Technology Trends in Bioprocessing Purification

Merck KGaA
Darmstadt, Germany
Takao Ito, Head of MSAT Japan & Korea
BIO KOREA International Convention
18 April 2019
Considerations for Continuous Processing and
Connected Polishing for mAb
Technology Trends
in Bioprocessing
Purification

01
02
03
04
05
Agenda
2
Market Trends and the Evolution of Next
Generation Processes
Intensified Capture Processing
Continuous Virus Inactivation
Connected Flow-Through Polishing
Summary

Therapeutic mAb Processing
Market Trends
*http://www.biophorum.com/category/resources/technology-roadmapping-resources/introduction/
1 2 3 4

Market Trends Require Step Change in Business Driver Performance
70% 10X 90% 90%

5
Therapeutic mAb processing
Factory of the Future
Facility Goals
FACTORY
OF THE
FUTURE
Enablers
Process
Intensification
Single Use Process Analytics
Software &
Automation

An Evolutionary Journey Across Many Disciplines
6
Today
Near-term
Mid-term
End state
Process
Batch
Intensified
Connected
Continuous
Format
Stainless
Hybrid
Single Use
SU/Closed
Analytics
QC
Manufacturing
At-Line
In-Line/
Real-Time
Sensing
Controls
Standalone
Control
Semi-
Centralized
Control
Full Process
Control
Predictive
Process
Control
Digital
Plant
Pre-Digital
Plant
Digital
Silos
Connected
Plant
Adaptive
Plant

BioContinuum™ Platform
Focus Areas:
(1) Cell Culture Media For Intensified Processes
(2) Integrated Perfusion Bioreactor
1 & 2 3
(3) Protein A for Intensified Capture
4
(4) In-Line Viral Inactivation
5 & 6
(5) Flow Through Polishing Toolbox
(6) Single-Pass UF/DF
Past
Current

Approach of mAb Downstream Process Intensification
Low
pH VI
Pool
CEX
Pool
AEX
Pool
VF
Pool
Traditional
Process
CEX B/E AEX FT VF with
prefiltrationPA B/E
VF
PoolTank Less
CEX B/E
AEX FT
VF with
prefiltration
PA B/E
VF
Pool
Continuous
Capture
(CMC, SMB)
CMC
CEX B/E
VF with
pre-filtration
AEX FT
Tandem
CMC
PA B/E
Connected
Flow-through
(post PA)
Low pH
VI Pool VF Pool
AC + AEX FT CEX + VF
In-line pH
Pro-A+AEX: M. Shamashkin et al.,
Biotechnol Bioeng, 110(10) 2013
Pro-A+CEX+AEX: Neil Soice, et. al.
ACS spring conference 2010
V. Warikoo et al., Biotechnol Bioeng,
109(12), 2012
R. Godawat et al., J Biotechnol. 213,
2015
AEX+HIC: J. Zhang et al., Eng Life
Sci. 17(2) 2017
Depth+AC+AEX: T Yamada et al., J
Chrom B 1061-1062 2017
EP 2574618 A1. 2012

01
02
03
04
05
Agenda
9
Summary

Improved resin utilization
Residence time operation: Higher or lower
What is Continuous Multi-Column Chromatography
Intensified Capture
2 column
Single loading
3 column
Loading in-series

Intensified Capture
What is Continuous Multi-Column Chromatography
▪ Better resin utilization
▪ Higher productivity
▪ Smaller columns
▪ Lower buffer consumption
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
0
10
20
30
40
50
60
70
80
1-Column
(1% BT)
2-Column
(1% BT)
DynamicCapacity(g/L)
Residence Time (min)
Protein conc. in column
3 min RT
1 min RT
0,6 min RT
0,4 min RT
Batch
Continuous

Intensified Capture
Quality and Yield of Continuous Multi-Column Chromatography
3 column continuous results
Total IgG loaded : 325mg (@RT 1min)
Washing : 25mg
Elution : 296mg 91%
Total Operation time: 8.3hr
Used protein A amount: 3ml
Batch results
Total IgG loaded : 480mg (@RT 7.2min)
Elution : 445mg 92%
Total Operation time: 24hr
Used protein A amount: 5ml
Data generated by Chiyoda
TechnoAce Co.,Ltd.
▪ Maintained performance
▪ Same robustness
▪ Comparable process yield
Protein A affinity resin: Eshmuno® A

