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High Throughput Accurate Mass Screening of Monoclonal Antibodies
•Jim Blasberg•April 28, 2011
sigma-aldrich.com
2Life Science and High Technology CenterLife Science and High Technology CenterSigmaSigma--Aldrich, St. LouisAldrich, St. Louis
3
Analytical R&D
4
Analytical R&D Objectives
• Serve as corporate center of excellence for mass spectrometry
• Provide analytical support to Research Biotech and SAFC product development teams
• Generate application data supporting marketing of new Biotech products and evaluation of new technologies for Business Development
• Provide high-end analytical characterization of proteins and antibodies
• Provide specialized testing in support of Quality Control
• Lead and/or support troubleshooting and problem-solving initiatives in support of high-value products
5
BulkSample
ReduceComplexity
SpecificStructural Details
Data Analysis
Intact MW analysis with all PTMSDS-PAGE
LC-MS
Peptide/Glycopeptide MappingLC-MS/MS
Glycan AnalysisMALDI/ESI
HPAECHPLC
Sialic Acid AnalysisHPLC-FLDHPAEC,
Colorimetry
Correlate Data from All Methods
Remove PTMEnzymaticallyChemically
Reduce & SeparateSEC-MS
Generalized Protein/Glycoprotein Characterization Workflow
6
Why Study Intact Protein Mass?
• Intact mass analysis can detect altered chemical and physical properties related to protein function
• Detection of PTMs– N/C-terminal variation
• Glycosylation state– 50 - 90% of proteins are glycosylated
• Confirm primary sequence • Evaluate stability
– Oxidation or degradation
7
Intact Protein Analysis – General Considerations
• ESI • We chose to focus on Electrospray Ionization or ESI as our technique of choice
for intact protein analysis
Infusion or RP-HPLCsample introduction
8
Intact Protein Analysis – General Considerations
• Deconvolution • How we get from multiply charged to zero-charge accurate mass• Time of Flight (TOF) mass analyzer – Waters LCT or QTof Premier• Multiply charged ions centered around 1000 m/z
– Good resolving power in this m/z range– Deconvolute to zero charge
A: The m/z values can beexpressed as follows:
m/z = (MW + nH+)/n
B: With 2 adjacent m/z valueswe can determine charge state and solve for MW
9
Intact Protein Analysis – General Considerations
• Sample Introduction - ESI
• Direct Infusion - MS– Low throughput– Good approach for small numbers of
relatively pure samples– Typically requires cleanup
• RP-HPLC - MS– Moderate throughput– Analyte dependent, MS compatible– Good for protein purity and intact
Infusion RP-HPLC
Sample Prep Requirement
Analyte Dependent
MS CompatibleSample Load Requirement
Throughput
10
Intact Protein Analysis – Direct Infusion• Desalting and Direct Infusion
• Request for intact mass of single protein sample• UF solvent exchange/desalting• Zero charge accuracy within 0.01%
Desalted BSA 1 mg/mL
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
10020110421_BSA_INFUSION_001 TOF MS ES+
TIC1.60e6
1.15
0.85
1.28
Desalted BSA 1 mg/mL
m/z1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100
%
0
10020110421_BSA_INFUSION_003 28 (0.519) Cm (17:29) TOF MS ES+
2401510.4850
1444.8199
1444.79191444.7218
1384.63661384.5267
1582.2905
1582.3198
1620.8247
1665.4830
1665.7242
TIC
MS Raw DataSum ~20 scans
Deconvoluted DataUsing MaxEnt1
Cysteinylated BSAΔ=119
Desalted BSA 1 mg/mL
mass66100 66200 66300 66400 66500 66600 66700 66800 66900 67000
%
0
10020110421_BSA_INFUSION_003 28 (0.519) M1 [Ev-72518,It25] (Gs,0.750,1322:1984,1.00,L50,R50); Cm (17:29) TOF MS ES+
6.66e366429
6654866461
+32
66429
66548
11
Intact Protein Analysis – RP-HPLC-MS• RP-HPLC of Intact Protein
• Purity and intact data – Murine EGF• Generic 0.1% TFA/ACN gradient
12-Jun-200819:19:18E1257 047K0440
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
%
54
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
AU
0.0
1.0e+1
2.0e+1
3.0e+1
4.0e+1
5.0e+1
6.0e+1
7.0e+1
061208_EGF_004 2: Diode Array Range: 7.979e+111.17
1.60
061208_EGF_004 1: TOF MS ES+ TIC
8.63e411.26
1.72
0.028.207.91 8.46
9.83
24.8924.56
22.62
20.6717.83
28.25
UV Trace
MS TIC
Sum across peak including potential impurities
12
Intact Protein Analysis – RP-HPLC-MS• RP-HPLC of Intact Protein
• Purity and intact data – Murine EGF• More than one isoform under main peak
Deconvoluted MS DataTheory = 6039.7Experimental = 6039.3
Minor Component B6039-5925=114Truncated N-terminal ASN?
