Addressing Analytical Challenges in Biopharmaceuticals Analysis & QC
Gurmil Gendeh, Ph.D. Director, Biopharmaceutical Segment Marketing
Agilent Technologies Santa Clara, California, USA
Outline
The big switch in Pharma
Biopharma customers have different challenges
The best kept secrets in Agilent - Bioanalyzer – The Lab-on-a-Chip - TapeStation – The Next-gen electrophoresis platform - OFFGEL Fractionator – A novel sample prep tool - CE and CE/MS in biopharmaceutical analysis - HPLC-Chip Technology
Biosimilars - Definitions & Regulations - How similar is similar enough - Case studies: Comparability data between a biosimilar and its innovator reference
Summary
Traditional Pharma business model is changing
It’s a mAb!
It’s a large Pharma!
Steven Kozlowski, FDA, WCBP 2010
Biologics manufacturing is highly complex & requires analysis at every step
Market Manufacturing
Centrifuge
Pre-clinical Clinical
Biomarker ID & validation
DNA Vector
HT Clone Screening & Selection
Chromatography Column #1
Chromatography Column #2
Chromatography Column #3
mAb Generation Platform
e.g. transgenic mice
Cell Culture Scale-up & Production
Downstream Processing & Purification Formulation
Human gene
Establishment of genetically engineered cells (e.g. CHO) that produce desired product
“the process is the product”
Market Manufacturing
QA/QC Centrifuge
Pre-clinical Clinical
Biomarker ID & validation
DNA Vector
HT Clone Screening & Selection
Chromatography Column #1
Chromatography Column #2
Chromatography Column #3
mAb Generation Platform
e.g. transgenic mice
Analytical Groups Develop analytical methods; analyze samples from other functions, methods transfer
Cell Culture Scale-up & Production
Downstream Processing & Purification Formulation
Methods
Samples
Samples Samples
Samples
Samples Samples
Human gene
Analytical groups are tasked with methods development, sample analysis & methods transfer
Establishment of genetically engineered cells (e.g. CHO) that produce desired product
Typical workflows in biopharma analytical labs — from cell culture harvest to clone selection
Cell culture fluid
Prep Protein A capture of mAb
Fraction collection
Neutralization
Dilution
Reject
Passed
Failed
Size Analysis
Fragment Analysis
Peptide Digestion
Peptide Mapping
Charge Variant
Analysis
C-Terminal Lys removal
by CpB
PTM (Oxidation,
Deamidation)
Stability Studies
Glycan Analysis
Glycan Release & Labeling
Intact Mass &
Glycoforms
Middle-Up, -Down
Analysis
Amino Acid Sequencing
Impurity Analysis
PTM Analysis
Glycan ID & Quant
Clone selection
Higher Order Structure
(HOS) Analysis
Titer check
Sample Purification LC/MS characterization
Papain, FabRICATOR
Pepsin
Sample Preparation LC Characterization
HOS Sample
Prep e.g. HDX
HOS Analysis
Samples from cell culture & purification process development, formulation, stability studies, QA/QC
SDS PAGE Bioanalyzer SEC FFF, MALS
RP SEC HIC Bioanalyzer
LC-UV LC-MS CE CE-MS
IEC cIEF Image cIEF CZE
RP IEC HIC
HILIC HPAE-PAD CE mAb Glyco Chip
CD HDX Crystallography NMR
Biopharma customers have different challenges
• Biopharma sample prep is complex - Too many timed-steps that tie scientist to bench
• Bioseparation methods are longer, product specific & require long method development time
• There are many steps & methods within workflows - Need to automate and streamline workflows and methods to meet
the demand without impacting quality
• Bioseparation methods uses corrosive buffers not amenable to LC/MS analysis
• MS are becoming important and the need to make them biologist friendly
Outline
• The big switch in Pharma • Biopharma customers have different challenges • The best kept secrets in Agilent
• Bioanalyzer – The Lab-on-a-Chip • TapeStation – The Next-gen electrophoresis platform • OFFGEL Fractionator – A novel sample prep tool • CE and CE/MS in biopharmaceutical analysis
• Biosimilars - Definitions & Regulations - How similar is similar enough - Case studies: Comparability data between a biosimilar and its innovator
reference
• Summary
Traditional Slab Gel Analysis The bottleneck in Protein, DNA and RNA analysis
• Manual process
• Difficult to automate
• Slow
• Not accurate enough
• Bad reproducibility
• No direct comparison
Agilent solutions offer the potential to address this
2100 Bioanalyzer: The Lab-on-a-Chip Approach
Increasing quality and speed of gel electrophoresis
Highly reproducible results
Qualitative & Quantitative in a single step
Sample volumes 1 -5 µl
Up to 12 samples depending on Assay