Is Multi-column Chromatography Productive?
Same operation for resin: B/E
• Same resin
• Same buffer
Batch
Multi Column
Changes:
• Reduction of resin and buffer
• Shorter residence time
• Increase cycle
• Interconnected operation
• Complex flow path for cycling

Intensified Capture
Operational Windows on Productivity
Batch 2-column switching 3-column cycling
Works around 20cm Bed
at 250-350 cm/hr
Productivity:
15-30 g/L/hr
Limitation of flow rate
Bed height at 5-15cm
25-40 g/L/hr
Operation point is determined
by 24hr run (perfusion)
3.5-4.5g/L/hr
2000L (Titer 5.4g/L)
Eshmuno® A resin
Least Squares Fit by response surface
(Predicted by JMP software)
ID 63cm ID 25cm ID 35cm
Different sweet spot of resin utilization

Intensified Capture
Capture Column Strategy for Upstream Processing
Fed-batch bioreactor
2kL, 5.4g/L
Single harvest
Perfusion bioreactor
(30days)
150L, 2.29g/L
Continuous harvest
Concentrated Fed-batch
500L, 21.4g/L
Single harvest
UF
MF
BPOG Next Gen Tech Roadmap Upstream Scenarios
Titers are referenced from Xu, S. et al., Biotechnol. Prog., 33(4) 2017
0%
50%
100%
Intensifie
dFB&
Batch
Intensifie
dFB&
CMC
HD
perfusion
&
Switching
Concentra
tedFB
&CMC
Buffer Total resin
0%
100%
200%
300%
Productivity
x1.7
1/4
x2.2
-97%
-50%
1
2
3
4

16
Intensified Capture
Impact on Long Duration Cycling Run
J. Pollock et al., J. Chromatography A 2013
Continuous
Batch
P
Perfusion Bioreactor
Cell Retention device,
breeding device
Protein A capture
Semi continuous and
continuous manufacturing
Centrifugation, ATF, TFF
Multicolumn cycling
In-line
perfusate
clarification
Limited impurity removal
Significantly more foulants
Steeper loss in DBC
D. Burgstaller et al., J. Chem.
Technol. Biotechnol. 2017
pDADMAC
Millistak® HC ProDesign with enough safety factor
Improved CIP
Additional perfusate clarification
Cycle Number
LossinDBC(%)

-5
0
5
10
15
20
-0.5
0
0.5
1
1.5
2
2.5
0 5 10 15 20 25 30 35 40 45 50 55 60 65
pHorConductivity/4
A280(AU)
Column Volumes
A280
Conductivity / 4
pH
wash
-5
0
5
10
15
20
-0.5
0
0.5
1
1.5
2
2.5
0 5 10 15 20 25 30 35 40 45
pHorConductivity/4
A280(AU)
Column Volumes
A280
Conductivity / 4
pH
Unconcentrated
mAb Harvest
1.3 g/L titer
24 CV load
SPTFF Concentrated
12.4 g/L titer
2.5 CV load
equil load elute re-equil CIP
Load time = 144 min
Cycle time = 4.25 hour
Load time = 14 min
Cycle time = 2 hour
SPTFF feed concentration increased capture step productivity by 115%
(7.2 g/L/hr → 15.5 g/L/hr)
Case Study: 1.6 L Eshmuno® A column for batch mAb capture
Improve Capture Chromatography with SPTFF
Intensified Capture

Flow Rate
Existing resins
The Drive For Higher Productivity Solutions
Residence times is the key
for future
Higher productivity enables:
✓ Increased speed
✓ Smaller footprint
→ Potential for Single Use
→ Manufacturing flexibility
✓ Reduced COGS
3-8 min.
1-3 min.
0.5-1 min.
2-10 sec.
Productivity(g/L/h)
Residence Time (min)
Intensified Capture
18
Conventional Column
Chromatography

Multi-column cycling optimizes resin utilization and enables continuous processing:
• Upstream culture strategy should be considered: Fed-batch or Perfusion
• Harvest type: Single or Continuous
• mAb concentration: SPTFF can boost productivity for Protein A capture
• Design consideration for multi cycling operation for higher loading:
Safety factor, Improved CIP, Additional perfusate clarification
Summary of intensified capture

01
02
03
04
05
Agenda
20
Summary

In-Line Virus Inactivation
ACID
→ Virus inactivation kinetics
→ Efficient incubation chamber design
PROTEIN A
CAPTURE
POLISHING
CHROM.
STEP #1
BATCH
Industry standard: 60 minute hold
Robust inactivation > 4 LRV
2 holding tanks required
Manual process
Continuous pH adjustment with dynamic hold
Eliminates large holding tanks
Automated
Enables continuous operation
60 min
HOLD
BASE
Applicability dependent upon:
CONTINUOUS

