agilent technologies april 7, 2015 1 · peptide mapping standard april 7, 2015 agilent technologies...
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April 7, 2015
Agilent Technologies
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April 7, 2015
Agilent Technologies
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Improving your laboratory HPLC analysis workflow with new Agilent BioHPLC Columns
Paul Dinsmoor
Technical Specialist, Bio-Columns
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April 7, 2015
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Tools for Therapeutic Protein Characterization
Size Exclusion Chromatography
• Aggregates and fragment analysis
• Molecular size analysis
Ion-Exchange Chromatography
• Charge variant analysis
2- 1-
+
Reversed-Phase Chromatography
• Primary structure analysis
• Post-translational modifications/ degradations
• Peptide mapping • Amino acid
analysis
Affinity Chromatography
• Titer and protein quantitation
Hydrophilic Interaction Chromatography (HILIC)
• Glycan mapping • Glycopeptide analysis H20
H20 H20
H20
H20
H20
H20 H20
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Agilent Bio-LC Column Portfolio
Agilent Bio-LC Columns
Affinity
Bio-Monolith Protein A
Multiple Affinity Removal System
Reversed Phase
AdvanceBio Peptide Mapping
ZORBAX RRHD 300A 1.8um
Poroshell 300
AdvanceBio RP mAb
ZORBAX 300SB
ZORBAX Amino Acid Analysis
PLRP-S
HILIC
AdvanceBio Glycan Mapping
ZORBAX RRHD 300-HILIC
Size Exclusion
Bio SEC-3
Bio SEC-5
ProSEC 300S
ZORBAX GF-250
ZORBAX GF-450
Ion Exchange
Bio-Monolith (QA, DEAE, SO3)
Bio mAb
Bio IEX (SAX, SCX, WAX, WCX)
PL SAX
PL SCX
April 7, 2015
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Products in red are new!
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AFFINITY CHROMATOGRAPHY
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Analytical Bio-Monolith Protein A Columns
Used for: • Fast screening of harvest cell culture
samples for IgG – process optimization
• Accurate analysis of mAb quantities to determine protein harvest
• Capture and purification of protein for further characterization
Features and benefits: • Bio-Monolith Protein A
(immunoaffinity) • Monolith type material for fast, flow
rate independent separations • Monolith material does not clog easily
with cell debris • Attaches easily to all LCs with
standard fittings
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2 Minute Analysis – Antibody Titer from Cell Culture Supernatant
April 7, 2015
Agilent Technologies
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min 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25
mAU
0
50
100
150
200
250
Lysate proteins containing mAb
wash elute re-equilibrate
IgG1 (2.5 µg) Flow through (8 µg)
Abso
rban
ce (m
AU
at 280 m
m)
Time (min)
IgG1
Column Agilent Bio-Monolith Protein A Sample: Cell lysate spiked with IgG1 Equilibration buffer: 50 mM NaPO4, pH 7.4 Elution buffer: 0.1 M citric acid, pH 2.8 Flow Rate: 1.0 mL/min Detector: UV 280nm System: 1200 Infinity
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REVERSED-PHASE BIOCHROMATOGRAPHY
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Characterization of Primary Structure – RP LC
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Agilent Technologies
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Levels of Characterization
Improve accuracy and resolution
Intact mAb
Reduction/Alkylation
Light and Heavy Chain
Enzymatic Digestion
Peptide Map
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Common Challenges with RP Bioseparations
• Poor characterization in the separation results in poor identification - Need best stationary phase to perfect the separation - Need high resolution/efficiency
• Lack of analytical consistency
