MSB 2012
Poster # P-104
Bioanalytical Characterization of Therapeutic Proteins by Electrophoresis Techniques
Suresh Babu CV1, Ravindra Gudihal1, Tobias Preckel2, Andreas Ruefer2, Christian Wenz2 and Martin Greiner2
1Agilent Technologies India Pvt. Ltd, Bangalore, India. 2Agilent Technologies R&D and Mktg. GmbH & Co.KG, Waldbronn, Germany
-100
0
100
200
300
400
500
600
700
800
10 15 20 25 30 35 40 45 50 [S]
[FU]
Lowermarker
UppermarkerLow MW
impurities
System peak
Light chain
Heavy chain
P80
-100
0
100
200
300
400
500
600
15 20 25 30 35 40 45 50
[FU]
[S]
Lowermarker
Uppermarker
Aggregates
System peak
Light chain
Heavy chain
P230
-50
0
50
100
150
200
250
300
350
400
15 20 25 30 35 40 45 50[S]
[FU]
Lowermarker
Aggregates
Light chain
Heavy chain
HSP-250
2100 BioAnalyzer (P80, P230, HSP 250 protein assay kits)
3100 OFFGEL
G7100 Capillary Electrophoresis (CE)
G7100 Capillary Electrophoresis – 6520 QTOF Mass
Spectrometry (CE-MS)
Introduction
Therapeutic proteins such as monoclonal antibodies
(mAbs) are major biopharmaceutical products with
clinical applications. Regulatory agencies demand
comprehensive protein characterization data hence
different analytical techniques are employed for the
analysis. Complexity of sample cannot be covered by a
single analytical technique but requires a suite of tools
to provide necessary quality of data. In this work, we
present a workflow based approach for the analysis of
therapeutic proteins like purity, accurate mass,
aggregation, peptide sequence, glycopeptide and glycans.
Initial screening for product purity, fragments of mAb,
protein PEGylation products were analyzed using
microfluidic electrophoresis as a QC tool. Capillary
electrophoresis (CE), liquid chromatography (LC), and
combinations of these with mass spectrometry (MS) are
applied for the analysis of intact protein (mAb), tryptic
digest, peptide mapping, glycopeptide/glycan and
accurate mass measurement. Further, mAb charge
heterogeneity was separated using liquid-phase
isoelectric focusing (IEF) technique followed by
microfluidic CE analysis. The result presented here shows
the utility of multiple analytical platforms for in-depth
protein characterization.
Instrumentation
Results and Discussion
Therapeutic protein analysis with the microfluidic-
based Bioanalyzer
-50
0
50
100
150
200
250
300
15 20 25 30 35 40 45 50 [S]
[FU]Lowermarker
ngAb
Upper marker
Intact antibody
P230
System peak
Mixture of Light and Heavy chain
-200
100
400
700
1000
1300
15 20 25 30 35 40 45 50 [S]
[FU]Lowermarker
ngAb
Intact antibody
HSP-250
Mixture of Light and Heavy chain
Aggregates
Figure 1A. Bioanalyzer analysis of IgG2 preparation under
reducing conditions
Figure 1B. Bioanalyzer analysis of IgG2 preparation under
non-reducing conditions
Analysis of antibody charge heterogeneity
Size
[kDa]
pH 3.0 pH 10.0
1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 15 16 17 18 19 20 21 22 23 24
LOA
D
LAD
DER
mAb
240-
150-
95-
63-
46-
28-15-
5-
Isoelectric point (pl)
Mo
lecu
lar
we
igh
t
240-
150-
95-
63-
46-
28-15-
5-
Size
[kDa]
pH 3.0 pH 10.0
1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 15 16 17 18 19 20 21 22 23 24
