-100 3 2- g0f/g0f 2- molecular weight isoelectricpoint (pl ... · msb 2012 poster # p-104 ....

MSB 2012

Poster # P-104

Bioanalytical Characterization of Therapeutic Proteins by Electrophoresis Techniques

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

-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

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

3100 OFFGEL

G7100 Capillary Electrophoresis (CE)

G7100 Capillary Electrophoresis – 6520 QTOF Mass

Spectrometry (CE-MS)

Introduction

Therapeutic proteins such as monoclonal antibodies

(mAbs) are major biopharmaceutical products with

clinical applications. Regulatory agencies demand

comprehensive protein characterization data hence

different analytical techniques are employed for the

analysis. Complexity of sample cannot be covered by a

single analytical technique but requires a suite of tools

to provide necessary quality of data. In this work, we

present a workflow based approach for the analysis of

therapeutic proteins like purity, accurate mass,

aggregation, peptide sequence, glycopeptide and glycans.

Initial screening for product purity, fragments of mAb,

protein PEGylation products were analyzed using

microfluidic electrophoresis as a QC tool. Capillary

electrophoresis (CE), liquid chromatography (LC), and

combinations of these with mass spectrometry (MS) are

applied for the analysis of intact protein (mAb), tryptic

digest, peptide mapping, glycopeptide/glycan and

accurate mass measurement. Further, mAb charge

heterogeneity was separated using liquid-phase

isoelectric focusing (IEF) technique followed by

microfluidic CE analysis. The result presented here shows

the utility of multiple analytical platforms for in-depth

protein characterization.

Instrumentation

Results and Discussion

Therapeutic protein analysis with the microfluidic-

based Bioanalyzer

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150

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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

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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

Figure 1A. Bioanalyzer analysis of IgG2 preparation under

reducing conditions

Figure 1B. Bioanalyzer analysis of IgG2 preparation under

non-reducing conditions

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

-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

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20

40

60

80

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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)


Figure 2. Monitoring antibody charge variants using a

combination of OFFGEL Fractionation by isoelectric point

and high sensitivity protein detection with Bioanalyzer


Ladd

er

0.5m

g/m

l

1 m

g/m

l

2 m

g/m

l

4 m

g/m

l

6 m

g/m

l

Cont

rol

PEGlyation reagent (pNP)

240.0

150.0

95.0

63.0

46.0

28.0

15.0

7.0

4.5

Size [kDa]

Characterization of PEGylated proteins

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

Figure 3. Bioanalyzer analysis of protein PEGylation.

PEGylating reagents: Methoxy PEG p-nitrophenyl carbonate

(mPEG pNP, MW 5000)


Conclusions

• 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.

• 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 protein characterization.

Acknowledgment

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.

CE-QTOF MS analysis of glycopeptide and glycans of

monoclonal antibodies

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

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

BPE

Figure 4. Base peak electropherogram (BPE) of a trypsin-

digested mAb and CE-MS/MS analysis

Δ1444.87

Δ2890.81

G0F/G0Fx 104

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

Figure 7. LC-MS analysis of mAb

Figure 5. Extracted ion electropherogram (EIE) and the

representative MS trace from CE-MS analysis of APTS

labeled neutral (A) and neutral/sialylated(B)glycans

4x 1 0

0

0 . 05

0 . 1

0 . 15

0 . 2

0 . 25

0 . 3

0 . 35

0 . 4

0 . 45

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0 . 7

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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 1 0

0

0 .1

0 .2

0 .3

0 .4

0 .5

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0 .7

0 .8

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1

1 .1

1 .2

1 .3

1 .4

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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

G2F

G2-

1NA

NA

G2-

2NA

NA

G2F

-2N

AN

A

G2F

-1N

AN

A

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 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-

Figure 6. CE-MS analysis of released glycans from a

glycoprotein

MSB 2012

Poster P012

For Research Use Only. Not for use in diagnostic procedures.

-100 3 2- g0f/g0f 2- molecular weight isoelectricpoint (pl ... · msb 2012 poster # p-104 ....

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