Application of Charged Aerosol HPLC Detection in Biopharmaceutical Analysis

Bill Kopaciewicz, David Thomas, Bruce Bailey, Qi Zhang, Marc Plante and Ian Acworth Thermo Fisher Scientifi c, Chelmsford, MA

Po

ster No

te 7180

3

Signal is directly proportional to the analyte quantity

1

2

3

4

5 6

7

8

9

Conclusions Charged Aerosol Detection provides a universal

response that does not require a chromophore

Glycans and other carbohydrates can be detected without the need for labeling

Virtually all types of excipients can be detected and quantified

CAD can be combined with UV, Mass Spec. or fluorescence detection to provide a more complete view of the sample

Although not shown here, CAD has also been used for the analysis of phospholipids in liposomes and the quantification of extractables.

© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

Application of Charged Aerosol HPLC Detection in Biopharmaceutical Analysis

Bill Kopaciewicz, David Thomas, Bruce Bailey, Qi Zhang, Marc Plante & Ian Acworth, Thermo Fisher Scientific, Chelmsford, MA

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0

-5.4

0.0

5.0 MONOSIALYLATED

DISIALYLATED

DIGEST REAGENTS TRISIALYLATED

TETRASIALYLATED

ALDITOLS STANDARD

FETUIN

a1-ACID GLYCOPROTEIN

Column: Thermo Scientific™ GlycanPac™ AXR-1 1.9 µm, 2.1 x 100 mm Column Temp: 30 °C Flow Rate: 0.4 mL/min Injection Vol.: 2 µL Mobile Phase A: Deionized water Mobile Phase B: 100 mM ammonium formate, pH 4.4 Gradient: Time, %B; 0, 4; 4, 4; 35, 39

FIGURE 3: Direct Charged Aerosol Detection of a1-Acid Glycoprotein and Bovine Fetuin N-linked Glycans

FIGURE 1: Charged Aerosol Detector and Principle of Operation

FIGURE 5: Label-free Fetuin Glycan Analysis using Charged Aerosol Detection and MS 128B, C18, 100 ug

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00Time0

100

%

0

100

%

AH08MAR0704 Channel 1An1

5.72e4

AH08MAR0704 TOF MS ES+ TIC

5.91e3

Glycopeptide #1

900 mL to CAD

100 mL to MS

Glycopeptide #2

• de-N glycosylate • -elimination • desalt (evap. and AG-50) • C18 cleanup • LC/CAD/MS

Monosaccharide Key

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

UV/Vis detection has been the workhorse of biopharmaceutical HPLC analysis because many proteins and other active biopharmaceutical ingredients typically possess a good chromophore. However, there are many instances where, although the biotherapeutic agent itself may be UV active, it has critical non-chromophoric chemical modifications and/or is formulated in UV-transparent excipients that must be detected and measured. In contrast to UV/Vis instruments, the Charged Aerosol Detector (CAD) is a mass-based instrument that provides a sensitive, near universal response for any nonvolatile analyte independently of chemical composition. Thus it can detects substances unseen by UV/Vis absorption providing valuable orthogonal data. CAD is well suited for the label free detection of analytes without chromophores and the quantification of unknowns. By virtual of its universal response, the charged aerosol detector can be used for many analytical applications in bioPharm ranging from the label free analysis released glycans to quantification of formulation excipients for quality control. This poster demonstrates several examples where the CAD can be applied to detect and quantify substances separated by reversed phase and hydrophilic interaction chromatography. Examples include: glycans, sialic acids, surfactants, adjuvants, proteins and amino acids. In many cases, detection limits are in the low nanogram range with good peak area precision.

FIGURE 7: Vaccine Adjuvants by CAD and UV

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 -100

0

100

200

Saponins

Cholesterol

0

20

40

60

80

Saponins

Lyso-PC

Cholesterol oxidation products

Cholesterol DPPC

Column: Thermo Scientific™ Accucore™ PFP 2.6 μm, 2.1 100 mm Column Temp.: 50 oC Flow Rate: 1.0 mL/min Injection Vol.: 0.2 μL Mobile Phase A: 0.1 % formic acid in deionized water Mobile Phase B: 0.1 % formic acid in 30:70 acetonitrile:2-propanol Gradient: 25-70-70% B at 0-4-9 min Evaporation Temp: 50 oC Detector: VCAD 2Hz, 5s, PF 1

FIGURE 10: Tween 80 Lot-to-Lot Variability

FIGURE 8: Surfactants - Quantitative

Triton X-100

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time [min]

0

20

30

40

Res

pons

e [p

A]

10

Column: Thermo Scientific™ Betasil™ C1, 5 μm, 3 x 100 mm Column Temp.: 30 ºC, StillAir mode Flow Rate: 0.8 mL/min Injection Vol.: 5 μL Mobile Phase A: Water Mobile Phase B: Acetonitrile:methanol:THF (28:70:2) Gradient: Time(min) Flow Rate %B

(mL/min) 0.0 1.00 0 0.5 1.00 0 0.7 1.35 100 0.8 1.50 100 1.5 1.50 100 1.7 1.00 0 3.5 1.00 0

Detector: VCAD, 70 ºC, 10 Hz, 5s, PF 1.0 Sample Prep.: Dissolve in deionized water

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0

5

10

15

20

25

30

Res

pons

e [p

A]

Tween 80

FIGURE 9: Polyethylene glycol - Qualitative

16 12 20

PEG (MW~300)

PEG (MW~400)

PEG (MW~600)

PEG (MW~1000)

0 8 4

0

80 Column: Thermo Scientific™ Acclaim™ Surfactant Plus, 3 µm, 3.0 x 150 mm Column Temp.: 30 °C Flow Rate: 0.6 mL/min Injection Vol.: 2 µL Gradient: Time Acetonitrile 0.1 M Ammonium (min) Acetate, pH 5 -8.0 2 98 0.0 2 98 20.0 20 80 Detection: Corona ultra Sample: (2.5 mg/mL each) PEG (MW~300) PEG (MW~400) PEG (MW~600) PEG (MW~1000)

FIGURE 6: Direct Detection of Released Sialic Acids from Human and Bovine Transferrin Using HPLC with Charged Aerosol Detection.

0.2 2.0 4.0 6.0 8.0 10.0 12.0 -5.0

0.0

10.0

20.0

25.0

Human Transferrin

Bovine Transferrin

Column: Thermo Scientific™ Acclaim™ Trinity P2 column, 3 µm, 2.1 x 100 mm Column Temp.: 30 °C Flow Rate: 0.3 mL/min Injection Vol.: 5 µL Mobile Phase: 65/25/10 ACN/water/100 mM Ammonium Formate, pH 4

Res

pons

e [p

A]

Time [min]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e

0

20

40

60

80

100

120

140

160

180

200

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

Tween 80 Column: C18, 1.7 µm 2.1 x 50 mm Column Temp: 40 oC Injection volume: 1 µL Mobile Phase A: DI Water Mobile Phase B: Acetonitrile Gradient: Time %B Flow Rate (min.) (mL/min.) 0 6 0.5 1 12 0.6 3 17 0.6 10 20 0.6 18 26 0.6 18.5 55 0.6 23.5 80 0.6 24 6 0.5 Corona ultra – 200pA; Filter = high; Neb Temp = 25 oC Sample: 30 mg/mL Tween 80 in water

Time [min]

Res

pons

e (m

V)

Time [min]

Res

pons

e [p

A]

Abso

rban

ce (m

Au)

0.0

0.5

1.0

1.5

2.0

2.5

0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00

Abso

rban

ce (m

Au)

UV 214 nm

His Phe

Trp

Time [min]

Met

4.00 8.00 12.00 16.00 20.00 24.00 28.00

0

50

100

150

200

250

0.00

Res

pons

e [m

V]

Gly

Arg

Glu

Phe Trp His

Charged Aerosol Detection

FIGURE 2: Direct Measurement of Amino Acids and BSA by Charged Aerosol and UV Absorbance Detection

Column: C18 4.6 x 250mm, 5µm Column Temp.: Ambient Flow Rate: 0.6 mL/min. Injection Vol.: 10 µL of 1 mg/mL Mobile Phase: 0.1% TFA in water (A) and ACN (B) Gradient: 5 min hold at 100% A, 0-40% B in 20 min

FIGURE 4: Label-free Detection of O-linked Glycans by HPLC-CAD Mucin Glycans Released by Reductive Beta Elimination

0.0 5.0 10.0 15.0 20.0 25.0 30.0 -1.0

0.0

5.0

10.0

15.0

Time [min]

Res

pons

e (p

A)

Column: Thermo Scientific™ GlycanPac™ AXH-1, 1.9 μm, 2.1 x 150 mm Column Temp.: 30 oC, StillAir mode Flow Rate: 0.5 mL/min Injection Vol.: 2 μL Mobile Phase A: 95:5 acetonitrile: DI water Mobile Phase B: 50 mM Ammonium Formate, pH 4.4 Gradient: 2 % B to 42 % B in 60 min Detector: VCAD, 50 oC, PF 1.0, 10 Hz, 5s

Column: Thermo Scientific™ Hypercarb™ Flow Rate: 0.9 mL/min CAD: 0.1 mL/min MS Injection Vol.: 20 µL Mobile Phase: Aqueous acetonitrile (containing 0.1% TFA) Gradient: 4% to 18% ACN in 40 min.

UV @ 210 nm

Charged Aerosol

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 -4.0 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0

UV 280nm

CAD

Time [min]

Res

pons

e

BSA Column: Thermo Scientific™ Accucore™ 150, C4 Column Temp.: 45 ºC Flow Rate: 0.4 mL/min Injection Vol.: 1 µL of 0.5 mg/mL std. Mobile Phase A: 0.2% Aqueous TFA Mobile Phase B: 0.1% TFA in ACN Gradient: 10–90% B in 20 min

Amino Acids and Proteins

Glycans, Sialic Acids and Adjuvants Excipients Introduction

Figure 1 depicts the operation of the charged aerosol detector. At the top left (1) the mobile phase from the LC column entering the detector is nebulized by combining with a concentric stream of nitrogen gas or air (2). The fine droplets carried by bulk gas flow to the heated evaporation sector (3) are desolvated to form dry particles (5) from any nonvolatile or semivolatile species. Any remaining large droplets drain away to waste (4). The dry analyte particles combine with another gas stream that has been charged by a high voltage Corona charger (6). The charged gas transfers positive charge to the analyte particle’s surface (7). The charged analyte particles pass through an ion trap (8) that removes any high mobility species and pass to a collector where they are measured by a sensitive electrometer. The signal produced (9) is directly proportional to the quantity of analyte.

