Handling High Titer Processes and Strategies for DSP Facility Fit. Originally presented at BioProcess International 2018 by Christopher Miller, Senior Scientist, Downstream Process Development, KBI Biopharma.

1. Christopher Miller

2. Confidential
2
KBI was the first CDMO in the world to implement 2,000L SUB for cell culture & associated
downstream equipment
Success track record in mAb and non-mAb drug substance manufacturing since 2010
KBI now has two cGMP manufacturing train housing three 2,000L SUBs
Train 1: Clinical Manufacture (200L and 2,000L SUBs)
Train 2: Commercial/Clinical Manufacture (Two 2,000L SUBs)
Recent KBI Cell Culture Manufacturing Experience
Over 40 different products manufactured to drug substance
Over 50 cGMP batches delivered to the clinic in the last 2 years
Mammalian Cell Culture cGMP Manufacturing

3. Facility fit challenges for a single-use CDMO biologics manufacturer primarily arise from:
All projects must fit into the manufacturing facility despite these challenges
AND meet desired manufacturing cadence
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Variety of molecules
- mAbs
- mAb fusions
- Bispecific mAbs
- Fc fusions
- mAb fragments
- Non-mAb glycoproteins
- Other
Wide range of titers
- << 1g/L
(Low titer, as low as 0.01g/L)
- 1-5g/L
(Common titer range)
- >> 5g/L
(High titer, as high as 10g/L)
Wide range of processing
conditions
- Many projects can be
platform-able
- Other projects do not fit a
platform due to molecule
type / titer / scale variety

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Low (<< 1 g/L) Average (1 – 5 g/L) High (>> 5 g/L)
- Material generation
- Scale limitations for MFG
- Off-platform development
- Scalability risk
- Molecule specific
- Often highly accelerated
- Processing time in MFG
- High mass facility fit limitations
(tank size, pump capacity,
space, filter surface area, etc)
- High protein concentrations
- Keep column loads low
- Understand smallest scale
limitations in MFG
- Use smaller ID columns, if
necessary
- Use platform development
- Test capacities necessary to
manufacture at scale
- Focus on impurity removal
- Move quickly into MFG
- DBC for all steps necessary
- Flow rate and volume
optimization
- Filter capacity testing
- Avoid steps requiring dilution /
extended elutions
- Less material available for
development work
- Off-platform processes require
additional work
- Higher risk for scale effects
with very small columns
- Generally low for well behaved
molecules
- Ensuring process
reproducibility for highly
accelerated programs
- Hard to maintain desired MFG
cadence
- Potential for more deviations
using complex pooling
strategies.
Development
strategyChallengesRisks

5. High (>> 5 g/L)
- Processing time in MFG
- High mass facility fit limitations
(tank size, pump capacity,
space, filter surface area, etc)
- High protein concentrations
- DBC for all steps necessary
- Flow rate and volume
optimization
- Filter capacity testing
- Avoid steps requiring dilution /
extended elutions
- Hard to maintain desired MFG
cadence
- Potential for more deviations
using complex pooling
strategies.
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Development
strategyChallengesRisks
• Traditional development / optimization strategies
will only get us so far in maintaining desired MFG
cadence.
• Operating near resin / filter max capacities
• Increasing column sizes / filter surface areas to max limit
for MFG plant
• Procuring larger tanks / scales to accommodate increased
product volume
• Other efficiency gains must be achieved to ensure
all programs will fit into plant
ProA+VI
Cycle 1
ProA+VI
Cycle 2
ProA+VI
Cycle 3
VIN
Pooling
FT step
2 cycles
B/E step
2 cycles
VF UFDF Bulk Fill
ProA+VI
Cycle 1
ProA+VI
Cycle 2
ProA+VI
Cycle 3
ProA+VI
Cycle 4
ProA+VI
Cycle 5
ProA+VI
Cycle 6
VIN
Pooling
FT step
2 cycles
FT step
2 cycles
B/E step
2 cycles
B/E step
2 cycles
VF
2 cycles
UFDF Bulk Fill
Unoptimized DS process for a 2g/L titer program
Optimized DS process for a 7g/L titer program
TargetMFG
completiontime

