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-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
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