• Method robustness
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Poroshell 300
• 300Å pore size
• StableBond and Extend chemistry
• Available in SB-C3, SB-C8, SB-C18, and Extend-C18
• 5 µm particle size
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High Flow Rates with 2.1 mm id Poroshell for High Resolution and Fast Separations
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Agilent Technologies
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12
34
5
67
8
0 0.5 1.0Time (min)
Columns: Poroshell 300SB-C18 2.1 x 75 mm, 5 µm MP: A: 0.1% TFA B: 0.07% TFA in ACN Gradient: 5 – 100% B in 1.0 min. Flow Rate: 3.0 mL/min. Temperature: 70 °C Pressure: 250 bar Detection: UV 215 nm
Sample: 1. Angiotensin II 2. Neurotensin 3. Rnase 4. Insulin 5. Lysozyme 6. Myoglobin 7.Carbonic Anhydrase 8.Ovalbumin
Pub No# 5989-9899EN for complete app note
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ZORBAX 300SB RRHD for Proteins
• Stablebond 300 silica/bonding
• C18, C8, C3, and Diphenyl bonded phase
• 1.8 µm particle size for high resolution
• 1200 Bar pressure limit for UHPLC
• 2.1 x 50 mm and 2.1 x 100 mm
April 7, 2015
Agilent Technologies
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C18 C8 C3 Diphenyl
Increasing protein size and hydrophobicity
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UHPLC Columns Increase Resolution and Increase Speed
April 7, 2015
Agilent Technologies
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Column Length (mm)
Resolving Power
N(5 µm)
Resolving Power
N(3.5 µm)
Resolving Power
N(1.8µm)
150 12,500 21,000 32,500
100 8,500 14,000 24,000
75 6000 10,500 17,000
50 4,200 7,000 12,000
30 N.A. 4,200 6,500
15 N.A. 2,100 2,500
Analysis Time
Peak Volume
Analysis Time*
-33%
-50%
-67%
-80%
-90% Solvent Usage
* Reduction in analysis time compared to 150 mm column
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Comparison C3 and Diphenyl Phases
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Agilent Technologies
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min 1 1.2 1.4 1.6 1.8 2 2.2 2.4
mAU
2
4
6
8
10
12
1.3
37
1.5
87
1.6
66
1.7
54
1.8
11
min 1 1.2 1.4 1.6 1.8 2 2.2 2.4
mAU
2
4
6
8
10
12 1.3
45
1.5
64
1.6
46
1.7
87
1.8
46
ZORBAX RRHD 300SB-C3, 1.8 µm
ZORBAX RRHD 300-Diphenyl, 1.8 µm
Ribonuclease A, Cytochrome C and Lysozyme (3 mg/mL)
Time (min)
%B
0 10
2.5 70
Arrows indicate better separation resolution of ZORBAX RRHD 300-Diphenyl
Pub No# 5990-9668EN for complete application note.
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Peptide Mapping: Common Challenges
• Analytical consistency
• Long analysis times to get full resolution
• Insufficient resolution and sensitivity
• Method robustness
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AdvanceBio Peptide Mapping Column for HPLC and UHPLC: • Peptide Maps in < 15 minutes • 2.7 µm Superficially Porous • 120Å pore size • C18 functionality • 600 bar pressure limit • 2 µm frit to reduce clogging
Greater analytical confidence: Each batch is tested with a rigorous peptide mix to ensure suitability and reproducibility Save Time: 2 to 3 times faster than fully porous particles Increased Flexibility: Highly compatible with TFA and formic acid mobile phases for efficient LC UV and LC/MS analysis
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Superficially Porous Particle Technology
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Agilent Technologies
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Decrease the diffusion time for macromolecules and limit the diffusion path!
The particle has a solid core (1.7 µm) and porous outer layer with a 0.5 µm diffusion path Reduces secondary interactions and enables selective separation for a wide range of peptides.
