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a Novartis company
Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®
Andreas Premstaller, Ph.D.Biopharmaceutical OperationsSan Francisco, January 14, 2009
2 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Outline
• Analytical tools for the characterization of somatropin• Comparability studies in the development of Omnitrope®:
Scope, approaches and execution• Case studies of comparability assessments:
− Biosimilarity− Process changes− Novel formulations
Analytical toolsComprehensive characterization of
somatropin
4 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
• Recombinant human growth hormone(rhGH) from modified E. coli
• Chemically identical to pituitary growth hormone, non-glycosylated protein
• 1 chain of 191 amino acids, 2 disulfide bridges, with 4 antiparallel α-helices
• C990H1528N262O300S7
• Mass 22125
Somatropin, the active ingredient of Omnitrope®
Source: PDB ID: 1HGUChantalat, L. Jones, N.D., Korber, F., Navaza, J., Pavlovsky, A.G. (1995) The crystal-structure of wild-type growth-hormone at 2.5 Angstrom resolution. Protein Pept. Lett. 2: 333-340
5 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Analytical methods to characterize somatropin in comparability studiesMolecular parameter Method
Edman sequencing
Peptide map with MS detection
Mass Mass spectrometry: MALDI-TOF, ESI-MS
Capillary electrophoresisCharge
Immunological tests Immunoblotting
Circular dichroism spectroscopy
1D {1H} NMR spectroscopy
Reversed phase chromatography
Isoelectric focusing
Ion exchange chromatography
Size exclusion chromatographySize
Gel electrophoresis
In-vivo bioassays
Cell proliferation assay
Primary structure
Spatial structure(secondary and tertiary)
Polarity
Biological activity
6 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Assessment of identity: Complete sequence coverage by combination of peptide maps
AA# 1 2 3 4 5 1 0 0 0 0 0 T1 T2 T4 T6 FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQT G1 G2 G3 G5 G6 AA# 1 5 6 7 8 9 0 1 0 0 0 0 0 T7 T8 T9 T10 SLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANS G7 G8 1 1 1 1 1 1 0 1 2 3 4 5 1 0 0 0 0 0 T11 T12 T13 T14 T15 LVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNS G13 AA# 1 1 1 1 1 5 6 7 8 9 1 0 0 0 0 T16 T18 T19 T20 T19 HNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF G18 G21 G22
Complete sequence coverage is obtained by combination of the detected peptides from digests with trypsin (blue) and Glu-C (orange).
7 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Considerations for analytical methods:Impact of matrix and sample preparation
Sample preparation and analytical methods must be developed to account for differences in sample matrices.
40 45 50 55 60
04-WHOOmnitrope
T4+T
5
T10
(T6+
T7)-S
-S-T
16T6
-S-S
-T16
T1-1 T1
0-2
T11
T4
40 45 50 55 60
04-WHOOmnitrope
T4+T
5
T10
(T6+
T7)-S
-S-T
16T6
-S-S
-T16
T1-1 T1
0-2
T11
T4Product 1
40 45 50 55 60
04-WHOOmnitrope
T4+T
5
T10
(T6+
T7)-S
-S-T
16T6
-S-S
-T16
T1-1 T1
0-2
T11
T4
40 45 50 55 60
04-WHOOmnitrope
T4+T
5
T10
(T6+
T7)-S
-S-T
16T6
-S-S
-T16
T1-1 T1
0-2
T11
T4Product 1
Digestion without buffer exchange
•Somatropin NIBSC 98/574•Somatropin, liquid formulation, pH 6.2
40 45 50 55 60
04-WHOOmnitrope, buffer exchange
T4+T
5
T10
(T6+
T7)-S
-S-T
16T6
-S-S
-T16
T1-1 T1
0-2
T11
T4
40 45 50 55 60
04-WHOOmnitrope, buffer exchange
T4+T
5
T10
(T6+
T7)-S
-S-T
16T6
-S-S
-T16
T1-1 T1
0-2
T11
T4
Product 1, buffer exchange
40 45 50 55 60
04-WHOOmnitrope, buffer exchange
T4+T
5
T10
(T6+
T7)-S
-S-T
16T6
-S-S
-T16
T1-1 T1
0-2
T11
T4
40 45 50 55 60
04-WHOOmnitrope, buffer exchange
T4+T
5
T10
(T6+
T7)-S
-S-T
16T6
-S-S
-T16
T1-1 T1
0-2
T11
T4
Product 1, buffer exchange
Digestion with buffer exchange
•Somatropin NIBSC 98/574•Somatropin, liquid formulation, after buffer exchange
Comparability studiesScope, common elements, differences
9 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Comparability studies in the development of Omnitrope®
• Biosimilarity: Comparison to a reference product• Process changes: Comparison between a pre- and post-change
product• Novel formulations: Comparison of different drug product
formulations, and assessment of potential differences in the profile of product-related variants
Scope
10 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Comparability studies in the development of Omnitrope®
• Science and knowledge based approach− Previous knowledge− Risk assessment with respect to potential impact of changes/
differences− Identification of Critical Quality Attributes (CQA), Critical Process
Parameters (CPP)• Focus on analytical methods to monitor CQA’s, CPP’s• Type and extend of studies (physicochemical, biological,
preclinical, clinical) is determined in a step-wise approach based on these consideration and the knowledge regarding product and process.
