Accelerated Path to Probe Biology through Deferred Cloning and Applying Platform Manufacturing Processes Rohini Deshpande, Ph.D.,
Executive Director Drug Substance Development, Amgen Thousand Oaks, CA
CMC Strategy Forum Europe May 7th, 2014
We need a product development strategy that meets the needs of a Tough Industry Environment
•Pressures on health care budgets
•Heightened payer focus on value
•Declining R&D productivity
•Global regulatory expectations
• Lengthy timelines for product development
•High cost to develop drugs due to low success rate
Objectives of Accelerated Process Development
• Reach ‘go/no go’ decisions early by probing biology in humans
• Enhance first-to-market probability
• Increase pipeline capacity
• Generate representative material efficiently
• Allow phase appropriate PD and Reg CMC investment
Can we shorten cycle time by moving to a dual cycle approach for cell line development?
Legacy Process: Opportunity for Phase Appropriate Approach
Molecule Assessment
FIH process development
Clinical Production
Phase I/II studies
Commercial Process
Development
Clinical Production
Pivotal studies
IND
Project Strategy Team
INDa
Cell Bank 1 and Process 1 Cell Bank 1 and Process 2
•Dual cycle investment for drug substance process development (P1 for FIH to P2 for commercial) •Single cycle approach for cell line development (commercial ready cell lines at FIH – opportunity?)
Generating phase appropriate clinical material earlier: Dual Cycle Cell Line Development
Transfections, selection and amplification
Cell Pools
Single cell cloning
Final clone selection
Toxicology and early phase
clinical studies
Pivotal studies and commercialization
Cell bank 1
Cell bank 2
Deferred Cloning
A
B
CA
B
C
A
B
CA
B
C
D
E
A
B
CA
B
CA
B
CA
B
C
A
B
CA
B
CA
B
CA
B
CA
B
CA
B
C
D
E
Compose and test
Cell line and
process selection
Purification
process definition
Database In silico designs
Process Dev. Platform and High Throughput Tools
Candidate selection to FIH: platform process for representative material
Platform verification/set point definition & supply
For Internal Use Only. Amgen Confidential. 7
Deferred Cloning and platform application reduces time to IND by ~8 months
Phase appropriate approach saves time for Tox and FIH material supply
Molecule, methods, process, and
formulation
Cell bank 1 and DS
manufacturing
Lead
Molecule selection
Cell bank1 and DS
manufacturing
Lead
Methods, process, and
formulation
Accelerated approach
Traditional approach
For Internal Use Only. Amgen Confidential. 8
Deferred Cloning & Comparability
•Is the product quality data for material generated from cell pools and clones comparable? •Does it pose a risk to comparability?
Phase I/II studies
CPD Clinical
Prod Pivotal studies
Molecule, process,
formulation, and methods
Tox and Clinical
Prod
IND PST
INDa
Accelerated approach
Process 1 and Cell Bank 1 • Material generated
from cell pools
Process 2 and Cell Bank 2 • Material generated
from clonal cell lines
For Internal Use Only. Amgen Confidential. 9
Cell bank 1 from cell pools: Measures for fit-for-use
•Plasmid quality is controlled and sequencing performed on single isolates •Multiple transfections are performed to ensure many parental cell pool options •Product quality attributes are tested using sensitive LC/MS/MS methods to detect microheterogeneity and confirm primary sequence •Product consistency is achieved by using defined population doublings for each run (one vial thaw/run) •ICH guidelines are followed for cell bank genetic characterization (DNA sequencing), release testing, and adventitious agent testing