0
0.2
0.4
0.6
0.8
1
0 0.5 1 1.5 2 2.5
CumulativeDistribution
V/V50
Incubation Chamber Design – Flow Distribution Characterization
Straight Tube
Coiled
Serpentine
Early virus
breakthrough
→ Larger safety
factor Long protein
exposure
C. Gillespie et al., Biotechnol J. 2019 Feb;14(2)

Inactivation
Kinetics
Virus type
PRV < XMuLV
Buffer type
acetate  glycine < citrate
Temperature
22ºC < 16ºC
mAb concentration
Minimal impact
Virus Inactivation Kinetics
Rapid inactivation kinetics (~2-3 min)
C. Gillespie et al., Biotechnol J. 2019 Feb;14(2)

• Virus inactivation at low pH is rapid and requires less than 5 minutes, implying
that this process can be run in continuous mode. The kinetics of inactivation
depend on pH, temperature, and buffer conditions, and are not dependent on
protein concentration.
• Technologies exist to move from batch to continuous virus inactivation. Efficient
incubation chamber design and control offers continuous flow systems shorter
processing times than traditional batch processes.
• Proper implementation could result in equivalent levels of virus inactivation in
batch and in-line systems.
Summary of in-line virus inactivation

01
02
03
04
05
Agenda
25
Summary

Toolbox Approach
Flow-through Polishing
Leached
Protein A
Aggregates
Virus
Host Cell DNA
Host Cell Protein
Others?
(Fragments,
Charge Variants)
Chemistries Matrices Devices

Flow-through Polishing
Existing Toolbox
▪ Eshmuno® CP-FT resin
Flow through aggregate removal at high
loading
5 – 10X buffer reduction
Easy implementation
▪ NatriFlo® HD-Q AEX membrane
Impurity clearance at high loading
High velocity operation (~1 sec RT)
▪ Millistak+® Pod CR40 depth filter
Activated carbon media
Binding: van der Waals interactions
Size-based selectivity
Impurity (MW) Capacity
Insulin 60 g/L
Methotrexate 75 g/L
Pluronic® F68
surfactant
75 g/L
Antifoam C > 16 g/L

Eshmuno® CP-FT resin
Benchmarking
Only Eshmuno® CP-FT resin was efficient for the removal of aggregates in the flow-
through mode under strong binding conditions that favor frontal chromatography
M. Stone et al., J Chrom A, 1599, 2019

Batch vs. Integrated Implementation of Flow-through Technology
Robust Performance with Significant Footprint Reduction
Flow Through Polishing Delivers:
Robust Purification across 8 molecules
 High product yield (83 – 91%)
 Aggregates: < 1%
 Fragments: up to 1 LRV
 HCP clearance: < 10 ppm
Significant Facility Footprint Reduction
 Lower media and buffer requirements
 Eliminate intermediate hold tanks
 Single-skid operation
Low pH
VI Pool
Virus
Filter
Pool
Carbon Depth Filter
+ FT-AEX
FT-CEX
+ Virus Filtration
In-line pH
Low pH
VI Pool
Depth
Filter
Pool
CEX
Pool
AEX
Pool
Virus
Filter
Pool
pH Adjust
& Dilution
Depth Filter Bind & Elute
CEX
FT-AEX Virus
Filtration
Traditional Batch Polishing
Flow Through Polishing

3-step Flow-through Polishing
100
98
96
94
92
3.0
2.5
2.0
1.5
1.0
0.5
0.0
VIA AC Feed FT-AC AEX Feed FT-AEX CEX Feed FT-CEX
HMW1
HMW2
LMW1
LMW2
Monomer
HMW,LMW(%)
Monomer(%)
10000
1000
100
10
1
DNA
DNA(pg/mg),HCP(ng/mg)
HCP3985
1888
27.8
512
0.15
0
0
2.47
0.25
0.01
0.01
0
1.7
0.07
1.57
0.03
1.68
0.03
98.22
94.22
<0.76
<0.25
0.00
FT-AC FTCEXFTAEX
pH5
3mS/cm
Activated carbon shows 3 log10 DNA clearance.
Robust clearance of HCP and DNA by FT-AEX.
Eshmuno® CP-FT reduces mAb-related HMW aggregates (Dimer).
mAb 10.9g/L
pH7
3mS/sm
T. Ichihara et al., mAbs J., 10, 2018

