Analytical methods for therapeutic antibody characterization, comparability, release, and stability

-Confidential-

Analytical Methods for Therapeutic Antibody Characterization, Comparability,

Release, and Stability

BioPharma Asia 2017Suntec Convention Center

SingaporeMarch 22, 2017

Thomas Jung, M.S.Vice President,

Business DevelopmentKBI Biopharma

Contract Services

Cell Line Development

Process Development

Analytical Method Development

Pre-formulation and Formulation Development

Mammalian API Manufacturing

Microbial API Manufacturing

Release testing and Stability Studies

Analytical Services

Analytical Methods Development

Mass Spectrometry

Method Qualification / Validation

Release and Stability

Reference Standard Characterization

Comparability

Analytical Work Progression

Method Development

Method Qualification

ICH Method Validation

Non-GMP Release,Stability cGMP Release,

ICH StabilityRef Standard

Characterization

Comparability Assessment

Development Tox Manufacturing

Ph I Manufacturing

Ph III Manufacturing

Comparability Assessment

Protein Structure

http://en.wikipedia.org/wiki/Protein_structure

•Primary – Amino Acid Sequence

•Secondary – Alpha Helix, Beta Sheet Sub-Structures

•Tertiary – 3-Dimensional Structure, Disulfide Bonding

•Quaternary – Multiple Subunits

Protein Analytical and Biophysical Methods

http://www.mimetibody.com/Antibody.gif

Monoclonal Antibody

Analytical Methods Portfolio• Protein Primary Structure

Peptide Sequencing via LC/MS/MS Amino Acid Analysis Peptide Mapping

• Biophysical Characterization CD, FTIR, DSC, DLS, fluorescence

spectroscopy

• Capillary and Slab Gel Electrophoresis CZE SDS-CGE cIEF and icIEF SDS-PAGE and IEF Western blot Microchip electrophoresis 2D gels and blots

• Glycan Analysis Oligosaccharide mapping Monosaccharide composition Sialic Acid Quantitation

• Process Residuals• ELISA (HCP, protein A etc.)• HPLC (antibiotics, IPTG, detergents, etc)• qPCR (DNA)

• HPLC• Size Exclusion (with MALLS)• Ion Exchange• Reverse Phase• Hydrophobic Interaction• Affinity

• Potency Assays• Binding Assays via ELISA, Biacore and

ForteBio• Cell Based Assays (e.g., proliferation,

cytokine release, etc.)

• Mass Spectrometry• Intact mass• Peptide mapping with LC/MS or

LC/MS/MS• Disulfide Mapping• Post translational modifications (e.g.,

oxidation, deamidation)• PEGylation site identification• Glycan Identification & site identification

Peptide Mapping

•Enzymatic digestion of full length protein•Protein is cleaved at predictable sites generating peptide fragments•95-100% of peptides are identified by mass analysis using LC-MS•N-terminal, C-terminal, or binding site specific regions may be sequenced by LC-MS/MS•Comparison of peptides generated for reduced vs. non-reduced protein can be used for disulfide bond characterization•Evaluation of peptide map generated from digestion of glycosylated protein can be used to characterize glycan at specific glycosylation sites

Amino Acid Analysis

• Procedure• Vapor Phase Acid Hydolysis• Derivatization of Hydrolyzed AA’s• Separation of Derivatized AA’s by RP-HPLC• Data Interpretation

• Determine Amino Acid Composition• Determine Actual Extinction Coefficient vs Theoretical• Allows for protein concentration determinations by A280

Biophysical MethodsMethod Use InstrumentDifferentialScanning Calorimetry (DSC)

Determine protein melting pointThermal stability analysis

Perkin Elmer Diamond (solidstate) , Microcal Capillary VP-DSC (high sensitivity)

Circular Dichroism(CD)

Secondary structure analysis (alpha helix vs. beta sheet)Identify changes in secondary structure

Jasco J-810

Dynamic LightScattering

Protein particle size analysisAggregation, degradation evaluation

Malvern Zetasizer Nano

Fourier Transform Infrared Spectroscopy (FTIR)

Secondary structure determinationIdentify changes in secondary structure

ABB Bomen Prota

FluorescenceSpectroscopy

Probe protein unfolding equilibrium and kinetics

Spex Fluoromax 3

Glycan AnalysisMethod Use InstrumentMonosaccharide Quantitation

Composition of sugar content

Agilent 1100 / 1200 HPLC

Oligosaccharide Profile Glycosylation Pattern Agilent 1100 / 1200 HPLC

Sialic Acid Sialic Acid End Capping Agilent 1100 / 1200 HPLC

Peptide Map Glycosylation Site Evaluation

Waters Acquity UPLC, Waters Xevo G2 QTOF LC/MS/MS, Xevo G2-S

HILIC UPLC-FLD/MS Separation, Detection, and Identification of Glycans

Waters Acquity UPLC, Waters Xevo G2 QTOF LC/MS/MS, Xevo G2-S

HPLC and UPLC

Ultra Performance Liquid ChromatographyFeatures:

•Operates at high pressure and high linear velocity•Utilizes very small resin particles (< 2 um)

UPLC Advantages Compared to HPLC:•Improved Resolution•Improved Speed (Shorter Run Times)•Greater Sensitivity

Method Use InstrumentSize Exclusion Chromatography (SEC)

Separation based primarily on size of molecule. Useful in aggregation, degradation analysis.

