addressing analytical challenges in biopharmaceuticals ......downstream processing &...

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



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




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

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

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

36

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)

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)

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

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Protigel P Advanced Analytical 10

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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

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

Instrument type : Agilent Q-TOF


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

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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(C) CE-MS and LC-MS comparison of peptide distributions. (A) Molecular weight plot (B) Peptide length plot (C) Isoelectric point plot


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)



Schematic overview of the glycoprofiling of mAb using CE/MS

Analysis of N-Glycans of a Monoclonal Antibody by CE/MS

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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

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CE/MS Analysis of N-Glycans Released from mAb

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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

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3286.04257

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3298.48630

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3370.90992

3372.32084

3376.21089

3442.44591

3450.90523

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3453.11476

3454.83932

3459.41343

3526.44880

3534.901543535.48525

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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)

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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 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


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


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


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


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


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


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


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


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


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


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


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

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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

http://www.chem.agilent.com/en-US/products-services/Instruments-Systems/Automated-Electrophoresis/Pages/cems_video.aspx

http://www.chem.agilent.com/en-US/products-services/Parts-Supplies/Electrophoresis/CE-CE-MS/Pages/video-metabolomics.aspx

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


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


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


Main Peak


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


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!

addressing analytical challenges in biopharmaceuticals ......downstream processing &...

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