EGF SequenceNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR
E1257 047K0440
m/z200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
%
0
100061208_EGF_004 614 (11.262) Cm (610:621-(633:648+580:600)) 1: TOF MS ES+
A4;1510.6832
A51208.7834
B51185.8752
1007.5003
B41482.2542
1213.6077
1510.884
1511.3408
1511.7428
A32014.0394
1513.5339
1516.4966B3
1976.1003
A: 6039.32±0.08B: 5925.13±0.06
A5
A4
A3
B5
B4
B3
13
Intact Protein Analysis – RP-HPLC-MS• RP-HPLC of Intact Protein
• Purity and intact data – Murine EGF• XICs demonstrate multiple isoforms are present
12-Jun-200819:19:18E1257 047K0440
Time10.20 10.40 10.60 10.80 11.00 11.20 11.40 11.60 11.80 12.00 12.20 12.40
%
0
10.20 10.40 10.60 10.80 11.00 11.20 11.40 11.60 11.80 12.00 12.20 12.40
%
0
10.20 10.40 10.60 10.80 11.00 11.20 11.40 11.60 11.80 12.00 12.20 12.40
%
1
10.20 10.40 10.60 10.80 11.00 11.20 11.40 11.60 11.80 12.00 12.20 12.40
%
2
061208_EGF_004 1: TOF MS ES+ 1486.1
32811.10
10.2010.14 10.64
11.3411.41 11.50
11.96 12.4212.22
061208_EGF_004 1: TOF MS ES+ 1514.71.07e3
11.04
11.32
061208_EGF_004 1: TOF MS ES+ 1510.87.43e3
11.26
061208_EGF_004 1: TOF MS ES+ 1482.21.89e3
11.37
oxEGF-N
oxEGF
EGF
EGF-N
3%
10%
69%
18%
14
Intact Protein Analysis – SEC-MS
• SEC-MS of Intact Protein• Traditionally not MS compatible• Developed MS compatible MP system, 45% ACN/0.1% TFA• 3 – 300 x 7.8 mm columns in series
Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00
%
40
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00
AU
0.0
5.0
1.0e+1
1.5e+1
2.0e+1
032207_JB_002 2: Diode Array Range: 2.359e+141.56
29.84
28.16
38.03
31.7150.58
032207_JB_002 Sm (Mn, 2x5); Sm (Mn, 2x5) 1: TOF MS ES+ TIC
1.26e441.9341.29
29.88
28.14
23.19
13.00
38.13
33.73
50.59
47.1543.57
Peak Analyte MW1 BSA 664292 Cytochrome C 123273 Aprotinin 65114 Insulin B 34965 LH-RH 11836 TRH 3627 Phenylalanine 165
1
2
34
5
6
7
SEC
Sample Prep Requirement
Analyte Dependent
MS Compatible
Sample Load Requirement
Throughput
SEC
Sample Prep Requirement
Analyte Dependent
MS Compatible
Sample Load Requirement
Throughput
15
Intact Protein Analysis – SEC-MS• SEC-MS of Intact Proteins
• Modify for general use, single column– Shorten run time– Divert small MW after analyte(s) elute– 8-minute run time, no re-equilibration required
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
%
0
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
%
0
10020110421_BSA_JB_QTOF_001 1: TOF MS ES+
TIC3.17e5
3.54
0.046.13
5.54
20110421_BSA_JB_QTOF_002 1: TOF MS ES+ TIC
4.19e53.41
BSA Non-Reduced
BSA Reduced
Divert
SEC
Sample Prep Requirement
Analyte Dependent
MS Compatible
Sample Load Requirement
Throughput
16
Intact Protein Analysis – SEC-MS
• SEC-MS of Reduced Antibody• Optimize for sample load, 2.0 and 4.6 mm id
Time1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75
%
53
1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75
AU 5.0e-3
1.0e-2
1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75
%
57
1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75
AU
0.0
2.5e-3
5.0e-3
20090527_SO57_JB_QTOF_007 2: Diode Array Range: 7.175e-33.52
4.15
20090527_SO57_JB_QTOF_007 1: TOF MS ES+ TIC
1.28e43.54
3.261.65 1.76 1.98 2.282.08 2.32 2.862.48 2.71 2.97
4.174.14 4.254.43
4.69 4.86 5.194.97 5.675.45 5.77
20090527_SO57_JB_QTOF_001 2: Diode Array Range: 1.02e-24.00
4.77
20090527_SO57_JB_QTOF_001 1: TOF MS ES+ TIC
