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Tosoh Bioscience
Gentle Bioanalysis of Proteins APPLICA 2017
Patrick Endres, Judith Vajda, Egbert Müller
Tosoh Bioscience GmbH September 7th, 2017
Tosoh Bioscience
Demand on chromatographic analysis • Fast
• Inexpensive (Column and buffer)
• Robust
• (Easy method development)
• (Non-denaturing)
à Is RPC always the best option? I think not!
2
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Small Molecules – large Molecules
• Convential API‘s Biologics
*EvaluatePharma World Preview 2014, Outlook to 2020
aspirin 180,16 g/mol IgG > 150.000 g/mol
Sizing factor 100-1000
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Biological Products are complex!
Quaternary structure Spherical arrangement of different domains
Amino acid sequence Secondary structure Alpha, beta
Tertiary structure Protein folding
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Plus: glycosylation, deamidation, hydroxylation… etc.
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Monoclonal Antibodies • >60 approved mAbs • 5 of the top 10 drugs are mAbs • #1: Humira® with 16 billion US $ (2016) • > 600 candidates in clinical trials
Characterization: • Size • Charge variants • Glycosylation • Peptides after enzymatic digest • Synthetic modifications
*Zahlen 2017, FDA & Wikipedia
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Fc
Fab Antigen binding
Biological activity
Glycosylation site
λ or κ light chain
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Chromatography Toolbox
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Characteristic LC method Size SEC (gel filtration)
Charge IEX Hydrophobicity RPC/HIC
Titer Analytical Protein A
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MAb Heterogeneity
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Characterization by Size Exclusion Chromatography
Aggregation
Fragmentation
Incorrect disulfide bonds
Met oxidation
C-terminal Lys truncation
N-terminal cyclization (Glu/Gln àpGlu)
Deamidation (AsnàAsp)
Isomerization (AspàisoAsp)
Glyco pattern
Artificial modifications (PEG, isotopes)
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Size Exclusion Chromatography
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• Separation of molecules according to the hydrodynamic radius • Larger molecules have no or limited pore access • Non-adsorptive
0,1
1
10
100
1000
6 8 10 12 14 16
MW
[kD
a]
retention time [min]
Column Calibration TSKgel SuperSW mAb HR
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Speed…or the applied flow rate
• Separation in SEC relies on pore diffusion • High flow rates decrease separation performance
mAb aggregate separation on TSKgel UP-SW3000 30 cm L
àthere is always a trade-off between performance and time
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y = 0,0565x + 0,0154 R² = 0,98805
0
0,01
0,02
0,03
0,04
0,05
0,06
0 0,2 0,4 0,6 0,8 H
[mm
] superficial velocity [mm/s]
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SEC-UHPLC
• UHPLC systems: • Smaller system dead volume • Optimized detector flow cells • Can be operated at pressures >1000 bars
• UHPLC columns: • Particle size ↓, ID ↘, superficial velocity ↑, backpressure ↑
• Two dimensions: • 4.6 mm ID x 15 cm à fast analysis and high throughput. • 4.6 mm ID x 30 cm à maximum performance. • Guard columns for direct coupling reduce dead volume
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Increase of efficiency: time-wise or performance-wise
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Charge Variants
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Aggregation
Fragmentation
Incorrect disulfide bonds
Met oxidation
C-terminal Lys truncation
N-terminal cyclization (Glu/Gln àpGlu)
Deamidation (AsnàAsp)
Isomerization (AspàisoAsp)
Glyco pattern
Artificial modifications (PEG, isotopes)
Various changes at amino acids and a varying sialinic acid content can lead to charge variants of a mAb.
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Specifications of IEX analytical Columns
• Hydrophilic polymerbeads (7 µm) are alkaline resistant • Non-porous particles have fast mass transfer properties • Innovative surface design à comparatively high capacity for a non-
porous resin • Fast, high-resolution power for the analysis of proteins and peptides • TSKgel CM-STAT – Carboxymethyl ligand (WCX) • TSKgel SP-STAT - Sulfopropyl ligand (SCX)
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- - - - - -
- - - - - -
- - - - - - - - -
- - -
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Example: C-terminal Lysine
Column: TSKgel CM-STAT (4.6 x 100 mm) Eluent A: 20 mmol/L MES (pH 6.0) Eluent B: 20 mmol/L MES + 0.5 mol/L NaCl (pH 6.0) Gradient: 0 min 10 %B
15 min 30 %B 15.1 min 100 %B 18 min 100 %B 18.1 min 10 %B
Flow rate: 1.0 mL/min Detection: UV 280 nm Temp.: 25 ℃ Inj. vol.: 20 µL Conc. : 0.5 g/L Samples: Therapeutic antibody, treated and
untreated with carboxypeptidase B Procedure: To a 35 µL of therapeutic antibody (10 g/L), 1 µL of carboxypeptidase B (Sigma C9584, 140 U/mg protein, 5 g/L in PBS) was added and incubated for 3 hr at 37 ℃. After adding 664 µL of 20 mmol/L MES (pH 6.0) to dilute the antibody concentration of 0.5 g/L, 20 µL of the diluted sample was injected.
min0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
mAU
-5
0
5
10
15
20
25
30
35
40
45
50
55
before digestion
digested with carboxypeptidase B
K KK
Varying number of positive charges at a mAb (lysine) can be resolved by CEX
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UHPLC system HPLC system
mAb separation (TSKgel SP-STAT 4.6 x 100 mm)
Comparison of Resolution and plate numbers
- - - - - -
- - - - - -
- - - - - - - - -
- - -
7 µm
Why are UHPLC systems beneficial for IEX?
