Thomas Jung, M.S. Vice President, Business Development BioPharma Asia 2017, Suntec Convention Center Singapore, March 22, 2017

1. -Confidential-
Analytical Methods for Therapeutic
Antibody Characterization, Comparability,
Release, and Stability
BioPharma Asia 2017
Suntec Convention Center
Singapore
March 22, 2017
Thomas Jung, M.S.
Vice President,
Business Development
KBI Biopharma

2. 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

3. Analytical Services
Analytical Methods Development
Mass Spectrometry
Method Qualification / Validation
Release and Stability
Reference Standard Characterization
Comparability

4. Analytical Work Progression
Method
Development
Method
Qualification
ICH Method
Validation
Non-GMP
Release,Stability
cGMP Release,
ICH Stability
Ref Standard
Characterization
Comparability
Assessment
Development
Tox
Manufacturing
Ph I
Manufacturing
Ph III
Manufacturing
Comparability
Assessment

5. 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

6. Protein Analytical and Biophysical Methods
http://www.mimetibody.com/Antibody.gif
Monoclonal Antibody

7. 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

8. 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

9. 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

10. Biophysical Methods
Method Use Instrument
Differential
Scanning
Calorimetry (DSC)
Determine protein melting
point
Thermal stability analysis
Perkin Elmer Diamond (solid
state) , 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 Light
Scattering
Protein particle size analysis
Aggregation, degradation
evaluation
Malvern Zetasizer Nano
Fourier Transform
Infrared
Spectroscopy
(FTIR)
Secondary structure
determination
Identify changes in
secondary structure
ABB Bomen Prota
Fluorescence
Spectroscopy
Probe protein unfolding
equilibrium and kinetics
Spex Fluoromax 3

11. Glycan Analysis
Method Use Instrument
Monosaccharide
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

12. HPLC and UPLC
Ultra Performance Liquid Chromatography
Features:
•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 Instrument
Size 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 cation
chemistries. 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

13. Electrophoresis and Isoelectric Focusing
Method Use Instrument
SDS-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 Coulter
PA800 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 iCE
280, 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 Elmer
Labchip GXII,
Biorad Esperion
Western Blot Identity by Antibody Binding Electrophoresis

14. Activity and Potency
Method 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 plasmon
resonance
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

15. Residual Host Cell and Process Contaminants
Method 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

16. Particulates Methods
Method Use Instrument
Dynamic 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

17. Particulates Methods / Size

18. Mass Spectrometry Core Facility

19. 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 Validation
System Suitability X
Specificity X X
Linearity X X
LOD X X
LOQ X X
Accuracy X X
Precision (Repeatability and Intermediate Precision) X X
Range X
Standards/Samples Stability X
Robustness X

20. Release Testing for mAb
Typical Drug Substance Release Assays
Assay Description
A280 Quantity / Concentration
SE-HPLC Purity & Heterogeneity
CE-SDS (R & NR) Purity & Heterogeneity
cIEF Identity, Purity & Heterogeneity
Binding ELISA Potency
Residual HCP Process derived impurities
Residual DNA (qPCR) Process derived impurities
Residual protein A Process derived impurities
Appearance (color, clarity) Quality
pH Quality
Endotoxin Safety
Bioburden Safety

21. Release Testing for mAb
Typical 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

22. Reference Standard Characterization
Typical Reference Standard Characterization Assays
Assay
A280
SE-HPLC
CE-SDS (R & NR)
cIEF
Binding ELISA
Residual HCP
Residual DNA (qPCR)
Residual protein A
Appearance (color, clarity)
pH
Endotoxin
Bioburden
Amino acid analysis
Molecular 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 assessment
Biophysical Characterization (DSC, CD, FTIR, Fluorescence)
Dynamic light scattering
SEC-MALS
Oligosaccharide Profile (N-linked)
RP-HPLC
CEX-HPLC

23. • 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

24. Stability Program
Drug Substance
Typical Drug Substance Stability 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
Bioburden (DS) – Annual
timepoints /end of study only
Safety
Stability Program Design – cGMP BDS
Storage
1M 3M 6M 9M 12M 18M 24M
Conditions
- 80o
C X X X X X X X
2 - 8°C X X X
25°C / 60% RH X X

25. Stability Program
Drug Product
Typical 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
Stability Program Design – cGMP Drug Product
Storage
1M 3M 6M 9M 12M 18M 24M 36M
Conditions
2-8o
C X X X X X X X X
25°C / 60% RH X X X

26. 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.

27. Acknowledgements
The following KBI senior scientific staff provided valuable content and
input for this presentation:
Wayne Yount, Ph.D. Vice President, Biopharmaceutical Development
Michael Nold, Ph.D. Director, Mass Spectrometry Core Facility
Jimmy Smedley, Ph.D. Director, Analytical Development
Amber Fradkin, Ph.D. Associate Director, R&D

28. 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
Development
1-630-518-2172
tjung@kbibiopharma.com
www.kbibiopharma.com

29. Thank you!