This document discusses strategies for orphan biopharmaceutical process development using contract development and manufacturing organizations (CDMOs). It notes that orphan biopharmaceuticals often require smaller scale and more flexible manufacturing. The document outlines considerations for clinical and commercial process development, including using single-use technologies, quality by design principles, and ensuring fidelity between clinical and commercial manufacturing processes. It emphasizes characterizing processes early and getting the process design right the first time for orphan drugs.

1. Orphan Biopharmaceuticals
&
the CDMO
(Contract Development and
Manufacturing Organization)
Abhinav A. Shukla, Ph.D.
Vice President, Process Development &
Manufacturing
KBI Biopharma, Durham NC
Presented at: World Orphan Drug Congress, Washington DC, April 9-11, 2013

2. Why are orphan biopharmaceuticals unique?
• Smaller material demand
• Fewer clinical batches  reduced large scale manufacturing
experience prior to BLA/MAA filing
Flexible manufacturing at a smaller scale (< 2000L cell
culture volumes) needed (Single-Use Manufacturing
Technologies)
Increased focus on process knowledge from scale-down
experimentation (QbD)
• Limited ability to do clinical bridging studies
• Process changes during clinical development are less
desirable since their clinical impact can often not be studied
readily
Getting the process right the first time (Building robustness
and scalability into the process right from the start)

3. -Confidential-
Biologics Commercialization
Pre-Clinical Phase I Phase II Phase III
Process Development
Process
Characterization
Process
Validation
Process Monitoring
& Improvement
FIH Process
• Deliver clinical process
quickly
• Platform process
• Clinical Supply
Submission &
Approval
Lifecycle
management
BLA Prep &
PAI
Commercial Process
• Deliver manufacturing process for
registrational trials and market
• Design keeping large-scale manufacturing in
mind
• Improve productivity, efficiency, robustness,
manufacturability, COGs
• Analytical characterization and method
development
Process Characterization and Validation
• Develop IPC strategy through understanding of process inputs and
outputs (design space)
• Scale-down characterization and validation studies
• Large-scale process validation to demonstrate process consistency
• BLA preparation
• Supporting documents for licensure inspections
• Post-commercial process improvements (CI)
• Post-commercial process monitoring
FIH process Commercial process
Gottschalk U., Brorson K., Shukla A. Nature Biotechnology, 30(6), 489-491, 2012

4. -Confidential-
Biologics Commercialization
Pre-Clinical Phase I Phase II Phase III
Process Development
Process
Characterization
Process
Validation
Process Monitoring
& Improvement
FIH Process
• Deliver clinical process
quickly
• Platform process
• Clinical Supply
Submission &
Approval
Lifecycle
management
BLA Prep &
PAI
Commercial Process
• Deliver manufacturing process for
registrational trials and market
• Design keeping large-scale manufacturing in
mind
• Improve productivity, efficiency, robustness,
manufacturability, COGs
• Analytical characterization and method
development
Process Characterization and Validation
• Develop IPC strategy through understanding of process inputs and
outputs (design space)
• Scale-down characterization and validation studies
• Large-scale process validation to demonstrate process consistency
• BLA preparation
• Supporting documents for licensure inspections
• Post-commercial process improvements (CI)
• Post-commercial process monitoring
FIH process Commercial process
A single development cycle
Robust and complete
process characterization
package
Commercial manufacturing at smaller scales

5. -Confidential-
SINGLE-USE
MANUFACTURING
TECHNOLOGIES

6. Why are single-use manufacturing
systems growing?
• Lower capital and utility costs (up to 40% reduction*)
• Increasing titers driving bioreactor scales smaller
• Single-use bioreactors now up to 2000L volume
• Increased universalization of biomanufacturing
• Co-location of manufacturing with markets
• Biosimilars (estimated $ 17 billion market by 2020)
• Smaller market sizes for novel drugs in niche/personalized
applications
• Market fragmentation making large single-product
manufacturing facilities redundant
• Single-use systems finding application in stainless
steel facilities for enhanced operational flexibility
Laukel et al, BioProcess International, May 2011 Supplement, pp. 14-21.

