Strategies for rapid acceleration of cell line, upstream and downstream process development. A presentation by Ying Huang, Ph.D., Associate Director of Cell Line Development at KBI Biopharma. Presented at World Orphan Drug Congress. Washington DC. (2014)

1. -Confidential-
One
and
Done
–
Ge+ng
Biopharmaceu5cal
Produc5on
Processes
Right
the
First
Time
Ying
Huang,
Ph.D.
Associate
Director,
Process
Development
KBI
Biopharma,
Durham
NC
Presented
at:
World
Orphan
Drug
Congress,
Washington
DC,
April
25,
2013

2. -Confidential-
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
Biologics
Commercializa5on
2

3. -Confidential-
Approaches
to
Get
Every
Step
Right
at
the
1st
Time?
• Screen
the
best
protein
candidate
construct
by
transient
transfec3on.
• Iden3fy
the
stable
high
producing
clones
within
a
short
3meline
• Pool
enrichment
with
FCAS
to
achieve
a
good
heterogeneous
pool
• Single
cell
cloning
with
ClonePix
to
improve
selec3vity
• Define
the
op3mized
cell
culture
process
by
using
high
throughput
microbioreactor
with
rapid,
accurate
3ter
and
product
quality
assessment.
• Develop
an
efficient
downstream
purifica3on
processes
using
selec3ve
column
washes.
3

4. -Confidential-
Cell
Line
Development
–
Ini5a5on
of
Biologic
Produc5on
4
Vector
Construc5on
Transfec5on
&
Selec5on
(Amplifica5on)
Pool
Enrichment
Clone
Isola5on
&
Screening
Stability
Assessment
Strong
vector
w/
enhance
element
Transient
Transfec5on
Gene
Stable
Pool
FACS
High
throughput
cloning
(FACS
or
ClonePix)
Earlier
material
generaHon
Stable
Cell
Line
GeneraHon
Gene
codon
op5miza5on
Valid
Clone
Screening
Process
CSI
Process
Development
Process
Development
Cell
Bank

5. -Confidential-
Decide
the
Right
Molecule
with
Transient
Gene
Expression
(TGE)
5
7
to
14
days
Large
Amount
of
Star5ng
Cells
(K-­‐Sep)
Large
Scale
FB
Culture
for
Produc5on
(Wave)
Benefit
of
TGE
Ø
Screening
candidate
molecules
–
lock
the
best
construct
Ø
Fast
material
genera3on
–
analy3c
method
development
0
1
2
3
4
5
6
7
8
Titer
Culture
Days
GOI_1
GOI_2
GOI_3

6. -Confidential-
Pool
Enrichment
with
FACS
6
#3.
2nd
Round
of
Enrichment
Sorting
#2.
1st
Round
of
Enrichment
Sorting
#1.
Original
Population
Gate1
Using
the
FACS
Jazz
instrument
to
sort
the
top
5%
of
the
popula5on
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Original
Round
1
Enriched
Round
2
Enriched
Fold
Increase
Pools
Pool
Produc5vity
Assessment
(
7-­‐day
batch
culture)
Time:
~
2
weeks
Ø FACS
allows
sor5ng
of
high
producing
cells
to
an
enriched
pool,
which
provides
a
be_er
pool
for
earlier
material
genera5on
and/or
cloning.

7. -Confidential-
• Automated
Colony
Picking
Robot
• High
throughput
• Asep3c
• Fluorescent
Technology
for
Selec3ve
Cloning
• Ranks
and
selects
only
the
highest
producing
clones
0
5000
10000
15000
20000
25000
30000
35000
40000
1G7
1E10
1C12
1F10
2B6
2A8
1D12
1B12
1A8
1A4
1G5
1F7
2B1
2B9
2A1
2A6
1G4
1G12
1B5
2A2
2A4
1B8
1H7
2G8
2A9
1F1
2B5
1B9
2G11
1A2
1A5
1F3
2D2
1C4
2F12
1F9
2B2
2E11
2G10
1F2
2A10
1E5
2E10
1H5
1E8
1F8
1C2
1B11
1E11
1A6
2F10
2E4
2C12
2E8
1F4
1H8
2F1
1F5
2G12
2F5
2G7
2G2
1F12
1B4
2A12
2A11
2E3
1E6
2C2
1C10
2F9
2F4
1G8
2F3
1E9
1E2
1C5
1H9
2F2
2B10
2E5
2E12
1G2
2E2
1E1
1E4
2E1
1H4
2F8
2E9
2B8
2F11
1A9
2A3
2H8
1C11
Colony
Titer(PDU/mLL)
Semi-­‐Solid
Matrix
with
Conjugated
An3body
Colony
(White
light)
Halo
(Florescent
light)
Image
Plane
High
throughput
Cloning
with
ClonePixTM
7

8. -Confidential-
100%
is
the
1ter
of
best
clone
from
ClonePixTM
ClonePix vs LDC
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Clone
RelativeRanking(%)
ClonePix Top 30
LDC Top 30
ClonePixTM
Can
Isolate
Be_er
Clones
with
Less
Effort
than
LDC
Clones
RelativeTiter(%)
ClonePixTM
Top
30
Clones
LDC
Top
30
Clones