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Peptide Mapping Standard
April 7, 2015
Agilent Technologies
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Peak # /Peptide MW 1 Bradykin frag (1-7) 756.85 2 Bradykin Acetate 1045.53 3 Angiotensin II 1060.21 4 Neurotensin 1296.48 5 Angiotensin I 1672.92 6 Renin 1759.01 7 {Ace-F-3,-2H-1] Angiotens-
inogen (1-14) 2231.61 8 Ser/Thr Protein Phosphotase
(15-31) 1952.39 9 [F14] Ser/Thr Protein Phosphotase
(15-31) 2099 10 Mellitin (Honey
bee venom) 2846.46
Each batch of AdvanceBio Peptide Mapping media is tested with the Agilent Peptide Standard (PN 5190-0583) to ensure batch to batch reproducibility
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Peptide Mapping by LC/MS
April 7, 2015
Agilent Technologies
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8 x10
0
0.75
1.75
2.75
3.75
+ESI TIC Scan Frag=200.0V igg023.d
Counts vs. Acquisition Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
40 min. Run, 2.1 x 100 mm 6x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
Cpd 154: B(357-366): + ECC Scan igg036-Agilent long run-peptides in -20.d
Cpd 154: B(357-366)
Counts vs. Acquisition Time (min)
18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 24.5
Native peptide
Deamidated form 2
Deamidated form 1
Critical Post Translational Modifications (PTM) Identified in Fast and Slow Analyses
7x10
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
0.95
1
Cpd 132: B(357-366): + ECC Scan igg035-Agilent short run-peptides in -20.d
Cpd 132: B(357-366)
Counts vs. Acquisition Time (min)
6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8 8.1 8.2 8.3 8.4 8.5
Native peptide
Deamidated form 2
Deamidated form 1
8 x10
0
0.6
1.6
2.6
3.6
4.6
433 bar 0.6 mL/min 10-40% B
+ESI TIC Scan Frag=200.0V igg011.d
Counts vs. Acquisition Time (min) 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13
140 bar 0.2 mL/min 10-40% B
Heavy Chain Peptide 357-366 and its two deamidated forms
conserved
14 min. Run, 2.1 x 100 mm
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New AdvanceBio RP mAb
Particle
• 3.5 μm SP particle
• 0.25 μm porous layer depth
• ρ value of 0.86
• 450 Å pore diameter
The optimum large molecule resolution for use with both HPLC and UHPLC systems
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Agilent Confidential
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3.5 um 3.0 um
0.25 um
Phases
• C4
• SB-C8
• Diphenyl The most popular phases for proteins, plus a unique selectivity
New
New
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Fast Intact mAb Analysis
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AdvanceBio RP-mAb provides superior peak shape at a lower pressure and resolves more fine detail than a UHPLC protein column from competitor
Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water:IPA (98:2) Mobile phase B: IPA:acetonitrile:MPA* (70:20:10) Flow rate: 1.0 mL/min
Gradient: 10-58% B in 4 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 5 μL injection of Humanized Recombinant Herceptin Variant IgG1 Intact from Creative Biolabs (1 mg/mL) Temperature: 80 °C Detection: UV @ 254 nm
AdvanceBio RP-mAb C4, 450Å, 3.5 μm 490 bar
Competitive C4, 300Å, 1.7 μm 910 bar
* MPA = Mobile Phase A
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Fast, High Resolution mAb Fragment Analysis
min2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25
mAU
0
200
DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...C_MD\AEM_PS450_FAB-FC_MD_4 2014-09-10 09-02-53\1443508-69-0005.D)
min2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25
mAU
0
200
DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...B-FC\AEM_PS450_IGG1_FAB-FC 2014-09-11 13-54-40\USRIT001297-003.D)
min2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25
mAU
0
200
DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...B-FC\AEM_PS450_IGG1_FAB-FC 2014-09-11 14-37-19\706785-1-000003.D)
min2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25
mAU
0
200
DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...B-FC\AEM_PS450_IGG1_FAB-FC 2014-09-10 11-45-32\CD4F123-0000003.D)