7. -Confidential-
Media and Feed preparation utilizing disposable
mixing, filtration and storage systems
Disposable shake flasks or
disposable spinner flasks
MCB or
WCB vial
Disposable expansion
reactor
Disposable
seed bioreactor
Disposable production
bioreactor
Disposable fluid path
centrifuge
Disposable depth
filtration system
0,2 µm
filter
Hold vessels
(Bags)
Hold vessel
(bag)
Disposable fluid path
purification system
Disposable
mixing tank
0,2 µm
filter
Retentate
Permeate
PD
Disposable fluid path
purification system
Disposable
mixing tank
0,2 µm
filter
BPC
Virus
filter
BPC
0,2 µm
filter
BPCBPC
Sterile bulk fill and
sampling bags
Buffer preparation utilizing disposable mixing,
filtration and storage systems
0,2 µm
filter
Disposable fluid path
UF/DF system
Aseptic
connection
Hold vessel
(bag)
Hold vessel
(bag)
Hold vessel
(bag)
Hold vessel
(bag)
Hold vessel
(bag)

8. Process Reproducibility
4 manufacturing runs in
Single Use Bioreactors
Highly consistent process

9. -Confidential-
Scalability
•4 different scales
•3L and 15L scales in
non-disposable
bioreactors
•Process performance
with different working
volumes is also
reproducible

10. Single-use technologies in downstream processing
• Centrifugation (kSep® Systems)
• Closed, continuous centrifuge with class VI product contact
surfaces
• Counteraction of Centrifugal force and fluid flow force
• Very low shear
• Continuous operation
• Reversal of flow direction
empties the chamber
• Up to 7.2 L/min

11. Single-use technologies in downstream processing
• Depth filtration:
• Harvest depth filters have traditionally been single-use except for
their holders
• Based on particle entrapment in a fibrous bed
• Can be used as the primary cell separation step for smaller cell
culture harvest volumes
• Millipore – POD® system
• Pall - Stax® system
• Sartorius – Sartoclear P ®
• Cuno – Zeta Plus ®
Pall – Stax System
Millipore - POD

12. Single-use technologies in downstream processing
• Chromatography
• Membrane adsorbers
• Mustang® (Pall), Sartobind® (Sartorius), Chromasorb® (Millipore),
Adsept® (Natrix),
• Q, S, HIC and salt-tolerant ion-exchange functionalities
• Most widely used for trace impurity removal in a flow-through mode
(DNA, endotoxin, viral clearance)
• Pre-packed chromatography columns
• ReadyToProcess (GE Healthcare), Opus (Repligen), GoPure (Life
Technologies)
• Monoliths
• CIM monoliths (BIA Separations), Uno monoliths (Biorad)
Up to 20 cm D
available

13. Clinical and commercial manufacturing
using single-use technologies
• Smaller material demand drives reduced scale for
commercial manufacturing
• Fidelity between clinical and commercial product
needed (ideally single facility that fits both needs)
• Single-use manufacturing technologies reduce costs
and reduce risk of cross-contamination

14. -Confidential-
Shukla, A., Mostafa, S., Wilson, M., Lange, D. Vertical Integration of Disposables in
Biopharmaceutical Drug Substance Manufacturing, Bioprocess International, 10(6), 34-47,
2012.
Gottschalk, U., Shukla, A. Single-use disposable technologies for biopharmaceutical
manufacturing, Trends in Biotechnology, 31(3), 147-154, 2013.

15. -Confidential-
QUALITY BY DESIGN (QBD)

16. -Confidential-
Quality by Design (QbD)
• “Quality by design means designing and developing
manufacturing processes during the product
development stage to consistently ensure a
predefined quality at the end of the manufacturing
process.” ICH Q10, FDA 2006
Process Design
(Process Development)
Process
Control
Strategy
Definition
Process
Validation
Continued Process
Verification

17. QbD
Critical Quality
Attributes (CQAs)
Process Design
Space
Linking CQAs to
Clinical outcome

18. -Confidential-
Process design space
Characterization Space
Control Space
Operating Range
Acceptable Range
Design Space
Process
Parameters
Key
Parameters
CPPs

19. -Confidential-
Integrative Approach
Each step is influenced by the preceding step
 Shake flask and seed bioreactor parameters may affect growth rate in the
seed bioreactor.
 Seed bioreactor and production bioreactor parameters may affect
productivity and critical quality attributes.
 Production bioreactor parameters may affect downstream steps.
 Characterization studies are linked.
Vial
Thaw
Shake Flasks Seed
Bioreactor
Production Bioreactor
Downstream Steps
Biotechnology and Bioengineering, 106(6), 894-905, 2010.