Agilent 1100 / 1200 HPLC

Reverse-Phase HPLC (RP-HPLC)

Useful for separation of peptides and proteins including purity, oxidation, and deamidation analysis.

Agilent 1100 / 1200 HPLC

Affinity Makes use of binding affinity of specific ligands such as Protein A for antibodies.

Agilent 1100 / 1200 HPLC

Ion Exchange Chromatography (IEX)

Separation based on charge including anion and cationchemistries. Useful to monitor changes in tertiary structure and overall charge.

Agilent 1100 / 1200 HPLC

Hydrophobic Interaction (HIC)

Makes use of hydrophobic interactions between the resin and the protein. Useful for hydrophobic molecules such as membrane proteins and monoclonal antibodies.

Agilent 1100 / 1200 HPLC

Electrophoresis and Isoelectric FocusingMethod Use InstrumentSDS-PAGE (Sodium Dodecyl Sulfate –Polyacrylimide Gel Electrophoresis)

Separation in polyacrylamide gel based on relative size useful for aggregation and degradation analysis

Electrophoresis

cIEF (Capillary Iso-Electric Focusing)

Capillary separation based on pI (isoelectric point) useful to monitor changes in the charge profile

Beckman CoulterPA800 Plus

icIEF(Imaged Capillary Iso-Electric Focusing)

Capillary separation based on pI using a whole column imaged detector useful to monitor formation of impurities with various mass to charge ratios

Protein Simple iCE280, iCE3

CZE (Capillary Zone Electrophoresis)

Separation in a buffer based on various mass to charge ratios to monitor various species

Electrophoresis

SDS-CGE (Sodium Dodecyl Sulfate –Capillary Gel Electrophoresis)

Separation based on various mass to charge ratios in a capillary filled with polyacrylamide gel useful in impurities profiling

Electrophoresis

Microchip Capillary Electrophoresis Separation based on mass to charge ratios in a micro channel/microchip format

Perkin ElmerLabchip GXII, Biorad Esperion

Western Blot Identity by Antibody Binding Electrophoresis

Activity and PotencyMethod Use Instrument

ELISA(Enzyme-Linked Immunosorbent Assay)

Binding activity using various ligand binding assays

Spectramax C

Biacore Binding affinity and kinetics using label-free surface plasmonresonance

Biacore T100, T200

ForteBio Receptor binding and kinetics using a label-free dip technique; high throughput anitibody titer determinations

Pall Octet QK, Octet QK 384

Cell Based Assay Potency assays such as cell profliferation assay, apoptosis assay, ADCC assay, stimulation or inhibition of cytokine production, with or without accompanying ELISA assay

Plate readers, Spectramax C

Residual Host Cell and Process ContaminantsMethod Use Instrument

HCP ELISA Residual Host Cell Protein Impurity Assessment

Spectramax 2

Protein A ELISA Residual Protein A Impurity Assessment

Spectramax 2

Residual DNA by QPCR Residual Host Cell DNA Impurity Assessment

Applied Biosystems 7500 Fast

Residual Detergent Residual Polysorbate 20, Polysorbate 80

Agilent 1100, 1200 HPLC

Particulates MethodsMethod Use InstrumentDynamic Light Scattering Qualitative method to determine size distribution of

particles in the submicron range of 0.001 – 1 um.Malvern Zetasizer

Size Exclusion Chromatography – Multiple Angle Light Scattering (SEC-MALS)

Measurement of molecular weights for each peak eluted can provide insight into association/disassociation behavior.

Wyatt Mini Dawn Treos

Resonant Mass Measurement

Orthogonal technique to MFI and LO recommended for detection of particles in the 0.5 to 5 um size range when solutions have silicon oil present.

Archimedes

Light Obscuration Compendial method which detects particles based on blockage of light, but does not characterize of identify the particles.

HIAC 9703 HRLD

Micro-Flow Imaging Orthogonal method to LO which captures bright field images as a solution is passed through a cell which can be used to classify and differentiate particles.

Protein Simple MFI DPA 4200, MFI 5200

Raman Microscopy Provides image analysis and Raman spectroscopy of particles >/= 3-5 um and provides positive chemical identity of particles.

