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Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, Ph. D. Senior Application Scientist Agilent Technologies

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Page 1: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Recent Advancement in Analytical Techniques

for Biologics Characterization

Ning Tang, Ph. D.

Senior Application Scientist

Agilent Technologies

Page 2: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Agenda

• Introduction to Agilent Biopharma portfolio

• Intact protein analysis

• Peptide Mapping

• PTM analysis

• CE/MS

Page 3: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 4: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 5: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 6: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 7: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Intact Protein Analysis

Accurate Mass TOF, QTOF

1290 Infinity HPLC, nano LC with HPLC-chip

Page 8: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 9: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Mass Spectrum of mAb

10 ng on Zorbax SB300-C18 HPLC-Chip

Page 10: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Deconvolution results10 ng mAb injected on HPLC-Chip

Page 11: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 12: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 13: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Peptide mapping and PTM

characterization Using Agilent Q-TOF

and BioConfirm Software

Accurate Mass TOF, QTOF

1290 Infinity HPLC, nano LC with HPLC-chip

Page 14: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

HPLC-Chip-Based Peptide Mapping - Base Peak

Chromatogram (BPC) of Trypsin Digested mAb

on C18 Chip

Page 15: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 16: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

MassHunter BioConfirm Software

Matching rules allow incomplete

digest, predicted modifications

and DNA point mutation

Page 17: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

BioConfirm Matches MS/MS Spectra

17

Product ion assignment

Product ion label

Page 18: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 19: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

MS/MS Interpretation Helps to Determine PTM Location

Page 20: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

MS/MS Helps to Determine PTM Locations

Page 21: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Analysis of Glycopeptide from mAb

Group/Presentation Title

Agilent Restricted

Page 21

BPC

Page 22: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 23: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Forced Oxidation Study

Using Agilent Q-TOF

Mass Profiler Pro and BioConfirm Software

Page 24: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

•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

Page 25: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Forced Oxidation Study

Trypsin

TrypsinOx Ox

LC/MS/MS

LC/MS/MS

Ox

Ox

Ox

t-BHP

varies conc.

Page 26: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Comparison at Intact Protein Level

Page 27: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Comparison at Peptide Digest Level

Page 28: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 29: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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)

Page 30: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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.

Page 31: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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.

Page 32: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 33: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Unmodified peptide

R

S

R

S

SR

Page 34: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Single oxidation

R

S

R

S

SR

Page 35: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Double Oxidation

R

S

R

S

SR

Page 36: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Using Agilent CE/Q-TOF for peptide

mapping and glycan analysis

Page 37: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 38: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 39: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

March 14, 2011

Agilent Confidential

39

Peptide Mapping Results from CE/MS

glycopeptides

Page 40: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 41: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

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

Page 42: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

A1F/A2F Glycans PVA Capillary, pH 9.2

Page 43: Recent Advancement in Analytical Techniques for … · Recent Advancement in Analytical Techniques for Biologics Characterization Ning Tang, ... • Bio-inert HPLC-UV, CE ... 2011

Acknowledgements

Agilent

Ravindra Gudihal

Sureshbabu CV

Martin Greiner

Tobias Preckel

Susanne Moyer

Greg Kilby

Xiaoling Wu

Taegan Clary

Keith Waddell

Thank you !