Acknowledgements We acknowledge the contribution of Mike Swartz who conducted the amino acid work in Figure 2 and Andy Hanniman who ran the CAD-MS experiment in Figure 5.

PO71803-EN 1015S

Signal is directly proportional to the analyte quantity

1

2

3

4

5 6

7

8

9

Conclusions Charged Aerosol Detection provides a universal

response that does not require a chromophore

Glycans and other carbohydrates can be detected without the need for labeling

Virtually all types of excipients can be detected and quantified

CAD can be combined with UV, Mass Spec. or fluorescence detection to provide a more complete view of the sample

Although not shown here, CAD has also been used for the analysis of phospholipids in liposomes and the quantification of extractables.

© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

Application of Charged Aerosol HPLC Detection in Biopharmaceutical Analysis

Bill Kopaciewicz, David Thomas, Bruce Bailey, Qi Zhang, Marc Plante & Ian Acworth, Thermo Fisher Scientific, Chelmsford, MA

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0

-5.4

0.0

5.0 MONOSIALYLATED

DISIALYLATED

DIGEST REAGENTS TRISIALYLATED

TETRASIALYLATED

ALDITOLS STANDARD

FETUIN

a1-ACID GLYCOPROTEIN

Column: Thermo Scientific™ GlycanPac™ AXR-1 1.9 µm, 2.1 x 100 mm Column Temp: 30 °C Flow Rate: 0.4 mL/min Injection Vol.: 2 µL Mobile Phase A: Deionized water Mobile Phase B: 100 mM ammonium formate, pH 4.4 Gradient: Time, %B; 0, 4; 4, 4; 35, 39

FIGURE 3: Direct Charged Aerosol Detection of a1-Acid Glycoprotein and Bovine Fetuin N-linked Glycans

FIGURE 1: Charged Aerosol Detector and Principle of Operation

FIGURE 5: Label-free Fetuin Glycan Analysis using Charged Aerosol Detection and MS 128B, C18, 100 ug

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00Time0

100

%

0

100

%

AH08MAR0704 Channel 1An1

5.72e4

AH08MAR0704 TOF MS ES+ TIC

5.91e3

Glycopeptide #1

900 mL to CAD

100 mL to MS

Glycopeptide #2

• de-N glycosylate • -elimination • desalt (evap. and AG-50) • C18 cleanup • LC/CAD/MS

Monosaccharide Key

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

UV/Vis detection has been the workhorse of biopharmaceutical HPLC analysis because many proteins and other active biopharmaceutical ingredients typically possess a good chromophore. However, there are many instances where, although the biotherapeutic agent itself may be UV active, it has critical non-chromophoric chemical modifications and/or is formulated in UV-transparent excipients that must be detected and measured. In contrast to UV/Vis instruments, the Charged Aerosol Detector (CAD) is a mass-based instrument that provides a sensitive, near universal response for any nonvolatile analyte independently of chemical composition. Thus it can detects substances unseen by UV/Vis absorption providing valuable orthogonal data. CAD is well suited for the label free detection of analytes without chromophores and the quantification of unknowns. By virtual of its universal response, the charged aerosol detector can be used for many analytical applications in bioPharm ranging from the label free analysis released glycans to quantification of formulation excipients for quality control. This poster demonstrates several examples where the CAD can be applied to detect and quantify substances separated by reversed phase and hydrophilic interaction chromatography. Examples include: glycans, sialic acids, surfactants, adjuvants, proteins and amino acids. In many cases, detection limits are in the low nanogram range with good peak area precision.

FIGURE 7: Vaccine Adjuvants by CAD and UV

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 -100

0

100

200

Saponins

Cholesterol

0

20

40

60

80

Saponins

Lyso-PC

Cholesterol oxidation products

Cholesterol DPPC

Column: Thermo Scientific™ Accucore™ PFP 2.6 μm, 2.1 100 mm Column Temp.: 50 oC Flow Rate: 1.0 mL/min Injection Vol.: 0.2 μL Mobile Phase A: 0.1 % formic acid in deionized water Mobile Phase B: 0.1 % formic acid in 30:70 acetonitrile:2-propanol Gradient: 25-70-70% B at 0-4-9 min Evaporation Temp: 50 oC Detector: VCAD 2Hz, 5s, PF 1

FIGURE 10: Tween 80 Lot-to-Lot Variability

FIGURE 8: Surfactants - Quantitative

Triton X-100

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time [min]

0

20

30

40

Res

pons

e [p

A]

10

Column: Thermo Scientific™ Betasil™ C1, 5 μm, 3 x 100 mm Column Temp.: 30 ºC, StillAir mode Flow Rate: 0.8 mL/min Injection Vol.: 5 μL Mobile Phase A: Water Mobile Phase B: Acetonitrile:methanol:THF (28:70:2) Gradient: Time(min) Flow Rate %B

(mL/min) 0.0 1.00 0 0.5 1.00 0 0.7 1.35 100 0.8 1.50 100 1.5 1.50 100 1.7 1.00 0 3.5 1.00 0

Detector: VCAD, 70 ºC, 10 Hz, 5s, PF 1.0 Sample Prep.: Dissolve in deionized water

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0

5

10

15

20

25

30

Res

pons

e [p

A]

Tween 80

FIGURE 9: Polyethylene glycol - Qualitative

16 12 20

PEG (MW~300)

PEG (MW~400)

PEG (MW~600)

PEG (MW~1000)

0 8 4

0

80 Column: Thermo Scientific™ Acclaim™ Surfactant Plus, 3 µm, 3.0 x 150 mm Column Temp.: 30 °C Flow Rate: 0.6 mL/min Injection Vol.: 2 µL Gradient: Time Acetonitrile 0.1 M Ammonium (min) Acetate, pH 5 -8.0 2 98 0.0 2 98 20.0 20 80 Detection: Corona ultra Sample: (2.5 mg/mL each) PEG (MW~300) PEG (MW~400) PEG (MW~600) PEG (MW~1000)

FIGURE 6: Direct Detection of Released Sialic Acids from Human and Bovine Transferrin Using HPLC with Charged Aerosol Detection.

0.2 2.0 4.0 6.0 8.0 10.0 12.0 -5.0

0.0

10.0

20.0

25.0

Human Transferrin

Bovine Transferrin

Column: Thermo Scientific™ Acclaim™ Trinity P2 column, 3 µm, 2.1 x 100 mm Column Temp.: 30 °C Flow Rate: 0.3 mL/min Injection Vol.: 5 µL Mobile Phase: 65/25/10 ACN/water/100 mM Ammonium Formate, pH 4

Res

pons

e [p

A]

Time [min]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e

0

20

40

60

80

100

120

140

160

180

200

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

Tween 80 Column: C18, 1.7 µm 2.1 x 50 mm Column Temp: 40 oC Injection volume: 1 µL Mobile Phase A: DI Water Mobile Phase B: Acetonitrile Gradient: Time %B Flow Rate (min.) (mL/min.) 0 6 0.5 1 12 0.6 3 17 0.6 10 20 0.6 18 26 0.6 18.5 55 0.6 23.5 80 0.6 24 6 0.5 Corona ultra – 200pA; Filter = high; Neb Temp = 25 oC Sample: 30 mg/mL Tween 80 in water

Time [min]

Res

pons

e (m

V)

Time [min]

Res

pons

e [p

A]

Abso

rban

ce (m

Au)

0.0

0.5

1.0

1.5

2.0

2.5

0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00

Abso

rban

ce (m

Au)

UV 214 nm

His Phe

Trp

Time [min]

Met

4.00 8.00 12.00 16.00 20.00 24.00 28.00

0

50

100

150

200

250

0.00

Res

pons

e [m

V]

Gly

Arg

Glu

Phe Trp His

Charged Aerosol Detection

FIGURE 2: Direct Measurement of Amino Acids and BSA by Charged Aerosol and UV Absorbance Detection

Column: C18 4.6 x 250mm, 5µm Column Temp.: Ambient Flow Rate: 0.6 mL/min. Injection Vol.: 10 µL of 1 mg/mL Mobile Phase: 0.1% TFA in water (A) and ACN (B) Gradient: 5 min hold at 100% A, 0-40% B in 20 min

FIGURE 4: Label-free Detection of O-linked Glycans by HPLC-CAD Mucin Glycans Released by Reductive Beta Elimination

0.0 5.0 10.0 15.0 20.0 25.0 30.0 -1.0

0.0

5.0

10.0

15.0

Time [min]

Res

pons

e (p

A)

Column: Thermo Scientific™ GlycanPac™ AXH-1, 1.9 μm, 2.1 x 150 mm Column Temp.: 30 oC, StillAir mode Flow Rate: 0.5 mL/min Injection Vol.: 2 μL Mobile Phase A: 95:5 acetonitrile: DI water Mobile Phase B: 50 mM Ammonium Formate, pH 4.4 Gradient: 2 % B to 42 % B in 60 min Detector: VCAD, 50 oC, PF 1.0, 10 Hz, 5s

Column: Thermo Scientific™ Hypercarb™ Flow Rate: 0.9 mL/min CAD: 0.1 mL/min MS Injection Vol.: 20 µL Mobile Phase: Aqueous acetonitrile (containing 0.1% TFA) Gradient: 4% to 18% ACN in 40 min.

UV @ 210 nm

Charged Aerosol

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 -4.0 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0

UV 280nm

CAD

Time [min]

Res

pons

e

BSA Column: Thermo Scientific™ Accucore™ 150, C4 Column Temp.: 45 ºC Flow Rate: 0.4 mL/min Injection Vol.: 1 µL of 0.5 mg/mL std. Mobile Phase A: 0.2% Aqueous TFA Mobile Phase B: 0.1% TFA in ACN Gradient: 10–90% B in 20 min

Amino Acids and Proteins

Glycans, Sialic Acids and Adjuvants Excipients Introduction

Figure 1 depicts the operation of the charged aerosol detector. At the top left (1) the mobile phase from the LC column entering the detector is nebulized by combining with a concentric stream of nitrogen gas or air (2). The fine droplets carried by bulk gas flow to the heated evaporation sector (3) are desolvated to form dry particles (5) from any nonvolatile or semivolatile species. Any remaining large droplets drain away to waste (4). The dry analyte particles combine with another gas stream that has been charged by a high voltage Corona charger (6). The charged gas transfers positive charge to the analyte particle’s surface (7). The charged analyte particles pass through an ion trap (8) that removes any high mobility species and pass to a collector where they are measured by a sensitive electrometer. The signal produced (9) is directly proportional to the quantity of analyte.