Feed condition:
mAb-A (8.2mg/mL)
Impurities: HCP = 567ng/mg IgG, DNA=7276 pg/mgIgG, HMW1 = 0.78%,
HMW2= 1.65%, Monomer = 96.53%, LMW1=0.93%, LMW2=0.1%.
pH6, 4mS/cm, 0.2mL/min
High Loading : 200mL (1640mg mAb)
Operation system : AKTAexplorer 100
Single equilibrium: 25mM sodium acetate buffer pH6
Single elution: 25mM Sodium acetate buffer pH6 w 1M NaCL
Evaluation: Connected flow-through
1. AEX(1mL) – CEX(0.5mL)
2. AC(0.5mL) – AEX(1mL) –CEX(0.5mL)
Connected Flow-through Purification
Proof of Concept Study from DOE Optimization
AC
CEX
T. Ichihara et al., mAbs J., 10, 2018
T. Ichihara et al., Eng. Life Sci., 19, 2019

(A) AEX - CEX(A) AEX - CEX
(B) AC - AEX - CEX
5
5.5
6
6.5
20 40 60 80 100 120 140 160 180 200 220 240
pH
0
500
1000
1500
2000
2500
3000
3500
0
A280(mAU)
Volume (mL)
5
5.5
6
6.5
7
0
500
1000
1500
2000
2500
3000
3500
0 20 40 60 80 100 120 140 160 180 200 220 240
pH
As80(mAU)
Volume (mL)
7
0
40
80
120
160
200
Conductivity(mS/sm)0
40
80
120
160
200
Conductivity(mS/sm) 0
0.2
0.4
0.6
0.8
1
MPa
0
0.2
0.4
0.6
0.8
1
MPaFigure 1. Typical chromatograms obtained from the in-series, connected flow-through
polishing steps. (A) AEX-CEX, (B) AC-AEX-CEX.
Load
Wash
Elute
Load
Wash
Elute
Evaluation of Connected Flow-through Purification
Results of Connected Flow-through Polishing
AEX-CEX
AC-AEX-CEX
Wash
Load
Elute
Wash
Load
Elute
90
92
94
96
98
100
5
8
11
○Monomer(%)
■mAbconc.
(mg/mL)
0
2000
4000
6000
8000
10000
0
100
200
300
400
500
0 1000 2000
△DNA(pg/mg)
◆HCP(ng/mg)
mAb loading (mg)
AEX-CEX
AC- AEX-CEX
mAb
conc.
Mono-
mer
HCP DNA
Chromatograms by in-series flow-through polishing
1/2 -1/3 reduction of processing time
Extremely high pressure of elution
mAb 100% breakthrough at >400mg lording
AC improves HCP and DNA clearance
T. Ichihara et al., Eng. Life Sci., 19, 2019

33
Value Modeling
What Value Does Flow Through Polishing Deliver?
Bioreactor Clarification Affinity
Chromatography
Virus
Inactivation
CEX Bind and
Elute
Flow Through AEX Viral
Clearance
Final
Filtration
Concentration &
Diafiltration
Traditional Batch
(Baseline)
Depth
Filtration
Four polishing steps run continuously without intermediate hold steps
▪ Lower buffer volume
▪ Higher loading on chromatography media and virus filter
▪ Smaller facility footprint for systems, columns, and tanks
Bioreactor Clarification Affinity
Chromatography
Virus
Inactivation
Final
Filtration
Concentration &
Diafiltration
Flow Through
Polishing
Activated Carbon Flow Through
CEX
Flow Through
AEX
Viral
Clearance
Bioreactor: 2k L fed-batch
Titer: 5 g/L Mab
Felo, Holistic Development of Intensified Mab Processes for Higher
Productivity and Improved Economics, BPI Boston, Sep 2018

Assumption Activated
Carbon
AEX FT CEX FT Virus
Filtration
Technology Millistak+®
Carbon
depth filter
Eshmuno® Q
resin
Eshmuno®
CP-FT resin
Viresolve®
Pro filter
Yield 93% 95% 95% 98%
Capacity 1500 g/m2 3000 g/L 1000 g/L 5000 g/m2
# Re-uses Single use 150 150 Single use
# Buffers 1 5 4 0
Activated Carbon Flow Through
CEX
Flow Through
AEX
Viral
Clearance
Value Modeling
Assumptions
Key to Output
• Single skid versus
four
• Yields matched to
baseline process
• Baseline process
assumptions per
BPOG costs &
assumptions
Titer: 5 g/L Mab
34 Felo, Holistic Development of Intensified Mab Processes for Higher

