SGS Life Sciences’ range of services dedicated to biopharmaceutical characterization focus on the most recent technical and regulatory advances. A biologic characterization strategy designed to confirm structural and physicochemical properties will be presented in accordance with a uniform set of internationally accepted principals for characterization of new and biosimilar biopharmaceutical products (ICH Q6B).

1. PEGS, May 6, 2014
BIOPHARMACEUTICAL CHARACTERIZATION
ACCORDING TO
ICHQ6B HARMONIZED GUIDELINES
Kenneth Warrington, PhD
Director, Biosafety Business Development
North America

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PEGS, May 6, 2014
DISCOVERY
PRE
SUBMISSION
POST
APPROVAL
Discovery that you have a
Bio-molecule that does
something.
This discovery phase is
growing in the University,
Small Company &Venture
capital supported space
Need to
 Characterize
molecule (chemical,
biophysical)
 Develop basic
assays for potency,
purity, identity
 Cell line selection
characterization and
purification
Proof of effectiveness,
efficacy and acceptable
safety profile
 Certified reference
material, and analytical
standards (Stability)
 Cell line optimization and
characterization
 Cell bank creation and
management for future
production
 Validation of analytical
methods for routine use
and testing of submission
batches, premarket
stability.
 Formulation development
and packaging selection
Regulatory approval and
license to market obtained.
 Routine testing with
validated methods.
 Management and re-
characterization of
reference materials &
standards
 Post-market stability studies
The development life cycle
may continue
 Development of new
strengths/more stable
formulations, different dose
formats, better analytical
methods
PRE-CLINICAL
Extended proof of concept
that the molecule is selective
 Further characterization
creation of reference
materials
 Development of fit for
purpose assays
 Tests in tissue culture and
animal models for activity
 Selection and
characterization of
producer cell lines
 Cell toxicity, Biomarker
analysis as indicators of
areas and specificity of
biological activity & effect
 Forced degradation studies
BIOPHARMACEUTICAL LIFE CYCLE

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WHY CHARACTERIZE?
 Product characterization is essential for product
development and regulatory acceptance
 Characterization is the basis of all knowledge and
understanding of the product and it’s structure/function
relationship
 Understanding the product structure is key to all aspects of
product and process development
 Process and Analytical Development (GLP and/or GMP)
 Understand the chemical structure, physical properties,
impurity profile and degradation pathways
 Determine the effect of process change on drug
substance
 Formulation
 GMP Manufacture
 Guide to select specification, QC & stability assays
 Comparability studies - e.g. changes pre- and post-
approval

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PEGS, May 6, 2014
 Co- and Post-Translational Modifications
 Microheterogeneities
 Immunogenicity
WHY ARE BIOPRODUCTS A CHALLENGE?
Acetylation
Acylation
Addition of lipid (palmitoylation)
Amidation (deamidation)
Carbamylation
Carboxylation
Formylation
Gla (gamma carboxyglutamic acid)
Glycosylation (N-linked, O-linked)
Glycation
Methylation
Norleucine
Phosphorylation
Sulphation
Proteolysis
Methionine Oxidation
Di-sulphide bond formation

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PEGS, May 6, 2014
WHAT REGULATIONS COVER
PHYSICOCHEMICAL CHARACTERIZATION?
 ICH Topic Q6B “Specifications: Test Procedures
and Acceptance Criteria for Biotechnological/Biological
Products”
 Structural characterization and confirmation
1. Amino acid sequence
2. Amino acid composition
3. Terminal amino acid sequence
4. Peptide map
5. Sulfhydryl group(s) and disulfide bridges
6. Carbohydrate structure
 Physicochemical properties
1. Molecular weight or size
2. Isoform pattern
3. Extinction coefficient
4. Electrophoretic pattern
5. Liquid Chromatographic pattern
6. Spectroscopic profiles

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PEGS, May 6, 2014
HOW? ANTIBODY CHARACTERIZATION
CASE STUDY
 Typical analyses performed
 Mass spectrometry of intact
protein & released L &H chains
 Amino Acid Composition
Analysis
 N-terminal sequencing
 Peptide “MAPPING” Analysis
(Sequence coverage: 100% LC
and 100% HC)
 Monosaccharide & sialic acid
analysis
 Oligosaccharide population
analysis
 SDS-PAGE analysis
 Circular Dichroism
 Analytical Ultracentrifugation