mAb
LOA
D
LAD
DER
Isoelectric point (pl)
Mo
lecu
lar
we
igh
t
Tween-20 conditions
Native conditions
-1
0
1
2
3
4
5
10 20 30 40 50 60
[FU]
Time [s]
Native, OFFGEL fraction 1
Native, OFFGEL fraction 2
Native, OFFGEL fraction 3
mAb variants (139 kDa)
-5
0
5
10
15
20
25
30
10 20 30 40 50 60
[FU]
Time [s]
Native, OFFGEL fraction 9
Native, OFFGEL fraction 10
mAb main product (142 kDa)
-0.5
0.5
1.5
2.5
3.5
10 20 30 40 50 60
[FU]
Time [s]
Load
mAb (142 kDa)
-5
0
5
10
15
20
25
30
10 20 30 40 50 60
[FU]
Time [s]
With Tween OFFGEL fraction 1
With Tween, OFFGEL fraction 2
With Tween, OFFGEL fraction 3
With Tween, OFFGEL fraction 4
With Tween, OFFGEL fraction5
mAb variants (144-147 kDa)
-20
0
20
40
60
80
100
120
10 20 30 40 50 60
[FU]
Time [s]
With Tween, OFFGEL fraction 9
With Tween, OFFGEL fraction 10
With Tween, OFFGEL fraction 11
mAb main product (142 kDa)
-2
0
2
4
6
8
10
10 20 30 40 50 60
[FU]
Time [s]
Load
mAb (142 kDa)
Results and Discussion
Figure 2. Monitoring antibody charge variants using a
combination of OFFGEL Fractionation by isoelectric point
and high sensitivity protein detection with Bioanalyzer
Results and Discussion
Ladd
er
0.5m
g/m
l
1 m
g/m
l
2 m
g/m
l
4 m
g/m
l
6 m
g/m
l
Cont
rol
PEGlyation reagent (pNP)
240.0
150.0
95.0
63.0
46.0
28.0
15.0
7.0
4.5
Size [kDa]
Characterization of PEGylated proteins
The Bioanalyzer P230 Assay for Protein PEGylation
Easy-to-use tool that provides high level of resolution
Allows efficient optimization of PEGylation reaction
conditions
Fast and quantitative monitoring of production batches
Figure 3. Bioanalyzer analysis of protein PEGylation.
PEGylating reagents: Methoxy PEG p-nitrophenyl carbonate
(mPEG pNP, MW 5000)
Results and Discussion
Conclusions
• Initial characterization of therapeutic protein/mAb is
achieved using the electrophoretic techniques such
as OFFGEL and microfluidc based electrophoresis.
This sets further stage for detail analysis of mAb by
advanced mass spectrometric techniques (CE-MS, LC-
MS).
• The combination of CE with Q-TOF MS is a valuable
tool for peptide mapping of small quantity
biopharmaceuticals.
• Highly sialylated glycans was more suited when CE-
MS was used as analysis tool while LC-MS seems to
be better adapted for analysis of neutral glycans.
• Combination of various electrophoretic and LC
techniques with mass spectrometry techniques was
demonstrated for protein characterization.
Acknowledgment
The study of protein PEGylation was in collaboration with GangaGen
Biotechnologies Pvt. Ltd, India. We would like to thank Sundaram M
Palaniswamy, Umamaheshwari S, Suneel Basingi for their direct
involvement in this project. We also acknolowdge the support received
from Dr.M.Jayasheela and Mrs Bharathi Sriram.
CE-QTOF MS analysis of glycopeptide and glycans of
monoclonal antibodies
x105
0.5
1
1.5
2
2.5
Counts vs. Acquisition Time (min)7 8 9 10 11 12 13 14 15 16 17 18 19 20
Glycopeptide
m/z 878.6812 - 14.284 min
3x10
0
0.2
0.6
1
1.4
1.8
2.2
2.6 204.0852
126.0539
138.0542168.0644
186.0742
366.1371
Counts vs. Mass-to-Charge (m/z)110 130 150 170 190 210 230 250 270 290 310 330 350 370 390
36.0345
162.1939
18.0145
Diagnostic ion at m/z 204.085
