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Critical Considerations for Analytical Similarity Assessment
Jennifer Liu
CASSS CMC Strategy Forum Japan 2015
Development of Biosimilars: Technical Aspects
Biologics Are Larger and Structurally More Complex Than Chemically Synthesized Drugs
2
Generic Biosimilars 1.0 Biosimilars 2.0
Same Structure Similarity in Structure
Monoclonal antibodies have multiple biological functions
Potency Reflective of
Target Binding and
Mechanism of Action
Binding Region for FcRn
(PK)
Binding Region for FcR,
Effector Function
(CDC, ADCC)
N-glycan
Heavy chain
Intra-chaindisulfide
Inter-chaindisulfide
Light chain
FcRn
EU
ABP
US
CDC
EU
ABP
US
ADCC
EU
ABP
US
Potency
Biological and functional assays are used to investigate structure/functional differences which are clinically meaningful for biosimilarity
Human
PK and PD
Non-clinical PK/PD and toxicology as appropriate
Match all predicted functions and confirm no enhancement of
functions absent
Demonstrate analytical similarity
Biosimilar product development begins with establishing target quality product profiles
Known mechanism of actions, biological functions, safety, and immunogenicity profiles
Define critical quality attributes for the
reference product
Characterize reference product to establish targets and ranges for critical product quality attributes
Establish product quality profiles based on the
target reference product
Match reference product profiles with greater emphasis on matching all biological functions
Develop biosimilar products to match the
target reference product
Additional clinical data
Demonstrate biosimilarity
in a step-wise manner
Biosimilar development is reverse
engineering of the reference product
5
Stability
Primary structure
Biological function
Particles and
aggregates
Process-related
impurities
General properties
and excipients
Higher order
structureProduct-related
substances and
impurities
Attributes related to the amino acid
sequence and all post-translational
modifications, including glycansBiological and functional
activities, including receptor binding
and immunochemical properties
Quantitative levels of
product variants and
their identities
Degradation profiles
denoting stability
Subvisible and submicron particles
and aggregates of various sizes
Integrity of the secondary, tertiary,
and quaternary structure
Properties of the finished
drug product, including
strength and formulation Impurities from host cells
and downstream process
Comprehensive analytical similarity is critical foundation for biosmilarity
Similarity assessment may use risk-based tier approach and statistical analysis
6
High
Low
Tsong, Y (FDA CDER) DIA/FDA Statistics Forum 2015
Consider criticality ranking of quality attributes with regard to their potential impact on activity, PK/PD, and immunogenicity. Christl, L (FDA CDER) FDA Oncologic Drugs Advisory Committee meeting, 2015
StatisticalRigor
CriticalityRanking
An approach to statistical equivalence testing for tier 1 attributes
Dong, X. (FDA) FDAMcCamish, M. (Sandoz)
This case study is provided for the sole purpose of illustrating FDA’s statistical approach for similarity assessment as presented at FDA ODAC meeting on Jan. 7 2015. No conclusion of the data is made by the presenter.