Acknowledgements We acknowledge the contribution of Mike Swartz who conducted the amino acid work in Figure 2 and Andy Hanniman who ran the CAD-MS experiment in Figure 5.

PO71803-EN 1015S

2 Application of Charged Aerosol HPLC Detection in Biopharmaceutical Analysis

Signal is directly proportional to the analyte quantity

1

2

3

4

5 6

7

8

9

Conclusions Charged Aerosol Detection provides a universal

response that does not require a chromophore

Glycans and other carbohydrates can be detected without the need for labeling

Virtually all types of excipients can be detected and quantified

CAD can be combined with UV, Mass Spec. or fluorescence detection to provide a more complete view of the sample

Although not shown here, CAD has also been used for the analysis of phospholipids in liposomes and the quantification of extractables.

© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

Application of Charged Aerosol HPLC Detection in Biopharmaceutical Analysis

Bill Kopaciewicz, David Thomas, Bruce Bailey, Qi Zhang, Marc Plante & Ian Acworth, Thermo Fisher Scientific, Chelmsford, MA

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0

-5.4

0.0

5.0 MONOSIALYLATED

DISIALYLATED

DIGEST REAGENTS TRISIALYLATED

TETRASIALYLATED

ALDITOLS STANDARD

FETUIN

a1-ACID GLYCOPROTEIN

Column: Thermo Scientific™ GlycanPac™ AXR-1 1.9 µm, 2.1 x 100 mm Column Temp: 30 °C Flow Rate: 0.4 mL/min Injection Vol.: 2 µL Mobile Phase A: Deionized water Mobile Phase B: 100 mM ammonium formate, pH 4.4 Gradient: Time, %B; 0, 4; 4, 4; 35, 39

FIGURE 3: Direct Charged Aerosol Detection of a1-Acid Glycoprotein and Bovine Fetuin N-linked Glycans

FIGURE 1: Charged Aerosol Detector and Principle of Operation

FIGURE 5: Label-free Fetuin Glycan Analysis using Charged Aerosol Detection and MS 128B, C18, 100 ug

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00Time0

100

%

0

100

%

AH08MAR0704 Channel 1An1

5.72e4

AH08MAR0704 TOF MS ES+ TIC

5.91e3

Glycopeptide #1

900 mL to CAD

100 mL to MS

Glycopeptide #2

• de-N glycosylate • -elimination • desalt (evap. and AG-50) • C18 cleanup • LC/CAD/MS

Monosaccharide Key

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

UV/Vis detection has been the workhorse of biopharmaceutical HPLC analysis because many proteins and other active biopharmaceutical ingredients typically possess a good chromophore. However, there are many instances where, although the biotherapeutic agent itself may be UV active, it has critical non-chromophoric chemical modifications and/or is formulated in UV-transparent excipients that must be detected and measured. In contrast to UV/Vis instruments, the Charged Aerosol Detector (CAD) is a mass-based instrument that provides a sensitive, near universal response for any nonvolatile analyte independently of chemical composition. Thus it can detects substances unseen by UV/Vis absorption providing valuable orthogonal data. CAD is well suited for the label free detection of analytes without chromophores and the quantification of unknowns. By virtual of its universal response, the charged aerosol detector can be used for many analytical applications in bioPharm ranging from the label free analysis released glycans to quantification of formulation excipients for quality control. This poster demonstrates several examples where the CAD can be applied to detect and quantify substances separated by reversed phase and hydrophilic interaction chromatography. Examples include: glycans, sialic acids, surfactants, adjuvants, proteins and amino acids. In many cases, detection limits are in the low nanogram range with good peak area precision.

FIGURE 7: Vaccine Adjuvants by CAD and UV

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 -100

0

100

200

Saponins

Cholesterol

0

20

40

60

80

Saponins

Lyso-PC

Cholesterol oxidation products

Cholesterol DPPC

Column: Thermo Scientific™ Accucore™ PFP 2.6 μm, 2.1 100 mm Column Temp.: 50 oC Flow Rate: 1.0 mL/min Injection Vol.: 0.2 μL Mobile Phase A: 0.1 % formic acid in deionized water Mobile Phase B: 0.1 % formic acid in 30:70 acetonitrile:2-propanol Gradient: 25-70-70% B at 0-4-9 min Evaporation Temp: 50 oC Detector: VCAD 2Hz, 5s, PF 1

FIGURE 10: Tween 80 Lot-to-Lot Variability

FIGURE 8: Surfactants - Quantitative

Triton X-100

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time [min]

0

20

30

40

Res

pons

e [p

A]

10

Column: Thermo Scientific™ Betasil™ C1, 5 μm, 3 x 100 mm Column Temp.: 30 ºC, StillAir mode Flow Rate: 0.8 mL/min Injection Vol.: 5 μL Mobile Phase A: Water Mobile Phase B: Acetonitrile:methanol:THF (28:70:2) Gradient: Time(min) Flow Rate %B

(mL/min) 0.0 1.00 0 0.5 1.00 0 0.7 1.35 100 0.8 1.50 100 1.5 1.50 100 1.7 1.00 0 3.5 1.00 0

Detector: VCAD, 70 ºC, 10 Hz, 5s, PF 1.0 Sample Prep.: Dissolve in deionized water

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0

5

10

15

20

25

30

Res

pons

e [p

A]

Tween 80

FIGURE 9: Polyethylene glycol - Qualitative

16 12 20

PEG (MW~300)

PEG (MW~400)

PEG (MW~600)

PEG (MW~1000)

0 8 4

0

80 Column: Thermo Scientific™ Acclaim™ Surfactant Plus, 3 µm, 3.0 x 150 mm Column Temp.: 30 °C Flow Rate: 0.6 mL/min Injection Vol.: 2 µL Gradient: Time Acetonitrile 0.1 M Ammonium (min) Acetate, pH 5 -8.0 2 98 0.0 2 98 20.0 20 80 Detection: Corona ultra Sample: (2.5 mg/mL each) PEG (MW~300) PEG (MW~400) PEG (MW~600) PEG (MW~1000)

FIGURE 6: Direct Detection of Released Sialic Acids from Human and Bovine Transferrin Using HPLC with Charged Aerosol Detection.

0.2 2.0 4.0 6.0 8.0 10.0 12.0 -5.0

0.0

10.0

20.0

25.0

Human Transferrin

Bovine Transferrin

Column: Thermo Scientific™ Acclaim™ Trinity P2 column, 3 µm, 2.1 x 100 mm Column Temp.: 30 °C Flow Rate: 0.3 mL/min Injection Vol.: 5 µL Mobile Phase: 65/25/10 ACN/water/100 mM Ammonium Formate, pH 4

Res

pons

e [p

A]

Time [min]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e

0

20

40

60

80

100

120

140

160

180

200

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

Tween 80 Column: C18, 1.7 µm 2.1 x 50 mm Column Temp: 40 oC Injection volume: 1 µL Mobile Phase A: DI Water Mobile Phase B: Acetonitrile Gradient: Time %B Flow Rate (min.) (mL/min.) 0 6 0.5 1 12 0.6 3 17 0.6 10 20 0.6 18 26 0.6 18.5 55 0.6 23.5 80 0.6 24 6 0.5 Corona ultra – 200pA; Filter = high; Neb Temp = 25 oC Sample: 30 mg/mL Tween 80 in water

Time [min]

Res

pons

e (m

V)

Time [min]

Res

pons

e [p

A]

Abso

rban

ce (m

Au)

0.0

0.5

1.0

1.5

2.0

2.5

0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00

Abso

rban

ce (m

Au)

UV 214 nm

His Phe

Trp

Time [min]

Met

4.00 8.00 12.00 16.00 20.00 24.00 28.00

0

50

100

150

200

250

0.00

Res

pons

e [m

V]

Gly

Arg

Glu

Phe Trp His

Charged Aerosol Detection

FIGURE 2: Direct Measurement of Amino Acids and BSA by Charged Aerosol and UV Absorbance Detection

Column: C18 4.6 x 250mm, 5µm Column Temp.: Ambient Flow Rate: 0.6 mL/min. Injection Vol.: 10 µL of 1 mg/mL Mobile Phase: 0.1% TFA in water (A) and ACN (B) Gradient: 5 min hold at 100% A, 0-40% B in 20 min

FIGURE 4: Label-free Detection of O-linked Glycans by HPLC-CAD Mucin Glycans Released by Reductive Beta Elimination

0.0 5.0 10.0 15.0 20.0 25.0 30.0 -1.0

0.0

5.0

10.0

15.0

Time [min]

Res

pons

e (p

A)

Column: Thermo Scientific™ GlycanPac™ AXH-1, 1.9 μm, 2.1 x 150 mm Column Temp.: 30 oC, StillAir mode Flow Rate: 0.5 mL/min Injection Vol.: 2 μL Mobile Phase A: 95:5 acetonitrile: DI water Mobile Phase B: 50 mM Ammonium Formate, pH 4.4 Gradient: 2 % B to 42 % B in 60 min Detector: VCAD, 50 oC, PF 1.0, 10 Hz, 5s

Column: Thermo Scientific™ Hypercarb™ Flow Rate: 0.9 mL/min CAD: 0.1 mL/min MS Injection Vol.: 20 µL Mobile Phase: Aqueous acetonitrile (containing 0.1% TFA) Gradient: 4% to 18% ACN in 40 min.

UV @ 210 nm

Charged Aerosol

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 -4.0 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0

UV 280nm

CAD

Time [min]

Res

pons

e

BSA Column: Thermo Scientific™ Accucore™ 150, C4 Column Temp.: 45 ºC Flow Rate: 0.4 mL/min Injection Vol.: 1 µL of 0.5 mg/mL std. Mobile Phase A: 0.2% Aqueous TFA Mobile Phase B: 0.1% TFA in ACN Gradient: 10–90% B in 20 min

Amino Acids and Proteins

Glycans, Sialic Acids and Adjuvants Excipients Introduction

Figure 1 depicts the operation of the charged aerosol detector. At the top left (1) the mobile phase from the LC column entering the detector is nebulized by combining with a concentric stream of nitrogen gas or air (2). The fine droplets carried by bulk gas flow to the heated evaporation sector (3) are desolvated to form dry particles (5) from any nonvolatile or semivolatile species. Any remaining large droplets drain away to waste (4). The dry analyte particles combine with another gas stream that has been charged by a high voltage Corona charger (6). The charged gas transfers positive charge to the analyte particle’s surface (7). The charged analyte particles pass through an ion trap (8) that removes any high mobility species and pass to a collector where they are measured by a sensitive electrometer. The signal produced (9) is directly proportional to the quantity of analyte.