Cell Bank 1 created from a cell pool will follow the same testing paradigm (no change is proposed)
Purpose Tests CB1
Identity Isoenzyme analysis X
Safety Sterility X
Mycoplasma X
In vitro viral assay X
In vivo viral assay X
Transmission electron microscopy of cells X
Mouse antibody production X
Hamster antibody production X
Cocultivation with reverse transcriptase and focus endpoints for retrovirus
detection
X
In vitro assay for bovine viruses* X
In vitro assay for porcine viruses* X
Genetic
stability
cDNA sequence confirmation X**
Expression system is designed to generate stable cell pools & clones
• Antibody and selection marker are produced as a single mRNA
• Only cells with functional selection marker survive • Provides a tight linkage between antibody light and heavy
chain genes and the survival gene i.e. they exist as part of the same mRNA
• Leads to the creation of highly productive and
homogeneous stable pools which can be used for large-scale production
Cell Pool Productivity & Homogeneity
*Flow cytometry Assay
GMD_30min.fcs
APC-A
Count
100
101
102
103
104
0
1
3
4
5
Non-expressing population
Expressing Cells
Freq
uen
cy
Antibody Secretion*
HC+LC
Amgen Expression System Alternate Expression System
LC
HC
APC-A
Count
100
101
102
103
104
0
16
32
47
63
Cell pool
Antibody Secretion*
Freq
uen
cy
Mock pool
Homogeneity of Cell Pools is an Enabler for the Accelerated Approach
Antibody Secretion
Similar FACS profile between cell pool and clones
Grey- Pool Red- Clone 1 Blue- Clone 2
Efficient generation of tox and GMP supply
0
4
8
12
16
GITR BAFF IL2-Mutein
(g
/L)
Titer
0
200
400
600
800
0 2 4 6 8 10 12 14 16 Culture Duration (Days)
VCD
High titers from cell pools allows use of a single lot for tox and FIH supply
Cell pools
•Charge variant
•Clipping
•Identity
•Glycans
•Chemical modification
•Disulfide integrity Total Ion Chromatogram
Extracted Ion Chromatograms
Modality independent platform analytical technology
Digest
Proteins
Analytical methodologies allow detection of sequence variants from cell pools
Multi-attribute method
• Change from profile to attributes • Utilize Orbitrap MS
• Detect low level protein micro-heterogeneity
• Quantify desired attributes • Detect Unknowns • Fully automated
• Suitable for product disposition
• Replaces conventional methods
• Fewer DS/DP release/stability testing
No sequence variants were detected in 3 pilot programs from material produced using cell pools
Cell pools & clones: similar PQA ranges %
a-f
uco
syla
ted
%
HM
W
% H
igh
Man
no
se
% G
alac
tosy
lati
on
(A
2G
1F)
Cell Pools Clones Cell Pools Clones
Cell Pools Clones Cell Pools Clones
Over 20 cell pools are generated for diversity and over hundred clones are screened to ensure comparability
Molecule 1: Comparable PQAs for material from cell pools & clones
Assay Attribute Cell pool Clone FIH Spec (DS/DP)
Purity
Size Exclusion
HMW 1.3 0.8 < 5%
Ion-exchange
Acidic 26.4 17.3
± 10% Main 60.4 76
Basic 13.1 6.6
rCE-SDS
LC+HC 97.3 97.3 ≥ 95%
LMW 0.1 0.1 N/A
NGHC 2.2 2.3 N/A
Glycan Afucosylation 2.5 1.6
N/A High Mannose 8.52 5.7
Impurity CHOP ng/mL 42 17 < 200 ppm
DNA pg/mg N/A < 0.2 10 ng/dose
Activity Bioassay Potency 75 88 60-140%
*Difference in CEX acid and main is from change in harvest operation
Molecule 1: Comparable Product Quality for material from cell pools & clones
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
Minutes