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INTACT MASS MEASUREMENT

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PEGS, May 6, 2014
N-Linked biantennary core fucosylated with varying number of galactose residuesIgG
Fuc Man – GlcNAc
Asn - GlcNAc-GlcNAc- Man Man - GlcNAc
- Gal
- Gal
Mab +2 x G0F
Mab +1 x G0F
+ 1 x G1F
Mab +2 x G1F
Mab +1 x G1F
+ 1 x G2F
G0F Mass shift = +1444 Da
G1F Mass shift = +162 Da
G2F Mass shift = +324 Da
INTACT MASS: MONITORING GLYCOSYLATION

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LC-MS: Heavy and light chain analysis
REF STD
BATCH 2
BATCH 1
 Additional peak in development material
 Mass 128 Da heavier than major HC component
(GOF)
 Basic from cIEF confirmed on C-terminal
INTACT MASS: MONITORING MODIFICATIONS

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PEGS, May 6, 2014
+128Da
+ Lysine at C-terminus
+162Da
+ Glycation
ON-LINE LC/ES-MS MASS MEASUREMENT
INTACT MASS: MONITORING MODIFICATIONS

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PEGS, May 6, 2014
ON-LINE LC/ES-MS TOTAL ION CURRENT
INTACT MASS: MONITORING MODIFICATIONS

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PEGS, May 6, 2014
INTACT MASS: MONITORING MODIFICATIONS
Light chain
Light chain SS Bridged to
Glutathione
Light chain-Cysteinylated
ON-LINE LC/ES-MS MASS MEASUREMENT

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PEGS, May 6, 2014
S S
SH
Disulphide bridged
protein
E
E
E
Enzymic/Chemical
digestion
S S SH
Mixture of
peptides
Identification by MS
Followed by reduction
And further MS
CHARACTERIZATION OF S-S BRIDGES

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PEGS, May 6, 2014
2971.0 2989.6 3008.2 3026.8 3045.4 3064.0
Mass (m /z)
0
2567.0
0
10
20
30
40
50
60
70
80
90
100
%Intensity
Voyager Spec #1 MC[BP = 3017.6, 2567]
3048.7
1154.0 1169.4 1184.8 1200.2 1215.6 1231.0
Mass (m /z)
1122.8
20
30
40
50
60
70
80
90
100
%Intensity
Voyager Spec #1=>SM5[BP = 1662.4, 7089]
1955 1970 1985 2000 2015 2030
Mass (m /z)
754.3
10
20
30
40
50
60
70
80
90
100
%Intensity
Voyager Spec #1=>SM5[BP = 1662.4, 7089]1062.6
1988.1
VTCVVVDISK
280 289
TCIVPEVSSVFIFPPKPK
252 269
KTCIVPEVSSVFIFPPKPK
KVTCVVVDISK
252 269
280 289
Reduction
CHARACTERIZATION OF S-S BRIDGES

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MAPPING WORKFLOW

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ANTIBODY ANALYSIS – GENERAL WORKFLOW

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PEGS, May 6, 2014
Q-TOF MS/MS
of 785 [M+2H]2+
MS/MS AMINO ACID SEQUENCING

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 2 basic types of glycosylation normally observed.
 N-linked to the amide of Asparagine (Asn) in the
consensus sequence …Asn-X-Ser/Thr…where X
is any AA except Pro.
 O-linked to the hydroxyl functions of Serine (Ser)
or Threonine (Thr).
 The populations of sugars attached to an individual
protein will depend on the cell type in which the
protein is expressed and on the physiological
status of the cell
 Glycoproteins are mixtures of glycoforms i.e. the
same polypeptide but different glycans
PROTEIN GLYCOSYLATION

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PROTEIN GLYCOSYLATION

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ICH Topic Q 6 B
Structural characterization and confirmation
6. Carbohydrate structure
“For glycoproteins, the carbohydrate content (neutral
sugars, amino sugars and sialic acids) is determined. In
addition, the structure of the carbohydrate chains, the
oligosaccharide pattern (antennary profile) and the
glycosylation site(s) of the polypeptide chain is
analysed, to the extent possible”
WHAT REGULATIONS COVER
GLYCOSYLATION CHARACTERIZATION?