Digital results in 30 minutes available
No extra waste removal needed
Disposable Chip, no crosscontamination
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P 80 Range 5 - 80 kDa Sensitivity: Coomassie Samples: 10 Samples -Antibodies (reduced) -Small Proteins
P 230 Range 14 - 230 kDa Sensitivity: Coomassie Samples: 10 Samples -Antibodies (all types) -Standard Proteins
Range: 10 - 250 kDa Sensitivity: 1 pg/µl BSA on Chip Samples #: 10 per Chip Chips #: 10 per Kit Labeling Conc: 1 ng – 1 µg /µl
Coomassie Range (5 ng/µL BSA)
HSP 250
Silver stain Range (200 pg/µL BSA)
Agilent Protein 80 kit Prod Number 5067-1515 Agilent Protein 230 kit Prod Number 5067-1517 Agilent High Sensitivity Protein 250 kit Prod Number 5067-1575
Feb. 2009
Bioanalyzer Protein Kit portfolio
90 kDa 160 kDa
intact antibody
16% half antibody
Determine the half antibody content in IgG preparations
Antibody analysis under reducing and non-reducing conditions
Ab reduced
Ab non-reduced
light chain
heavy chain
intact antibody
light + heavy chain
Absolute Quantitation of IgG samples
Quality Control of Antibodies
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Impurity analysis The High Sensitivity Protein 250 kit is perfectly suited to measure minor impurities besides dominant main compounds. With highest sensitivity and a 4 orders of magnitude range for linear quantification the 2100 can reliably analyze up to 10 samples in 40 minutes
Quantification and Purity Analysis
IgG Stress Tests
RP-HPLC Bioanalyzer (SDS)
A Wakankar, Y Chen, Y Gokarn & F Jacobson (Genentech)
Analysis of Reduced ADC fragments
Bioanalyzer (SDS)
Compliance Services
Declaration of Conformity
(consumables)
Application Notes
21 CFR part 11 compliant Security
Pack Software
2100 Bioanalyzer – 21 CFR Part 11 Compliance
Eas
e of
use
Gold Standard for NGS sample and library QC
Automation & Throughput
2100 Bioanalyzer
2200 TapeStation
• 96-well compatible • 1min/sample run time • Fully scalable throughput • Ready-to-use ScreenTape • 1µl - 2µl of sample
Agilent 2200 TapeStation – Extension of Agilent’s Electrophoresis Platforms
ScreenTapes
Co-extruded & formed polymer
16 individual acrylamide gel & buffer filled channels
Pre-packaged (no reagent or chip priming)
Barcoded to trace batch and usage
Run 2 to 16 samples
Unused lanes can used within 2 weeks
Only 1-2ul sample required
4 month shelf life at 4ºC
Various ScreenTape types available
Monitoring mAb Stability
Monitoring Purification
Schemes
2200 TapeStation
Agilent 3100 OFFGEL Fractionator IEF of Peptides and Proteins
OFFGEL Electrophoresis
What does the instrument do? It sorts proteins or peptides in a pH gradient (isoelectric focusing, IEF) = It separates proteins or peptides according to their isoelectric points (pI) Isoelectric point: pH at which the net charge of the protein is zero (can be calculated from the number of the basic and acidic side chains)
+
- -
- -
+ +
+
pH < pI pH = pI pH > pI positively charged balanced negatively charged
+ - (anode) (cathode)
pH gradient Low pH High pH
Isoelectric Focusing IEF
OFFGEL Principle pI-Based Fractionation
OFFGEL Electrophoresis
• after rehydration the IPG gel seals tightly against the compartment frame • the diluted sample is distributed across all wells in the strip • after fractionation the liquid fractions containing can be removed with a pipette Number of fractions 12 or 24 Samples proteins or peptides Fraction volume 150 ul Resolution 0.1/0.6 pH Loading capacity 50 µg – 5 mg per sample Fractionation time 8 - 24 h Typical recovery 70% for proteins, 90% for peptides
OFFGEL Used to Isolate Antibody Charge Heterogeneity Observed by Capillary IEF
Workflow Protein purification Native protein OFFGEL fractionation Fraction clean-up Fraction analysis: • LC/MS-MS (TOF), native & reduced mass • Peptide fingerprinting • cIEF (control)
Sample Preparation for Charge Variant Identification of Protein Drugs by OFFGEL
Figure 1. Representative cIEF profile of the antibody of interest for characterization using preparative IEF Figure 2. cIEF profiles of fractions
obtained from an optimized OFFGEL preparative IEF fractionation of the antibody show isolation of the basic, main, and acidic peaks across 12 fractions compared to the starting material.
Figure 4. Detailed view of the deconvoluted heavy chain spectra for fractions 14, 18, and 19 from the reduced mass analysis shows the enrichment of a +770 Da species in fractions 18 and 19 compared to the main peak in fraction 14.