10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00 125.00 130.00 135.00 140.00 145.00 150.00 155.00 160.00 165.00
Cell pool clone
• Peaks aligned for visualization • No significant new peaks or peak changes. • Slight differences in missed cleavage, minor PTMs, and glycosylation
Assay Attribute Amplified pool
(3-150) Clone*1 Clone*2
FIH Spec (DS/DP)
Purity
Size Exclusion HMW 1.2 2.1 2.7 < 5%
Ion-exchange
Acidic 13.2 15.2 15.7
± 10% Main 74.0 72.0 67.9
Basic 12.8 11.6 14.1
rCE-SDS
LC+HC 98.1 97.8 97.6 ≥ 95%
LMW 0.0 0.0 0.0 N/A
NGHC 0.8 0.8 1.0 N/A
nrCE-SDS
Pre-Peaks 5.5 5.5 4.8 < 5%
%HH 2.1 1.4 2.2 N/A
%HHL 1.6 1.9 1.7 N/A
Glycan
% Afucosylation 1.5 3.0 3.9
N/A % High Mannose 4.0 3.8 4.2
% Galactosylation 7.1 8.6 9.0
Impurity CHOP ng/mg 50 < 200 ppm
DNA pg/mg 0.7 < 10 ng/dose
Potency Binding % 107 60-140%
Molecule 2: Comparable PQAs for material from cell pools & clones
Assay Attribute Cell pool Clone 1 Clone 2 FIH Spec (DS/DP)
Purity
Size Exclusion
HMW 4.1 3.5 3.4 < 5%
Ion-exchange
Acidic 11.9 11.2 24.5
± 10% Main 65.8 65.3 54.6
Basic 22.3 23.4 20.8
rCE-SDS LC+HC 97.9 98.5 98.3 ≥ 95%
nrCE-SDS Pre-Peaks 1.7 3.2 3.1 < 5%
%Main 98.3 96.8 96.9 N/A
Glycan
% Afucosylation 1.6
1.3 1.74
N/A % High Mannose 6.4
7.4 10.7
% Galactosylation 13.1
11.9 20.8
Molecule 3: Comparable PQAs for material from cell pools & clones
0
1
2
3
4
5
6
PreMCB MCB Start of Production Bioreactor
End of Production Bioreactor
Titer
VCD
High Mannose
Galactosylation
Molecule 3: similar PQA profile from vial thaw to end of production bioreactors for cell pool and clones
A-Fucosylation
HMW Pool Clone
• Both cell sources performed similarly: • Cell Culture Process
• Purification Process
• Product Quality Attributes
• Purity /Impurity
• Stability
• Manufacturing and Quality controls are used to ensure no/low risk to patient safety and product consistency
• Next steps –
• Engagement and feedback from the regulatory agencies
• Application to molecules amenable to platform
• Molecules with less complex product quality attributes
• Risk based approached to comparability
In Summary, Product Quality from Cell Pools is comparable to clones
Biosimilars
Different:
Facility and equipment
Cell-line, vector and culture media
Fermentation/culture conditions
Downstream
processing/purification
Formulation and container/closure
Out of scope of comparability guidance (ICH Q5E)
Covered by EMA, FDA, and WHO Biosimilar Guidelines
[Adapted from presentation by G. Grampp (Amgen); Manufacturing Changes in the Era of Biosimilars,
BioProcess International Conference, November 2, 2011, Long Beach, CA]
Biosimilars
Change
filter
Raw
material
supplier
Replace
equipment
Site
Change
Change
cell
culture
media
Cell line
or
formulation
change
Nature of
Process
Change
Risk & Data
Requirements Low Risk
Commonly
implemented
- Analytical data
- Process studies
Moderate Risk
- Analytical data
- Process studies
- Stability data
High Risk
Less commonly implemented
- Analytical data
- Process studies
- Stability data
- Clinical data
Governed by comparability guidance (ICH Q5E), Para 2.2 of CHMP1
Innovator Process Change
Biosimilar
development requires
most if not all changes
“Abbreviated”
comparability
“Comprehensive” comparability
Biosimilar Development Transcends the Highest Risk Category of Process Change; experience can be applied to advancing NMEs
Acknowledgements
Discovery Research
Translational Sciences
Clinical Operations &
Operations Technology
Regulatory Affairs (CMC)
Product Quality
Questions