native, offgel fraction 9 load applications of ... pharm 2011_suresh.pdfce pharm 2011 poster # p-104...

$: Native, OFFGEL fraction 9 Load Applications Of ... Pharm 2011_Suresh.pdfCE Pharm 2011 Poster # P-104 Applications Of Electrophoretic Techniques For The Characterization Of Therapeutic$
CE Pharm 2011

Poster # P-104

Applications Of Electrophoretic Techniques For The Characterization Of Therapeutic Biomolecules Suresh Babu CV1, Ravindra Gudihal1, Tobias Preckel2, Andreas Ruefer2, Christian Wenz2 and Martin Greiner2 1Agilent Technologies India Pvt. Ltd, Bangalore, India. 2Agilent Technologies R&D and Mktg. GmbH & Co.KG, Waldbronn, Germany

The characterization of therapeutic proteins such as monoclonal antibody

(mAb) during different stages of manufacturing is crucial for timely and

successful product release. Electrophoresis-based techniques and liquid

chromatography (LC) either standalone or coupled to mass spectrometry (MS)

are at the forefront for the in-depth analysis of protein purity, isoforms,

stability, aggregation, posttranslational modifications, PEGylation, etc.

In this presentation, a combination of various electrophoretic techniques such

as liquid-phase isoelectric focusing, microfluidic and capillary-based

electrophoresis (CE) and combinations of those with mass spectrometry

techniques will be discussed. We present a workflow based approach to the

analysis of therapeutic proteins. In successive steps critical parameters like

purity, accurate mass, aggregation, peptide sequence, glycopeptide and

glycan analysis are analyzed. In brief, the workflow involved proteolytic

digestion of mAb for peptide mapping, N-Glycanase and chemical labeling

reaction for mAb glycan analysis, liquid-phase isoelectric focusing for

enrichment of charge variants followed by a very detailed analysis using state

of the art methods such as CE-MS and LC-MS. For the analysis of glycans, we

use combinations of CE-MS and LC-MS to highlight the sweet spots of these

techniques. CE-MS is found to be more useful in analysis of highly sialylated

glycans (charged glycans) while nano LC-MS seems to be better adapted for

analysis of neutral glycans. These two techniques can be used to get

complementary data to profile all the glycans present in a given protein. In

addition, microfluidic electrophoresis was used as a QC tool in initial

screening for product purity, analysis of papain digestion fragments of mAb,

protein PEGylation products, etc. The described workflow involves multiple

platforms, provides an end to end solution for comprehensive protein

characterization and aims at reducing the total product development time.

Therapeutic protein analysis with the microfluidic-based Bioanalyzer

Introduction Result and Discussion

-100

0

100

200

300

400

500

600

700

800

10 15 20 25 30 35 40 45 50 [S]

[FU]

Lowermarker

UppermarkerLow MW

impurities

System peak

Light chain

Heavy chain

P80

-100

0

100

200

300

400

500

600

15 20 25 30 35 40 45 50

[FU]

[S]

Lowermarker

Uppermarker

Aggregates

System peak

Light chain

Heavy chain

P230

-50

0

50

100

150

200

250

300

350

400

15 20 25 30 35 40 45 50[S]

[FU]

Lowermarker

Aggregates

Light chain

Heavy chain

HSP-250

The study of protein PEGylation was in collaboration with GangaGen Biotechnologies Pvt. Ltd, India. We would like to thank Sundaram M Palaniswamy, Umamaheshwari S, Suneel Basingi for their direct involvement in this project. We also acknolowdge the support received from Dr.M.Jayasheela and Mrs Bharathi Sriram.

-50

0

50

100

150

200

250

300

15 20 25 30 35 40 45 50 [S]

[FU]Lowermarker

ngAb

Upper marker

Intact antibody

P230

System peak

Mixture of Light and Heavy chain

-200

100

400

700

1000

1300

15 20 25 30 35 40 45 50 [S]

[FU]Lowermarker

ngAb

Intact antibody

HSP-250

Mixture of Light and Heavy chain

Aggregates

Instrumentation

Result and Discussion Result and Discussion

2100 BioAnalyzer (P80, P230, HSP 250 protein assay kits)

3100 OFFGEL

G7100 Capillary Electrophoresis (CE)

G7100 Capillary Electrophoresis – 6520 QTOF Mass Spectrometry (CE-MS)

Analysis of IgG2

preparation under

reducing (above)

and non-reducing

(below)conditions

Lad

de

r

0.5

mg

/ml

1 m

g/m

l

2 m

g/m

l

4 m

g/m

l

6 m

g/m

l

Co

ntr

ol

PEGlyation reagent (pNP)

240.0

150.0

95.0

63.0

46.0

28.0

15.0

7.0

4.5

Size [kDa]

The Bioanalyzer P230 Assay for Protein PEGylation

Easy-to-use tool that provides high level of resolution

Allows efficient optimization of PEGylation reaction

conditions

Fast and quantitative monitoring of production batches

Analysis of antibody charge heterogeneity

Size

[kDa]

pH 3.0 pH 10.0

1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 15 16 17 18 19 20 21 22 23 24