1.29e43.95
3.671.65 3.602.172.131.95 2.802.582.28 3.193.00 3.45
4.06
4.234.644.41 4.67 4.97 5.10 5.17
5.41 5.47 5.67
2.0 mm0.7 µg
4.6 mm3.5 µg
SEC
Sample Prep Requirement
Analyte Dependent
MS Compatible
Sample Load Requirement
Throughput
17
Antibody Analysis – HT Development
• SAFC Interests – Glycoprofile and Protein Quality
• Media Development
• Raw Material Characterization
• Cell Line Engineering
• Ultimately the ability to tune glycoprofile
• Standard workflows not amenable to high throughput
18
Antibody Analysis – HT Development
• Typical Mammalian Antibody Glycan Structures
Galactose
N-Acetylglucosamine
Mannose
Fucose
G2F
G1F
G0F
19
• SEC-MS Intact Protein Methodology• Analysis of intact and reduced mAb
22-Apr-200908:07:04SO57 1127-62 4.3 mg/mL Reduced
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25
%
4
042209_SO57_003 1: TOF MS ES+ TIC
2.32e53.84
4.45
SO57 1127-62 4.3 mg/mL Reduced
m/z400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800
%
0
100042209_SO57_003 207 (3.836) Cm (200:218-162:189) 1: TOF MS ES+
9611440.7675
1363.0123
1327.1686
1293.1565
1260.8181
1230.1190
1200.8855
1172.9645
1483.1755
1528.1180
1575.8411
1626.6205
1680.7927
1738.6687
1800.8450
1867.4794
1939.2255
SO57 1127-62 4.3 mg/mL Reduced
mass48800 49000 49200 49400 49600 49800 50000 50200 50400 50600 50800 51000
%
0
100042209_SO57_003 207 (3.836) M1 [Ev-138428,It32] (Gs,0.750,1031:2437,1.00,L33,R33); Cm (200:218-162:189) 1: TOF MS ES+
1.85e550395.0000
48949.0000 50361.0000
50557.0000
50585.0000
Heavy Chain
Deglycosylated
G0F
G1F
G2F
22-Apr-200907:33:02SO57 1127-62 4.3 mg/mL Intact
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25
%
21
042209_SO57_002 1: TOF MS ES+ TIC
4.27e43.56
0.06
4.39
SO57 1127-62 4.3 mg/mL Intact
m/z400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800
%
0
100042209_SO57_002 192 (3.559) Cm (185:209) 1: TOF MS ES+
492997.66892876.6860
2824.68462574.0740
2405.3655
3059.9355
3128.4614
3267.7783
3342.3911
3350.5981
SO57 1127-62 4.3 mg/mL Intact
mass145800 146000 146200 146400 146600 146800 147000 147200 147400 147600 147800 148000 148200 148400
%
0
100042209_SO57_002 192 (3.559) M1 [Ev-107673,It13] (Gs,0.750,2006:4000,1.00,L33,R33); Cm (185:209) 1: TOF MS ES+
627146660.0000
146513.0000146462.0000
146238.0000145761.0000
146823.0000
146990.0000
147131.0000
147289.0000
147574.0000 147691.0000 147929.0000
148543.0000
Intact Antibody 49
961 185000
627G0F/G0F
G0F/G1F
G0F/G2FG1F/G1F
Antibody Analysis – HT Development
MS TIC Raw Specta Deconvoluted Data
20
• Option 1 – Direct from media
Transfer clarified mediato 96-well plate
• Fast and simple workflow• Spiked pristine media ok• Real samples precipitated and gave no signal in SEC-MS
Reduce directIn-media
Analyze by SEC-MS
Process Datain BPL
Antibody Analysis – HT Development
21
• Option 2 – 96 well UF membrane
Transfer clarified mediato a 96-well UF plate andwash to remove low MW
• Two more steps than Option 1• Still fast and simple• Spiked pristine media showed only polymer, likely Pluronic
Reconstitute and reduce
Analyze by SEC-MS
Process Datain BPL
Antibody Analysis – HT Development
22
• Option 3 – Protein-A purification
Transfer Protein-A resinto a 96-well filter plate
• Several more steps than Option 1 or 2• A bit more involved but still reasonable time-wise, 2-3 hrs/plate• To-date has been successful for numerous media samples of suitable titer