35
Baseline Scenario vs. Flow Through Polishing
• Most significant savings capital, consumables, and labor
• Buffer reduction due primarily from switching CEX to flow through mode
10% COGS reduction
47% Buffer reduction
43% COGS reduction
87% Buffer reduction
Polishing alone… Total process…
Value Modeling
COGS Savings from Reduced Buffer Use, Intermediate Storage Tanks,
and Process Time/Labor
Titer: 5 g/L Mab
Ito BioKorea
2019 | 18-
Apr-2019,
Copyright
Merck 2019 Felo, Holistic Development of Intensified Mab Processes for Higher

36
Ito BioKorea
2019 | 18-
Apr-2019,
Copyright
Merck 2019
Pre-concentrate to Boost Eshmuno® Q Resin HCP Clearance
SPTFF with Flow-through Polishing
0
1
2
3
4
5
6
0 20 40 60 80 100 120
Improvementfactorabove
non-SPTFFfeed
mAb feed concentration (mg/mL)
Improvement vs. mAb feed concentration
Mass Loading (g/L resin)
▪SPTFF pre-concentration can boost Eshmuno® Q resin mass loading by a factor of 4x.
▪ Reduced loading at high feed concentrations, possibly due to mAb-HCP interactions.
▪In addition to load improvements, SPTFF concentration reduces process load time,
thus increasing productivity by a factor of 5x.
0
1
2
3
4
5
6
0 20 40 60 80 100 120
Improvementfactorabove
non-SPTFFfeed
mAb feed concentration (mg/mL)
Improvement vs. mAb feed concentration
Mass Loading (g/L resin) Productivity (g/L resin/hr)
Pre-Concentrate
Protein
SPTFF
Pellicon®
AEX
Eshmuno® Q
T Elich et al., ACS conference spring Apr 2017

Continuous diafiltration for buffer exchange
Tank
I
Tank
II
Recovery
Tank To next
process step
From previous
process step
UF
0
1
2
3
4
5
6
7
NormalizedtoBatch
All cases: no UF, 9 diavolumes
■ Batch: 3h process, 6LMM crossflow
■ CDF low area: 19h process, 6LMM crossflow
■ CDF low pump passes: 19h process, 1LMM crossflow
■ ILDF: 19h process
30
35
40
45
0 2 4 6 8 10 12 14 16 18 20 22 24
Cycle Number
Control Chart: DF Cycle Time
Average = 36.9 min, σ = 1.8 min
E. Goodrich et al.,
Recovery of biological
Products, Oct 2018

Comparable performance between the batch purification process and the pool-less
process configuration.
• Simple operation: Single system operation and removal of the intermediate hold tank
• Substantial quality: higher overall mAb yield and impurity clearance
• Small processing: Higher process loading = Process compression, reduced buffer usage
• Shorter process time: Single loading step
• COGS reduction: ~10% across full process
• SPTFF pre-concentration can boost AEX resin mass loading
Summary of Flow-through Polishing

01
02
03
04
05
Agenda
39
Summary

Intensified Downstream Processing
Summary
Industry trends are forcing bio-manufacturers to adopt
a more strategic view of manufacturing
− Process Intensification
− Single Use
− Process Analytical Technologies and controls
Process design and optimization requires an “holistic view”
− Upstream and Downstream
− Tool box for connected impurity clearance
Next generation technologies can support intensification
efforts of existing batch processes while enabling future
connected and continuous processing
Collaborations between academics, industry, and regulatory
agencies are critical for successfully moving the industry
forward in next generation processing
1
2
3
4

Acknowledgements
Christopher Gillespie
Kevin Galipeauc
Yasuhiko Kurisu
Yasuhiro Kawakami
Miyuki Koyama
Prof. Shuichi Yamamoto (Yamaguchi univ.)
Takamitsu Ichihara(Astellas)
Shuhei Kondo (Chiyoda TechnoAce)
Michael Phillips
Romas Skudas
Mikhail Kozlov
Lars Peeck
Ajish Potty
Matthew Stone
Alex Xenopoulos
Renaud Jacquemart
Danny Wu
Thomas Elich
Mochao Zhao
Michael Felo
Joseph Geringer
Elizabeth Goodrich

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