20. Production Bioreactor

21. -Confidential-
Establishing A Process Control Strategy

22. -Confidential-
Application of Quality by Design (QbD):
downstream resin lot variability
Biotechnology and Bioengineering, 107(6), 989-1001, 2010.

23. -Confidential-
Evolving expectations in Process Validation
• Q7A definition: “Process validation is the documented evidence that the
process, operated within established parameters can perform effectively and
reproducibly to produce an intermediate or API meeting its predetermined
specifications and quality attributes”
• FDA guidance, Jan 2011: “The collection and evaluation of data, from the
process design stage through commercial production, which establishes
scientific evidence that a process is capable of consistently delivering quality
product”
• Process validation is now viewed as a process that occurs throughout the
lifecycle of a product
Process Design
(Process Development)
Process
Control
Strategy
Definition
Process
Qualification
Continued Process
Verification

24. Scale-Down Process Validation Studies
• Scale-down validation studies in addition to large-
scale process validation (conformance lots)
• Probe extremes in the process and demonstrate them
to be acceptable
• Examples
• Reprocessing validation – combine hold times with process
conditions that create the greatest stress on the protein
• Intermediate hold times – combine hold times and
demonstrate releasable drug substance
• Viral clearance studies
• Impurity clearance studies

25. -Confidential-
Validation of Host Cell Protein Clearance
Harvest
Column 1
Column 2
Column 3
Worst-case
C1 eluate
Worst-case
C2 eluate
Harvest
Column 1
Column 2
Column 3
Harvest
Column 1
Column 2
Column 3
Spiking Strategy
• Some CHOP species in harvest
may not be encountered by C2
and C3 in Mfg
• LVR could be overstated for C2
and C3
Worst-case Strategy
• CHOP species in eluate is relevant
to the next step
• More accurate evaluation of LRV
• Need process characterization to
identify worst-case condition
By-pass Strategy
• HCP species in load are relevant to
that process step in case the
previous step is by-passed (e.g.
“resin bed channeling”)
• Represents most “challenged”
scenario
Biotechnol. Progr., 24(3), 615 – 622, 2008
Worst-case
harvest

26. Development Phase
• Utilizing the right set of analytical tools for in-process
testing and release
• Characterization assays are equally important
• Utilizing a broad set of tools up front gives the best
chance of determining CQAs & linking them to the
process

27. -Confidential-
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
• Particle measurements
• Visible & sub-visible particles
Comprehensive Analytics

28. -Confidential-
THE RIGHT SCIENCE FROM
THE START

29. Designing more efficient HCP clearance
into the downstream process
• Most current chromatographic steps are designed to
remove impurities based on differential binding to the
stationary phase surface
• Conventional wisdom: wash conditions are between
binding and elution conditions
• Orthogonal approach  disrupt impurity-product
interactions
Washes that
disrupt
protein-protein
interactions
Conventional washes

30. 30
Enhancing HCP clearance across Protein A
• HCPs form a diverse set of impurities
• HCP clearance is a key concern in biopharmaceutical
separation processes

31. -Confidential-
Washes can be developed to disengage HCPs from the product
rather than disrupt product-Protein A ligand interactions
96
11635
9243
34655
935491
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Null supernatant MAbSelect
eluate (load =
null
supernatant)
MAbSelect
eluate (load =
null supernatant
+ product)
Prosep A eluate
(load = null
supernatant)
Prosep A eluate
(load = null
supernatant +
product)
HostCellProteins(ng/mL)
Normalized Yield vs. normalized CHOP for a
variety of washes on MAbSelect Protein A
0%
20%
40%
60%
80%
100%
120%
140%
0% 20% 40% 60% 80% 100% 120%
Yield normalized to control experiment
CHOP(ppm)normalizedto
controlexperiment
Direction of
desired
trend
Biotechnology Progress, 24, 1115-1121, 2008.
Do HCPs co-elute with the product or co-associate with the
product?
Enhancing HCP clearance across Protein A