Balland, A., et al, 2010, Characterization of Antibody Charge Heterogeneity Resolved by Preparative Immobilized pH Gradients, Anal. Chem. 2010, 82, 3510–3518
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May 12, 2015
Confidentiality Label
37
CE and CE/MS in Biopharmaceutical analysis
Capillary Electrophoresis Applications
• Separation based on compound mobility (mass/charge) in an electrical field • High resolution separations (usually >> 40,000 plates) • Fast separation (few minutes) • Smallest sample volumes (few µL) • Less sample prep required (no stationary phase, just an open glass tube) • Low consumption of sample and buffer (green method)
• Orthogonal technique complementing HPLC
Ideal Ideal
Charged Compounds
Biocompatibility
High Resolution
Small sample Volume
Large Molecules
Sample Capacity
Reduced Sample Prep
Cost of Ownership
Neutral Compounds
Mainstream Technique
Sensitivity Reproducibility
Sample Capacity
Detector Options
Biocompatibility
Green Methods
Cost of Ownership
Green Methods
Separation Principles of Capillary Electrophoresis (CE)
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Analytical Tasks and Instrumentation
Small polar compounds
Proteins & Peptides
Metabolites
Glycans
Ions Anion and Cation analysis. Quantitation and ID of ions
ID and quantification of polar or charged compounds
ID and quantification of Amines, Organic Acids, Nucleosides, ...
Native or denatured protein analysis, charge states, sizing
Profiling of complex and linked Glycans
Modes: CZE
Detectors: UV-DAD, CCD
Modes: CZE
Detectors: UV-DAD, CCD
Modes: CZE
Detectors: UV-DAD, MS
Modes: CZE, cIEF, cGE
Detectors: UV-DAD, LIF, MS
Modes: CZE
Detectors: LIF, MS
Nucleic Acids Sizing and quantification of Oligonuclotides or dsDNA
Modes: CZE, cGE
Detectors: LIF, MS
Abbreviations: CZE-Capillary Zone Electrophoresis; MECC-Micellar ElectroKinetic Chromatography; CEC-Capillary ElectroChromatography; cIEF-CapillaryIsoeElectric Focussing; cGE-Capillary GelElectrophoresis
• Different resolution and separation than reversed phase LC • Avoids retention problems of Ions or polar compounds with RP-columns • No column packing materials – less adsorption, easy cleaning by capillary flush • Offers non-denaturing separations of bio molecules (e.g. Proteins) • Many separation modes on one instrument CZE, MECC, CEC, cIEF, cGE,...
CE in Biopharma Application
Slim housing
Sensitive UV diode array detector based on the Agilent 1200 Series LC
Quick access cassette type
Vial sensor for sample tray
Fast Agilent 1200 electronics
Low volume replenishment system
Improved pressure/vacuum pumps
Agilent Open LAB CDS software LabAdvisor Diagnostic tools
Performance Highest sensitivity for UV
Handling Quick, direct and easy
Automation Agilent replenishment system
Flexibility All modes, open to external detectors
CE/MS Complete single vendor solution
Economic Reducing cost of ownership
41
Agilent 7100 CE
2009 Agilent Innovations Seminar
Page 42
1) The whole capillary is filled by a mix of sample and ampholytes.
2) Ampholytes will create a pH gradient after an electrical field is applied.
3) Focusing step all sample compounds will migrate to reach their distinct pI value were they get uncharged and stop moving.
4) Detection, moving content of the capillary via pressure or chemical mobilization through the detection window
Agilent Application Notes (Publication 5991-1142EN) (Publication 5991-2885EN)
Application of cIEF Separate Peptides & Proteins Based on Their Isoelectric Point (pI)
Page 43
cIEF analysis of commercially available mAb samples on fluorocarbon coated capillaries Electropherograms of samples containing mouse IgG1-k and either cIEF gel (red) or 0.5 % MC (blue) are shown in (A) The resolution of main mAb isoform peaks was 2.74 ± 0.05 with the cIEF gel and 2.66 ± 0.09 with 0.5 % MC (n = 5). Samples containing an mouse anti-a1-antitrypsin mAb (B) or a rat anti-DYKDDDDK mAb (C) were analyzed in presence of 0.5 % MC. Rat mAb, electropherograms and peaks labeled from 1 to 8 obtained on: new capillary --- blue line after more than 200 injections --- red line
Agilent Application Note (Publication 5991-2885EN)
cIEF Analysis using Agilent µSIL FC capillaries
The Potential of CZE for Charge Variant Analysis!
Charge Variant Analysis – CZE vs. IEC vs. cIEF
SDS Gel Buffer Beckman Coulter
10
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100
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Protigel P Advanced Analytical 10
20
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100
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Agilent Application Note (Publication 5990-7976EN)
Protein Sizing by cGE Use of Commercial Capillary Gel-Electrophoresis Kits
48
Standard Agilent 7100 CE instrument
Detection inside of Agilent capillary cassette (full temperature control)
Picometrics Zetalif LED using fiber optical connection to the CE
Agilent OpenLAB CDS ChemStation Edition software
Laser-induced fluorescence detection (LIF) offers one way to achieve very high sensitivity and compound specificity in capillary electrophoresis. A tailored and seamless solution for CE-LIF is possible by combining Agilent 7100 CE instruments with LIF detectors from Picometrics*.