9. -Confidential-
Run
specific
assays
for
protein
glycan
and
protein
charge
variant
for
harvest
samples
with
turnaround
of
1-­‐2
days.
§ Use
24
miniaturized
Bioreactors
(10-­‐15mL
working
volume).
§ DOE
design
of
factors
(pH,
DO,
temperature,
feeds
volume
and
frequency).
§ Perform
daily
analysis
to
monitor
cell
growth
and
key
metabolites.
ambrTM
ProA-­‐based
3ter
analysis
of
in
process
cell
culture
samples
with
quick
turnaround
of
same
day.
Ø Cell
growth,
cell
viabili3es,
metabolite
profiles
For
each
tested
condi5on
Ø Titers
and
Product
Quality
+
Selec3on
of
best
process
condi3on(s)
with
respect
to
cell
growth,
produc3vity
and
product
quality.
Integrated
High
throughput
Process
Development
and
Analy5cal
Support
9
forteBIO
Octet
Caliper
LabChip
GX
II

10. -Confidential-
Cell
Culture
Process
Op5miza5on
with
ambrTM
–
Growth
Profile
10
Ø Real
5me
monitor
of
cell
growth
profiles
by
tes5ng
mul5ple
culture
condi5ons
like
pH,
temperature,
DO
level
and
feeds.

11. -Confidential-
Cell
Culture
Process
Op5miza5on
with
ambrTM
–
Produc5vity
11
Ø
Produc5vity
and
protein
quality
(data
not
shown)
reported
by
high
throughput
assays
with
a
quick
turnaround
5me.

12. -Confidential-
A
CHO
cell
line
producing
a
recombinant
glycoprotein
Cell Growth
Titers Product Quality Attributes
Scalable
Model
Micro-­‐bioreactor
:
ambrTM
12
Ø The
process
decisions
and
results
from
ambrTM
were
reproducible
to
other
tradi5onal
bioreactor
scales
(10
L
and
200
L).

13. -Confidential-
Taking
Advantage
of
Modulators
in
Downstream
Purifica5on
• Incorpora3on
of
modulators
into
process
can
help
increase
selec3vity
and
purity
of
product
• Combina3ons
of
modulators
can
further
enhance
process
step
• Goal:
U3lize
mobile
phase
modulators
to
decrease
HCP
levels
during
Capto
MMC
process
step
for
an3body
purifica3on

14. -Confidential-
Incorpora5ng
Modulators
into
Wash
steps
• Case
Study:
» Target
molecule:
E.
coli
derived
recombinant
protein
» Process
step:
Phenyl
Sepharose
FF
» Ini1al
product
yield:
88.8%
» Result:
– Individual
modulators
showed
some
selec3vity
enhancement
but
also
product
loss
– A
combina3on
of
urea,
sodium
thiocyanate
and
glycerol
in
the
wash
step
increased
product
purity
to
>95%
Shukla AA, et al., 2002. Biotechnol Prog 18: 556–564.

15. -Confidential-
0.50
0.70
0.90
1.10
1.30
1.50
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
Normalized
HCP
Recovery
HCP
vs.
Recovery
aier
Intermediate
Wash
for
Capto
MMC
Capture
baseline
During
Capture
step
Wolfe LS, et al., 2014. J. Chromatogr. A 1340: 151-156.

16. -Confidential-
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
Normalized
HCP
Recovery
HCP
vs.
Recovery
aier
Intermediate
Wash
for
Capto
MMC
Polishing
baseline
During
Polishing
step
Wolfe LS, et al., 2014. J. Chromatogr. A 1340: 151-156.

17. -Confidential-
Process
Impact
on
HCP
Clearance
of
Using
Selec5ve
Column
Washes
• Inclusion
of
an
intermediate
wash
using
Tris,
0.1M
NaCl,
50mM
arginine,
5%
ethylene
glycol,
pH
7.0
resulted
in
2-­‐fold
lower
HCP
levels
when
compared
to
process
where
a
modulator
was
not
u3lized.

18. -Confidential-
Conclusions
• Orphan
biopharmaceu3cal
development
needs
par3cular
emphasis
on
• Genera3ng
a
high
producing
cell
line
with
short
3meline
» Earlier
material
genera1on
» Iden1fying
high
producer
clones
with
an
integrated
approach
• Developing
a
process
with
the
end
in
mind
(licensure
filing)
to
avoid
mul3ple
changes
along
the
way
» Op1mizing
cell
culture
process
with
high
throughput
technologies
• U3lizing
mixed
mode
chromatography
to
improve
product
purity
and
maintain
process
step
yield
• A
dedicated
CDMO
with
the
right
knowledge
and
capabili3es
can
help
smooth
the
development
pathway

19. -Confidential-
Acknowledgments
• Abhinav
Shukla,
Ph.D.
Vice
President,
Process
Development
&
Manufacturing
• Michael
Cavanaugh
Vice
President,
Business
Development
• Sigma
Mostafa,
Ph.D.
Director,
Process
Development
• Shahid
Rameez,
Ph.D.
Process
Development
Scien3st
II,
Upstream
Process
Development
• Leslie
Wolfe,
Ph.D.
Process
Development
Scien3st
II,
Downstream
Process
Development
• Rich
Harper,
B.S.
Process
Development
Scien3st
I,
Cell
Line
Development