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Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water Mobile phase B: n-propanol/acetonitrile/MPA (80/10/10) Flow rate: 0.8 mL/min
Gradient: 5-40% B in 5 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 1 μL injection of Fc/Fab, Papain Digested Humanized Recombinant Herceptin Variant IgG1 from Creative Biolabs (2 mg/mL) Temperature: 60 °C Detection: UV @ 220nm
AdvanceBio RP-mAb provides superior peak shape and resolution than other columns designed for protein separations
AdvanceBio RP-mAb C4, 450Å, 3.5 μm
Competitor A Protein C4, 400Å, 3.4 μm
Competitor B WIDEPORE C4, 200Å, 3.6 μm
Competitior C C4-30, 300Å, 2.6 μm
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Fast Intact mAb Analysis AdvanceBio RP-mAb Diphenyl resolves additional fine detail - the Diphenyl phase is unique to Agilent
Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water/IPA (98/2) Mobile phase B: IPA/acetonitrile/MPA* (70/20/10) Flow rate: 1.0 mL/min
Gradient: 10-58% B in 4 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 5 μL injection of Humanized Recombinant Herceptin IgG1 Intact from Creative Biolabs (1 mg/mL) Temperature: 80 °C Detection: UV @ 254nm
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Agilent Confidential
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min1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5
mAU
0
20
40
60
80
100
120
140
DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-21 08-02-26\1443508-52-0006.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-20 09-06-44\1435601-25-0037.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-19 15-36-09\DIP143501-3-047.D)
min1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5
mAU
-4
-2
0
2
4
6
8
10
12
14
DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-21 08-02-26\1443508-52-0006.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-20 09-06-44\1435601-25-0037.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-19 15-36-09\DIP143501-3-047.D)
AdvanceBio RP-mAb C4 AdvanceBio RP-mAb SB-C8 AdvanceBio RP-mAb Diphenyl
* MPA = Mobile Phase A
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Fast, High Resolution mAb Fragment Analysis
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Agilent Confidential
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Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water Mobile phase B: n-propanol/acetonitrile/MPA* (80/10/10) Flow rate: 0.8 mL/min
Gradient: 5-40% B in 5 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 1 μL injection of Fc/Fab, Papain Digested Humanized Recombinant Herceptin IgG1 from Creative Biolabs (2 mg/mL) Temperature: 60 °C Detection: UV @ 220nm
min2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25
mAU
0
100
200
300
400
DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...C_MD\AEM_PS450_FAB-FC_MD_4 2014-09-10 09-02-53\1443508-69-0005.D)
AdvanceBio RP-mAb provides sharp peaks and good resolution in less than 5-minutes
AdvanceBio RP-mAb C4
* MPA = Mobile Phase A
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AdvanceBio RP-mAb C4 Separtes Proteins with small differences: Biosimilars in development
Agilent-RIC collaboration biopharma - Nov 2014
26
Using 1.0 % B/mL gradient
Remicade Remicade clone
Using 2.1 % B/mL gradient
Remicade Remicade clone
Shift of Fc
Annotated shift of the Fc part implicates differences in hydrophobicity of the Fc part due to a 2-point mutation in the AA sequence of the biosimilar compared to the originator. The shift is observed with either a fast or slow gradient.
Shift of Fc
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RP Summary
Agilent Column Positioning
AdvanceBio RP-mAb • Designed for mAb separations • Fast, high resolution HPLC and UHPLC analysis of intact mAbs and mAb
fragments
ZORBAX RRHD 300SB • High resolution UHPLC analysis of proteins, including intact mAbs, and protein fragments
Poroshell 300 • Fast, HPLC analysis of large intact proteins, including intact mAbs
ZORBAX 300SB • High resolution HPLC analysis of proteins, including intact mAbs, and protein fragments
PLRP-S • Polymeric for high pH stability and alternate selectivity.
