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Global BioPharma Summit

Ion Chromatography based analytical strategies for unravelling glycan complexity in biologics

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High Complexity, High Risk

• Glycans represent the most complex post-

translational modifications to determine

• They have the largest impact upon efficacy,

safety and stability

• Regulatory guidelines mean glycans have to be

fully profiled

• No single analytical technique can provide all the

necessary information required

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Glyco-engineering to improve biopharmaceuticals

T1/2 = 19h T1/2 = 32h

EPO:

Therapeutic antibodies: Fc glycans determine function

Fc

Fab

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ICH (Q6B) recommended 6 test approaches for characterization

and confirmation of biological products:

• Amino acid sequence

• Amino acid composition

• Terminal amino acid sequence

• Peptide map

• Sulfhydryl group(s) and disulfide bridges

• Carbohydrate structure

• “For glycoproteins, the carbohydrate content and Structure (neutral

sugars, amino sugars, and sialic acids) is determined.”

Characterization and Confirmation of Biological Products

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HPAE-PAD for Carbohydrate Analysis

HPAE-PAD refers to a technique where non-derivatized carbohydrates (ranging from simple sugars

to complex carbohydrates) are analyzed by High-Performance Anion-Exchange (HPAE)

chromatography coupled with Pulsed Amperometric Detection (PAD).

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HPAE-PAD for Carbohydrate Analysis

Basics of HPAE

• Carbohydrates are separated as oxyanions at high pH (>12).

• These separations are achieved using sodium hydroxide or sodium acetate gradients in sodium hydroxide.

• Separations of carbohydrate ranging from monosaccharides to oligosaccharides are achieved using our diverse column portfolio

• For more information : www.thermofisher.com

Basics of PAD

• Non-derivatized carbohydrates are detected on a Au working electrode (WE) at high pH by pulsed amperometric detection (PAD)

• PAD applies a series of potentials (a waveform) to a WE

• Pulsed amperometry detects analytes containing functional groups which get oxidized at the applied detection voltage

• For more information : www.thermofisher.com

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Monosaccharides & Sialic

Acids

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Glycoprotein Monosaccharide Analysis by HPAE-PAD

Monosaccharide Analysis Workflow

1. Hydrolyze the sample

2. Remove acid and dry the sample

3. Reconstitute the sample in water

4. Directly inject it for analysis

Benefits of HPAE-PAD

1. Monosaccharide composition can screen for changes in

glycosylation

2. Sensitive detection with no derivatization

3. Allows measurement of total sugars and amounts of specific

monosaccharides

4. Same system can analyze variety of carbohydrates

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Simple Workflow using Ion Chromatography Systems

Data and System Management

High-Pressure Non-Metallic Pump

Eluent (OH– )Generator

Waste

Sample Inject(Autosampler)

EGC

H20

CR-TC ED

Separation Column

Recycle Mode

Electrochemical

Detector

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Column: Dionex CarboPac PA20 +

Thermo Scientific™ Dionex™ AminoTrap™ column

Eluent: 10 mM KOH

Flow Source: EG50 + CR-ATC

Flow Rate: 0.5 mL/min

Inj. Volume: 10 µL (2 µg)

Detection: PAD (Au) Disposable

Waveform A (TN21)

Temperature: 30 °C

Sample: A) 6N HCI hydrolyzate

B) 4N TFA hydrolyzate

Peaks: 1. Fucose

2. Glucosamine

3. Galactose

4. Mannose

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20

20

38

1

2

3 4

1

2

3

4

nC

nC

0 3 6 9 12Minutes

A – HCl –For aminosugars

B – TFA – For neutral sugars

Monosaccharide Compositional Analysis of hIgG

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1. Hydrolyze under mildly acidic conditions or treat with a neuraminidase

2. Remove acid and dry the sample

3. Dissolve the dried the sample in DI water and inject or just inject for the enyzme digest

4. Separate on either a CarboPac PA20 column set or a Fast Sialic Acid CarboPac PA20 column and detect by PAD

Basic HPAE-PAD Glycoprotein Sialic Acid Analysis

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Simple Workflow using Ion Chromatography Systems

Data and System Management

High-Pressure Non-Metallic Pump

Sample Inject(Autosampler)

ED

Separation Column Electrochemical Detector

Eluent

Reservoirs

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Separation of Sialic Acids

min1.0 2.0 3.019

31

0.0

nC

Column: Dionex CarboPacPA20 Fast Sialic Acid, 3 x 30 mmEluent: 70-300 mM acetate in 100 mM NaOH Temperature: 30 °CFlow Rate: 0.5 mL/minInj. Volume: 4.5 µL (full loop)Detection: PAD

Samples: A) human 1-acid glycoprotein, 23 ng proteinB) fetuin, 18 ng proteinC) s. 1-acid glycoprotein, 7.9 ng protein

Peaks: A) B) C)1. Neu5Ac 13 5.6 6.1 pmol2. Neu5Gc ---- 0.20 1.2

A)

B)

C)

1

2

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Column: Dionex CarboPac PA20 guard, 3 x 30 mmDionex CarboPac PA20, 3 x 150 mm

Eluent: 70-300 mM acetate in 100 mM NaOH from 0-7.5 min, 300 mM acetate in 100 mM NaOH from 7.5-9.0 min, 300-70 mM acetate from 9.0-9.5 min. 7 min of equilibration at 70 mM acetate in 100 mM NaOHTemperature: 30 °CFlow Rate: 0.5 mL/minInj. Volume: 10 µLDetection: PAD, Au (Disposable)

Samples: A) b. apo-transferrin, B) h. transferrin, C) fetuin,

D) s. 1-acid glycoprotein, E) h. 1-acid glycoproteinSample Prep: acid hydrolysis followed by lyophilization and dissolution

