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Recent Advancement in Analytical Techniques
for Biologics Characterization
Ning Tang, Ph. D.
Senior Application Scientist
Agilent Technologies
Agenda
• Introduction to Agilent Biopharma portfolio
• Intact protein analysis
• Peptide Mapping
• PTM analysis
• CE/MS
Agilent’s Tools and Technologies for
BioPharmaceutical Analyses
• Liquid chromatography
• LC/MS
• Capillary electrophoresis
• Ion analysis (CE and HPLC)
• Isoelectric focusing
• Lab-on-a-Chip
• Evaporative light scattering
• Gas chromatography
• GC/MS
• qPCR
• LC column technology (reversed phase, ion exchange and size exclusion)
• Protein standards and protein processing kits
Agilent’s 1290 HPLC and 6530/6540 Q-TOF- Infinitely Better for LC/MS
Lowest Delay Volume
Highest Precision
Highest Mass Accuracy
Best Dynamic Range
Best A/S Performance
Greatest Productivity
High Resolution at fast
Acquisition Rate
4
Agilent Biopharma Training, AFO NPT 2010
Agilent 1290 Infinity LC
Agilent 6540 Ultra High
Definition QTOF
Enhanced performance• Zero dead volume for better
chromatographic performance
• LC/MS sensitivity
• Improved run-to-run reproducibility and
chip life-time
Robustness & ease-of-use• No clogging of spray needle
• Plug-&-Play replacement
• Improved surface characteristics for
optimal contact and sealing
Agilent’s nano HPLC and HPLC-chip Q-TOF – fully integrated solution for nano spray
The Agilent Toolbox for Biologic Characterization
Intact Antibody characterization (accurate mass)
• Instrumentation: Q-TOF/TOF with 1290 HPLC or nano LC+ HPLC-Chip, CE, CE-MS
• HPLC Columns: Poroshell 300, Zorbax SB 300, HPLC-Chip
Peptide mapping (resolution, resolution per time, Bioconfirma SW)
• Q-TOF/TOF with 1290 HPLC or nano LC+ HPLC-Chip, RRLC-UV, CE-MS
• HPLC Columns: Eclipse plus or Poroshell 120, HPLC-Chip
Charge variants (resolution of charge variants)
• Bio-inert HPLC-UV, CE
• Bio HPLC Columns: Bio Mab WCX, Bio IEX
Aggregate analysis (resolution of monomers and multimers)
• Bio-inert HPLC UV, CE
• HPLC Columns: Bio SEC
Glycan analysis (confirmation of glycan pattern, detection and separation of variant)
• mAB-Glyco-Chip, LC-MS, LC, CE,CE-MS
6
Intact Protein Analysis
Accurate Mass TOF, QTOF
1290 Infinity HPLC, nano LC with HPLC-chip
mAb Structure
Light Chain
Fc
Fab
Antigen
binding
Hinge
Glycosylation
siteTruncation
(lysine)
Disulfide
shuffling
Pyroglutamate
Deamidation/oxidation
Heavy Chain
Fucose – 146 Da
Mannose – 162 Da
N-Acetylglucosamine – 203 Da
Galactose – 162 Da
1299.3G0
1445.4G0F
1607.5G1F
1769.6G2F
Average massStructureGlycan
Mass Spectrum of mAb
10 ng on Zorbax SB300-C18 HPLC-Chip
Deconvolution results10 ng mAb injected on HPLC-Chip
MassHunter BioConfirm Software
•More than 80 predefined modifications in the chemical data dictionary
•Allows user defined modifications
•Allows C13, N15, O18, D labeled amino acid
Accurate Mass Measurement on Agilent TOF/Q-TOF
Mcalc Mexp Error (ppm)
Intact
major glycoform148,811.95 148,812.81 5.8
deglycosylated 145,923.24 145,924.41 8
Light chain 23,746.63 23,746.50 5.5
Heavy chain
major glycoform50,675.47 50,675.58 2.2
Fc
major glycoform52,755.62 52,755.64 0.4
FAB 48,046.18 48,046.09 1.8
Peptide mapping and PTM
characterization Using Agilent Q-TOF
and BioConfirm Software
Accurate Mass TOF, QTOF
1290 Infinity HPLC, nano LC with HPLC-chip
HPLC-Chip-Based Peptide Mapping - Base Peak
Chromatogram (BPC) of Trypsin Digested mAb
on C18 Chip
Peptide Mass Accuracy
Page 15
light chain A(112-130) 2101.1245 2101.1208 1.76
light chain A(113-130) 1945.0199 1945.0197 0.09
light chain A(131-146) 1796.8922 1796.888 2.38
light chain A(150-173) 2676.2669 2676.2627 1.55
light chain A(150-187) 4160.01 4160.0033 1.61