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AdvanceBio RP-mAb is an addition to the market leading Agilent reversed-phase bio-column portfolio and complements existing columns
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How the New Columns Fit: Agilent Positioning
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Match The Column To System Pressure Capabilities
Agilent Column Particle Pressure Rating Phases
AdvanceBio RP-mAb SPP, 3.5 μm, 450Å 600 bar SB-C8, C4, Diphenyl
ZORBAX RRHD 300SB TPP, 1.8 μm, 300Å 1200 bar SB-C18, SB-C8, SB-C3, Diphenyl
Poroshell 300 SPP, 5 μm, 300Å 400 bar SB-C18, SB-C8, SB-C3, Extend-C18
ZORBAX 300SB TPP, 3.5 & 5 μm, 300Å 400 bar SB-C18, SB-C8, SB-C3, SB-CN
PLRP-S TPP, 3, 5, 8 um 100, 300, 1000, and 4000A 400 bar NA
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HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY(HILIC)
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Glycan Analysis – Common Challenges
• Very long analysis times
• Instrument limitations
• Difficulty achieving reproducible results
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N-Glycan Mapping
Glycoprotein
N-Glycan
2-AB labelled glycan
HILIC column LC/MS
HILIC column LC/FLD (-MS)
Majority process
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Glycan Analysis Workflow
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Glycoprotein
N-Glycans
2-AB Labelled N-glycans
Deglycosylation
Work-up
2-AB Labelling Work-up
HILIC FLD / MS
Deglycosylation Kit
Description (24 or 96 samples)
Reaction buffer 5X
Denaturant
Detergent
PNGase F
Deglycosylation Work-up
Description (24 or 96 samples)
SPE cartridges
2-AB Labeling Kit
Description (24 or 96 samples)
2-AB solution
Reductant solution
2-AB Labeling Work-up
Description (24 or 96 samples)
SPE cartridges
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Glycan Analysis Workflow
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Deglycosylation
Work-up
2-AB Labelling Work-up
HILIC FLD / MS
Glycoprotein
N-Glycans
2-AB Labelled N-glycans
Unlabelled Standards
Labelled Standards
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AdvanceBio Glycan Mapping Column
• Super-fast glycan analysis
• 1.8 µm fully porous for highest performance
• 2.7 µm superficially porous for lower pressures
• Unique hydrophilic bonding
High-speed, high-resolution performance Ideal for all UHPLC/HPLC instruments Batch tested with the Agilent IgG glycan standard to ensure reproducibility
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Glycan Analysis by LC-FLD
1260 Infinity Bio-inert HPLC
AdvanceBio Glycan Mapping,
2.7 um +
AdvanceBio Glycan Mapping,
1.7 um +
1290 Infinity UHPLC
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HILIC Summary
Characterization Product Features Advantage Benefit Key Challenge
Glycan Mapping AdvanceBio Glycan Mapping
1.8um FPP, bonded phase
High efficiency, right selectivity
Improved accuracy and
reproducibility of data – reliable results, cost
saving
Resolution 2.7um SPP,
bonded phase
1.8um FPP, bonded phase Efficiency at
higher flow rate, fast gradients
Improved analysis efficiency, cost
saving Throughput
2.7um SPP, bonded phase
FPP: Fully Porous Particle, SPP: Superficially Porous Particle
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SIZE EXCLUSION CHROMATOGRAPHY
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Common SEC challenges
• Insufficient/incorrect pore sizes can reduce resolution
• Non-specific interactions contribute to loss of sample, lead to inconsistent results, rework
• SEC is typically slow
• Poor pressure stability creates rework and increased cost
• Consistent and robust results
• High salt conditions puts excessive wear on instrument, parts
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SEC Column Choice: Resolving Ranges
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Pore Size Comparison
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Eluent: 50 mM NaH2PO4 + 0.15M NaCl, pH6.8 Columns: Agilent Bio SEC-3, 4.6 x 300 mm Flow: 0.35 mL/min Detector: UV @ 220 nm System: Agilent 1260 Infinity Bio-inert LC System Sample: BioRad Gel Filtration Standards Mix
1. Thyroglobulin Aggregates 2. Thyroglobulin 3. IgA 4. γ-globulin 5. Ovalbumin 6. Myoglobin 7. Vitamin B12
300Å
150Å
100Å
1
2
3
4 5 6
7 Best starting point for mAbs
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Improved Efficiency With Smaller Particles
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Column: Bio SEC-3 300Å and Bio SEC-5 300Å Buffer: 150 mM Phosphate buffer, pH 7 Flow rate: 1.0 mL/min for 7.8 x 300 mm Temperature: Ambient (~23 °C) Detection: UV 214 nm Injection: 10 µL (3 µL for 4.6 x 300 mm) Sample: 1) Thyroglobulin (1.0 mg/mL), 670 kD; 2) BSA dimer, 132 kD; 3) BSA (1.0 mg/mL), 66 kD; 4) Ribonuclease A (1.0 mg/mL), 13.7 kD, and 5) Uracil (2.5 µg/mL), 120 D.