Peaks: A) B) C) D) E)1. Neu5Ac 1.7 4.4 18 15 37 pmol2. Neu5Gc 2.1 ND 0.39 2.6 ND

A 10% signal offset has been applied.ND = Not Detected0 3 6 9.5

35

65

min

nC

A)

B)

C)

D)

E)

1

1

1

1

1 2

2

2

Separation of Glycoprotein Acid Hydrolyzates

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Unlabeled glycans

• O-linked & N-linked

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Simple Workflow using Ion Chromatography Systems

Data and System Management

High-Pressure Non-Metallic Pump

Sample Inject(Autosampler)

ED

Separation Column Electrochemical Detector

Eluent

Reservoirs

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Unlabeled N-Glycan Analysis using HPAE-PAD

• Direct detection for profiling released glycans

• New, rapid workflow to release glycans from 200μg of glycoproteins with excellent reproducibility

• Sensitive separations based on charge, linkage, position, and fucosylation

Benefits of Ion Chromatography

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Rapid workflow for native glycan preparation for analysis

Digestion

- 200 µg glycoprotein- HEPES buffer at pH 7.9- TCEP- Proprietary detergent

-S-S-

Denaturation, reduction at 90 C for 5 min

Digestion with PNGase F at 50 C for 15 min

Protein removal on Hypersep Hypercarb Plate

Elute MeCN concentration isadjusted to 90 % and load onto Hypersep Diol Plate

Elution in 50 % MeCN and 0.1 TFAElution of glycans in water

Analysis of native glycansusing IC S-5000+ coupled toQ Exactive Mass Spectrometer

Clean Up

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0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

min

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A) B) C) D)

Sensitive HPAE-PAD Analysis Based on Charge

TrisialylatedDisialylatedMonosialylatedNeutral

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Sensitive HPAE-PAD Analysis Based on Charge and Linkage

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0

min

12

3

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5

6

78

9

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3031

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A) B) C) D)

Monosialylated glycans terminated by Neu5Ac

Monosialylated glycans terminated by Neu5Gc and disialylated glycans terminated by Neu5Ac

Disialylated glycans terminated with one Neu5Ac and one Neu5Gc

Disialylated glycans terminated by Neu5Gc

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0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0min

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5152

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A) B) C) D) E) F)

Sensitive separations based on charge, position, and fucosylation

Disialylated glycans with α-1,3 outer arm fucose

Disialylated glycans without fucose

Trisialylated glycans with α-1,3 outer arm fucose

Trisialylatedglycans without fucose

Tetrasialylatedglycans with α-1,3 outer arm fucose

Tetrasialylated glycans without fucose

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Unlabeled N-Glycan Analysis using HPAE-PAD/MS

Glycoproteins Released Glycans Ion Chromatography Orbitrap

• HPAE-PAD/MS is an excellent tool to profile and annotate complex mixture of native glycans released from different glycoproteins

• Easy interface with MS provides in-depth characterization of all released glycans including low abundant N-linked glycans

• Excellent reproducibility of peak area distribution of glycans released from denatured mAb.

Benefits of Ion Chromatography

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Unlabeled N-Glycan Analysis using HPAE-PAD/MS

Data and System Management

High-Pressure Non-Metallic Pump

Sample Inject(Autosampler)

ED

Separation Column

Electrochemical Detector

Eluent

Reservoirs

MS

Structural information by comparing with known glycan standards

Structural IdentificationQE Mass Spectrometer

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Label-free Analysis of N-linked Glycans by HPAE-PAD-MS

Time (min)

0.0

20.0

30.0

40.0

50.0

60.0

70.0

10.0

25 30 35 40 45 50 55 60 65 70 750

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100

Relat

iveAb

unda

nce

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A

Disialylated with outer arm α-1,3 Fucose

Disialylated withoutouter arm α-1,3 Fucose

nC

Trisialylated with outer arm α-1,3 Fucose

Trisialylated withoutouter arm α-1,3 Fucose

Tetrasialylated with outer arm α-1,3 Fucose

Tetrasialylated withoutouter arm α-1,3 Fucose

A)

B)

PAD (A) and MS base peak (B) chromatograms of hAGP glycans

HPAE-PAD is able to separate highly complex glycan mixtures based on charge, linkage, & fucose

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Label-free Analysis of N-linked Glycans by HPAE-PAD-MS

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IC for BioPharma Applications

Sensitive carbohydrate analysis of recombinant glycoproteins, biosimilars, antibiotics, and glycoconjugate vaccines

Analytes ranging –

• Carbohydrate (mono-, di-, polysaccharide) analysis

• Sialic acid analysis

• Asn (N-)-linked oligosaccharides

• Ser/Thr (O-)-linked oligosaccharide

• Sugar phosphates (mannose-6-phosphate)

• Sugar alcohols

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Benefits of IC

• Speed

• Reproducibility

• Accuracy

• Safety

• Cost effectiveness

Ion Chromatography for Pharmaceutical Analysis

Applications

• Counterion determinations

• Impurity analysis

• Drug substance assay

• Measuring excipients in drug products

One solution for all drug development stages

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Universal

Safe

Fast

Simple

Sensitive Detection

Reliable

Green

Ion Chromatography – Making It Easier To Get Results

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Conclusions

• HPAE-PAD is a fast carbohydrate analysis method

• Directly quantify non-derivatized carbohydrates with

high sensitivity and selectivity

• HPAE-PAD is the one system for monosaccharides,

sialic and other sugar acids, sugar phosphates, sugar

alcohols, sulfate sugars, aminoglycoside antibiotics,

oligosaccharides (charged and neutral), and small

polysaccharides

• With innovations, such as HPIC and CarboPac

columns, carbohydrates are analyzed in as little as 5

minutes

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Questions?

•THANK YOU