LOA

D

LAD

DER

mAb

240-

150-

95-

63-

46-

28-15-

5-

Isoelectric point (pl)

Mo

lecu

lar

we

igh

t

240-

150-

95-

63-

46-

28-15-

5-

Size

[kDa]

pH 3.0 pH 10.0

1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 15 16 17 18 19 20 21 22 23 24

mAb

LOA

D

LAD

DER

Isoelectric point (pl)

Mo

lecu

lar

we

igh

t

Tween-20 conditions

Native conditions

Native conditions

Separation of different structural variants of mAb

Major forms: fractions 9-10

Charge variants: fractions 1-3

Fractions can directly be applied to downstream

LC/MS analysis

Lower protein recovery (< 50%)

Tween-20 conditions

Separation of different structural variants of mAb

Major forms: fractions 9-11

Charge variants: fractions 1-5

Need to remove Tween-20 before downstream LC/MS analysis

Enhanced protein recovery (>70%)

Tween-20 enhances protein labeling efficiency with HSP-250 kit

Characterization of PEGylated proteins

PEGylating reagents: Methoxy PEG p-nitrophenyl carbonate (mPEG pNP, MW 5000)

Acknowledgment

-1

0

1

2

3

4

5

10 20 30 40 50 60

[FU]

Time [s]

Native, OFFGEL fraction 1



mAb variants (139 kDa)

-5

0

5

10

15

20

25

30

10 20 30 40 50 60

[FU]

Time [s]



mAb main product (142 kDa)

-0.5

0.5

1.5

2.5

3.5

10 20 30 40 50 60

[FU]

Time [s]

Load

mAb (142 kDa)

-5

0

5

10

15

20

25

30

10 20 30 40 50 60

[FU]

Time [s]

With Tween OFFGEL fraction 1

With Tween, OFFGEL fraction 2



With Tween, OFFGEL fraction5

mAb variants (144-147 kDa)

-20

0

20

40

60

80

100

120

10 20 30 40 50 60

[FU]

Time [s]




mAb main product (142 kDa)

-2

0

2

4

6

8

10

10 20 30 40 50 60

[FU]

Time [s]

Load

mAb (142 kDa) Conclusion

Deconvoluted spectrum

of intact mAb

Δ1444.87

Δ2890.81

G0F/G0Fx104

0

1

2

3

4

5

6

6.5

148812.81

145922.00

147367.94

146329.69

146816.21

147719.65

Counts vs. Deconvoluted Mass (amu)

145500 146000 146500 147000 147500 148000 148500 149000 149500 150000 150500 151000 151500

5.5

4.5

3.5

2.5

1.5

0.5

Δ 162.16 hexose unit

x104

0

1

2

3

4

5

6

7

8

148812.81

148974.97148840.65148916.37

148765.43

C ounts vs. Deconvoluted Mass (amu)

148750 148800 148850 148900 148950

G0F/G0F

G0F/G1F

Theoretical: 148811.9 5Da

Mass accuracy: 5.7 ppm

5x10

0

1

2

3

4

5

23746.50

23727.8223762.96


23540 23560 23580 23600 23620 23640 23660 23680 23700 23720 23740 23760 23780 23800 23820 23840 23860 23880 23900 23920 23940 23960 23980

Theoretical: 23746.63 Da

Mass accuracy: -5.5 ppm


of Heavy chain


of Light chain

Data base search

Match tolerance 10ppm

EIE

LC-MS analysis of monoclonal antibody (mAb)

Overlay of all glycan structures found

in the antibody

Compound list for the identified

glycans

CE-QTOF MS analysis of glycopeptide - monoclonal antibody (mAb)

Glycan analysis using mAb-Glyco chip (HPLC-Chip/MS)

3x10

0

0.2

0.6

1

1.4

1.8

2.2

2.6 204.0852

126.0539

138.0542168.0644

186.0742

366.1371

Counts vs. Mass-to-Charge (m/z)110 130 150 170 190 210 230 250 270 290 310 330 350 370 390

36.0345

162.1939

18.0145

Diagnostic ion at m/z 204.085

Base peak electropherogram (BPE)

Electrophoretic resolution of a BPE of

trypsin digested mAb

CE-MS/MS of Glycopeptide

Glycopeptide was confirmed with

intense sugar oxonium fragment ions

CE-MS spectrum of Glycopeptide

Peptide EEQYNSTYR with the assigned

glycan structures

CE-QTOF MS analysis of glycans- Glycoprotein

4x 1 0

0

0 . 05

0 . 1

0 . 15

0 . 2

0 . 25

0 . 3

0 . 35

0 . 4

0 . 45

0 . 5

0 . 55

0 . 6

0 . 65

0 . 7

0 . 75

0 . 8

0 . 85

0 . 9

0 . 95

1

1 . 05

1 . 1

1 . 15

1 . 2

1 . 25

1 . 3

-

1

Co unts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0

G0 G0F Man5 G1 G1F G2 G2F

3x 10

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. 75

5

5. 25

5. 5

5. 75

6

6. 25

6. 5

6. 75

7

7. 25

7. 5

7. 75

8

8. 25

C ounts vs. Mass-to-Charge (m/z)