light chain A(154-173) 2134.9657 2134.9615 1.99
light chain A(193-211) 2140.0778 2140.0735 1.99
light chain A(195-211) 1874.9254 1874.9197 3.06
Sequence
NameLabel Mass
Target
Sequence
Mass
Match
Difference
(ppm)
light chain A(1-19) 1996.088 1996.0841 1.98
light chain A(26-47) 2438.1986 2438.1979 0.31
light chain A(48-56) 992.5656 992.5655 0.15
light chain A(64-93) 3388.5238 3388.5194 1.31
light chain A(94-107) 1538.7191 1538.7154 2.43
Sequence Name Label MassTarget Sequence
Mass
Match Difference
(ppm)
heavy chain A(1-19) 1880.0511 1880.048 1.65
heavy chain A(20-38) 2126.9587 2126.9554 1.56
heavy chain A(39-67) 2927.4492 2927.4414 2.68
heavy chain A(39-65) 2714.3216 2714.3188 1.05
heavy chain A(44-65) 2233.0584 2233.0539 2
heavy chain A(44-67) 2446.1802 2446.1765 1.51
heavy chain A(66-72) 835.4663 835.4664 -0.11
heavy chain A(68-72) 622.3431 622.3439 -1.28
heavy chain A(73-87) 1768.8812 1768.8778 1.93
heavy chain A(77-87) 1324.6816 1324.6809 0.51
heavy chain A(88-98) 1317.5937 1317.5911 2
heavy chain A(99-105) 714.4018 714.4024 -0.94
heavy chain A(106-129) 2531.2534 2531.2479 2.2
heavy chain A(107-129) 2375.1509 2375.1468 1.74
heavy chain A(130-141) 1185.6426 1185.6394 2.72
heavy chain A(142-155) 1320.6705 1320.6708 -0.21
heavy chain A(156-218) 6712.309 6712.3072 0.27heavy chain A(156-221) 7054.4995 7054.4975 0.28
heavy chain A(156-222) 7182.5962 7182.5925 0.52
heavy chain A(227-256) 3333.643 3333.6349 2.45
heavy chain A(231-256) 2843.4544 2843.4503 1.45
heavy chain A(257-263) 834.4274 834.4269 0.55
heavy chain A(264-282) 2138.0249 2138.0202 2.22
heavy chain A(283-296) 1676.7985 1676.7947 2.25
heavy chain A(297-309) 3115.3418 3115.3315 2.13
heavy chain A(310-325) 1807.0038 1806.9992 2.51
heavy chain A(310-328) 2227.2034 2227.2001 1.5
heavy chain A(335-342) 837.496 837.496 0
heavy chain A(347-368) 2509.3347 2509.3289 2.32
heavy chain A(349-368) 2310.1993 2310.1968 1.08
heavy chain A(353-363) 1285.6677 1285.6667 0.79
heavy chain A(353-368) 1871.9648 1871.9629 1.03
heavy chain A(369-378) 1160.6228 1160.6223 0.4
heavy chain A(379-400) 2543.1289 2543.1241 1.9
heavy chain A(401-417) 1872.9184 1872.9146 2.06
heavy chain A(423-447) 3043.3964 3043.393 1.12
heavy chain A(425-447) 2800.2679 2800.2598 2.89
heavy chain A(448-454) 659.3488 659.349 -0.35
Average Error = 1.3ppm
MassHunter BioConfirm Software
Matching rules allow incomplete
digest, predicted modifications
and DNA point mutation
BioConfirm Matches MS/MS Spectra
17
Product ion assignment
Product ion label
MS score contribution
MS/MS score contribution
Scored peak intensity /
total peak intensity
Each scored MS/MS
fragment is counted as 1
Contribution to the MS/MS score
MS/MS Helps to Determine PTM Locations
MS/MS Interpretation Helps to Determine PTM Location
MS/MS Helps to Determine PTM Locations
Analysis of Glycopeptide from mAb
Group/Presentation Title
Agilent Restricted
Page 21
BPC
Sugar Oxonium Ion Helps to Identify Glycopeptide
Group/Presentation Title
Agilent Restricted
Page 22
EIC m/z 204.084 (GlcNac)
BPCMass spectrum of the glycopeptide
TKPREEQYNSTYR
Fucose – 146 Da
Mannose – 162 Da
N-Acetylglucosamine – 203 Da
Galactose – 162 Da
Fucose – 146 Da
Mannose – 162 Da
N-Acetylglucosamine – 203 Da
Galactose – 162 Da366 528 1040
366
-
H2
O
-
2H2
O
MS/MS
Forced Oxidation Study
Using Agilent Q-TOF
Mass Profiler Pro and BioConfirm Software
•FDA Guidance for Industry. Analytical Procedures and Methods
Validation (draft guidance), August 2000.
•ICH guideline Q1A (R2). Stability Testing of New Drug Substances and
Products , November 2003.Drug Delivery Technology June 2010 Vol 10 No5
Forced Oxidation Study
Trypsin
TrypsinOx Ox
LC/MS/MS
LC/MS/MS
Ox
Ox
Ox
t-BHP
varies conc.