ReduceAnalyze bySEC-MS
Process Datain BPL
Transfer clarified media to the 96-well filter plateWash Equilibrate/
Wash
Add elution buffer equilibrate and collect
Antibody Analysis – HT Development
23
HT Application – Clone Screening
• Purification Procedure• 50-µL P3476 Protein A Agarose
Fast Flow per well, wash w/EQ buffer
• 750-µL clarified spent media containing target IgG preferably <100 µg/mL
• Equilibrate 10-min, wash 2x w/EQ, add 100-µL ELUT buffer equilibrate 10-min
• Collect eluted antibody in a 96-well plate
• Add 5-µL 1M ABC and 1M DTT, incubate 0.5 hr at RT
• SEC-MS Procedure• Waters Acquity UPLC• Column: Toso Haas TSK Gel
SW3000XL, 300 x 2.0 mm, 4 µm• MP: 0.1% TFA 45% CH3CN• Flow Rate: 0.125 ml/min• Inj. Vol.: up to 20 µL • Run time: 8 minutes• Flow Divert: 4.6-7.9 min• MS: Waters QToF Premier (ESI+)• Capillary: 3.2 kV• Sample Cone: 40.0 V• RF Lens: 600.0 V• Extraction Cone: 3.0 V• Desolvation: 240 °C• Source: 120 °C• Scan Range: 400-4000
24
HT Application – Clone Screening• Purification Procedure
• Typical Plate Layout• Real throughput
– 84 spent media samples complete overnight– Data analysis day 2
Purified mAb Reference Spent Media Control
Clone Screening Assay – Day 7Biological duplicate samples
Clone Screening Assay – Day 10Biological duplicate samples
25
• BiopharmLynx Data Processing• Automated deconvolution of data• Set up BPL method with appropriate sequence and modifications• Automated deconvolution of 96 samples in <20 min• Manual inspection of results with final summary in Excel
HT Application – BPL Output
26
• Protein-A + SEC-MS + Data Processing• Holistic assay variability evaluation• Coaching expectations
HT Application – Reproducibility
Model Antibody - Reference StandardModifiers Average SD %RSDG0F 66.3 66 66.5 66.3 0.25 0.4%G1F 22.1 22 21.7 21.9 0.21 0.9%Non-Glycosylated 5.4 5.7 5.3 5.5 0.21 3.8%G2F 2.7 2.9 2.7 2.8 0.12 4.2%G0 2.4 2.5 2.6 2.5 0.10 4.0%Man5 1.1 1 1.2 1.1 0.10 9.1%
Model Antibody - Spent Media ControlModifiers Average SD %RSDG0F 72.5 72.7 72.2 72.5 0.25 0.3%G1F 20 19.9 20 20.0 0.06 0.3%Non-Glycosylated 2.5 2.6 2.6 2.6 0.06 2.2%G2F 2.5 2.4 2.4 2.4 0.06 2.4%G0 1.7 1.6 1.8 1.7 0.10 5.9%Man5 0.8 0.8 1 0.9 0.12 13.3%
Relative Abundance %
Relative Abundance %
27
• Clone Screen Data Processing• Evaluate glycoprofile of various clones• Biological replicates look good• Discovered widely variable clonal Man5 levels
Analyte: 20100525_IgG2B_JB_QTOF_071.raw
50374 51468Average Mass50600 50800 51000 51200 51400
% (m
ax =
532
0.6
Cou
nts)
0
25
50
75
100A
50846.64
A51008.54
A51170.10A
50620.7350947.02
51120.3850788.4951271.93 51402.58
A : IgG 2B HC N-Term Q No K : 50846.29
T
Min Intensity Threshold : 0 counts
Control: 20100525_IgG2B_JB_QTOF_069.raw
50374 51468Average Mass
50600 50800 51000 51200 51400
% (m
ax =
225
5.1
Cou
nts)
0
25
50
75
100A
51008.44
A50846.41
A50619.07
A51171.4750703.85 50950.17
A51299.73
51213.00 51428.3950561.22
A : IgG 2B HC N-Term Q No K : 50846.29
T
Min Intensity Threshold : 0 counts
Clone 1
Clone 2
Man
5
G0F
G1F
G2F
HT Application – Clone Screening
28
• Man5 Content• Export data from BPL to Excel• Average biological replicates• Normalize Man5 to G0F and compare Man5 relative abundance
Man5 Content
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39
Assay Replicate (1/2, 3/4, etc.)
