400
500
2.5
20
min
0.5 1 1.5 2 2.5 3 3.5
mA
U
0
100
200
300
400 2.5
17
Innovator
rituximab
Biosimilar
rituximab
Intact mAb analysis on a AdvanceBio RP-mAb C4, 2.1 x 50 mm, 3.5µm column
0.5 1 1.5 2 2.5 3 3.5
0
50
100
150
200
250
300 1.9
63
mA
U
Innovator
rituximab
0.5 1 1.5 2 2.5 3 3.5
0
50
100
150
200
250
300 1.9
61
mA
U
Biosimilar
rituximab
Biosimilar
rituximab
Innovator
rituximab
LCHC
LCHC
Reduced mAb analysis on a AdvanceBio RP-mAb column
Diphenyl, 2.1 x 50 mm, 3.5µm
C4.1 x 50 mm, 3.5µm
Biosimilar
rituximab
Innovator
rituximab
LC HC
LC HC
Intact mAb mass analysis on a AdvanceBio RP-mAb C4, 2.1 x 100 mm, 3.5µm column
7x10
0
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 10
mAb1
Co
un
ts
4x10
0
0.4
0.8
1.2
1.6
2
2.4
2200 2400 2600 2800 3000 3200 3400 3600 3800
Co
un
ts
5x10
0
0.4
0.8
1.2
1.6
2
2.4 148225.23
148063.92148389.73
148545.04
147917.97
147800 148000 148200 148400 148600
G0/G0F
G0F/G0F
G0F/G1F
G1F/G1F
G1F/G2F
Co
un
ts %
7x10
0
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 10
mAb 2
Co
un
ts
4x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
2200 2400 2600 2800 3000 3200 3400 3600 3800
Co
un
ts
5x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
147244.07 147405.32
147079.63
147564.57
147000 147200 147400 147600
G0F/G0F
G0F/G1F G0F/G2F
G1F/G2F
Co
un
ts %
7x10
0
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10
mAb3 (antibody drug conjugated )
Co
un
ts
Acquisition Time (min)
3x10
0
0.4
0.8
1.2
1.6
2
2.4
2.8
2200 2400 2600 2800 3000 3200 3400 3600 3800
Co
un
ts
3x10
0
0.4
0.8
1.2
1.6
2
2.4
2.8153018.05151101.04
152058.74
149183.80
150143.59
153970.66
154928.31
156046.34
148225.52
148000 150000 152000 154000 156000 158000
DAR 0
DAR 1
DAR 2
DAR 3DAR 4
DAR 5
DAR 6
DAR 7
DAR 8Co
un
ts %
Mass-to-Charge (m/z) Deconvoluted Mass (amu)
mAb fragment analysis on AdvanceBio RP-mAb C4, 2.1 x 100 mm, 3.5µm column
8x10
0
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9 10
Acquisition Time (min)
Coun
ts LC
HC
8x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
2 3 4 5 6 7 8 9 10 11
Acquisition Time (min)
Coun
ts
2 * Fab
2 * Fc
9x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
2 3 4 5 6 7 8 9 10 11 12
Acquisition Time (min)
Coun
ts
F(ab’)2
ScFc
7x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
23036.20
23058.1823074.94
23093.61
Deconvoluted Mass (amu)
22980 23020 23060 23100 23140
Coun
ts %
5x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4 50673.74
50511.62
50836.01
Counts vs. Deconvoluted Mass (amu)
50400 50600 50800 51000
Coun
ts %
4x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
25646.24
25484.18
25808.33
Counts vs. Deconvoluted Mass (amu)
25350 25450 25550 25650 25750 25850 25950
Co
un
ts %
Co
un
ts %
Counts vs. Deconvoluted Mass (amu)
6x10
0
0.4
0.8
1.2
1.6
2
2.4
2.8
3.225200.6225362.81
24997.3625524.86
24835.3524631.9825815.9924469.80 25654.04
24400 24600 24800 25000 25200 25400 25600 25800
6x10
0
1
2
3
4
5
6
7
8
996715.02
96808.97
96874.21
96400 96600 96800 97000 97200
Co
un
ts %
Counts vs. Deconvoluted Mass (amu)
mAb1
mAb2
mAb3 (antibody drug
conjugate)
LC HC
2 * Fc
6x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.647179.76
47342.31
Counts vs. Deconvoluted Mass (amu)
46900 47000 47100 47200 47300 47400 47500 47600
Cou
nts
%
2 * Fab
ScFc F(ab’)2
Publication Number 5991-6626EN
