High Throughput Accurate Mass Screening of Monoclonal ......10 Intact Protein Analysis – Direct...

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


• 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


• 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


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


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


• 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


• 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


Analyte Dependent

MS Compatible

Sample Load Requirement

Throughput

SEC


Analyte Dependent

MS Compatible


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


Analyte Dependent

MS Compatible


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


Analyte Dependent

MS Compatible


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


• 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


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


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


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


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


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

%


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


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

High Throughput Accurate Mass Screening of Monoclonal ......10 Intact Protein Analysis – Direct...

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Transcript of High Throughput Accurate Mass Screening of Monoclonal ......10 Intact Protein Analysis – Direct...