Acknowledgements We acknowledge the contribution of Mike Swartz who conducted the amino acid work in Figure 2 and Andy Hanniman who ran the CAD-MS experiment in Figure 5.

PO71803-EN 1015S

Signal is directly proportional to the analyte quantity

1

2

3

4

5 6

7

8

9

Conclusions Charged Aerosol Detection provides a universal

response that does not require a chromophore

Glycans and other carbohydrates can be detected without the need for labeling

Virtually all types of excipients can be detected and quantified

CAD can be combined with UV, Mass Spec. or fluorescence detection to provide a more complete view of the sample

Although not shown here, CAD has also been used for the analysis of phospholipids in liposomes and the quantification of extractables.

© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

Application of Charged Aerosol HPLC Detection in Biopharmaceutical Analysis

Bill Kopaciewicz, David Thomas, Bruce Bailey, Qi Zhang, Marc Plante & Ian Acworth, Thermo Fisher Scientific, Chelmsford, MA

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0

-5.4

0.0

5.0 MONOSIALYLATED

DISIALYLATED

DIGEST REAGENTS TRISIALYLATED

TETRASIALYLATED

ALDITOLS STANDARD

FETUIN

a1-ACID GLYCOPROTEIN

Column: Thermo Scientific™ GlycanPac™ AXR-1 1.9 µm, 2.1 x 100 mm Column Temp: 30 °C Flow Rate: 0.4 mL/min Injection Vol.: 2 µL Mobile Phase A: Deionized water Mobile Phase B: 100 mM ammonium formate, pH 4.4 Gradient: Time, %B; 0, 4; 4, 4; 35, 39

FIGURE 3: Direct Charged Aerosol Detection of a1-Acid Glycoprotein and Bovine Fetuin N-linked Glycans

FIGURE 1: Charged Aerosol Detector and Principle of Operation

FIGURE 5: Label-free Fetuin Glycan Analysis using Charged Aerosol Detection and MS 128B, C18, 100 ug

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00Time0

100

%

0

100

%

AH08MAR0704 Channel 1An1

5.72e4

AH08MAR0704 TOF MS ES+ TIC

5.91e3

Glycopeptide #1

900 mL to CAD

100 mL to MS

Glycopeptide #2

• de-N glycosylate • -elimination • desalt (evap. and AG-50) • C18 cleanup • LC/CAD/MS

Monosaccharide Key

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

UV/Vis detection has been the workhorse of biopharmaceutical HPLC analysis because many proteins and other active biopharmaceutical ingredients typically possess a good chromophore. However, there are many instances where, although the biotherapeutic agent itself may be UV active, it has critical non-chromophoric chemical modifications and/or is formulated in UV-transparent excipients that must be detected and measured. In contrast to UV/Vis instruments, the Charged Aerosol Detector (CAD) is a mass-based instrument that provides a sensitive, near universal response for any nonvolatile analyte independently of chemical composition. Thus it can detects substances unseen by UV/Vis absorption providing valuable orthogonal data. CAD is well suited for the label free detection of analytes without chromophores and the quantification of unknowns. By virtual of its universal response, the charged aerosol detector can be used for many analytical applications in bioPharm ranging from the label free analysis released glycans to quantification of formulation excipients for quality control. This poster demonstrates several examples where the CAD can be applied to detect and quantify substances separated by reversed phase and hydrophilic interaction chromatography. Examples include: glycans, sialic acids, surfactants, adjuvants, proteins and amino acids. In many cases, detection limits are in the low nanogram range with good peak area precision.

FIGURE 7: Vaccine Adjuvants by CAD and UV

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 -100

0

100

200

Saponins

Cholesterol

0

20

40

60

80

Saponins

Lyso-PC

Cholesterol oxidation products

Cholesterol DPPC

Column: Thermo Scientific™ Accucore™ PFP 2.6 μm, 2.1 100 mm Column Temp.: 50 oC Flow Rate: 1.0 mL/min Injection Vol.: 0.2 μL Mobile Phase A: 0.1 % formic acid in deionized water Mobile Phase B: 0.1 % formic acid in 30:70 acetonitrile:2-propanol Gradient: 25-70-70% B at 0-4-9 min Evaporation Temp: 50 oC Detector: VCAD 2Hz, 5s, PF 1

FIGURE 10: Tween 80 Lot-to-Lot Variability

FIGURE 8: Surfactants - Quantitative

Triton X-100

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time [min]

0

20

30

40

Res

pons

e [p

A]

10

Column: Thermo Scientific™ Betasil™ C1, 5 μm, 3 x 100 mm Column Temp.: 30 ºC, StillAir mode Flow Rate: 0.8 mL/min Injection Vol.: 5 μL Mobile Phase A: Water Mobile Phase B: Acetonitrile:methanol:THF (28:70:2) Gradient: Time(min) Flow Rate %B

(mL/min) 0.0 1.00 0 0.5 1.00 0 0.7 1.35 100 0.8 1.50 100 1.5 1.50 100 1.7 1.00 0 3.5 1.00 0

Detector: VCAD, 70 ºC, 10 Hz, 5s, PF 1.0 Sample Prep.: Dissolve in deionized water

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0

5

10

15

20

25

30

Res

pons

e [p

A]

Tween 80

FIGURE 9: Polyethylene glycol - Qualitative

16 12 20

PEG (MW~300)

PEG (MW~400)

PEG (MW~600)

PEG (MW~1000)

0 8 4

0

80 Column: Thermo Scientific™ Acclaim™ Surfactant Plus, 3 µm, 3.0 x 150 mm Column Temp.: 30 °C Flow Rate: 0.6 mL/min Injection Vol.: 2 µL Gradient: Time Acetonitrile 0.1 M Ammonium (min) Acetate, pH 5 -8.0 2 98 0.0 2 98 20.0 20 80 Detection: Corona ultra Sample: (2.5 mg/mL each) PEG (MW~300) PEG (MW~400) PEG (MW~600) PEG (MW~1000)

FIGURE 6: Direct Detection of Released Sialic Acids from Human and Bovine Transferrin Using HPLC with Charged Aerosol Detection.

0.2 2.0 4.0 6.0 8.0 10.0 12.0 -5.0

0.0

10.0

20.0

25.0

Human Transferrin

Bovine Transferrin

Column: Thermo Scientific™ Acclaim™ Trinity P2 column, 3 µm, 2.1 x 100 mm Column Temp.: 30 °C Flow Rate: 0.3 mL/min Injection Vol.: 5 µL Mobile Phase: 65/25/10 ACN/water/100 mM Ammonium Formate, pH 4

Res

pons

e [p

A]

Time [min]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e

0

20

40

60

80

100

120

140

160

180

200

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

Tween 80 Column: C18, 1.7 µm 2.1 x 50 mm Column Temp: 40 oC Injection volume: 1 µL Mobile Phase A: DI Water Mobile Phase B: Acetonitrile Gradient: Time %B Flow Rate (min.) (mL/min.) 0 6 0.5 1 12 0.6 3 17 0.6 10 20 0.6 18 26 0.6 18.5 55 0.6 23.5 80 0.6 24 6 0.5 Corona ultra – 200pA; Filter = high; Neb Temp = 25 oC Sample: 30 mg/mL Tween 80 in water

Time [min]

Res

pons

e (m

V)

Time [min]

Res

pons

e [p

A]

Abso

rban

ce (m

Au)

0.0

0.5

1.0

1.5

2.0

2.5

0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00

Abso

rban

ce (m

Au)

UV 214 nm

His Phe

Trp

Time [min]

Met

4.00 8.00 12.00 16.00 20.00 24.00 28.00

0

50

100

150

200

250

0.00

Res

pons

e [m

V]

Gly

Arg

Glu

Phe Trp His

Charged Aerosol Detection

FIGURE 2: Direct Measurement of Amino Acids and BSA by Charged Aerosol and UV Absorbance Detection

Column: C18 4.6 x 250mm, 5µm Column Temp.: Ambient Flow Rate: 0.6 mL/min. Injection Vol.: 10 µL of 1 mg/mL Mobile Phase: 0.1% TFA in water (A) and ACN (B) Gradient: 5 min hold at 100% A, 0-40% B in 20 min

FIGURE 4: Label-free Detection of O-linked Glycans by HPLC-CAD Mucin Glycans Released by Reductive Beta Elimination

0.0 5.0 10.0 15.0 20.0 25.0 30.0 -1.0

0.0

5.0

10.0

15.0

Time [min]

Res

pons

e (p

A)

Column: Thermo Scientific™ GlycanPac™ AXH-1, 1.9 μm, 2.1 x 150 mm Column Temp.: 30 oC, StillAir mode Flow Rate: 0.5 mL/min Injection Vol.: 2 μL Mobile Phase A: 95:5 acetonitrile: DI water Mobile Phase B: 50 mM Ammonium Formate, pH 4.4 Gradient: 2 % B to 42 % B in 60 min Detector: VCAD, 50 oC, PF 1.0, 10 Hz, 5s

Column: Thermo Scientific™ Hypercarb™ Flow Rate: 0.9 mL/min CAD: 0.1 mL/min MS Injection Vol.: 20 µL Mobile Phase: Aqueous acetonitrile (containing 0.1% TFA) Gradient: 4% to 18% ACN in 40 min.

UV @ 210 nm

Charged Aerosol

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 -4.0 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0

UV 280nm

CAD

Time [min]

Res

pons

e

BSA Column: Thermo Scientific™ Accucore™ 150, C4 Column Temp.: 45 ºC Flow Rate: 0.4 mL/min Injection Vol.: 1 µL of 0.5 mg/mL std. Mobile Phase A: 0.2% Aqueous TFA Mobile Phase B: 0.1% TFA in ACN Gradient: 10–90% B in 20 min

Amino Acids and Proteins

Glycans, Sialic Acids and Adjuvants Excipients Introduction

Figure 1 depicts the operation of the charged aerosol detector. At the top left (1) the mobile phase from the LC column entering the detector is nebulized by combining with a concentric stream of nitrogen gas or air (2). The fine droplets carried by bulk gas flow to the heated evaporation sector (3) are desolvated to form dry particles (5) from any nonvolatile or semivolatile species. Any remaining large droplets drain away to waste (4). The dry analyte particles combine with another gas stream that has been charged by a high voltage Corona charger (6). The charged gas transfers positive charge to the analyte particle’s surface (7). The charged analyte particles pass through an ion trap (8) that removes any high mobility species and pass to a collector where they are measured by a sensitive electrometer. The signal produced (9) is directly proportional to the quantity of analyte.