Aver
age
G0
Nor
mal
ized
%
HT Application – Clone Screening
29
BulkSample
ReduceComplexity
SpecificStructural Details
Data Analysis
Intact MW analysis with all PTMSDS-PAGE
LC-MS
Peptide/Glycopeptide MappingLC-MS/MS
Glycan AnalysisMALDI/ESI
HPAECHPLC
Sialic Acid AnalysisHPLC-FLDHPAEC,
Colorimetry
Correlate Data from All Methods
Remove PTMEnzymaticallyChemically
Reduce & SeparateSEC-MS
Generalized Protein/Glycoprotein Characterization Workflow
30
RT: 0.00 - 91.02
0 10 20 30 40 50 60 70 80 90Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
20.83
84.2431.70 35.12 64.2130.65 82.0868.4348.44 60.9120.278.691.2220.83
84.3238.90 45.66 48.4421.49 67.87 83.6965.80 75.7837.1919.98
NL: 1.90E6m/z= 1317.85-1318.20 F: FTMS + p NSI Full ms [350.00-1600.00] MS 2010709_IgG2B_JB_LTQFT_10
NL: 4.68E5m/z= 1203.80-1204.11 F: FTMS + p NSI Full ms [350.00-1600.00] MS 2010709_IgG2B_JB_LTQFT_10
• Man5 Confirmation• Confirm Man5 by glycopeptide analysis on the LTQ-FT• Tryptic peptide EEQYNSTYR-Man5 (2405.9349)
2010709_IgG2B_JB_LTQFT_10 #2166-2228 RT: 20.64-21.21 AV: 7 NL: 7.11E5F: FTMS + p NSI Full ms [350.00-1600.00]
1150 1200 1250 1300 1350 1400 1450 1500 1550m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1318.03
1399.06
1203.98
1216.49 1358.041244.50 1480.091375.761297.52 1346.54
1261.26 1406.05 1439.32 1533.101511.371176.44 1562.47
G0F XIC (+2)1.9E6
Man5 XIC (+2)4.7E5 (25% G0F)
Man
5
G0F
G1F
G2F
+2 Glycopeptides
HT Application – Clone Screening
31
• Component Titration Experiment – Product Quality Impact• Same Protein-A - SEC-MS - BPL workflow
HT Application – Media Component Titration
RMC.01.SOY Glycan Distribution
0.0
10.0
20.0
30.0
40.0
50.0
60.0
8H02
84B - 0
8H02
84B - 5
8H02
84B - 1
5
9K00
48 - 0
9K00
48 - 5
9K00
48 - 1
5
8E02
58 - 0
8E02
58 - 5
8E02
58 - 1
5
6L03
59 - 0
6L03
59 - 5
6L03
59 - 1
06L
0359
- 15
9C02
30 - 0
9C02
30 - 5
9C02
30 - 1
5
Lot - g/L Soy
% T
otal
Gly
can
Glycosylation G0F N(1) Glycosylation G1F N(1) Glycosylation G2F N(1) Non-glycosylated Glycosylation G0 N(1) Glycosylation Man5 N(1)
32
Acknowledgements
• Sigma Analytical R&D• Kevin Ray• Ben Cutak• Jim Walters• Gordon Nicol• Janet Irungu• Mark Angeles
• SAFC Biosciences• Nan Lin• Joaquina Mascarenhas• Andrew Christie• Chas Hernandez
• Supelco• Tracy Ascah