Common approach
11 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Comparability studies in the development of Omnitrope
• Setting acceptance criteria:− Considering the previous experience with the product at the
biosimilar developer and the information in the public domain− Biosimilarity: Considering the batch-to-batch variability of the
reference product− Process changes: Based on the historical performance of the
process • Differences need to be assessed in appropriate studies and
scientifically justified to exclude an impact on efficacy and safety of the product
− Classification of the product variants into product-related substances or impurities (ICH Q6B)
Execution
Comparability studiesBiosimilarity
13 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
In a representative indication (growth hormone deficient children)
Comprehensive analysis of protein
Comparative PK/PD in healthy volunteers
14- day repeat toxicity (rats)local tolerance (rabbits)
In-vitro and in-vivo bioassays
Development according to relevant guidelines
Com
para
bilit
y
Clinical efficacy and safety
PK/PD
Preclinical
Biologicalcharacterization
Physicochemical characterization
Comparability of Omnitrope® with the reference product was established at all stages
Complete stand-alone product and target-directed process development
14 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Controlling the product profile of somatropin:Detecting potential product-related variants
Variant Polarity (RP-HPLC)
Charge (CZE, IEF, IEX)
Size(SEC)
Deamidated X X -
-
-
-
-
-
Thioether - - - X
Aggregates –Covalent dimer
X - X X
X
X
Succinimide X X -
Isoaspartate X - x
Oxidized X - X
Truncated and clipped variants
X X X
Trisulfide X - X
Non-covalent aggregates – Dimer, oligomers
- - -
Peptide mapping
X…resolved and detected; -…not resolvable with this method
15 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
A thioether variant of somatropin:Background
Disulfide bridges in somatropin:• Cys(53)-Cys(165)• Cys(182)-Cys(189)
Thioether variant:• Cys(182)-Cys(189)*
* Datola, A. et al., ChemMedChem, 2 (2007) 1181-1189.
IVQCRSS
SVEGSCGF
IVQCRS
SVEGSCGF
16 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
684.83
685.34
685.84
678 680 682 684 686 688 690 692 694 696 698 700 702 704 706m/z
0
20
40
60
80
100 700.82
701.32
701.82
702.32
A thioether variant of somatropin:Detection and identification
Time (min)17 18 19 20 21
Reference product
Omnitrope
Somatropin X
T20-
SS-T
21
T20-
S-T2
1
T20-S-S-T21, (M+2H)2+
Expected mass: 700.82Found mass: 700.82
Detection by peptide mapping Identification by high resolution mass spectrometry
T20-S-T21, (M+2H)2+
Expected mass: 684.83Found mass: 684.83
17 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Phase I PK/PD study program
Protocol Design Objective No. Subj. Dose
EP2K99PhI S USA Double-blind, randomised, 2-way cross-over, placebo-controlled study
Phase I safety study in healthy volunteers to assess the safety, tolerance and pharmacokinetics of Omnitrope lyophilised powder (API Covance).Feasibility study to demonstrate that continuous i.v infusion of octreotide is effective to suppress endogenous GH secretion in healthy subjects
12 5 mg single dose s.c. injection
EP2K99PhI USA Double-blind, randomised, 2-way cross-over, controlled study
Phase I study in healthy volunteers to compare the pharmacokinetics, pharmacodynamics and safety between Omnitrope lyophilised powder (API Covance) and Genotropin
25 5 mg single dose s.c. injection
EP2K00PhI AQ Double-blind, randomised, 2-way cross-over, controlled study
Phase I study in healthy volunteers to compare the pharmacokinetics, pharmacodynamics and safety between Omnitrope lyophilised powder (API Covance) and Omnitrope 3.3 mg/ml solution f.i. (API Kundl)
24 5 mg single dose s.c. injection
EP00-104 Double-blind, randomised, 3-way cross-over, controlled study
Phase I study in healthy volunteers to compare the pharmacokinetics, pharmacodynamics and safety between Omnitrope 5mg/ml powder f.s.f.i., Omnitrope 3.3 mg/ml solution f.i. and Genotropin
36 5 mg single dose s.c. injection
EP00-105 Double-blind, randomised, 3-way cross-over, controlled study
Phase I study in healthy volunteers to compare the pharmacokinetics, pharmacodynamics and safety between Omnitrope 5mg/ml powder f.s.f.i., Omnitrope 6.7 mg/ml solution f.i. and Genotropin
36 5 mg single dose s.c. injection
18 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Phase III Clinical Program to confirm comparable safety and efficacy
Protocol Design Objective No. Subj. Dose
EP2K99PhIII
EP2K00PhIIIFo
EP2K00PhIIIAQ part A
EP2K00PhIIIAQ part B
EP2K02PhIIILyo
Randomized, controlled, open, multicenter study in GHD children
Efficacy and safety -Demonstrate therapeutic equivalence between Omnitrope Lyophilized Powder and Genotropin
89 Omnitrope Lyophilized Powder vs. Genotropin0.03 mg/kg daily, sc
Controlled, open, multicenter study in GHD children