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PEGS, May 6, 2014
COOH2HN
S---S
S---S
N-Glycans
O-Glycans
Intact Mass by MALDI or
ES MS
Monosaccharide
Composition Analysis
(LC & MS)
Reduction Carboxymethylation
COOH2HN
S-CM S-CMS-CMS-CM
Reductive
elimination
Specific Protease Digest
PNGase F
Sep-pak
0% 20% 40%
Permethylation MALDI,
Nanospray-MS/MS & Linkage analysis
LC & MS methods
Monosaccharide Composition
Glycan Population Screening
Glycan Antennary Profile
Glycosylation Site
Linkage Analysis
ANALYSIS OF GLYCOSYLATION

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Column: CarboPac PA10
Eluent: 18 mM Sodium hydroxide
Flow Rate: 1.5 mL/min
Detection: Pulsed amperometry,
gold electrode
Sample: MAb 2 M TFA
Hydrolysate
Peaks: 1. Fucose
2. Rhamnose
3. Glucosamine
4. Galactose
5. Mannose
1
2
3
4
5
Minutes
0 10 20 30
nC
MONOSACCHARIDE COMPOSITION ANALYSIS
BY HPAEC-PAD

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PEGS, May 6, 2014
Column: CarboPac™ PA-100
Eluent: 0–250 mM Sodium acetate over
110 min in 100 mM Sodium hydroxide
Flow Rate: 1 mL/min
Detection: Pulsed amperometry, gold electrode
Minutes
25
150
nA
25
190
nA
49%
35%
10%
19%
35%
14%
28%
A
B
1
2
3
4
1
2
3
5
0 10 20 30 40 50
25
300
nA
44%
37%
10%
C
1
2
3
 Possibility of semi-quantitative analysis
 Isomers could, in very specific conditions,
be separated
 Possibility of batch to batch comparison
N-glycan population profiling analysis of three different antibodies
OLIGOSACCHARIDE POPULATION ANALYSIS
BY HPAEC-PAD

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OLIGOSACCHARIDE POPULATION ANALYSIS
BY MALDI-TOF MS
From CFG data (http://functionalglycomics.org)

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FLD chromatogram
TIC chromatogram
 One molecule of N-glycan = one
tag (response independent from
N-glycan structural features)
 Isomers could, in very specific
conditions, be separated
 Possibility of batch to batch
comparison based on profile
 Glycan structural identification
could be obtained through
coupling with MS
2-AB labelling and HPLC-FLD for profiling Oligosaccharide population
Example of IgG N-glycans
OLIGOSACCHARIDE PROFILING:
LC- AND MS-BASED METHOD

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TIC chromatogram
Annotations based on MS data
2-AB labelling and HPLC-FLD for profiling Oligosaccharide population coupled with ESI-MS
Example of IgG N-glycans
OLIGOSACCHARIDE PROFILING:
LC- AND MS-BASED METHOD

27. 27
PEGS, May 6, 2014 From Dell et al. Comprehensive Glycoscience, 2006
GLYCAN ANTENNAE PROFILING ANALYSIS
BY MS/MS

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PEGS, May 6, 2014
Sample: Fetuin N-glycans linkage
Acquired on: 17-Sep-2002 at 11:56:11 Job No: MS02 Sample No: MS02M-Scan Ltd.
10.000 11.000 12.000 13.000 14.000 15.000 16.000 17.000 18.000 19.000 20.000
rt0
100
%
16.752
14.001
13.171
13.471 14.941
Scan EI+
117+118+129+159
3.33e6
RT
FETNLIN
t-Gal
2-Man
3-Gal
6-Gal
3,6-Man
4-GlcNAc
2,4-Man
4,6-GlcNAc
LINKAGE ANALYSIS BY GC-MS

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Imaging cIEF of
Monoclonal Antibodies
COMPARABILITY ON BASIS OF CHARGE

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 Spectroscopic method measuring the absorption of left and
right handed circularly polarized light
 Information on secondary structure such as α-helices and
sheets
HIGHER ORDER STRUCTURE: CD
Far UVNear UV
Monitor unfolding in presence of heat or denaturants
260-190nm320-250nm

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Measures melting points (Tm’s) of IgG regions
Good indicator of thermal stability
HIGHER ORDER STRUCTURE: DSC