Min 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Agilent Bio SEC-3, 300Å, 7.8 x 300 mm 93 bar
Agilent Bio SEC-5, 300Å, 7.8 x 300 mm 45 bar
Peak Protein Efficiency Gain
SEC-3, 300Å (7.8x300mm)
SEC-5, 300Å (7.8x300mm)
1 Thyroglobulin 2.2X 2460 1120
2 BSA Dimer 1.9X 5100 2720
3 BSA 2.0X 13090 6590
4 Ribonuclease A 2.0X 22000 11160
5 Uracil 1.4X 38500 27860
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Fast monomer/dimer separation using the Agilent Bio SEC-3 300Å 7.8 x 150 mm column
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Flow Rate Resolution Monomer/Dimer
Monomer Efficiency
Percentage Dimer
1.0 mL/min 1.53 3,510 0.64
1.5 mL/min 1.43 2,502 0. 47
2.0 mL/min 1.13 1,917 0.64
monomer
2.0 1.5 1.0 mL/min mL/min mL/min
dimer
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Size Exclusion Summary
Characterization Product Features Advantage Benefit Key Pain Point
Aggregation Bio SEC
3um particles, porosities
High efficiency, right selectivity
Improved accuracy and
reproducibility of data – reliable results, cost
saving
Resolution High efficiency, right MW range
3um particles High efficiency Improved analysis
efficiency, cost saving
Throughput
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ION-EXCHANGE CHROMATOGRAPHY
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Charge Variant Analysis
Common challenges
• Resolution can be limited and inconsistent – can require troubleshooting and rework
- Need capability to handle complex analyses consistently - mAbs present special challenges, due to their complexity
• Column contamination can lead to early column failure and produce incomplete sample recovery
• Method development is time-consuming - Need capability for faster, systematic method development with
different buffer strengths
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Some Guidelines for IEX
1.The General Rule for choosing a Bio IEX column - Acidic proteins: SAX or WAX - Basic proteins: SCX or WCX 2. Consider the isoelectric point (pI) of your protein when choosing the pH
of your mobile phase: - If pH>pI, your protein will have a net negative charge - If pH<pI, your protein will have a net positive charge 3. The pH of your starting buffer should be 0.5 to 1 pH unit from your pI - Above pI for anion-exchange - Below pI for cation-exchange
4. If your pI is unknown - Start with pH 6 for cation-exchange - Start with pH 8.0 for anion-exchange
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Ion Exchange – Product Families
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Particle Porosity Functionalities Particle Sizes Pore Size Application
Agilent Bio-IEX Polymer Non-porous SAX, WAX, SCX, WCX
1.7um, 3um, 5um 10um
N/A Peptides, proteins
Agilent Bio MAb Polymer
Non-Porous WCX 1.7um, 3um, 5um 10um
N/A IgG
PL-SAX PS/DVB Fully Porous SAX 5um, 8m, 10um, 30um
1000A, 4000A Peptides, oligos, proteins. Larger column sizes
PL-SCX PS/DVB Fully Porous SCX 5um, 8m, 10um ,30um
1000A, 4000A Peptides, proteins. Larger column sizes
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1. Non-porous particles for high efficiency analytical separations
2. Porous particles for scale up to purification
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Ion-Exchange Columns
• Non-porous PS/DVB particles (polystyrene divinylbenzene) • Uniform polymeric coating with SCX, WCX, SAX, WAX layers, designed
for protein and peptide separations • Available in 10, 5, 3, 1.7 µm particle sizes • High surface area • High capacity