Acknowledgements We acknowledge the contribution of Mike Swartz who conducted the amino acid work in Figure 2 and Andy Hanniman who ran the CAD-MS experiment in Figure 5.

PO71803-EN 1015S

Signal is directly proportional to the analyte quantity

1

2

3

4

5 6

7

8

9

Conclusions Charged Aerosol Detection provides a universal

response that does not require a chromophore

Glycans and other carbohydrates can be detected without the need for labeling

Virtually all types of excipients can be detected and quantified

CAD can be combined with UV, Mass Spec. or fluorescence detection to provide a more complete view of the sample

Although not shown here, CAD has also been used for the analysis of phospholipids in liposomes and the quantification of extractables.

© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

Application of Charged Aerosol HPLC Detection in Biopharmaceutical Analysis

Bill Kopaciewicz, David Thomas, Bruce Bailey, Qi Zhang, Marc Plante & Ian Acworth, Thermo Fisher Scientific, Chelmsford, MA

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0

-5.4

0.0

5.0 MONOSIALYLATED

DISIALYLATED

DIGEST REAGENTS TRISIALYLATED

TETRASIALYLATED

ALDITOLS STANDARD

FETUIN

a1-ACID GLYCOPROTEIN

Column: Thermo Scientific™ GlycanPac™ AXR-1 1.9 µm, 2.1 x 100 mm Column Temp: 30 °C Flow Rate: 0.4 mL/min Injection Vol.: 2 µL Mobile Phase A: Deionized water Mobile Phase B: 100 mM ammonium formate, pH 4.4 Gradient: Time, %B; 0, 4; 4, 4; 35, 39

FIGURE 3: Direct Charged Aerosol Detection of a1-Acid Glycoprotein and Bovine Fetuin N-linked Glycans

FIGURE 1: Charged Aerosol Detector and Principle of Operation

FIGURE 5: Label-free Fetuin Glycan Analysis using Charged Aerosol Detection and MS 128B, C18, 100 ug

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00Time0

100

%

0

100

%

AH08MAR0704 Channel 1An1

5.72e4

AH08MAR0704 TOF MS ES+ TIC

5.91e3

Glycopeptide #1

900 mL to CAD

100 mL to MS

Glycopeptide #2

• de-N glycosylate • -elimination • desalt (evap. and AG-50) • C18 cleanup • LC/CAD/MS

Monosaccharide Key

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

UV/Vis detection has been the workhorse of biopharmaceutical HPLC analysis because many proteins and other active biopharmaceutical ingredients typically possess a good chromophore. However, there are many instances where, although the biotherapeutic agent itself may be UV active, it has critical non-chromophoric chemical modifications and/or is formulated in UV-transparent excipients that must be detected and measured. In contrast to UV/Vis instruments, the Charged Aerosol Detector (CAD) is a mass-based instrument that provides a sensitive, near universal response for any nonvolatile analyte independently of chemical composition. Thus it can detects substances unseen by UV/Vis absorption providing valuable orthogonal data. CAD is well suited for the label free detection of analytes without chromophores and the quantification of unknowns. By virtual of its universal response, the charged aerosol detector can be used for many analytical applications in bioPharm ranging from the label free analysis released glycans to quantification of formulation excipients for quality control. This poster demonstrates several examples where the CAD can be applied to detect and quantify substances separated by reversed phase and hydrophilic interaction chromatography. Examples include: glycans, sialic acids, surfactants, adjuvants, proteins and amino acids. In many cases, detection limits are in the low nanogram range with good peak area precision.

FIGURE 7: Vaccine Adjuvants by CAD and UV

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 -100

0

100

200

Saponins

Cholesterol

0

20

40

60

80

Saponins

Lyso-PC

Cholesterol oxidation products

Cholesterol DPPC

Column: Thermo Scientific™ Accucore™ PFP 2.6 μm, 2.1 100 mm Column Temp.: 50 oC Flow Rate: 1.0 mL/min Injection Vol.: 0.2 μL Mobile Phase A: 0.1 % formic acid in deionized water Mobile Phase B: 0.1 % formic acid in 30:70 acetonitrile:2-propanol Gradient: 25-70-70% B at 0-4-9 min Evaporation Temp: 50 oC Detector: VCAD 2Hz, 5s, PF 1

FIGURE 10: Tween 80 Lot-to-Lot Variability

FIGURE 8: Surfactants - Quantitative

Triton X-100

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time [min]

0

20

30

40

Res

pons

e [p

A]

10

Column: Thermo Scientific™ Betasil™ C1, 5 μm, 3 x 100 mm Column Temp.: 30 ºC, StillAir mode Flow Rate: 0.8 mL/min Injection Vol.: 5 μL Mobile Phase A: Water Mobile Phase B: Acetonitrile:methanol:THF (28:70:2) Gradient: Time(min) Flow Rate %B

(mL/min) 0.0 1.00 0 0.5 1.00 0 0.7 1.35 100 0.8 1.50 100 1.5 1.50 100 1.7 1.00 0 3.5 1.00 0

Detector: VCAD, 70 ºC, 10 Hz, 5s, PF 1.0 Sample Prep.: Dissolve in deionized water

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0

5

10

15

20

25

30

Res

pons

e [p

A]

Tween 80

FIGURE 9: Polyethylene glycol - Qualitative

16 12 20

PEG (MW~300)

PEG (MW~400)

PEG (MW~600)

PEG (MW~1000)

0 8 4

0

80 Column: Thermo Scientific™ Acclaim™ Surfactant Plus, 3 µm, 3.0 x 150 mm Column Temp.: 30 °C Flow Rate: 0.6 mL/min Injection Vol.: 2 µL Gradient: Time Acetonitrile 0.1 M Ammonium (min) Acetate, pH 5 -8.0 2 98 0.0 2 98 20.0 20 80 Detection: Corona ultra Sample: (2.5 mg/mL each) PEG (MW~300) PEG (MW~400) PEG (MW~600) PEG (MW~1000)

FIGURE 6: Direct Detection of Released Sialic Acids from Human and Bovine Transferrin Using HPLC with Charged Aerosol Detection.

0.2 2.0 4.0 6.0 8.0 10.0 12.0 -5.0

0.0

10.0

20.0

25.0

Human Transferrin

Bovine Transferrin

Column: Thermo Scientific™ Acclaim™ Trinity P2 column, 3 µm, 2.1 x 100 mm Column Temp.: 30 °C Flow Rate: 0.3 mL/min Injection Vol.: 5 µL Mobile Phase: 65/25/10 ACN/water/100 mM Ammonium Formate, pH 4

Res

pons

e [p

A]

Time [min]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e

0

20

40

60

80

100

120

140

160

180

200

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

Tween 80 Column: C18, 1.7 µm 2.1 x 50 mm Column Temp: 40 oC Injection volume: 1 µL Mobile Phase A: DI Water Mobile Phase B: Acetonitrile Gradient: Time %B Flow Rate (min.) (mL/min.) 0 6 0.5 1 12 0.6 3 17 0.6 10 20 0.6 18 26 0.6 18.5 55 0.6 23.5 80 0.6 24 6 0.5 Corona ultra – 200pA; Filter = high; Neb Temp = 25 oC Sample: 30 mg/mL Tween 80 in water

Time [min]

Res

pons

e (m

V)

Time [min]

Res

pons

e [p

A]

Abso

rban

ce (m

Au)

0.0

0.5

1.0

1.5

2.0

2.5

0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00

Abso

rban

ce (m

Au)

UV 214 nm

His Phe

Trp

Time [min]

Met

4.00 8.00 12.00 16.00 20.00 24.00 28.00

0

50

100

150

200

250

0.00

Res

pons

e [m

V]

Gly

Arg

Glu

Phe Trp His

Charged Aerosol Detection

FIGURE 2: Direct Measurement of Amino Acids and BSA by Charged Aerosol and UV Absorbance Detection

Column: C18 4.6 x 250mm, 5µm Column Temp.: Ambient Flow Rate: 0.6 mL/min. Injection Vol.: 10 µL of 1 mg/mL Mobile Phase: 0.1% TFA in water (A) and ACN (B) Gradient: 5 min hold at 100% A, 0-40% B in 20 min

FIGURE 4: Label-free Detection of O-linked Glycans by HPLC-CAD Mucin Glycans Released by Reductive Beta Elimination

0.0 5.0 10.0 15.0 20.0 25.0 30.0 -1.0

0.0

5.0

10.0

15.0

Time [min]

Res

pons

e (p

A)

Column: Thermo Scientific™ GlycanPac™ AXH-1, 1.9 μm, 2.1 x 150 mm Column Temp.: 30 oC, StillAir mode Flow Rate: 0.5 mL/min Injection Vol.: 2 μL Mobile Phase A: 95:5 acetonitrile: DI water Mobile Phase B: 50 mM Ammonium Formate, pH 4.4 Gradient: 2 % B to 42 % B in 60 min Detector: VCAD, 50 oC, PF 1.0, 10 Hz, 5s

Column: Thermo Scientific™ Hypercarb™ Flow Rate: 0.9 mL/min CAD: 0.1 mL/min MS Injection Vol.: 20 µL Mobile Phase: Aqueous acetonitrile (containing 0.1% TFA) Gradient: 4% to 18% ACN in 40 min.

UV @ 210 nm

Charged Aerosol

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 -4.0 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0

UV 280nm

CAD

Time [min]

Res

pons

e

BSA Column: Thermo Scientific™ Accucore™ 150, C4 Column Temp.: 45 ºC Flow Rate: 0.4 mL/min Injection Vol.: 1 µL of 0.5 mg/mL std. Mobile Phase A: 0.2% Aqueous TFA Mobile Phase B: 0.1% TFA in ACN Gradient: 10–90% B in 20 min

Amino Acids and Proteins

Glycans, Sialic Acids and Adjuvants Excipients Introduction

Figure 1 depicts the operation of the charged aerosol detector. At the top left (1) the mobile phase from the LC column entering the detector is nebulized by combining with a concentric stream of nitrogen gas or air (2). The fine droplets carried by bulk gas flow to the heated evaporation sector (3) are desolvated to form dry particles (5) from any nonvolatile or semivolatile species. Any remaining large droplets drain away to waste (4). The dry analyte particles combine with another gas stream that has been charged by a high voltage Corona charger (6). The charged gas transfers positive charge to the analyte particle’s surface (7). The charged analyte particles pass through an ion trap (8) that removes any high mobility species and pass to a collector where they are measured by a sensitive electrometer. The signal produced (9) is directly proportional to the quantity of analyte.