Morphologi G3-ID

Particulates Methods / Size

Mass Spectrometry Core Facility

Method Qualification / Validation

• Qualification of non-compendial methods for Phase I / II• Full ICH Validation of methods prior to:

• using methods for process validation• conformance lot manufacture• use of DS or DP for Phase III clinical studies.

Parameters Qualification ValidationSystem Suitability XSpecificity X XLinearity X XLOD X XLOQ X XAccuracy X XPrecision (Repeatability and Intermediate Precision) X XRange XStandards/Samples Stability XRobustness X

Release Testing for mAbTypical Drug Substance Release Assays

Assay DescriptionA280 Quantity / ConcentrationSE-HPLC Purity & HeterogeneityCE-SDS (R & NR) Purity & HeterogeneitycIEF Identity, Purity & HeterogeneityBinding ELISA PotencyResidual HCP Process derived impuritiesResidual DNA (qPCR) Process derived impuritiesResidual protein A Process derived impuritiesAppearance (color, clarity) QualitypH QualityEndotoxin SafetyBioburden Safety

Release Testing for mAbTypical Drug Product Release Assays

Assay Description

A280 Quantity / Concentration

SE-HPLC Purity & Heterogeneity

CE-SDS (R & NR) Purity & Heterogeneity

cIEF Identity, Purity & Heterogeneity

Binding ELISA Potency

Appearance (color, clarity) Quality

pH Quality

Osmolality Quality

Endotoxin Safety

Particulate matter Quality

Sterility (outsourced) Safety

Reference Standard CharacterizationTypical Reference Standard Characterization Assays

AssayA280 SE-HPLCCE-SDS (R & NR)cIEFBinding ELISAResidual HCP Residual DNA (qPCR)Residual protein AAppearance (color, clarity)pHEndotoxinBioburdenAmino acid analysisMolecular Mass via LC/MS (Intact / reduced & deglycosylated)Peptide mapping and protein sequence via LC-UV/MS/MS including N- and C-terminal evaluation and post-translational modification assessmentBiophysical Characterization (DSC, CD, FTIR, Fluorescence)Dynamic light scatteringSEC-MALSOligosaccharide Profile (N-linked)RP-HPLCCEX-HPLC

• Ensure Drug Substance / Drug Product Stability• Accelerated Stability and Real Time Stability• All ICH Stability Conditions• Continuous Rees Monitoring• Backup Chambers• Backup Power

ICH Stability Services

Stability ProgramDrug Substance

Typical Drug Substance Stability Assays

Assay DescriptionA280 Quantity / ConcentrationSE-HPLC Purity & HeterogeneityCE-SDS (R & NR) Purity & HeterogeneitycIEF Identity, Purity & HeterogeneityBinding ELISA Potency

Appearance (color, clarity) Quality

pH Quality

Bioburden (DS) – Annual timepoints /end of study only Safety

Stability Program Design – cGMP BDS

Storage1M 3M 6M 9M 12M 18M 24M

Conditions

- 80oC X X X X X X X

2 - 8°C X X X

25°C / 60% RH X X

Stability ProgramDrug Product

Typical Drug Product Release AssaysAssay DescriptionA280 Quantity / ConcentrationSE-HPLC Purity & HeterogeneityCE-SDS (R & NR) Purity & HeterogeneitycIEF Identity, Purity & HeterogeneityBinding ELISA PotencyAppearance (color, clarity) QualitypH QualityOsmolality QualityEndotoxin SafetyParticulate matter QualitySterility (outsourced) Safety

Stability Program Design – cGMP Drug Product

Storage1M 3M 6M 9M 12M 18M 24M 36M

Conditions

2-8oC X X X X X X X X

25°C / 60% RH X X X

Conclusions

Protein Therapeutics have complex chemical, structural, and thermal properties which determine their biologic activity.

Maintaining the correct biological activity requires sophisticated analytical, biophysical, and potency methods for monitoring those critical characteristics.

Choose a development partner that understands the complexity of protein therapeutics and which can apply the necessary analytical and biophysical tools at appropriate stages in the development of your protein therapeutic or biosimilar.

Acknowledgements

The following KBI senior scientific staff provided valuable content and input for this presentation:

Wayne Yount, Ph.D. Vice President, Biopharmaceutical DevelopmentMichael Nold, Ph.D. Director, Mass Spectrometry Core FacilityJimmy Smedley, Ph.D. Director, Analytical DevelopmentAmber Fradkin, Ph.D. Associate Director, R&D

Further Discussion…

We would be glad to discuss analytical support for your monoclonal antibody or other therapeutic protein programs.

Please visit KBI stand E17A in the Exhibit Hall.

Please contact:Thomas Jung, M.S.Vice President, Business Development1-630-518-2172tjung@kbibiopharma.comwww.kbibiopharma.com

Thank you!