Efficacy and safety -Follow-up study to demonstrate therapeutic equivalence between Omnitrope Lyophilized Powder and Genotropin
86 Omnitrope Lyophilized Powder vs. Genotropin0.03 mg/kg daily, sc
Controlled, open, multicenter study in GHD children
Efficacy and safety -Demonstrate therapeutic equivalence between Omnitrope Lyophilized Powder and Omnitrope Liquid Formulation
86 Omnitrope Lyophilized Powder vs. Omnitrope Liquid Formulation0.03 mg/kg daily, sc
Open, multicenter study in GHD children
Demonstrate long term safety and efficacy of Omnitrope Liquid Formulation
86 Omnitrope Liquid Formulation0.03 mg/kg daily, sc
Pivotal safety study
Open, multicenter study in GHD children
Safety and efficacy -Confirmation of low immunogenicity of Omnitrope Lyophilized Powder
51 Omnitrope Lyophilized Powder 0.03 mg/kg daily, sc
Pivotal efficacy study
19 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Biosimilarity: Comparability with the reference product
• A biosimilar product is designed to meet the criteria of the reference product with regards to quality, safety and efficacy.
• Biosimilars development requires complete product and process development plus comparative testing at all relevant levels.
• The comparability of Omnitrope® to the reference product was shown using a comprehensive array of test methods.
• Pre-clinical and clinical assessment confirmed similarity and clinical equivalence.
Targeted development/
Quality by Design
Quality
Module 3
Physico-chemical and
biological comparability
Comparative preclinical
studies
Comparative clinical studies
Risk Management
Plan
Comparative
Reference product
Biosimilar product
Comparative
Comparability studiesProcess changes
21 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Comparability assessment for process changes
• Acceptance criteria for comparability: − Based on the historical performance of the process (batch
history) and understanding of the product.− Data from reference product may be used as supportive data in
assessment of the change• Approaches:
− Submission of the entire comparability study− Submission of a comparability protocol ahead of implementation
of changes• Extend of the studies:
− Varies with extend of change− For scale or site changes to a well-controlled and understood
process physicochemical and biological data were found appropriate and sufficient
Comparability studiesNovel formulations
23 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Liquid formulations of somatropin
Stable liquid formulation, containing buffer system, tonicity modifying excipients, surface active excipients, antimicrobial preservation agent
Lyophilized and liquid formulation after submission to mechanical stress:Particle profile in the subvisible range
Particle count512 channels
0.5 µm – 450 µm
Dynamic Light Scattering
0.1 nm – 1 µm
0
5
10
15
0.1 1 10 100 1000
Inte
nsity
(%)
Size (d.nm)
Size Distribution by Intensity
0
5
10
15
20
0.1 1 10 100 1000
Inte
nsity
(%)
Size (d.nm)
Size Distribution by Intensity
Reconstituted powder Liquid formulation
24 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Qualification of a liquid formulation of Omnitrope®
Efficacy and safety of profile of product-related variants confirmed in Phase III studies
Equivalence to lyophilized formulation and reference product in Phase I studies
Toxicology and local tolerance studies, potency in animal model of fresh and aged product
Biological characterization of aged product and product-related variants
Stability studies;characterization of aged product and of product-related variants at end of shelf life
Biological characterization
Preclinical
PK/PD
Clinical safety & efficacy
Physicochemical characterization
25 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Conclusions
• Assessments of comparability are crucial components in the development of any biopharmaceutical product, and specifically so for biosimilar development.
• The extend of a comparability study needs to be based on the magnitude of the introduced change and the knowledge of product and process.
• A set of orthogonal analytical tools is essential to overcome the limitations of single methods and to obtain a complete picture of the quality of the product – biosimilar candidate and reference product.
• The efficacy and safety and the comparability of Omnitrope® with the reference product were shown combining data from physico-chemical, biological, preclinical and clinical studies.
• Data from all these levels were used to demonstrate that the liquid formulations of Omnitrope® have consistent quality characteristics, and are efficacious and safe formulations of somatropin.
26 Comparability assessment in the development of follow-on protein products: Case studies from Omnitrope®/ AP / 2009.01.14
Acknowledgements
Technical team• Sabine Fürtinger
• Bernt Pragl
• Hansjörg Toll
Development concepts and registration• Ajaz Hussain
• Martin Schiestl
• Ingrid Schwarzenberger
• Jörg Windisch