* Picometrics Technologies SAS (Toulouse, France)
Combined CE-LIF Solution with Picometrics Technologies Zetalif LED
The Zetalif LED detector is a sensitive solution and offers a range of wavelengths: 450, 480, 530 or 640 nm.
• Easy-to-access cassette type (no liquids or sealants) • Multiple detectors at a time (e.g., direct LIF-MS) • Full software control of LIF through RC.net driver • Signal transfer into Agilent OpenLAB CDSChemStation
• Complete solution with easy setup
• Small footprint of solution + flexible combinations
• Reduced capital cost for LIF detection
► CE-LIF, CE-LIF-MS, HPLC-LIF, ...
► detection inside or outside of CE, A/D converter included
► long-life LED based LIF, flexible use of modular devices
The Agilent 7100 CE is the most flexible CE instrument to host external detectors.
Advantage of Agilent-Picometrics CE-LIF
50
Separation of non-reduced mAb spiked with cholera toxin B (CTB)
Quantification of cholera toxin B (CTB)
Application of CE-LIF Impurity Analysis
Typical Applications • Small Molecules / Metabolites • Peptide mapping • Protein ID and characterization • mAb and mAb conjugates
Screening
Target Analysis
MS choice: MSD/QQQ
MS choice: TOF/QTOF
Whole solution • CE hardware • MS hardware • CE/MS Interface • Integrated Software for LC/MS & CE/MS
CE/MS A Fully Integrated Solution
Single point SW control - 7100 CE instrument - MSD Single quadrupole - LC make-up flow
The Agilent CE/MS Advantage
Single vendor solution: direct and competent support Sheath Liquid Interface: robust and reliable, offering efficient control on chemistry Capillary outlet on ground: no compromises on voltages for CE or ESI-MS
Adapter (interface) Kit (G1603A)
Sheath Liquid Pump
Nitrogen nebulizing gas PC-system controlling
- 7100 CE system - MS system - Sheath liquid pump
CE-capillary
MS source JetStream or ESI On request also APPI / APCI
Sprayer Kit (G1607B)
Agilent CE/MSD Setup OpenLAB CDS (ChemStation edition)
The Agilent CE/MS Advantage
Single vendor solution: direct and competent support Sheath Liquid Interface: robust and reliable, offering efficient control on chemistry Capillary outlet on ground: no compromises on voltages for CE or ESI-MS
Single point SW control - 7100 CE instrument - TOF,QTOF,QQQ - LC make-up flow
Agilent CE/MS Setup Agilent MassHunter Software Control
Advantages of Sheath Liquid interface: - High stability & reproducibility for routine analysis - Decoupling chemistry (CE separation / MS ionization) - Constant flow rates during runs and sequences - No modification of capillary / columns required
Sheath liquid is added to the CE eluent by a software controlled LC pump at a rate of typically 1 - 5 µL/min. It often consists of a mix of water, methanol or isopropanol, adjusted for desired pH range by volatile acids or bases) Besides controlling flow rate and chemical conditions for ESI ionization of molecules it allows grounding of the non-conductive fused silica capillary to the metal tube of the spray needle
Agilent interface for CE/MS Sheath-Liquid Type
Page 55
MassHunter versions B.05.01 and higher are integrating and controlling Capillary Electrophoresis for CE/MS analysis as a single software package under Windows 7 (64 bit)
Graphical UI
MS parameters
MS online spectra
MS TIC and EIC
Agilent interface for CE/MS Agilent MassHunter software for LC-MS & CE/MS
Anion & Amino Acids Detection: Indirect UV Method: CZE
Amino Acid analysis Detection: TOF Method: CZE-ESI
CE/TOF-MS analysis
CE/UV-DAD analysis Amino Acids
Page 57
Instrument type : Agilent Q-TOF
Agilent Application Note (Publication 5991-2583EN)
CE/MS and LC/MS peptide map of BSA Among the total number of peptides identified (114), 37 peptides are unique to CE/MS and 33 peptides are unique to LC/MS. Each of the techniques contained 44 common peptides.