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Benefits of a UHPLC System - IEX
1 mL/min, A: 10 mM phosphate, pH 7.0, B: A + 1 M NaCl, gradient: 0-50 % B in 25 min, 5 µL Injection volume.
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Peaks are sharper: higher resolution and plate numbers
Due to smaller dead volume: Elution occurs earlier à Increase Gradient delay volume to keep the same integration method
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Artificial Modifications
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Aggregation
Fragmentation
Incorrect disulfide bonds
Met oxidation
C-terminal Lys truncation
N-terminal cyclization (Glu/Gln àpGlu)
Deamidation (AsnàAsp)
Isomerization (AspàisoAsp)
Glyco pattern
Artificial modifications (PEG, ADCs)
Artifical modifications can be introduced for diagnostic use, to prolong half-life or to increase potency of a therapy
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Lysine Conjugation Cysteine Conjugation Site directed conjugation
*Genentech, World ADC Summit US 2011
Modern MAb formats
Antibody - Drug - Conjugates
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Hydrophobic Interaction Chromatography
• Analogue to reversed phase in biochromatography
• Ligands comparable to RPC, often shorter alkyl chains and lower ligand density
• Mild conditions • Hydrophobic interactions induced by high
salt concentrations • Elution in a decreasing salt gradient
• TSKgel Butyl-NPR: • Non-porous base material • C4 ligand
18
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ADC – Drug/Antibody Ratio (DAR)
• mAbs (e.g. Herceptin) are usually characterized by SEC • BUT: conjugates are often too small to resolve ADC from mAb but are
hydrophobic (Drug Size: ~750 Da) • RPC (small molecules) - HIC (proteins)
Herceptin
AD CDAR=0
DAR=2 DAR=4
DAR=6
DAR=8
Column: TSKgel Butyl-NPR (2.5 µm, 4.6 x 100 mm)
Eluent: A) 25 mmol/L phosphate (pH 7.0),1.5 mol/L ammonium sulfate B) 25 mmol/L phosphate (pH 7.0) / 2-propanol = 8 / 2
Gradient: 0 - 100 % B (20 minutes) Flow rate: 0.5 mL/min Detection:: UV @ 280 nm Injection: 10 µL Sample: Herceptin; 0.24 g/L,
ADC 2.2 g/L
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Things to consider • Low pressure or high pressure gradient?
20 http://www.ddbst.com/en/EED/VE/Images/VE0%202-Propanol;Water_001.png
Flowrate is not constant with high pressure gradient due to excess volume
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Temperature
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40°C
40°C 40°C
50°C
30°C 40°C
Band broadening due to laminar flow profile
Sharper peak due to combined effect of laminar flow profile and viscosity
Temperature Laminar flow profile Combined effect
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Analytical Protein A Chromatography • Most of the monoclonal antibody biotherapeutics on the market today
are based on IgG1. Interest in IgG2 and IgG4 is rapidly growing.
• These samples must be screened for mAb titer; affinity protein A columns are often employed for this purpose.
• With many samples to be screened for different purposes, a reliable and high throughput column is needed for this workflow.
23
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High Flow Rates for High Throughput Analysis
20 µL of CHO cell supernatant spiked with polyclonal antibody (0.5 mg/mL)
• TSKgel Protein A-5PW column shows similar recovery of IgG up to 4.0 mL/min. • Less than 1 minute analysis was available at 4.0 mL/min with similar peak profile.
Tosoh Bioscience 25
Dynamic Range and Linearity
Sample: Purified polyclonal IgG
TSKgel Protein A-5PW column shows a wide dynamic range from 0.1 - 10 g/L (2 - 200 µg) with good linearity (R2 > 0.999) for IgG.
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• The column can be used with high flow rate while still maintaining peak area consistency with RSD of 1.7%
• The column was cleaned after 1230 injections using a stepwise cleaning protocol
26
Cleaning protocol reversed flow at 0.5 mL/min for 20 CV:
a. 0.1 mol/L NaOH b. DI Water c. 1 mol/L acetic acid
normal flow for 20 CV: a. DI water b. 0.5 mol/L sodium phosphate, pH 6.5
50 CV: 20mm sodium phosphate pH 7.4
Durability study using CHO crude Feedstock containing IgG1
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Summary
• Points to consider for method transfer from HPLC to UHPLC of biomolecules
• mAb aggregate analysis can be accomplished in 4 min! • UHPLC systems are beneficial for separation efficiency • Charge variants can rapidly be analyzed with non-porous
stationary phases connected to UHPLC systems • Antibody-drug-conjugates (ADC) can be analyzed with HIC • Titer determenation with analytical Protein A Chromatography
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Acknowledgements • Dr. Judith Vajda (All Data besides Protein A)
• Keegan Gyke (Protein A Data)
• PD Dr. Egbert Müller
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Questions?
http://www.separations.eu.tosohbioscience.com/ E-mail: [email protected] Phone: +49 6155 7043700 Mail: Im Leuschnerpark 4
64347 Griesheim Germany