Ion Chromatography Based Analytical Strategies for ...
Transcript of Ion Chromatography Based Analytical Strategies for ...
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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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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
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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
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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
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nC
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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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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
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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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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)
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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
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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