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 Common problem encountered during manufacture and
storage of proteins
 Undesirable due to potential immunogenicity (small
aggregates) or problems with administration (large
aggregates)
 Regulatory authorities requesting Size Exclusion
Chromatography (SEC) plus a column free technique such
as Analytical Centrifugation (AUC) or Dynamic Light
Scattering (DLS) to cross check data obtained from SEC
as aggregates can potentially be lost by non-specific
binding to an SEC column
 Field Flow Fractionation (FFF) is also being used
increasingly for analysis of protein aggregates
PROTEIN AGGREGATION

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Combination of UV, RI and MALS detection allow
an overview of the aggregation state.
Advantages
Easy method to establish
High throughput
Straightforward data analysis
Good resolution.
Minimal sample preparation required
Disadvantages
Potential for loss of aggregates by non specific
binding to the column
Also potential for breaking aggregates during
significant dilution effect following injection
MONITORING AND QUANTIFYING
AGGREGATION
SEC-MALS: SIZE EXCLUSION CHROMATOGRAPHY
WITH MULTI-ANGLE LASER LIGHT SCATTERING

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Matrix free platform to qualify aggregation based in the
interaction of scattered light with molecules of different
size.
Monitors the size of molecules and presence of
hydrodynamic species other than the monomer
(aggregates) with high molecular weight
Advantages
Non invasive/ Matrix free (avoidance of loss of aggregates
by non specific binding to the column).
Great sensitivity (trace concentrations)
No sample prep required (only buffer filtration)
Potential for moderate throughput (screening)
Disadvantages
Qualitative
Lack of specificity
Poor resolution
MONITORING AND QUANTIFYING
AGGREGATION
0
1
2
3
4
5
6
7
8
0.01 0.1 1 10 100 1000 10000
Intensity(%)
Size(d.nm)
Size Distribution by Intensity
Record 13: 100734 1 Record 14: 100734 2 Record 15: 100734 3
Size (radius nm)
Distribution by Volume
Formulation C - 45 days
Intensity(%)
0
2
4
6
8
10
12
14
16
18
0.01 0.1 1 10 100 1000 10000
Volume(%)
Size(d.nm)
Size Distribution by Volume
Record 4: 100732 1 Record 5: 100732 2 Record 6: 100732 3
Size (radius nm)
Distribution by Intensity
Formulation A Formulation B Formulation C - 0 days
Intensity(%) DLS: DYNAMIC LIGHT SCATTERING

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PEGS, May 6, 2014
SV-AUC provides a matrix free platform to quantify
aggregation
Monitors concentration distribution of solutes
(migration pattern) in a centrifuge cell as a function
of radius at different times
Advantages
Matrix free (avoidance of loss of aggregates by
non specific binding to the column).
Good resolution.
No sample preparation required.
Disadvantages
Low throughput. Complicated data analysis.
5 10 15 20
0.0
0.5
1.0
1.5
2.0 85.05 ± 0.35%
C(s)distribution
Sedimentation coeficient (s)
Cell 1
Cell 2
Cell 3
14.95 ± 0.35%
Formulation A
MONITORING AND QUANTIFYING
AGGREGATION
SV-AUC: ANALYTICAL ULTRACENTRIFUGATION
SEDIMENTATION VELOCITY

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PEGS, May 6, 2014
 Analytical characterisation is essential throughout all
stages of biopharmaceutical development.
 Advances in MS instrumentation and Proteomic/Glycomic
strategies enable rapid identification of protein products
and their PTMs, including glycosylation.
 MS techniques alone are not enough and other orthogonal
methods should also be included.
SUMMARY

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LABORATORY SERVICES
- FROM BIOMARKERS TO BATCH ANALYSIS -

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BIOPHARMACEUTICAL CHARACTERIZATION

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PEGS, May 6, 2014
LABORATORY SERVICES
DETAIL OF BIOPHARMACEUTICAL ANALYSIS

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PEGS, May 6, 2014
Life Science Services Kenneth Warrington, Jr., PhD
Director, Biosafety Business Development
North America
Phone: +1 (716) 796 4595
E-mail : kenneth.warrington@sgs.com
Web : www.sgs.com/lifescience
THANK YOU FOR YOUR ATTENTION