Peptide Mapping: CE/MS vs. LC/MS Comparison of Tryptic Digests
Page 58
Separation comparison of same set of BSA tryptic peptides (A) CE-MS (B) LC-MS List of peptides: 1 . LCVLHEK, 2. HLVDEPQNLIK, 3. NYQEAK, 4. QTALVELLK 5. YLYEIAR, 6. ECCHGDLLECADDR 7. LVTDLTK 8. LVNELTEFAK 9. AEFVEVTK 10. LCVLHEKTPVSEK 11. LVVSTQTA 12. DDSPDLPK
Due to different physical separation principles the elution order is totally different, reducing the risk of overlapping peaks if both methods are applied
Orthogonal techniques: CE and LC
CE/MS vs. LC/MS Comparison of Tryptic Peptide Maps
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LC-MS
CE-MS
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LC-MS
CE-MS
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10152025303540
Num
ber
of p
epti
des
Isoelectric point (pI)
LC-MS
CE-MS
(A) (B)
(C) CE-MS and LC-MS comparison of peptide distributions. (A) Molecular weight plot (B) Peptide length plot (C) Isoelectric point plot
CE/MS vs. LC/MS Comparison of Tryptic Peptide Maps
Page 60
CE-QTO MS (6520) LC-QTOF MS (Chipcube-6540)
Sample injected 44nl (0.34pmole) 2ul (15pmole)
Peptide elution window 30 min 16 min
Sequence coverage 80% 81%
Total peptides identified 82 78
Distinct peptides ID’ed 37 33
Selectivity & resolution Change in elution order of few peptide – shows the complementary value of two techniques
Selectivity CE-MS is shows the best separation/ionization for hydrophilic peptides
Peptide distribution
• Shorter peptides are represented (1-5 amino acid peptide length)
• Identified peptides starting with 3 amino acid length
• Low MW peptides are well presented (<500Da)
• Acidic peptides (pI 3-4) are well represented
• Shorter peptides are less represented (1-5 amino acid peptide length) and also cover wide range of peptide length identified
• Identified peptides starting with 4 amino acid length
• Low MW peptides are less represented (<500Da)
CE/MS vs. LC/MS Comparison of Tryptic Peptide Maps
Agilent Application Note (Publication 5991-1020EN)
Schematic overview of the glycoprofiling of mAb using CE/MS
Analysis of N-Glycans of a Monoclonal Antibody by CE/MS
4x10
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1 1
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
G0 G0F Man5 G1 G1F G2 G2F
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Counts vs. Acquisition Time (min)7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5
G2
G2F
G2-
1NA
NA
G2-
2NA
NA
G2F
-2N
AN
A
G2F
-1N
AN
A
Mixture of neutral glycan & sialylated glycans
Mixture of neutral glycan
Galactose Mannose Fucose N-acetylglucosamine sialic acid
CE/MS of APTS Labeled Glycan Standards
Page 62
CE/MS of APTS labeled N-glycans released from mAb
x10
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Acquisition Time (min)
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
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G2F+1NANA
G0
G0F
G1
G1F
G2
Total Compound Electropherogram
G2F
Cou
nts
CE/MS Analysis of N-Glycans Released from mAb
2x10
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0.42
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0.62
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2328.743392331.505342369.143792409.28741
2449.706172492.05640
2535.72220
2581.35545
2628.30008
2676.90812
2727.57101
2780.02722
2834.69878
2891.40256
2950.46904
3011.85876
3076.09781
3142.97359
3212.82610
3286.04257
3294.91650
3298.48630
3362.34069
3370.90992
3372.32084
3376.21089
3442.44591
3450.90523
3452.58853
3453.11476
3454.83932
3459.41343
3526.44880
3534.901543535.48525
3535.86933
3536.85840
3614.70596
3623.26985
3623.85178
3626.22695
3707.60875
3716.85722
3721.33959
3805.39120
3816.02521
3816.82275
3908.59680
4017.47575
4132.83522
4255.11324
4384.59041
4522.486814669.97454
4827.043104996.22281
5179.07487
Counts (%) vs. Mass-to-Charge (m/z)
2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 3800 3900 4000 4100 4200 4300 4400 4500 4600 4700 4800 4900 5000 5100 5200 5300 5400
2x10
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0.98
1
1.023526.44880
3614.70596
Counts (%) vs. Mass-to-Charge (m/z)
3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600 3610 3620 3630 3640 3650 3660 3670
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0
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0.425
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0.575
0.6
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0.725
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0.825