32. Enhancing HCP clearance across Protein A
• Use washes at high pH (pH > 7) to preserve Protein A –
mAb interactions
• Include selective modulators (moderate concentrations of
urea, ethylene glycol, salts, arginine) in washes to disrupt
HCP-mAb interactions
Shukla, A., Hinckley, P. Host cell protein clearance during Protein A resin chromatography: development of an
Improved wash step, Biotechnology Progress, 24, 1115-1121, 2008.
Evaluation of intermediate washes at pH > 7.0
0%
20%
40%
60%
80%
100%
120%
140%
0% 20% 40% 60% 80% 100% 120%
Normalized yield % of control
NormalizedCHOP
(%ofcontrol)

33. Mixed Mode Chromatography
• Takes advantage of more than one type of interaction
• Can reduce process steps
• Provides enhanced selectivity, “pseudo-affinity”
• Several mixed mode resins have recently been developed with:
» Increased loading capacities
» Higher ionic strength tolerance
+
+ +
+
+Mixe
d
Mode
GE Healthcare, Capto MMC ligand
Ionic interactions
Hydrophobic interactions
Hydrophobic interactions
Ionic interactions
GE Healthcare, Capto Adhere ligand

34. Log k’ vs Log [NaCl]
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
2.60 2.80 3.00 3.20 3.40 3.60
Logk'
Log [NaCl]
Lysozyme
pH 7.0
1M urea
5% ethylene glycol
50mM arginine
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.50 2.00 2.50
Logk'
Log [NaCl]
RNase
pH 7.0
1M urea
5% ethylene glycol
50mM arginine
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
2.10 2.30 2.50 2.70
Logk'
Log [NaCl]
Monoclonal antibody
pH 7.0
1M urea
5% ethylene glycol
50mM arginine

35. Wash development on mixed mode
0
50
100
150
200
250
300
350
400
450
500
0.0% 20.0% 40.0% 60.0% 80.0% 100.0%
HCP(ppm)
Recovery
Capto MMC HCP Clearance
25mM Tris pH 7.0 (baseline)
25mM Tris pH 7.0, 5% ethylene glycol
25mM Tris pH 7.0, 50mM arginine
25mM Tris pH 7.0, 50mM NaSCN
25mM Tris pH 7.0, 1M urea
25mM Tris pH 7.0, 1M ammonium sulfate
25mM Tris pH 7.0, 0.1M NaCl
25mM Tris pH 7.0, 0.5M ammonium sulfate
25mM Tris pH 7.0, 0.1M NaCl, 1M urea
25mM Tris pH 7.0, 0.1M NaCl, 1M urea, 5% ethylene glycol
25mM Tris pH 7.0, 0.1M NaCl, 1M urea, 5% glycerol
• Selective wash strategies can eliminate one
chromatographic step in non-mAb processes
• Designing quality into the process

36. Designing processes with the end in mind
• Having the right analytical methods and product quality
profile in mind from the start
• Keeping issues that can be encountered in large-scale
manufacturing in mind from the beginning
Process yields &
robustness
Titer & downstream yields
Reproducibility
Column loading and
buffer needs
Column loading drives
costs!
Raw material selection
Potential for variability
Supply assurance
Compatible with cGMP
Process impact
Transfer ready
processes
Processes that can be
compatible with many
scales and facilities

37. Conclusions
• Orphan biopharmaceutical development needs
particular emphasis on
• Developing a process with the end in mind (licensure filing)
to avoid multiple changes along the way
• Manufacturing costs
• Demonstrating process robustness without recourse to an
extensive manufacturing history
• A dedicated CDMO with the right knowledge and
capabilities can help smooth the development
pathway