Comparison at Intact Protein Level
Comparison at Peptide Digest Level
Comparison of Oxidized and Unoxidized Met
Control
0.6% t-BHP
Unmodified peptide
Oxidized peptide
3% t-BHP
Control
0.6% t-BHP
3% t-BHP
Unoxidized MetOxidized Met
Forced Oxidation ResultsMet Oxidation Percentage vs. Oxidizing Reagent Concentration
0
10
20
30
40
50
60
70
80
90
100
0 0.5 1 1.5 2 2.5 3
%P
ep
tid
e O
xid
ati
on
% t-BHP
DLTMISR (Met 256)
SRWQQGNVFSCSVMHEALHNHYTQK (Met 432)
NTLYLQMSSLR (Met 84)
Molecular Structure of mAb
M.Graille et al. Crystal structure of the
complex between the monomeric form of
Toxoplasma gondii surface antigen 1
(SAG1) and a monoclonal antibody that
mimics the human immune response. J
Mol Biol, (2005), 354, 447-458.
P.Sondermann et al. The 3.2-A crystal
structure of the human IgG1 Fc
fragment-Fc gammaRIII complex.
Nature, (2000) 406, 267-273.
Using Mass Profiler Pro to Analyze Large Batch of Samples
PCA plot of different oxidation conditions• Principal component
analysis (PCA) is a
clustering tool often applied
to reduce the
dimensionality of complex
data sets.
• The result for the oxidation
samples demonstrates
correct grouping of the
technical replicates and
clear separation between
different concentrations of t-
BHP treated mAbs.
• It is clearly observed that
control (cyan) and 0.1%
(red) are well separated as
compared with other
oxidized samples.
Using BioConfirm Qualitative Comparison to
Compare Sample One to One
Common
FeaturesReference Sample
R
S
R
S
SR
mirror
Table
MS spectra
MS/MS spectraChromatograms
Unmodified peptide
R
S
R
S
SR
Single oxidation
R
S
R
S
SR
Double Oxidation
R
S
R
S
SR
Using Agilent CE/Q-TOF for peptide
mapping and glycan analysis
Page 37
• Widest choice of detctors
DAD, LIF, CCD, Single-Q, IT, TOF, QQQ, QTOF, ICP-MS
• Only vendor for plug & spray ESI-MS
complete CE-MS portfolio with a single interface solution
• Easy change from LC-MS to CE-MS and back
quick and robust ESI by MS-adaptor & spray needle kit
using Agilent Chemstation software
• Combined detection option
UV-DAD plus MS detection, LIF plus MS detection
• Grounded CE capillary endNo compromises between separation- or ESI-spray voltage
Agilent 7100 Capillary ElectrophoresisThe CE-MS advantage
CE-Electrospray is a powerful method for high resolution separation and identification for a
wide variety of molecules. Additionally it is an orthogonal separation technique to HPLC
The Agilent interface combines the advantage of a grounded CE capillary end with a robust
and easy to use spray needle that fits into the whole portfolio of Agilent MS instruments
5x10
0.10.20.30.40.50.60.70.80.9
11.11.21.31.41.51.61.71.81.9
22.12.22.32.42.52.6
Counts vs. Acquisition Time (min)7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20
Base peak electropherogram (BPE)
of trypsin digested mAb
5x10
0.20.40.60.8
11.21.41.61.8
22.22.42.62.8
33.23.43.63.8
44.24.4
Counts vs. Acquisition Time (min)
7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20
Extracted ion electropherogram
(EIE) of some peptides from mAb
light chain and heavy chain
March 14, 2011
Agilent Confidential
38
Peptide Mapping Using CE/MS
March 14, 2011
Agilent Confidential
39
Peptide Mapping Results from CE/MS
glycopeptides
5x10
0.10.20.30.40.50.60.70.80.9
11.11.21.31.41.51.61.71.81.9
22.12.22.32.42.52.6
Counts vs. Acquisition Time (min)7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20
Base peak electropherogram (BPE) of trypsin digested mAb
Glycopeptide
m/z 878.6812 - 14.284 min
3x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
Counts vs. Acquisition Time (min)7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20
Extracted ion electropherogram (EIE) MS (all)
at m/z 204.085 for the trypsin-digested mAb
March 14, 2011
Agilent Confidential
40
CE-MS and MS/MS of Glycopeptide
4x10
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
4.75712.9930
879.0147
1068.9844
1318.0174
1686.6600 2071.8270
Counts vs. Mass-to-Charge (m/z)400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200
EEQYNSTYR
[M+3H]+3
[M+2H]+2
March 14, 2011
Agilent Confidential
41
CE-MS of Glycopeptide
3x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6204.0852
126.0539
138.0542
168.0644186.0742
366.1371
Counts vs. Mass-to-Charge (m/z)110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390
36.0345
162.1939
18.0145
MS
MS/MS
A1F/A2F Glycans PVA Capillary, pH 9.2
Acknowledgements
Agilent
Ravindra Gudihal
Sureshbabu CV
Martin Greiner
Tobias Preckel
Susanne Moyer
Greg Kilby
Xiaoling Wu
Taegan Clary
Keith Waddell
Thank you !
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