0.85
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0.95
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1
1.025
1.05
1.075
3223.40851
3297.69055
3371.78153
3375.58194
3379.22014
3453.65533
3459.68488
3538.16701
3544.03966
3626.32746
3632.56065
3633.45481
3636.79913
3719.71241
3725.58642
3817.92859
3823.67043
3921.39446
3927.09863
4030.41533
4145.40893
4267.47519
4398.84019
Counts (%) vs. Mass-to-Charge (m/z)
2900 3000 3100 3200 3300 3400 3500 3600 3700 3800 3900 4000 4100 4200 4300 4400 4500 4600 4700 4800 4900
2x10
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0.82
0.84
0.86
0.88
0.9
0.92
0.94
0.96
0.98
1
1.02
3719.71241
3725.58642
3817.92859
3823.67043
Counts (%) vs. Mass-to-Charge (m/z)
3685 3690 3695 3700 3705 3710 3715 3720 3725 3730 3735 3740 3745 3750 3755 3760 3765 3770 3775 3780 3785 3790 3795 3800 3805 3810 3815 3820 3825 3830 3835 3840 3845 3850 3855 3860
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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
1.05
1.1
1.15
1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
1.6
Response vs. Acquisition Time (min)
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46
5x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
Response vs. Acquisition Time (min)
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
3x10
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
1.05
1.1
1.15
1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
1.6
1.65
1.7
1.75
1.8
144483.56
144644.67
144322.96
144804.19
144894.40
145041.98
144111.35
145172.83
143912.21
Counts vs. Deconvoluted Mass (amu)
143900 144000 144100 144200 144300 144400 144500 144600 144700 144800 144900 145000 145100 145200
G1F/G1FG0F/G1F
G0F/G0F
2x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
2.5
2.6
145108.09144946.07
145266.97
144787.44
145378.96
145528.17
144656.92 145679.93
144496.85
Counts vs. Deconvoluted Mass (amu)
144200 144400 144600 144800 145000 145200 145400 145600 145800 146000
G0F/G1F
G2F/G1F
G1F/G1F
G0F/G0F
Deconvoluted spectrum Mass spectrum Expanded view of charge states TIC
IgG2
IgG1
CE/MS of Intact mAb
CE/MS of Reduced mAb
May 12, 2015
Confidentiality Label
67
2x10
0
0.025
0.05
0.075
0.1
0.125
0.15
0.175
0.2
0.225
0.25
0.275
0.3
0.325
0.35
0.375
0.4
0.425
0.45
0.475
0.5
0.525
0.55
0.575
0.6
0.625
0.65
0.675
0.7
0.725
0.75
0.775
0.8
0.825
0.85
0.875
0.9
0.925
0.95
0.975
1
1.025
1.05
1.075
1.1
1.125
1.15
1.175
1.2
1.225
1.25
855.21413
882.90070
918.18117
921.99856949.17303949.44092
956.37803
962.01820
997.91809
1043.232271092.86363
1147.45478
1207.795801274.83590
1349.76603
1351.54775
1434.06996
1529.59632
1638.79896
Counts (%) vs. Mass-to-Charge (m/z)
600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850
3x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
4.4
4.6
4.8
5
5.2
5.4
5.6
5.8
6
22930.01
22965.65
23057.3322872.72 23197.67 23270.9022769.33
Counts vs. Deconvoluted Mass (amu)
22400 22450 22500 22550 22600 22650 22700 22750 22800 22850 22900 22950 23000 23050 23100 23150 23200 23250 23300 23350 2340
2x10
0
0.025
0.05
0.075
0.1
0.125
0.15
0.175
0.2
0.225
0.25
0.275
0.3
0.325
0.35
0.375
0.4
0.425
0.45
0.475
0.5
0.525
0.55
0.575
0.6
0.625
0.65
0.675
0.7
0.725
0.75
0.775
0.8
0.825
0.85
0.875
0.9
0.925
0.95
0.975
1
1.025
1.05
1.075
1.1
1.125
1.15
1.175
1.2
1.225
1.25
855.23522
949.13718949.43208
949.68064956.39858
962.01115
997.92131
1043.22984
1092.87037
1147.453701207.79293
1274.839601349.77420
1434.06723
1529.59916
1638.78062
Counts (%) vs. Mass-to-Charge (m/z)
800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
3x10
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
2.75
3
3.25
3.5
3.75
4
4.25
4.5
4.75
5
5.25
5.5
5.75
6
6.25
6.5
6.75
7
7.25 22930.01
21820.10 22763.00 24023.95 24243.3422303.83
Counts vs. Deconvoluted Mass (amu)
21600 21800 22000 22200 22400 22600 22800 23000 23200 23400 23600 23800 24000 24200 24400 24600
5x10
0
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
Response vs. Acquisition Time (min)
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 4
5x10
0
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
Response vs. Acquisition Time (min)
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
2x10
0
0.025
0.05
0.075
0.1
0.125
0.15
0.175
0.2
0.225
0.25
0.275
0.3
0.325
0.35
0.375
0.4
0.425
0.45
0.475
0.5
0.525
0.55
0.575
0.6
0.625
0.65
0.675
0.7
0.725
0.75
0.775
0.8
0.825
0.85
0.875
0.9
0.925
0.95
0.975
1
1.025
1.05
1.075
1.1
1.125