Acknowledgements We acknowledge the contribution of Mike Swartz who conducted the amino acid work in Figure 2 and Andy Hanniman who ran the CAD-MS experiment in Figure 5.

PO71803-EN 1015S

PN71803-EN 1015S

Signal is directly proportional to the analyte quantity

1

2

3

4

5 6

7

8

9

Conclusions Charged Aerosol Detection provides a universal

response that does not require a chromophore

Glycans and other carbohydrates can be detected without the need for labeling

Virtually all types of excipients can be detected and quantified

CAD can be combined with UV, Mass Spec. or fluorescence detection to provide a more complete view of the sample

Although not shown here, CAD has also been used for the analysis of phospholipids in liposomes and the quantification of extractables.

© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

Application of Charged Aerosol HPLC Detection in Biopharmaceutical Analysis

Bill Kopaciewicz, David Thomas, Bruce Bailey, Qi Zhang, Marc Plante & Ian Acworth, Thermo Fisher Scientific, Chelmsford, MA

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0

-5.4

0.0

5.0 MONOSIALYLATED

DISIALYLATED

DIGEST REAGENTS TRISIALYLATED

TETRASIALYLATED

ALDITOLS STANDARD

FETUIN

a1-ACID GLYCOPROTEIN

Column: Thermo Scientific™ GlycanPac™ AXR-1 1.9 µm, 2.1 x 100 mm Column Temp: 30 °C Flow Rate: 0.4 mL/min Injection Vol.: 2 µL Mobile Phase A: Deionized water Mobile Phase B: 100 mM ammonium formate, pH 4.4 Gradient: Time, %B; 0, 4; 4, 4; 35, 39

FIGURE 3: Direct Charged Aerosol Detection of a1-Acid Glycoprotein and Bovine Fetuin N-linked Glycans

FIGURE 1: Charged Aerosol Detector and Principle of Operation

FIGURE 5: Label-free Fetuin Glycan Analysis using Charged Aerosol Detection and MS 128B, C18, 100 ug

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00Time0

100

%

0

100

%

AH08MAR0704 Channel 1An1

5.72e4

AH08MAR0704 TOF MS ES+ TIC

5.91e3

Glycopeptide #1

900 mL to CAD

100 mL to MS

Glycopeptide #2

• de-N glycosylate • -elimination • desalt (evap. and AG-50) • C18 cleanup • LC/CAD/MS

Monosaccharide Key

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

UV/Vis detection has been the workhorse of biopharmaceutical HPLC analysis because many proteins and other active biopharmaceutical ingredients typically possess a good chromophore. However, there are many instances where, although the biotherapeutic agent itself may be UV active, it has critical non-chromophoric chemical modifications and/or is formulated in UV-transparent excipients that must be detected and measured. In contrast to UV/Vis instruments, the Charged Aerosol Detector (CAD) is a mass-based instrument that provides a sensitive, near universal response for any nonvolatile analyte independently of chemical composition. Thus it can detects substances unseen by UV/Vis absorption providing valuable orthogonal data. CAD is well suited for the label free detection of analytes without chromophores and the quantification of unknowns. By virtual of its universal response, the charged aerosol detector can be used for many analytical applications in bioPharm ranging from the label free analysis released glycans to quantification of formulation excipients for quality control. This poster demonstrates several examples where the CAD can be applied to detect and quantify substances separated by reversed phase and hydrophilic interaction chromatography. Examples include: glycans, sialic acids, surfactants, adjuvants, proteins and amino acids. In many cases, detection limits are in the low nanogram range with good peak area precision.

FIGURE 7: Vaccine Adjuvants by CAD and UV

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 -100

0

100

200

Saponins

Cholesterol

0

20

40

60

80

Saponins

Lyso-PC

Cholesterol oxidation products

Cholesterol DPPC

Column: Thermo Scientific™ Accucore™ PFP 2.6 μm, 2.1 100 mm Column Temp.: 50 oC Flow Rate: 1.0 mL/min Injection Vol.: 0.2 μL Mobile Phase A: 0.1 % formic acid in deionized water Mobile Phase B: 0.1 % formic acid in 30:70 acetonitrile:2-propanol Gradient: 25-70-70% B at 0-4-9 min Evaporation Temp: 50 oC Detector: VCAD 2Hz, 5s, PF 1

FIGURE 10: Tween 80 Lot-to-Lot Variability

FIGURE 8: Surfactants - Quantitative

Triton X-100

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time [min]

0

20

30

40

Res

pons

e [p

A]

10

Column: Thermo Scientific™ Betasil™ C1, 5 μm, 3 x 100 mm Column Temp.: 30 ºC, StillAir mode Flow Rate: 0.8 mL/min Injection Vol.: 5 μL Mobile Phase A: Water Mobile Phase B: Acetonitrile:methanol:THF (28:70:2) Gradient: Time(min) Flow Rate %B

(mL/min) 0.0 1.00 0 0.5 1.00 0 0.7 1.35 100 0.8 1.50 100 1.5 1.50 100 1.7 1.00 0 3.5 1.00 0

Detector: VCAD, 70 ºC, 10 Hz, 5s, PF 1.0 Sample Prep.: Dissolve in deionized water

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0

5

10

15

20

25

30

Res

pons

e [p

A]

Tween 80

FIGURE 9: Polyethylene glycol - Qualitative

16 12 20

PEG (MW~300)

PEG (MW~400)

PEG (MW~600)

PEG (MW~1000)

0 8 4

0

80 Column: Thermo Scientific™ Acclaim™ Surfactant Plus, 3 µm, 3.0 x 150 mm Column Temp.: 30 °C Flow Rate: 0.6 mL/min Injection Vol.: 2 µL Gradient: Time Acetonitrile 0.1 M Ammonium (min) Acetate, pH 5 -8.0 2 98 0.0 2 98 20.0 20 80 Detection: Corona ultra Sample: (2.5 mg/mL each) PEG (MW~300) PEG (MW~400) PEG (MW~600) PEG (MW~1000)

FIGURE 6: Direct Detection of Released Sialic Acids from Human and Bovine Transferrin Using HPLC with Charged Aerosol Detection.

0.2 2.0 4.0 6.0 8.0 10.0 12.0 -5.0

0.0

10.0

20.0

25.0

Human Transferrin

Bovine Transferrin

Column: Thermo Scientific™ Acclaim™ Trinity P2 column, 3 µm, 2.1 x 100 mm Column Temp.: 30 °C Flow Rate: 0.3 mL/min Injection Vol.: 5 µL Mobile Phase: 65/25/10 ACN/water/100 mM Ammonium Formate, pH 4

Res

pons

e [p

A]

Time [min]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e

0

20

40

60

80

100

120

140

160

180

200

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

Tween 80 Column: C18, 1.7 µm 2.1 x 50 mm Column Temp: 40 oC Injection volume: 1 µL Mobile Phase A: DI Water Mobile Phase B: Acetonitrile Gradient: Time %B Flow Rate (min.) (mL/min.) 0 6 0.5 1 12 0.6 3 17 0.6 10 20 0.6 18 26 0.6 18.5 55 0.6 23.5 80 0.6 24 6 0.5 Corona ultra – 200pA; Filter = high; Neb Temp = 25 oC Sample: 30 mg/mL Tween 80 in water

Time [min]

Res

pons

e (m

V)

Time [min]

Res

pons

e [p

A]

Abso

rban

ce (m

Au)

0.0

0.5

1.0

1.5

2.0

2.5

0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00

Abso

rban

ce (m

Au)

UV 214 nm

His Phe

Trp

Time [min]

Met

4.00 8.00 12.00 16.00 20.00 24.00 28.00

0

50

100

150

200

250

0.00

Res

pons

e [m

V]

Gly

Arg

Glu

Phe Trp His

Charged Aerosol Detection

FIGURE 2: Direct Measurement of Amino Acids and BSA by Charged Aerosol and UV Absorbance Detection

Column: C18 4.6 x 250mm, 5µm Column Temp.: Ambient Flow Rate: 0.6 mL/min. Injection Vol.: 10 µL of 1 mg/mL Mobile Phase: 0.1% TFA in water (A) and ACN (B) Gradient: 5 min hold at 100% A, 0-40% B in 20 min

FIGURE 4: Label-free Detection of O-linked Glycans by HPLC-CAD Mucin Glycans Released by Reductive Beta Elimination

0.0 5.0 10.0 15.0 20.0 25.0 30.0 -1.0

0.0

5.0

10.0

15.0

Time [min]

Res

pons

e (p

A)

Column: Thermo Scientific™ GlycanPac™ AXH-1, 1.9 μm, 2.1 x 150 mm Column Temp.: 30 oC, StillAir mode Flow Rate: 0.5 mL/min Injection Vol.: 2 μL Mobile Phase A: 95:5 acetonitrile: DI water Mobile Phase B: 50 mM Ammonium Formate, pH 4.4 Gradient: 2 % B to 42 % B in 60 min Detector: VCAD, 50 oC, PF 1.0, 10 Hz, 5s

Column: Thermo Scientific™ Hypercarb™ Flow Rate: 0.9 mL/min CAD: 0.1 mL/min MS Injection Vol.: 20 µL Mobile Phase: Aqueous acetonitrile (containing 0.1% TFA) Gradient: 4% to 18% ACN in 40 min.

UV @ 210 nm

Charged Aerosol

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 -4.0 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0

UV 280nm

CAD

Time [min]

Res

pons

e

BSA Column: Thermo Scientific™ Accucore™ 150, C4 Column Temp.: 45 ºC Flow Rate: 0.4 mL/min Injection Vol.: 1 µL of 0.5 mg/mL std. Mobile Phase A: 0.2% Aqueous TFA Mobile Phase B: 0.1% TFA in ACN Gradient: 10–90% B in 20 min

Amino Acids and Proteins

Glycans, Sialic Acids and Adjuvants Excipients Introduction

Figure 1 depicts the operation of the charged aerosol detector. At the top left (1) the mobile phase from the LC column entering the detector is nebulized by combining with a concentric stream of nitrogen gas or air (2). The fine droplets carried by bulk gas flow to the heated evaporation sector (3) are desolvated to form dry particles (5) from any nonvolatile or semivolatile species. Any remaining large droplets drain away to waste (4). The dry analyte particles combine with another gas stream that has been charged by a high voltage Corona charger (6). The charged gas transfers positive charge to the analyte particle’s surface (7). The charged analyte particles pass through an ion trap (8) that removes any high mobility species and pass to a collector where they are measured by a sensitive electrometer. The signal produced (9) is directly proportional to the quantity of analyte.

Acknowledgements We acknowledge the contribution of Mike Swartz who conducted the amino acid work in Figure 2 and Andy Hanniman who ran the CAD-MS experiment in Figure 5.