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• Options for ensuring all high titer programs are able to fit into a facility within desired cadence:
Manufacturing:
1) Increase allotted time for
processing X
2) Additional similar scale facilities
3) New, larger facility
4) Efficiency optimization
Process Development:
1) Alternative high capacity resin / gel
separation methods
2) Non-chromatographic separations
3) Continuous chromatography
4) Fully integrated continuous
processing

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•Process more product mass in downstream from high titers
•Process more volume in downstream from perfusion
bioreactor applications
•Provide additional options / flexibility to clients
Expand Plant
Capacity
•Save on expensive resin(s)
•Save on column hardware
•Save on buffer chemicals and WFI
Reduce COGs
•Use smaller columns
•Use smaller tank sizes
Minimize MFG
Footprint
•More automation
•Less manual operator error
•Perform parallel operations
Improve
Efficiency

8. Continuous chromatography for capture chromatography evaluated
• Goals:
• Determine the extent of potential efficiency gains for a high titer project
• Identify potential hurdles for future implementation
• Approach:
• Select chromatography skid à Pall BioSMB single-use
• Select target molecule à High titer IgG1 mAb
• Select chrom step and resin à Protein A (most costly and time consuming) using Amsphere A3 resin
• Develop SMB methods based on current process conditions
• Execute SMB evaluation studies
• Analyze process and product quality data
• Model actual and predicted efficiency gains
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• Batch chrom load to 80% of 10% BT
• Continuous chrom load to 100% BT,
but capture FT on 2nd column
• Additional capacity can be achieved
• Use a series of smaller columns to
purify product continuously
• Requires different chrom skids to make
this work
Wash,
Elution,
Regeneration
Equilibration

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CadenceBioSMBProcessandPDdisposablevalveblocks

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Resin Vendor Matrix Ligand
Modified Protein A
domain
Mean
particle
size (µm)
MabSelect SuReTM GE Healthcare Highly cross-linked agarose
Alkali-stabilized
rProtein A
B domain 85
MabSelect SuReTM LX GE Healthcare Highly cross-linked agarose
Alkali-stabilized
rProtein A
B domain 85
ToyopearlTM AF-rProtein
A HC-650F†
Tosoh Polymethacrylate
Alkali-stabilized
rProtein A
C domain 45
EshmunoTM A† EMD Millipore Cross-linked Polyvinyl Ether
Alkali-stabilized
rProtein A
C domain 50
AmsphereTM A3† JSR Life Sciences Polymethacrylate
Alkali-stabilized
rProtein A
C domain 50
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
mAb2
DBC(g/L)
Residence Time (min)
MabSelect SuRe
MabSelect SuRe LX
rProtein A HC-650F
Eshmuno A
Amsphere A3
mAb1 mAb2 mAb3 mAb4
0
1000
2000
3000
8000
9000
10000
HCPLevel(ppm)
MabSelec SuRe
MabSelect SuRe LX
rProtein A HC-650F
Eshmuno A
Amsphere A3
• High capacity, caustic tolerant
resin
• Small bead size enables fast
mass transfer kinetics
• Faster loading flow rates
possible while saturating resin
• 5cm bed heights (vs 20cm) used
in continuous capture will keep
pressure low
• Peak DBC at 300 – 500 cm/hr
• Even faster flow rates can be
used with flow through capture
on 2nd
column in series
• Lower cost than other Protein
A resins
• Improves process economics
even further than other
competitive Protein A resins

12. • Batch capture: 1 eluate per cycle
• Ex. 1 x 6 cycles = 6 eluates
• VI for each eluate
• Continuous capture: 1 eluate per column
until all clarified harvest processed
• Ex. 1 x 5 col x 25 = 125 eluates
• Pool subset of eluates for VI
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13. • 76g/L loading achieved at <2 min residence time without any breakthrough
detected.
• Purity of neutralized eluates was on par or better than current batch process
• Yield lower than normal, due to unoptimized elution collection strategy (requires
dev’t for CVs of eluate collection w/out UV)
• Specific productivity significantly improved over batch processing in current
optimized process
NOTES:
- Pressure not an issue for A3 resin at high flow rate due to 5cm BH (vs 20cm)
- Very high A3 resin capacity
- A3 resin is ideal for use in continuous capture chromatography
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14. • Comparison between:
1) High titer mAb run on existing batch MFG process
2) High titer mAb run on BioSMB process
• Conclusion (productivity):
• Significantly higher productivity using BioSMB for
all titer scenarios (as expected)
» Full resin capacity utilization
» A3 resin well suited to high flow rate
» Parallel operations
• Evaluation phase at KBI still on going
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0
10
20
30
40
50
60
70
80
90
100
0.1 1 4 7 10
CaptureProductivity(g/L/hr)
Bioreactor Titer (g/L)
Batch Capture Chromatography SMB Capture Chromatography