1.15
1.175
1.2
922.00553
966.90569
998.10656
1006.33747
1028.867781043.20457
1049.18731
1071.969341075.39461
1094.92292
1099.228691120.60099
1124.19518
1147.14659
1175.70051
1202.51304
1207.19372
1232.60722
1236.60474
1263.99956
1268.20440
1274.83638
1297.334421301.61282
1349.75290
Counts (%) vs. Mass-to-Charge (m/z)
750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800
2x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
4.4
4.6
4.8
5
5.2
49259.49
49420.06
49579.58
49505.92
49339.6049054.2549676.56
49189.4949769.53
Counts vs. Deconvoluted Mass (amu)
49000 49050 49100 49150 49200 49250 49300 49350 49400 49450 49500 49550 49600 49650 49700 49750 49800
G0F
G1F
G2F*
2x10
0
0.025
0.05
0.075
0.1
0.125
0.15
0.175
0.2
0.225
0.25
0.275
0.3
0.325
0.35
0.375
0.4
0.425
0.45
0.475
0.5
0.525
0.55
0.575
0.6
0.625
0.65
0.675
0.7
0.725
0.75
0.775
0.8
0.825
0.85
0.875
0.9
0.925
0.95
0.975
1
1.025
1.05
1.075
1.1
1.125
1.15
1.175
1.2
1.225
925.67377
1035.075811053.626071057.16537
1076.65376
1080.12265
1092.76440
1100.66727
1104.06076
1125.680891129.16193
1153.25047
1178.71936
1182.87669
1197.20163
1207.76879
1211.68220
1237.93926
1241.93162
1269.47872
1273.86600
1303.00044
1307.26660
1338.15486
1342.55822
1346.88213
1367.90057
1375.33055
1379.82112
1414.57359
1419.20766
1434.04083
1456.15794
1460.90690
1500.23489
1505.17877
1547.16584
1552.15828
1596.95472
1602.21033
1650.14900
1655.58128
1712.63564
Counts (%) vs. Mass-to-Charge (m/z)
800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
3x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
2.5
2.6
49638.41
49476.72
49798.40
49272.74 49734.4949556.92 49905.95
49407.1649115.44 50049.51 50434.3748713.07 50234.45
Counts vs. Deconvoluted Mass (amu)
48700 48800 48900 49000 49100 49200 49300 49400 49500 49600 49700 49800 49900 50000 50100 50200 50300 50400 50500 50600
*
G0F
G1F
G2F
Deconvoluted spectrum Mass spectrum
TIC
Deconvoluted spectrum Mass spectrum
TIC
IgG2
IgG1
Light Chain
Heavy Chain
Light Chain
Heavy Chain
* n-acetyl glucosamine
Characterization of Small Immunoconjugates (< 40 kDa) Using CE-MS
5591-4433EN
Agilent is the only sole vendor to provide a completely integrated robust and sensitive CE/MS solution for research and for routine analysis Full Agilent series 6000 MS portfolio available – single quad, QQQ, TOF, and QTOF
Triple-tube interface to optimize individually separation and MS ionization – no compromises
Range of ion sources available - standard ESI and Agilent JetStream (APPI and APCI on demand)
Flexibility on additional detectors – UV-DAD, LIF, and CCD in parallel to MS
iFunnel-Sensitivity for small molecules down to the ppt range
Agilent MassHunter software control – one software, one workstation
Single-vendor solution – integrated system and single-source support
More information at: www.agilent.com/chem/cems
Summary 7100 CE/MS Systems
Visit our webpage at to find helpful documentation on
Product • 5991-1511EN Brochure • 5990-3962EN Data Sheet • 5990-3822EN CE consumables catalog • 5990-3980EN CE Partner CD
Basics on CE methodology and Application data
• 5990-3777EN Primer tutorial • 5990-5244EN Ion Analysis Compendium • WebPage go: Applications
Videos on CE/MS Agilent 7100 CE/MS Metabolomics by CE/MS (Keio University, Japan)
http://www.agilent.com/chem/ce
http://www.agilent.com/chem/cems
Information on Agilent 7100 CE
New trends in Biopharma presents new analytical challenges • Biosimilars
- Need to demonstrate similarity/comparability between biosimilar to its innovator molecule
• Antibody Drug Conjugates (ADCs) - Increased analytical complexity due conjugate, linker & conjugation
chemistries
BIOSIMILARS
Definitions
• Innovator biologic - Novel clinically-validated biologic on which biosimilars or biobetters are designed
• Biosimilar - Biologic molecule with identical primary amino acid sequence as innovator
biologic and developed with intention to be as close to the innovator product as possible
• Biobetter - Biologic molecule based on the innovator molecule but with improvements intended to
increase efficacy, potency, marketability, safety, or patient compliance
• Next-generation - Biologic molecule based on same validated target as innovator biologic, but with novel
VH/VL chains and (typically) different epitope, with intent of making an improved biologic against the validated target
Biologics are falling off the patent cliff too!
What does extensive structural and functional characterization means?
