Comprehensive characterization of

Comprehensive characterization of three IgG forms using CESI-MS

Bryan Fonslow1, Olga V. Friese2, and K. Steven Cook2

1SCIEX, Brea, CA and 2Pfizer, Chesterfield, MO

mAbs, ADCs, biosimilars, and biobetters are generated from IgG molecules

Beck et al., Nature Reviews Immunology, 2010, 10, 345-352.

Capillary electrophoresis with mass spectrometry is ideally-suited for IgG characterization

High separation efficiency of biomolecules

High ionization efficiency of biomolecules with reduced ion suppression

CESI “The integration of Capillary Electrophoresis (CE) with Electrospray Ionization (ESI) into a single dynamic process within the same device”

Maltotetroase Intensity Neurotensin Intensity

Evaluation of CESI-MS for the characterization of different IgG forms

IgG1, IgG2, & IgG4

Peptide mapping

Reduce, alkylate, & digest

Disulfide mapping Alkylate & digest

Intact IgG Charge Heterogeneity & Reduced Analysis

IgG1

IgG2

IgG4

Reduced IgG4

Non-reduced IgG4

Intact IgG1


IgG1, IgG2, & IgG4

Peptide mapping

Reduce, alkylate, & digest ~25 ng of digest analyzed

Separation and identification of small and large peptides

TISK (448.2766 Da ; +1) DYFPEPVTVSWNSGALTSGVHTFPAVLQS

SGLYSLSSVVTVPSSSLGTQTYICNVNHK

(6763.2573 Da ; +5)

VDK

(361.2082 Da ; +1)

SLSLSPG

(660.3563 Da;

+1)

QAPGK (500.2827 Da ; +2)

CESI separation and sensitivity contribute to comprehensive peptide mapping coverage of IgGs

Trypsin digestion only

~25 ng of digest analyzed

Identified Potential Critical Quality Attributes: • Glycosylation • Deamidation (Asp and IsoAsp) • Pyroglutamate formation • Methionine oxidation • C-terminal lysine

heterogeneity • Lysine glycation

Separation and relative quantification of glycopeptides

IgG1

G1F

G0F Man5

G2 G1

G0 G2F

G1F-linked peptide MS/MS spectra

MS/MS identification of glycan and peptide sequence

Separation and relative quantification of glycopeptides

IgG1

IgG2 IgG4

G1F

G0F Man5

G2 G1

G0 G2F

G1F

G0F A2G1F

A2G2

G2F

Man5

A2G1

G0

A2G2

G1F

G0F

Man7

G1

G2F

G0

Man5

Man5

Man6

Naturally peptide-“labeled” glycans as ionization and separation tags


IgG1, IgG2, & IgG4

Disulfide mapping

Alkylate & digest

~25 ng of digest analyzed

Disulfide linkages identified on IgG4

LC88-LC23

HC

20

3-H

C3

21

HC

20

3-H

C1

47

HC

13

4-L

C1

94

HC

13

4-H

C1

47

HC134-LC194

HC261-HC321 HC226-HC229

LC134-LC194

Additional potential usages – positional disulfide isomers and more highly-linked peptides