820 830 840 850 860 870 880 890 900 910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 1130

877.7188

950.7522

958.7443

1031.7785

836.6937

1113.31291039.7870

[M-2H]2-

[M-2H]2-

[M-2H]2-

[M-2H]2-

[M-2H]2-

[M-2H]2- [M-2H]2-

3x 1 0

0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

0 .9

1

1 .1

1 .2

1 .3

1 .4

1 .5

1 .6

1 .7

1 .8

1 .9

2

C o u nts vs. Acquisiti on Ti me (m in)

7 7 .5 8 8 .5 9 9 .5 1 0 1 0 .5 1 1 1 1 .5 1 2 1 2 .5 1 3 1 3 .5 1 4 1 4 .5 1 5 1 5 .5 1 6 1 6 .5 1 7 1 7 .5 1 8 1 8 .5 1 9 1 9 .5 2 0 2 0 .5

G2

G2

F

G2

-1N

AN

A

G2

-2N

AN

A

G2

F-2

NA

NA

G2

F-1

NA

NA

3x 10

0

0. 025

0. 05

0. 075

0. 1

0. 125

0. 15

0. 175

0. 2

0. 225

0. 25

0. 275

0. 3

0. 325

0. 35

0. 375

0. 4

0. 425

0. 45

0. 475

0. 5

0. 525

0. 55

0. 575

0. 6

0. 625

0. 65

0. 675

0. 7

0. 725

0. 75

0. 775

0. 8

0. 825

0. 85

0. 875

0. 9

0. 925

0. 95

0. 975

1

1. 025

1. 05

1. 075


1040 1060 1080 1100 1120 1140 1160 1180 1200 1220 1240 1260 1280 1300 1320 1340 1360 1380 1400 1420

1113.3210[M-2H]2-

1185.8394[M-2H]2-

1258.8677[M-2H]2- 1331.3880

[M-2H]2-

1404.4208[M-2H]2-

1040.3042[M-2H]2-

2x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

Counts (%) vs. Acquisition Time (min)

8.8 9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8 13 13.2

G3-5NANA

G3-4NANA

G3-3NANA

G2-2NANA

G3-2NANA

G2-1NANA

G3-1NANA

3x10

0

0.25

0.5

0.75

1

1300.4115

3x10

0

1

2

31203.3829

901.9766

3x10

0

2

4

6

1106.0137

829.2007

3x10

0

2

4

1331.3964887.2662

3x10

0

0.5

1

1.51008.9831

1513.9684

3x10

0

0.25

0.5

0.75

1

1185.8510

2x10

0

1

2

1368.4168


850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800

[M-3H]3-

[M-3H]3-

[M-4H]4-

[M-3H]3-

[M-3H]3-

[M-4H]4-

[M-2H]2-

[M-3H]3- [M-2H]2-

[M-2H]2-

[M-2H]2-

Extracted ion electropherogram (EIE) and the representative MS trace from

CE-MS analysis of APTS labeled neutral (A) and neutral/sialylated(B)glycans

CE-MS analysis of released glycans from a glycoprotein

Primary characterization of mAb

BPE

EIE

EIE

MS spectrum

MS spectrum

EIE MS spectrum

(A)

(B)

(A)

(B)

Initial characterization of therapeutic protein/mAb is achieved using the

electrophoretic techniques such as OFFGEL and microfluidc based

electrophoresis. This sets further stage for detail analysis of mAb by

advanced mass spectrometric techniques (CE-MS, LC-MS).

The combination of CE with Q-TOF MS is a valuable tool for peptide

mapping of small quantity biopharmaceuticals, especially in analysis of

glycoproteins/peptides.

Highly sialylated glycans was more suited when CE-MS was used as

analysis tool while LC-MS seems to be better adapted for analysis of

neutral glycans.

Combination of various electrophoretic and LC techniques with mass

spectrometry techniques was demonstrated for comprehensive protein

characterization.

x105

0.5

1

1.5

2

2.5

Counts vs. Acquisition Time (min)7 8 9 10 11 12 13 14 15 16 17 18 19 20

Glycopeptide

m/z 878.6812 - 14.284 min Δ1446.6

Δ162.7

4x10

0

1

2

3

4

5 50675.58

49228.96

50838.33

49435.14 51023.4350121.1649920.8549009.52


48600 48800 49000 49200 49400 49600 49800 50000 50200 50400 50600 50800 51000 51200

Mcalc: 50675.47

Mexp: 50675.58

Error: 2.2ppm

native, offgel fraction 9 load applications of ... pharm 2011_suresh.pdfce pharm 2011 poster # p-104...

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