BPE
Figure 4. Base peak electropherogram (BPE) of a trypsin-
digested mAb and CE-MS/MS analysis
Δ1444.87
Δ2890.81
G0F/G0Fx 104
0
1
2
3
4
5
6
6.5
148812.81
145922.00
147367.94
146329.69
146816.21
147719.65
Counts vs. Deconvoluted Mass (amu)
145500 146000 146500 147000 147500 148000 148500 149000 149500 150000 150500 151000 151500
5.5
4.5
3.5
2.5
1.5
0.5
Δ 162.16 hexose unit
x104
0
1
2
3
4
5
6
7
8
148812.81
148974.97148840.65148916.37
148765.43
C ounts vs. Deconvoluted Mass (amu)
148750 148800 148850 148900 148950
G0F/G0F
G0F/G1F
Theoretical: 148811.9 5Da
Mass accuracy: 5.7 ppm
Figure 7. LC-MS analysis of mAb
Figure 5. Extracted ion electropherogram (EIE) and the
representative MS trace from CE-MS analysis of APTS
labeled neutral (A) and neutral/sialylated(B)glycans
4x 1 0
0
0 . 05
0 . 1
0 . 15
0 . 2
0 . 25
0 . 3
0 . 35
0 . 4
0 . 45
0 . 5
0 . 55
0 . 6
0 . 65
0 . 7
0 . 75
0 . 8
0 . 85
0 . 9
0 . 95
1
1 . 05
1 . 1
1 . 15
1 . 2
1 . 25
1 . 3
-
1
Co unts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0
G0 G0F Man5 G1 G1F G2 G2F
3x 1 0
0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
0 .7
0 .8
0 .9
1
1 .1
1 .2
1 .3
1 .4
1 .5
1 .6
1 .7
1 .8
1 .9
2
C o u nts vs. Acquisiti on Ti me (m in)
7 7 .5 8 8 .5 9 9 .5 1 0 1 0 .5 1 1 1 1 .5 1 2 1 2 .5 1 3 1 3 .5 1 4 1 4 .5 1 5 1 5 .5 1 6 1 6 .5 1 7 1 7 .5 1 8 1 8 .5 1 9 1 9 .5 2 0 2 0 .5
G2
G2F
G2-
1NA
NA
G2-
2NA
NA
G2F
-2N
AN
A
G2F
-1N
AN
A
3x 10
0
0. 25
0. 5
0. 75
1
1. 25
1. 5
1. 75
2
2. 25
2. 5
2. 75
3
3. 25
3. 5
3. 75
4
4. 25
4. 5
4. 75
5
5. 25
5. 5
5. 75
6
6. 25
6. 5
6. 75
7
7. 25
7. 5
7. 75
8
8. 25
C ounts vs. Mass-to-Charge (m/z)
820 830 840 850 860 870 880 890 900 910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 1130
877.7188
950.7522
958.7443
1031.7785
836.6937
1113.31291039.7870
[M-2H]2-
[M-2H]2-
[M-2H]2-
[M-2H]2-
[M-2H]2-
[M-2H]2- [M-2H]2-
3x 10
0
0. 025
0. 05
0. 075
0. 1
0. 125
0. 15
0. 175
0. 2
0. 225
0. 25
0. 275
0. 3
0. 325
0. 35
0. 375
0. 4
0. 425
0. 45
0. 475
0. 5
0. 525
0. 55
0. 575
0. 6
0. 625
0. 65
0. 675
0. 7
0. 725
0. 75
0. 775
0. 8
0. 825
0. 85
0. 875
0. 9
0. 925
0. 95
0. 975
1
1. 025
1. 05
1. 075
C ounts vs. Mass-to-Charge (m/z)
1040 1060 1080 1100 1120 1140 1160 1180 1200 1220 1240 1260 1280 1300 1320 1340 1360 1380 1400 1420
1113.3210[M-2H]2-
1185.8394[M-2H]2-
1258.8677[M-2H]2- 1331.3880
[M-2H]2-
1404.4208[M-2H]2-
1040.3042[M-2H]2-
2x10
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
0.95
1
1.05
Counts (%) vs. Acquisition Time (min)
8.8 9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8 13 13.2
G3-5NANA
G3-4NANA
G3-3NANA
G2-2NANA
G3-2NANA
G2-1NANA
G3-1NANA
3x10
0
0.25
0.5
0.75
1
1300.4115
3x10
0
1
2
31203.3829
901.9766
3x10
0
2
4
6
1106.0137
829.2007
3x10
0
2
4
1331.3964887.2662
3x10
0
0.5
1
1.51008.9831
1513.9684
3x10
0
0.25
0.5
0.75
1
1185.8510
2x10
0
1
2
1368.4168
C ounts vs. Mass-to-Charge (m/z)
850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800
[M-3H]3-
[M-3H]3-
[M-4H]4-
[M-3H]3-
[M-3H]3-
[M-4H]4-
[M-2H]2-
[M-3H]3- [M-2H]2-
[M-2H]2-
[M-2H]2-
Figure 6. CE-MS analysis of released glycans from a
glycoprotein
MSB 2012
Poster P012
For Research Use Only. Not for use in diagnostic procedures.