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Agilent Bio IEX Columns Comparing Separations on Each Particle Size
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Min 0 2 4 6 8 10 12 14 16 18 20 22
1.7 µm
3 µm
5 µm
10 µm
Column: Bio WCX, 4.6 x 50 mm Buffer A: 20 mM PBS Buffer B: A+1.0 M NaCl Gradient: 0-100%B (20 min) Flow rate: 1.0 mL/min for 10 µm, 5 µm, 3 µm 0.75 mL/min for 1.7 µm Sample: 1) Ribonuclease A 2) Cytochrome C 3) Lysozyme Concentration: 1.0 mg/mL Detector: 280 nm Average N ~80,000 for WCX 1.7 µm
Peak N
Peak N
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Ion-Exchange Columns – Special for mAbs
• Non-porous PS/DVB particles with a uniform polymeric coating • Particle has a dense, weak cation-exchange (WCX) mono-layer with excellent
selectivity for basic monoclonal antibodies • Available in 10, 5, 3, 1.7 µm particle sizes • Small particles offering higher resolution than the commonly used 10 µm
particles
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Ion Exchange Chromatography Charge Isoform Analysis of Monoclonal Antibodies
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Min 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
A
B
C
D
E
Optimization of method conditions for the isoform characterization of a monoclonal antibody. Changes in the buffer conditions, pH and gradient
conditions sharpen peaks and increase resolution of acidic and basic isoforms.
Columns: Agilent Bio MAb, 10 µm, 4.6 x 250 mm Mobile phase: A, 10 mM phosphate, pH 7.5 B, A + 0.1 M NaCl Gradient: A) 15-75% B in 30 min B) 15-65% B in 30 min C) 15-55% B in 30 min D) 15-47.5% B in 30 min E) 15-40% B in 30 min Flow rate: 0.8 mL/min Sample: Monoclonal Antibody Injection: 10 µL (1.5 mg/mL) Temperature: 25 oC Detection: UV 214 nm
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Analytical Bio-Monolith Ion Exchange Columns
• Polymer based monolithic discs • Fast, high resolution ion-exchange • Key applications are for large proteins and
biomolecules (virus particles, pDNA, antibodies [IgG and IgM])
• Agilent Bio-Monolith QA (strong anion-exchanger) • Agilent Bio-Monolith DEAE (weak anion-exchanger) • Agilent Bio-Monolith SO3 (strong cation-exchanger)
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www.agilent.com/chem/getbioguides Application-focused Brochures
“How To” Guides
Keys for Enabling Optimum Peptide Characterizations: A Peptide Mapping “How To” Guide 5991-2348EN Protein Identification
and Impurity Profiling using Reversed-Phase HPLC/UHPLC 5991-0625EN
Resolve Protein Aggregates and Degradants with Speed and Confidence 5991-2898EN
Reversed-Phase
Affinity
Ion-Exchange
Characterize Charged Variants of Proteins with Speed and Confidence 5991-2449EN
Size Exclusion Chromatography for Biomolecule Analysis: A “How To” Guide 5991-3651EN
Selection Guide
Your Reference Guide to the Analysis of Biopharmaceuticals and Biomolecules 5990-9384EN
Ion-Exchange Chromatography for Biomolecule Analysis: a “How to” Guide 5991-3775EN
Characterize Charged Variants of Proteins with Speed and Confidence 5991-2449EN
Size Exclusion
Resources for More Information
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BioHPLC Columns on the Agilent Website
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To learn more and order online visit www.agilent.com/chem/biocolumns
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55
Thank You Any Questions?