IgG1, IgG2, & IgG4


Intact IgG1

Reduced IgG1

Minutes

21 22 23 24 25 26 27

AU

0.050

0.075

0.100

0.125

0.150

AU

0.050

0.075

0.100

0.125

0.150

8.7

08

0 8.6

18

0

8.5

13

0

7.6

48

0

7.4

83

0

7.3

16

0

7.2

14

0

UV - 280nm

1 mg/mL Pfizer IgG1 w/ markers

Quality

Minutes

17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5

AU

0.050

0.075

0.100

0.125

0.150

AU

0.050

0.075

0.100

0.125

0.150

10

.10

20

9.5

42

0

6.7

14

0

5.3

72

0

4.3

71

0

UV - 280nm


Quality

Minutes

23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5

AU

0.04

0.06

0.08

0.10

0.12

0.14

0.16

AU

0.04

0.06

0.08

0.10

0.12

0.14

0.16

7.5

22

0

7.4

76

0

7.3

59

0

7.2

96

0

7.2

44

07

.22

10

7.1

87

0

7.1

49

0

7.0

38

0

6.9

17

0

6.6

87

0

UV - 280nm


Quality

Minutes

24.5 25.0 25.5 26.0 26.5 27.0 27.5

AU

0.050

0.075

0.100

0.125

0.150

0.175

AU

0.050

0.075

0.100

0.125

0.150

0.175

7.4

39

0

7.4

11

0

7.2

63

0

7.2

28

0

7.1

78

0

7.1

31

07

.11

80

7.0

96

0

7.0

41

0

6.9

75

0

6.8

96

06

.87

20

6.8

50

0

6.8

24

0

6.7

60

0

UV - 280nm

1 mg/mL Pfizer IgG2

Quality

cIEF charge heterogeneity analysis of IgGs with UV detection

IgG1 with pI markers

IgG1 IgG4

IgG2

pI 7.22 pI 8.65

pI 7.11

pI range – 0.29

pI range – 1.68

pI range – 0.56

Charge heterogeneity analysis of IgG1 by CESI-MS

Minutes

21 22 23 24 25 26 27 28

AU

0.02

0.04

0.06

0.08

0.10

AU

0.02

0.04

0.06

0.08

0.10

UV - 280nm


pI 8.65

pI range – 1.68

~ 10 mg/mL sample desalted into 50 mM ammonium acetate, pH 4 BGE – 3% acetic acid (tITP-CZE mode) ~3.5 nL injection ~ 35 ng injected

cIEF-UV

CESI-TripleTOF® 6600 MS






Extracted ion electropherograms

of single charge states


Extracted ion electropherograms of single charge states

Reduced IgG1 CESI-MS analysis verifies heavy and light chain sequences

Unknown higher MW species (impurities?)

G0F

G1F

LC HC

Identification of main IgG1 charge isoform peaks

-2 deamidations +1 oxidation

Higher MW impurity? +4097 Da

Identification of IgG1 heavy chain dimer peak

Minutes

23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5

AU

0.04

0.06

0.08

0.10

0.12

0.14

AU

0.04

0.06

0.08

0.10

0.12

0.14

UV - 280nm


Quality


pI 7.22

pI range – 0.56

cIEF-UV CESI-TripleTOF® 6600 MS

~ 10 mg/mL sample desalted into 50 mM ammonium acetate pH 4 BGE – 3% acetic acid (tITP-CZE mode) ~3.5 nL injection ~ 35 ng injected

CESI-TripleTOF® 6600 MS

Minutes

23 24 25 26 27 28 29 30

AU

0.04

0.06

0.08

0.10

0.12

0.14

0.16

AU

0.04

0.06

0.08

0.10

0.12

0.14

0.16UV - 280nm

1 mg/mL Pfizer IgG2

pI range – 0.29

pI 7.11


G0F-GlcNAc G1F

Deamidated Oxidized

Lys loss (2)



Different glycosylation and modification profiles



Deamidations



Deamidations



Deamidations and oxidation


IgG1, IgG2, & IgG4

Peptide mapping

Reduce, alkylate, & digest

Disulfide mapping Alkylate & digest


IgG1

IgG2

IgG4

Reduced IgG4

Non-reduced IgG4

Intact IgG1

Combining CE & ESI-Mass Spectrometry

CESI 8000 High Performance Separation-ESI Module

TripleTOF® 6600 System High resolution, ultra-low flow rate CE separations coupled with high resolution, high sensitivity MS

Software analysis of CESI-TripleTOF® MS data • Peptide mapping experiments

– BioPharmaViewTM software and ProteinMetrics Byonic

• Disulfide mapping experiments – BioPharmaViewTM software and ProteinMetrics Byonic

• Intact charge heterogeneity experiments – BioPharmaViewTM software

Conclusions

• CESI-TripleTOF® MS analyses provides new and orthogonal information about IgG molecules

• Peptide and disulfide mapping achieve: – 100% sequence coverage from ~25 ng and single digest

– Characterization of potential critical quality attributes including glycosylation and disulfide bonds

• CZE-based charge heterogeneity analysis provides: – Separation resolution of major charge variants

– Charge variant-specific identification and correlation from deconvoluted MS spectra

Acknowledgements

• SCIEX

– Separations

• Chitra Ratnayake

• Rajeswari Lakshmanan

• Marcia Santos

• Clarence Lew

• Andras Guttman

– BioPharma

• Eric Johansen

• St John Skilton

• Pfizer

– Michael Jones

– James Carroll

• ProteinMetrics

– Eric Carlson

– Chris Becker

– Wilfred Tang

Documents

Comprehensive characterization of