PO71803-EN 1015S

Signal is directly proportional to the analyte quantity

1

2

3

4

5 6

7

8

9

Conclusions Charged Aerosol Detection provides a universal

response that does not require a chromophore

Glycans and other carbohydrates can be detected without the need for labeling

Virtually all types of excipients can be detected and quantified

CAD can be combined with UV, Mass Spec. or fluorescence detection to provide a more complete view of the sample

Although not shown here, CAD has also been used for the analysis of phospholipids in liposomes and the quantification of extractables.

© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

Application of Charged Aerosol HPLC Detection in Biopharmaceutical Analysis

Bill Kopaciewicz, David Thomas, Bruce Bailey, Qi Zhang, Marc Plante & Ian Acworth, Thermo Fisher Scientific, Chelmsford, MA

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0

-5.4

0.0

5.0 MONOSIALYLATED

DISIALYLATED

DIGEST REAGENTS TRISIALYLATED

TETRASIALYLATED

ALDITOLS STANDARD

FETUIN

a1-ACID GLYCOPROTEIN

Column: Thermo Scientific™ GlycanPac™ AXR-1 1.9 µm, 2.1 x 100 mm Column Temp: 30 °C Flow Rate: 0.4 mL/min Injection Vol.: 2 µL Mobile Phase A: Deionized water Mobile Phase B: 100 mM ammonium formate, pH 4.4 Gradient: Time, %B; 0, 4; 4, 4; 35, 39

FIGURE 3: Direct Charged Aerosol Detection of a1-Acid Glycoprotein and Bovine Fetuin N-linked Glycans

FIGURE 1: Charged Aerosol Detector and Principle of Operation

FIGURE 5: Label-free Fetuin Glycan Analysis using Charged Aerosol Detection and MS 128B, C18, 100 ug

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00Time0

100

%

0

100

%

AH08MAR0704 Channel 1An1

5.72e4

AH08MAR0704 TOF MS ES+ TIC

5.91e3

Glycopeptide #1

900 mL to CAD

100 mL to MS

Glycopeptide #2

• de-N glycosylate • -elimination • desalt (evap. and AG-50) • C18 cleanup • LC/CAD/MS

Monosaccharide Key

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

UV/Vis detection has been the workhorse of biopharmaceutical HPLC analysis because many proteins and other active biopharmaceutical ingredients typically possess a good chromophore. However, there are many instances where, although the biotherapeutic agent itself may be UV active, it has critical non-chromophoric chemical modifications and/or is formulated in UV-transparent excipients that must be detected and measured. In contrast to UV/Vis instruments, the Charged Aerosol Detector (CAD) is a mass-based instrument that provides a sensitive, near universal response for any nonvolatile analyte independently of chemical composition. Thus it can detects substances unseen by UV/Vis absorption providing valuable orthogonal data. CAD is well suited for the label free detection of analytes without chromophores and the quantification of unknowns. By virtual of its universal response, the charged aerosol detector can be used for many analytical applications in bioPharm ranging from the label free analysis released glycans to quantification of formulation excipients for quality control. This poster demonstrates several examples where the CAD can be applied to detect and quantify substances separated by reversed phase and hydrophilic interaction chromatography. Examples include: glycans, sialic acids, surfactants, adjuvants, proteins and amino acids. In many cases, detection limits are in the low nanogram range with good peak area precision.

FIGURE 7: Vaccine Adjuvants by CAD and UV

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 -100

0

100

200

Saponins

Cholesterol

0

20

40

60

80

Saponins

Lyso-PC

Cholesterol oxidation products

Cholesterol DPPC

Column: Thermo Scientific™ Accucore™ PFP 2.6 μm, 2.1 100 mm Column Temp.: 50 oC Flow Rate: 1.0 mL/min Injection Vol.: 0.2 μL Mobile Phase A: 0.1 % formic acid in deionized water Mobile Phase B: 0.1 % formic acid in 30:70 acetonitrile:2-propanol Gradient: 25-70-70% B at 0-4-9 min Evaporation Temp: 50 oC Detector: VCAD 2Hz, 5s, PF 1

FIGURE 10: Tween 80 Lot-to-Lot Variability

FIGURE 8: Surfactants - Quantitative

Triton X-100

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time [min]

0

20

30

40

Res

pons

e [p

A]

10

Column: Thermo Scientific™ Betasil™ C1, 5 μm, 3 x 100 mm Column Temp.: 30 ºC, StillAir mode Flow Rate: 0.8 mL/min Injection Vol.: 5 μL Mobile Phase A: Water Mobile Phase B: Acetonitrile:methanol:THF (28:70:2) Gradient: Time(min) Flow Rate %B

(mL/min) 0.0 1.00 0 0.5 1.00 0 0.7 1.35 100 0.8 1.50 100 1.5 1.50 100 1.7 1.00 0 3.5 1.00 0

Detector: VCAD, 70 ºC, 10 Hz, 5s, PF 1.0 Sample Prep.: Dissolve in deionized water

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0

5

10

15

20

25

30

Res

pons

e [p

A]

Tween 80

FIGURE 9: Polyethylene glycol - Qualitative

16 12 20

PEG (MW~300)

PEG (MW~400)

PEG (MW~600)

PEG (MW~1000)

0 8 4

0

80 Column: Thermo Scientific™ Acclaim™ Surfactant Plus, 3 µm, 3.0 x 150 mm Column Temp.: 30 °C Flow Rate: 0.6 mL/min Injection Vol.: 2 µL Gradient: Time Acetonitrile 0.1 M Ammonium (min) Acetate, pH 5 -8.0 2 98 0.0 2 98 20.0 20 80 Detection: Corona ultra Sample: (2.5 mg/mL each) PEG (MW~300) PEG (MW~400) PEG (MW~600) PEG (MW~1000)

FIGURE 6: Direct Detection of Released Sialic Acids from Human and Bovine Transferrin Using HPLC with Charged Aerosol Detection.

0.2 2.0 4.0 6.0 8.0 10.0 12.0 -5.0

0.0

10.0

20.0

25.0

Human Transferrin

Bovine Transferrin

Column: Thermo Scientific™ Acclaim™ Trinity P2 column, 3 µm, 2.1 x 100 mm Column Temp.: 30 °C Flow Rate: 0.3 mL/min Injection Vol.: 5 µL Mobile Phase: 65/25/10 ACN/water/100 mM Ammonium Formate, pH 4

Res

pons

e [p

A]

Time [min]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e

0

20

40

60

80

100

120

140

160

180

200

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

Tween 80 Column: C18, 1.7 µm 2.1 x 50 mm Column Temp: 40 oC Injection volume: 1 µL Mobile Phase A: DI Water Mobile Phase B: Acetonitrile Gradient: Time %B Flow Rate (min.) (mL/min.) 0 6 0.5 1 12 0.6 3 17 0.6 10 20 0.6 18 26 0.6 18.5 55 0.6 23.5 80 0.6 24 6 0.5 Corona ultra – 200pA; Filter = high; Neb Temp = 25 oC Sample: 30 mg/mL Tween 80 in water

Time [min]

Res

pons

e (m

V)

Time [min]

Res

pons

e [p

A]

Abso

rban

ce (m

Au)

0.0

0.5

1.0

1.5

2.0

2.5

0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00

Abso

rban

ce (m

Au)

UV 214 nm

His Phe

Trp

Time [min]

Met

4.00 8.00 12.00 16.00 20.00 24.00 28.00

0

50

100

150

200

250

0.00

Res

pons

e [m

V]

Gly

Arg

Glu

Phe Trp His

Charged Aerosol Detection

FIGURE 2: Direct Measurement of Amino Acids and BSA by Charged Aerosol and UV Absorbance Detection

Column: C18 4.6 x 250mm, 5µm Column Temp.: Ambient Flow Rate: 0.6 mL/min. Injection Vol.: 10 µL of 1 mg/mL Mobile Phase: 0.1% TFA in water (A) and ACN (B) Gradient: 5 min hold at 100% A, 0-40% B in 20 min

FIGURE 4: Label-free Detection of O-linked Glycans by HPLC-CAD Mucin Glycans Released by Reductive Beta Elimination

0.0 5.0 10.0 15.0 20.0 25.0 30.0 -1.0

0.0

5.0

10.0

15.0

Time [min]

Res

pons

e (p

A)

Column: Thermo Scientific™ GlycanPac™ AXH-1, 1.9 μm, 2.1 x 150 mm Column Temp.: 30 oC, StillAir mode Flow Rate: 0.5 mL/min Injection Vol.: 2 μL Mobile Phase A: 95:5 acetonitrile: DI water Mobile Phase B: 50 mM Ammonium Formate, pH 4.4 Gradient: 2 % B to 42 % B in 60 min Detector: VCAD, 50 oC, PF 1.0, 10 Hz, 5s

Column: Thermo Scientific™ Hypercarb™ Flow Rate: 0.9 mL/min CAD: 0.1 mL/min MS Injection Vol.: 20 µL Mobile Phase: Aqueous acetonitrile (containing 0.1% TFA) Gradient: 4% to 18% ACN in 40 min.

UV @ 210 nm

Charged Aerosol

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 -4.0 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0

UV 280nm

CAD

Time [min]

Res

pons

e

BSA Column: Thermo Scientific™ Accucore™ 150, C4 Column Temp.: 45 ºC Flow Rate: 0.4 mL/min Injection Vol.: 1 µL of 0.5 mg/mL std. Mobile Phase A: 0.2% Aqueous TFA Mobile Phase B: 0.1% TFA in ACN Gradient: 10–90% B in 20 min

Amino Acids and Proteins

Glycans, Sialic Acids and Adjuvants Excipients Introduction

Figure 1 depicts the operation of the charged aerosol detector. At the top left (1) the mobile phase from the LC column entering the detector is nebulized by combining with a concentric stream of nitrogen gas or air (2). The fine droplets carried by bulk gas flow to the heated evaporation sector (3) are desolvated to form dry particles (5) from any nonvolatile or semivolatile species. Any remaining large droplets drain away to waste (4). The dry analyte particles combine with another gas stream that has been charged by a high voltage Corona charger (6). The charged gas transfers positive charge to the analyte particle’s surface (7). The charged analyte particles pass through an ion trap (8) that removes any high mobility species and pass to a collector where they are measured by a sensitive electrometer. The signal produced (9) is directly proportional to the quantity of analyte.

Acknowledgements We acknowledge the contribution of Mike Swartz who conducted the amino acid work in Figure 2 and Andy Hanniman who ran the CAD-MS experiment in Figure 5.