• Pass equivalence testing if 90% confidence interval of the true mean difference is within equivalence margins
• Equivalence margins = 1.50σ where σ is the true standard deviation of the RP
• Statistical power (sensitivity to detect difference) dependent on number of lots
Number of biosimilar batches
Po
wer
Challenges in the tier 1 equivalence approach
• Panel of Tier 1 quality attributes should be consistent for all sponsors seeking biosimilar product approval to the same original product
• Relationship between quality attributes and safety/efficacy in patients may not be completely understood
• Specific roles for effector function as part of the clinical mechanism of action are not entirely clear
• Sufficient number of lots are required for meaningful statistical analysis
• Small number of biosimilar batches reduce statistical power to detect difference
• Small number of reference product lots can not estimate true variability
• The true variability (standard deviation) in the reference product is difficult to estimate
• Different reference product lots may come from the same drug substance lots → underestimating variability
• Differences due to manufacturing process changes → overestimating variability
Use appropriate characterization techniques in well-designed analytical studies is critical
9Slide from Kozlowski, S (CDER) presentation at 2014 Biomanufacturing Technology Summit, Rockville, MD, June 13, 2014
Reliability - Methods should be qualified and suitable for intended purposes
Relevance - Methods provide meaningful information and may predict clinical performance
Resolution - Methods should be sensitive and capable of resolving differences
Material age - Attributes impacted by material age should be considered in the assessment
Biosimilar
Reference product
Critical
Glycans(e.g. high-Man)
Understanding the biological relevance of measured quality attributes is important
10
Confidence in similarity
Measuring the quality attributes by retaining the important structural characteristics of the attributes
Increase understanding for the biological relevance of different glycan attributes and ensure the critical attributes are controlled properly in order to match biological functions
Glycan
Map
profile
Monosaccharide
analysis
Re
leva
nce
Reliability
0.0
10.0
20.0
30.0
4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0
min
A2
G0
F
A1
G0
F
M5
A2
G0
A1
G1
F
A2
G1
F(a
)A
2G
1F
(b)
M6
A2
G2
F
M7
Monosaccharide Analysis
Oligosaccharide Analysis (Glycan Map)
Re
leva
nce
Reliability
NK92
ADCC
FcgRIIIa
binding
PBMC
ADCC
Glycan
map
Confidence in manufacturing
Knowing the correlation of orthogonal methods helps develop a robust control strategy
11
High resolution and reliable methods suited for process control can guide the process and product development.
Orthogonal assays with increasing biological relevance to discern clinically meaningful differences and confirm functional similarity
Response (LU))
0.0
10.0
20.0
30.0
4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0
min
A2G
0F
A1G
0F
M5
A2G
0
A1G
1F
A2G
1F
(a)
A2G
1F
(b)
M6 A2G
2F
M7
Chung, mAbs, 326-340, May 2012
ADCC vs. Afucosylated glycan
Critical glycan attributes are modality and molecule dependent
12
Sensitivity to difference
Impact toepoetins
Glycan TypeImpact to
mAbSensitivity to
difference
HighIncrease
clearanceNo glycan No ADCC Low to medium
Low to mediumIncrease
clearanceBisecting GN Increase ADCC Low to medium
MediumIncrease
clearanceHigh mannose and ADCC Medium
HighIncrease
clearanceTerminal Gal Increase CDC Low
HighDecrease clearance
Sialic acidAnti-
inflammatory and ADCC
Low
N/A N/A Afucosylated Increase ADCC High
0.0
10.0
20.0
30.0
4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0
min
A2G
0F
A1G
0F
A2G
1F
(a)
A2G
1F
(b)
M6
A2G
2F
M7
M5
A2G
0
A1G
1F
0
50000
100000
150000
200000
250000
300000
350000
400000
5 7 8 9 10 11 12 13 14
[U/m
g]
Isoform Number
In Vivo Bioactivity
Isoform1011121314
Biosimilar epoetins: how similar are they?
RP RP
Schellekens H. Eur J Hosp Pharm Sci. 2004;3:43–47.
Fingerprint-like similarity versus Fingerprint-like methodsFDA Definition of Fingerprint-like:
a term to describe integrated, multi-parameter approaches that are extremely sensitive in identifying analytical differences.
FDA DRAFT GUIDANCE “Clinical Pharmacology Data to Support a Demonstration of Biosimilarity to a Reference Product”
13
Koslowski, presentation at CASSS WCBP Conference, Washington DC, Jan, 31 2013
Analytical methods which may provide fingerprint-like profiles
• Methods investigate the overall conformational integrity• 1-D NMR
• 2-D NMR
• Crystallography
• Antibody conformation array
• H/D exchange
• Methods investigate the heterogeneous characteristics• Peptide map
• Glycan map
14
Can any one of these methods provide prove fingerprint-like similarity in its own right?