• All need to be evaluated as part of analytical similarity studies
Amino acid sequence Charge hydrophobicity
Helix, β sheet, turns & loops
Folded polypeptide chain, disulfide bonds, forces
Subunits, protein-protein interactions, aggregates, higher order structure
Comparison of follow on biologics to an innovator mAb by HPLC, SEC and Peptide Mapping Samples:
• Innovator – Ristova (Rituximab/Roche)
• Biosimilar – _______ (Rituximab/Indian Manufacturer) - Samples purchased from local Pharmacy in Bangalore, India
Analytical tools: • The Agilent 1260 Bio-inert LC • Biocolumns • Match Compare Software
RP HPLC of Biosimilar and Innovator mAb Agilent 1260 Infinity Bio-inert LC using Poroshell 120 SB C18 4.6x150 mm, 2.7 µm column
min 2 4 6 8 10 12 14
mAU
0
50
100
150
200 DAD1 B, Sig=280, Innovator
5.28
1
8.46
3 8
.615
min 2 4 6 8 10 12 14
mAU
0
50
100
150
200 DAD1 B, Sig=280, Biosimilar 5
.264
8.45
1 8
.608
min 5 6 7 8 9 10 11
mAU
0
50
100
150
200 DAD1 B, Sig=280, Biosimilar DAD1 B, Sig=280, Innovator
min 2 4 6 8 10 12 14
mAU
0 250
750
1250 1500
DAD1 A, Sig=210, Reduced Ristova (Innovator) 6.00
7
7.85
9
min 2 4 6 8 10 12 14
mAU
0 200
600
1000
1400
DAD1 A, Sig=210, Reduced Biosimilar 6.05
2
7.88
0
500
1000
400
800
1200
min 2 4 6 8 10 12 14
mAU
-1500 -1000 -500
0 500 1000 1500 DAD1 A, Sig=210, Biosimilar
DAD1 A, Sig=210, Innovator
RP HPLC of reduced biosimilar and innovator mAb Agilent 1260 Infinity Bio-inert LC using Poroshell 120 SB C18 4.6x150 mm, 2.7 µm column
Intact SEC of Biosimilar and Innovator mAb Agilent 1260 Infinity Bio-LC using a Bio SEC-3, 300Å, 7.8x300 mm, 3 μm
SEC of Reduced Biosimilar and Innovator mAb Agilent 1260 Infinity Bio-LC using a Bio SEC-3, 300Å, 7.8x300 mm, 3 μm
Peptide mapping of biosimilar and innovator mAb Agilent 1260 Infinity Bio-LC using a Poroshell 120 SB C18 4.6x150 mm, 2.7 µm column
Peptide mapping of Biosimilar and innovator mAb Zoom in of chromatogram; 5 – 20 min
Peptide mapping of Biosimilar and innovator mAb Zoom in of chromatogram; 20 – 40 min
Zoom in of four representative peaks across the chromatogram to show separation reproducibility
Charge Variant Profile of Innovator and Biosimilar Agilent BioMAb Column, 4.6x250mm, 5um
min2.5 5 7.5 10 12.5 15 17.5
mAU
0
2
4
6
8
10
12
10.7
11 11.2
0411
.452 11
.880
12.1
6112
.545
13.1
1113
.771
Acidic variants Basic variants
Main Peak
Biosimilar – Reditux
min2.5 5 7.5 10 12.5 15 17.5
mAU
0
5
10
15
20
25
30
35
40
10.8
3611
.196
11.4
3911
.896
12.7
12
Acidic variants Basic variants
Main Peak
Innovator – Ristova
min2.5 5 7.5 10 12.5 15 17.5
mAU
0
5
10
15
20
25
30
35
40
45
671 2
3Acidic variants
Basic variants
Main Peak
Innovator – Ristova Biosimilar – Reditux
(A)
(B)
(C)
CpB Treatment of Innovator and Biosimilar Rituximab
min0 2 4 6 8 10 12 14 16 18
mAU
0
10
20
30
40
50
60
min0 2 4 6 8 10 12 14 16 18
mAU
0
10
20
30
40
Innovator – Ristova
Biosimilar – Reditux
Untreated
CPB treated
(A)
(B)
Untreated
CPB treated
min2 4 6 8 10 12 14 16 18
0
5
10
15
20
25
30
35
40 Innovator – Ristova Biosimilar – Reditux
Charge Variant Profile of Innovator and Biosimilar IEC vs. Agilent CZE
min2.5 5 7.5 10 12.5 15 17.5
mAU
0
2
4
6
8
10
12
10.7
11 11.2
0411
.452 11
.880
12.1
6112
.545
13.1
1113
.771
Acidic variants Basic variants
Main Peak
Biosimilar – Reditux
min2.5 5 7.5 10 12.5 15 17.5
mAU
0
5
10
15
20
25
30
35
40
10.8
3611
.196
11.4
3911
.896
12.7
12
Acidic variants Basic variants
Main Peak
Innovator – Ristova (A)
(B)
CE parameters Conditions Capillary: PVA, 56 cm, 50 μm id Sample: Rituximab Innovator and Biosimilar mAbs Injection: 5s @ 50 mbar Buffer: 400 mM EACA–acetic acid pH 5.7+0.05 % HPMC+2 mM TETA Voltage: 30 kV Temperature: 20 °C DAD: 214 nm
Agilent E-Book on Biosimilars
Webinar on Protein Biopharmaceuticals & Biosimilar Characterization
Summary
• The big switch in Pharma • Biopharma customers have different challenges • The best kept secrets in Agilent
• Bioanalyzer – The Lab-on-a-Chip • TapeStation – The Next-gen electrophoresis platform • OFFGEL Fractionator – A novel sample prep tool • CE and CE/MS in biopharmaceutical analysis • HPLC-Chip Technology
• Biosimilars - Definitions & Regulations - How similar is similar enough - Case studies: Comparability data between a biosimilar and its innovator
reference
• Summary
Thank you for your attention!