PO71803-EN 1015S

Signal is directly proportional to the analyte quantity

1

2

3

4

5 6

7

8

9

Conclusions Charged Aerosol Detection provides a universal

response that does not require a chromophore

Glycans and other carbohydrates can be detected without the need for labeling

Virtually all types of excipients can be detected and quantified

CAD can be combined with UV, Mass Spec. or fluorescence detection to provide a more complete view of the sample

Although not shown here, CAD has also been used for the analysis of phospholipids in liposomes and the quantification of extractables.

© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

Application of Charged Aerosol HPLC Detection in Biopharmaceutical Analysis

Bill Kopaciewicz, David Thomas, Bruce Bailey, Qi Zhang, Marc Plante & Ian Acworth, Thermo Fisher Scientific, Chelmsford, MA

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0

-5.4

0.0

5.0 MONOSIALYLATED

DISIALYLATED

DIGEST REAGENTS TRISIALYLATED

TETRASIALYLATED

ALDITOLS STANDARD

FETUIN

a1-ACID GLYCOPROTEIN

Column: Thermo Scientific™ GlycanPac™ AXR-1 1.9 µm, 2.1 x 100 mm Column Temp: 30 °C Flow Rate: 0.4 mL/min Injection Vol.: 2 µL Mobile Phase A: Deionized water Mobile Phase B: 100 mM ammonium formate, pH 4.4 Gradient: Time, %B; 0, 4; 4, 4; 35, 39

FIGURE 3: Direct Charged Aerosol Detection of a1-Acid Glycoprotein and Bovine Fetuin N-linked Glycans

FIGURE 1: Charged Aerosol Detector and Principle of Operation

FIGURE 5: Label-free Fetuin Glycan Analysis using Charged Aerosol Detection and MS 128B, C18, 100 ug

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00Time0

100

%

0

100

%

AH08MAR0704 Channel 1An1

5.72e4

AH08MAR0704 TOF MS ES+ TIC

5.91e3

Glycopeptide #1

900 mL to CAD

100 mL to MS

Glycopeptide #2

• de-N glycosylate • -elimination • desalt (evap. and AG-50) • C18 cleanup • LC/CAD/MS

Monosaccharide Key

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

N-acetyl neuraminic Acid (NeuAc)

N-Acetylglucosamine (GlcNAc)

Galactose (Gal)

Mannose (Man)

Fucose (Fuc)

N-glycoyl neuraminic acid (NeuGc)

N-Acetylgalactosamine (GlcNAc)

UV/Vis detection has been the workhorse of biopharmaceutical HPLC analysis because many proteins and other active biopharmaceutical ingredients typically possess a good chromophore. However, there are many instances where, although the biotherapeutic agent itself may be UV active, it has critical non-chromophoric chemical modifications and/or is formulated in UV-transparent excipients that must be detected and measured. In contrast to UV/Vis instruments, the Charged Aerosol Detector (CAD) is a mass-based instrument that provides a sensitive, near universal response for any nonvolatile analyte independently of chemical composition. Thus it can detects substances unseen by UV/Vis absorption providing valuable orthogonal data. CAD is well suited for the label free detection of analytes without chromophores and the quantification of unknowns. By virtual of its universal response, the charged aerosol detector can be used for many analytical applications in bioPharm ranging from the label free analysis released glycans to quantification of formulation excipients for quality control. This poster demonstrates several examples where the CAD can be applied to detect and quantify substances separated by reversed phase and hydrophilic interaction chromatography. Examples include: glycans, sialic acids, surfactants, adjuvants, proteins and amino acids. In many cases, detection limits are in the low nanogram range with good peak area precision.

FIGURE 7: Vaccine Adjuvants by CAD and UV

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Saponins

Cholesterol

0

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Lyso-PC

Cholesterol oxidation products

Cholesterol DPPC

Column: Thermo Scientific™ Accucore™ PFP 2.6 μm, 2.1 100 mm Column Temp.: 50 oC Flow Rate: 1.0 mL/min Injection Vol.: 0.2 μL Mobile Phase A: 0.1 % formic acid in deionized water Mobile Phase B: 0.1 % formic acid in 30:70 acetonitrile:2-propanol Gradient: 25-70-70% B at 0-4-9 min Evaporation Temp: 50 oC Detector: VCAD 2Hz, 5s, PF 1

FIGURE 10: Tween 80 Lot-to-Lot Variability

FIGURE 8: Surfactants - Quantitative

Triton X-100

0.0 0.5 1.0 1.5 2.0 2.5 3.0

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Res

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Column: Thermo Scientific™ Betasil™ C1, 5 μm, 3 x 100 mm Column Temp.: 30 ºC, StillAir mode Flow Rate: 0.8 mL/min Injection Vol.: 5 μL Mobile Phase A: Water Mobile Phase B: Acetonitrile:methanol:THF (28:70:2) Gradient: Time(min) Flow Rate %B

(mL/min) 0.0 1.00 0 0.5 1.00 0 0.7 1.35 100 0.8 1.50 100 1.5 1.50 100 1.7 1.00 0 3.5 1.00 0

Detector: VCAD, 70 ºC, 10 Hz, 5s, PF 1.0 Sample Prep.: Dissolve in deionized water

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FIGURE 9: Polyethylene glycol - Qualitative

16 12 20

PEG (MW~300)

PEG (MW~400)

PEG (MW~600)

PEG (MW~1000)

0 8 4

0

80 Column: Thermo Scientific™ Acclaim™ Surfactant Plus, 3 µm, 3.0 x 150 mm Column Temp.: 30 °C Flow Rate: 0.6 mL/min Injection Vol.: 2 µL Gradient: Time Acetonitrile 0.1 M Ammonium (min) Acetate, pH 5 -8.0 2 98 0.0 2 98 20.0 20 80 Detection: Corona ultra Sample: (2.5 mg/mL each) PEG (MW~300) PEG (MW~400) PEG (MW~600) PEG (MW~1000)

FIGURE 6: Direct Detection of Released Sialic Acids from Human and Bovine Transferrin Using HPLC with Charged Aerosol Detection.

0.2 2.0 4.0 6.0 8.0 10.0 12.0 -5.0

0.0

10.0

20.0

25.0

Human Transferrin

Bovine Transferrin

Column: Thermo Scientific™ Acclaim™ Trinity P2 column, 3 µm, 2.1 x 100 mm Column Temp.: 30 °C Flow Rate: 0.3 mL/min Injection Vol.: 5 µL Mobile Phase: 65/25/10 ACN/water/100 mM Ammonium Formate, pH 4

Res

pons

e [p

A]

Time [min]

Time [min]

Res

pons

e [p

A]

Time [min]

Res

pons

e [p

A]

Time [min]

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pons

e

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160

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200

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0

Tween 80 Column: C18, 1.7 µm 2.1 x 50 mm Column Temp: 40 oC Injection volume: 1 µL Mobile Phase A: DI Water Mobile Phase B: Acetonitrile Gradient: Time %B Flow Rate (min.) (mL/min.) 0 6 0.5 1 12 0.6 3 17 0.6 10 20 0.6 18 26 0.6 18.5 55 0.6 23.5 80 0.6 24 6 0.5 Corona ultra – 200pA; Filter = high; Neb Temp = 25 oC Sample: 30 mg/mL Tween 80 in water

Time [min]

Res

pons

e (m

V)

Time [min]

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pons

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A]

Abso

rban

ce (m

Au)

0.0

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Abso

rban

ce (m

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UV 214 nm

His Phe

Trp

Time [min]

Met

4.00 8.00 12.00 16.00 20.00 24.00 28.00

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Res

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Gly

Arg

Glu

Phe Trp His

Charged Aerosol Detection

FIGURE 2: Direct Measurement of Amino Acids and BSA by Charged Aerosol and UV Absorbance Detection

Column: C18 4.6 x 250mm, 5µm Column Temp.: Ambient Flow Rate: 0.6 mL/min. Injection Vol.: 10 µL of 1 mg/mL Mobile Phase: 0.1% TFA in water (A) and ACN (B) Gradient: 5 min hold at 100% A, 0-40% B in 20 min

FIGURE 4: Label-free Detection of O-linked Glycans by HPLC-CAD Mucin Glycans Released by Reductive Beta Elimination

0.0 5.0 10.0 15.0 20.0 25.0 30.0 -1.0

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A)

Column: Thermo Scientific™ GlycanPac™ AXH-1, 1.9 μm, 2.1 x 150 mm Column Temp.: 30 oC, StillAir mode Flow Rate: 0.5 mL/min Injection Vol.: 2 μL Mobile Phase A: 95:5 acetonitrile: DI water Mobile Phase B: 50 mM Ammonium Formate, pH 4.4 Gradient: 2 % B to 42 % B in 60 min Detector: VCAD, 50 oC, PF 1.0, 10 Hz, 5s

Column: Thermo Scientific™ Hypercarb™ Flow Rate: 0.9 mL/min CAD: 0.1 mL/min MS Injection Vol.: 20 µL Mobile Phase: Aqueous acetonitrile (containing 0.1% TFA) Gradient: 4% to 18% ACN in 40 min.

UV @ 210 nm

Charged Aerosol

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 -4.0 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0

UV 280nm

CAD

Time [min]

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BSA Column: Thermo Scientific™ Accucore™ 150, C4 Column Temp.: 45 ºC Flow Rate: 0.4 mL/min Injection Vol.: 1 µL of 0.5 mg/mL std. Mobile Phase A: 0.2% Aqueous TFA Mobile Phase B: 0.1% TFA in ACN Gradient: 10–90% B in 20 min

Amino Acids and Proteins

Glycans, Sialic Acids and Adjuvants Excipients Introduction

Figure 1 depicts the operation of the charged aerosol detector. At the top left (1) the mobile phase from the LC column entering the detector is nebulized by combining with a concentric stream of nitrogen gas or air (2). The fine droplets carried by bulk gas flow to the heated evaporation sector (3) are desolvated to form dry particles (5) from any nonvolatile or semivolatile species. Any remaining large droplets drain away to waste (4). The dry analyte particles combine with another gas stream that has been charged by a high voltage Corona charger (6). The charged gas transfers positive charge to the analyte particle’s surface (7). The charged analyte particles pass through an ion trap (8) that removes any high mobility species and pass to a collector where they are measured by a sensitive electrometer. The signal produced (9) is directly proportional to the quantity of analyte.

Acknowledgements We acknowledge the contribution of Mike Swartz who conducted the amino acid work in Figure 2 and Andy Hanniman who ran the CAD-MS experiment in Figure 5.

PO71803-EN 1015S

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