1H NMR provides fingerprint pattern
15
L. Poppe, Anal. Chem. 2013, 85, 9623-9629
V. Visser et. al, BioDrugs, 07 May 2013
Similarity is assessed based on pattern similarity and little information can be discerned on the differences and their impact to purity, safety, and efficacy
• Applications for monoclonal antibodies and less complex proteins have been shown
• Visual profile comparison could be subjective. An objective comparison for spectrum similarity would require complex mathematical algorithm
2D-NMR has been applied for smaller non-glycosylated protein
16
• Several publications on filgrastim (small non-glycosylated protein of ~ 19,000 Dalton) 1H-15N 2D-NMR spectral (pattern) similarity
• Provides higher resolution in structural information compared to 1D-NMR down to the single amino acid level
May not be suitable for glycoprotein and monoclonal antibodies which is structurally more complex than filgrastim
Sandoz presentation to ODAC, 07 Jan. 2015M. Levy, Anal. Bioanal. Chem. 22 Nov 2013 R. Brinson, AAPS, 2013D. Hodgson, WCBP, 2012
Crystallography has been applied to investigate sub-domains of large proteins
17
Does not investigate heterogeneous populations or discern sub-populations which may have different biological activities
• Typically require changing formulation to allow crystallization of protein of sub-domains of mAb
• Crystallization process generally serves as a purification step selecting the most “homogeneous” population, and could miss low abundant sub-populations
S. K. Jung, mAbs 6:5, 1163--1177; September/October 2014V. Visser et. al, BioDrugs, 07 May 2013
H/D Exchange provides site-specific information on structural differences
18
Interpretation of complex results requires experienced subject matter experts
• H/D exchange require long experimental time with multiple testing time points.
• Investigate differences in the exchange rate comparing two products under the same
condition. Results could be informative in regards to detailed structural differences at
specific locations
Z. Zhang Anal. Chem. 2012, 84, 4942−4949
Antibody conformation array could identify new epitopes due to changes in conformation
19
S. K. Jung, mAbs 6:5, 1163--1177; September/October 2014
X. Wang, CHI’s 6th Annual Biotherapeutics Analytical SummitComparability for Biologics & Biosimilars March 13, 2015
• Evidences of regional conformational change may predict impact to biological activities mediated through specific antigen or receptor recognition
• Reagents maybe high cost and experiment is relatively low throughput
Orthogonal to biological assays in investigating structure/functional differences
Capabilities and gaps of these fingerprint-like methods
20
Technique Capabilities Gaps
1D-[1H]-NMR Sensitive to detect differences for both small and large proteins including monoclonal antibodies
Difficult to interpret results and primarily rely on pattern similarity.May not determine specific site and levels of differences
2D-[1H-15N] HSQCNMR
Sensitive to detecting differences down to amino acid levels for small proteins
May not be suitable for complex biologics, such as glycoprotein and monoclonal antibodies
X-ray crystallography Primary application for smaller protein or fragments of monoclonal antibody. Assess sub-populations which are homogenous in nature
Limited resolution to structural differences for large protein. May omit minor components with unknown safety or efficacy impact.
H/D exchange Provide detailed structural differences at specific locations
Long experimental time. Interpretation of complex results requires experienced subject matter experts
Ab conformational array
Data analysis is less complex compared to the other fingerprinting methods.Provides regional structural information
Require product-specific reagents which could be costly or may not be available
These methods might provide a partial 'fingerprint' of higher order structure
but are not sufficient to demonstrate 'fingerprint-like similarity’.
Summary
• Comprehensive and well-designed similarity assessment is the foundation for biosimilarity
• US FDA recommends risk-based approach for tier assignment and statistical analyses
• Challenges associated with the current approach require further guidance
• Biosimilar product need to demonstrate similar functions using biological assays relevant to potential clinical mechanism of action
• There is no one method alone capable of indicating “fingerprint-like similarity”; it requires a collection of methods, including bioactivity assays.
• “Fingerprint similarity” presumably is matching all structural/functional assessments while using comprehensive sensitive methods
21
Simon Hotchin
Margaret Karow
Sundar Ramanan
Richard Markus
Gino Grampp
Acknowledgements
22
Leszek Poppe
Pavel Bondarenko
Zhongqi Zhang
John Gabrielson
Brad Jordan
Izydor Apostol
Linda Narhi