15. • Model other scenarios for hypothetical manufacturing runs
• For predictive models to be valid, some data-based assumptions are made:
• 70 g/L load with no breakthrough can be achieved
» Conservative as 76g/L achieved in evaluation without breakthrough
» Molecule specific
• 500 cm/hr can be achieved during loading phase on pump for modeled column ID
• Amsphere A3 resin used
» Fast mass transfer kinetics allow for full resin capacity utilization most efficiently in continuous mode
» High capacity resin
» No pressure issues w/ short 5 cm bed heights
• No impact of load titer on binding efficiency / capacity
• No impact of extensive cycling (relative to batch processing)
» No pressure increases
» No column fouling
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• Modeled 7g/L titer, 2000L scale process
results in significant savings using BioSMB:
• Processing Time
• A3 ProA resin
• Column hardware
• Buffer Volume
BioSMB Batch
Max load (g/L) 70 45
Column ID (cm) 20 60
Column BH (cm) 5 20
CV per column (L) 1.6 56.5
# of columns 5 1
Total CV (L) 7.9 56.5
Max load / cycle 550 2543
Cycle time (hr) 0.9 3.2
# cycles req'd 26 6
Total time (hr) 24 19
Buffer vol (L) 5307 8817
Resin vol (L) 9 68
Column hardware ($) 50 250
Productivity (g/L/hr) 75 13
A balanced or targeted approach to savings
should be based on MFG goals / limitations.
- Limited suite time for DS operations
- Min/max flow rates on chrom skid
- COG limits for campaign
- Space for buffer drums / columns
- Etc

17. • BioSMB process can be tailored to achieve
different goals.
• Consider different ways to save:
1) Balance savings across all categories
- Balance column ID to achieve all goals
2) Focus on time savings
- Maximize column ID
- Limited by 350L/hr max flow rate on BioSMB)
3) Focus on resin and buffer savings
- Mininimize column ID
• Balance of savings depends on:
1) MFG cadence
2) Importance of resin cost
3) Available space in MFG suite
4) Buffer COGs
5) Facility fit / MFG limitations
6) Molecule stability (perfusion)
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Focused Savings for 4g/L titer
Balanced Time Buffer / Resin
Time -7% 50% -323%
Buffer 48% 45% 49%
Resin 86% 69% 97%
Hardware 80% 80% 80%
Productivity 570% 538% 582%
Focused Savings for 7g/L titer
Balanced Time Buffer / Resin
Time -24% 43% -148%
Buffer 40% 38% 41%
Resin 86% 69% 93%
Hardware 80% 80% 80%
Productivity 480% 459% 492%
Focused Savings for 10g/L titer
Balanced Time Buffer / Resin
Time 39% 39% -169%
Buffer 34% 34% 36%
Resin 69% 69% 93%
Hardware 80% 80% 80%
Productivity 426% 426% 447%
SavingsusingBioSMBforcapturevsbatchprocessing

18. • Evaluation of BioSMB at PD scale enabled modeling of efficiency improvements in MFG
• Significant efficiency improvements can be realized using continuous capture chromatography
• Reduce processing / MFG suite time
• Reduce resin volume
• Reduce column hardware size / cost
• Increase in plant capacity
• Additional flexibility for clients
• Overall improved efficiency
• Overall reduction in MFG costs
• Using BioSMB in single-use MFG facility for capture chromatography would allow for all past and
current programs to be manufactured within current desired cadence.
» A more ambitious cadence may be implemented.
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• Pall Life Sciences
• Marc Bisschops, PhD
• Krista Kern
• Eric Gershenow
• Sandhya Manjunath
• Julie Grace
• Downstream Development
• Yuan Chang, PhD – Director DSPD
• Sigma Mostafa, PhD – VP PD
• Carnley Norman, PhD – VP Manufacturing
• Abhinav Shukla, PhD
• Analytical Development
• Brendan Peacor, PhD – Scientist 2 AD
• Upstream Development
• Niket Bubna, PhD – Prinicipal Scientist USPD