Watch this webinar here: https://bit.ly/3fFRXDb This webinar illustrates a customer case study about the challenges related to the removal of Host Cell Proteins from bioreactor harvest, the selected fast-track approach and outcome. Our customer had one month to reduce the level of HCPs in the bioreactor harvest prior to a production run to supply drug substance for a scheduled Phase 1 clinical trial. The high level of HCPs (1,000,000 in the harvest and 700 ppm at the end of purification) unfavorably impacted the planned clarification process and subsequent downstream steps. The goal was to reduce the level of HCPs to maximum of 300 ppm at the end of process purification and ensure clarification of the entire 2000L harvest. In this webinar, you will learn about: - Challenges with Host Cell Proteins - Fast-Track Approach using caprylic acid precipitation followed by filtration using Clarisolve® filters - The impact of Design of Experiment

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Fast-track implementation of
innovative early downstream process
solutions to address challenges with
Host Cell Proteins (HCPs)
Magali Toueille, Ph.D. & Guillaume Plane
BioReliance® End-to-End Solutions
August 6, 2020

2. The life science business
of Merck KGaA, Darmstadt,
Germany operates as
MilliporeSigma in the U.S.
and Canada

3. Agenda
Reduce HCPs in one month
Case study
1
2
3
Challenges with HCPs in
Bioprocessing
About BioReliance® End-to-
End Solutions

4. Challenges with
HCPs in
Bioprocessing

5. Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins
From DNA to Clinic
5
A path paved with pitfalls

6. Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins
Process steps & Manufacturing yields
6
Clarification
Affinity
Chromatography
Ultrafiltration
Sterile filtration
Cex
Chromatography
Bulk
Drug
Substance
Bioreactor
Virus removal
Aex
Chromatography

7. Case study

8.  After process fitting for 2000L scale
− Too high level of HCPs at the end
of a 3 chromatography steps DSP
process (over 600ppm in Drug
Substance vs 300ppm target)
− Too low filterability on clarification
filter (not scalable at 2000L)
 Start study 1 month before GMP
production run at 2000L for a phase
I clinical trial
 Increase clarification filterability
 Reduce HCP levels to reach
specification in the Drug Substance
 Optimize chromatography / add
chromatography step
− Too long development
− Process would become too long
and costly
− Clarification filterability would not
be improved
 Decrease HCP level during harvest
− More easily/quickly optimized
− No lengthy step added
− Would improve clarification
filterability
Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins8
Case Study
Reduce the level of HCPs in the bioreactor harvest in one month
Challenge Objective & Constraints Options
Implementation of a fast-track development : Combine
caprylic acid precipitation with innovative depth filtration
device

9. Principle
 Direct introduction of caprylic acid in the harvest
→ Formation of large impurities aggregates triggered by caprilyc acid addition
 Removal of clumps thanks to depth filtration device
Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins9

10. Determination of caprylic acid precipitation conditions
DOE approach (1/2)
DoE with 2 main parameters
 Caprylic acid concentration
 Temperature
Other parameter values
[Caprylicacid]
Temperature
Low Medium High
High
Medium
Face-Centered Central Composite Design
Parameter Range
Working volume (mL) Fixed
Mixing time (h) Fixed
Mixing Speed (rpm) Fixed
pH adjusted with acetic acid Fixed
Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins10

11. Determination of caprylic acid precipitation conditions
DoE approach (2/2)
Responses
 Target molecule recovery
 Clarification filterability
 HCP removal
 Molecule integrity
[Caprylicacid]
Temperature
Low Medium High
High
Medium
Face-Centered Central Composite Design
Harvest
Harvest
precipitated
Clarisolve+ B1HC
clarification
Harvest Titration
Clarified Harvest
titration
Precipitated Harvest
Titration
Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins11

12. ➔ Loss of recovery during clarification
➔ Evaluation of 40MS versus 20MS device
➔ Improved recovery using 40MS
Precipitation Recovery/Clarification Recovery
➔ Caprylic acid concentration and temperature have an impact on
precipitation recovery
➔ In addition, caprylic acid concentration impacts the Harvest clarified
recovery
➔ Low caprylic acid concentration and temperature should be selected
Low Caprylic acid concentration
and low temperature = 80 %
recovery expected
value
value
n
10
16
22
28
34
40
0.5
0.8
1.1
1.4
1.7
2
0
20
40
60
80
100
precipitationrecovery(%)
A: caprylic acid concentration (%)
B: Temperature (°C)
Low
High
Low
Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins12

13. 13
➔ Significant HCP removal upon caprylic acid addition and clarification: no impact of temperature and limited impact of
caprylic acid concentration
HCP removal
Harvest clarified
dicted value
dicted value
10
16
22
28
34
40
0.5
0.8
1.1
1.4
1.7
2
50
60
70
80
90
100
SPP(%)
A: [Ac] (%)
B: Température (°C)
Post precipitation
High
Low
High
High
Low
High
Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins

14. Filterability
➔ No impact of Caprylic acid concentration and temperature on
the filterability
➔ Max filterability expected 90 L/m² for 5H process time at
2000 L scale
➔ The clarification of a 2000L scale is scalable with a
Clarisolve® 20 MS.
➔ 40MS depth filter provides better recovery
➔ 40MS depth filter allows higher filtrability
➔ 40MS selected for production scale to ensure better
performance
Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins14
t® Software
g: Actual
/m²)
nts above predicted value
nts below predicted value
lic acid concentration
erature
10
16
22
28
34
40
0.5
0.8
1.1
1.4
1.7
2
0
40
80
120
160
filterability(L/m²)
A: caprylic acid concentration (%)
B: Temperature (°C)
High
High
Low

15. Molecule integrity
 No impact of caprylic acid
treatment on molecule integrity
Process modification can be directly implement in developed process to
reduce final HCP level down to target value
Molecule activity
 Slight impact of caprylic acid
concentration on molecule
activity→ Caprylic acid
concentration maintained at
low levels
Impact on purification
process
 No impact of precipitation and
implementation of new
clarification on subsequent
steps of the process
 No need for further process
modification
Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins15
Molecule integrity and impact on overall process

16. 16
Process
parameters tested
Process
parameters
selected
Temperature
(°C)
Low-High Low-medium
Caprylic acid
concentration
Low-High Low
pH Fixed Fixed
Precipitation
incubation time
Fixed Fixed
Clarification
Filter
Clarisolve®
40MS/20MS
Clarisolve® 40MS
pH post filtration Fixed Fixed
Elimination of HCPs in Harvest
Final design and expected performance
Expected
Performance
Recovery 70 %
Filterability 300 L/m²
Quality
Reduction of HMW
Activity and molecule
integrity maintained
HCP removal 80 %
Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins

17. Process Scale up and GMP production
• Clarification scheme and sizing validated from 2L to 2000L scale
• Robustness of the step assessed for HCP reduction and filterability
• Acceptance criteria [HCP] in Drug Substance <300 ppm met from 2 to 2000L scale
• 3 Drug Substance released for phase I clinical assays
Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins17

18. Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins18
Conclusions
Fast-track implementation of HCP
reduction in harvest
Combination of classic approach
with innovative depth filtration
technology
From DoE to GMP production

19. About
BioReliance®
End-to-End Solutions

20. Quality, Regulatory Compliance
Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins20
Pre-clinical Phase I Phase II Phase III Commercial
Cell line development
Any mammalian cells & CHOZN®
Analytical development, Life cycle management
Process development
Templated or customized
GMP Manufacturing
Single-Use 50 to 2,000 L scale
Facility design, equipment commissioning
Process validation, scale up, TT
Cell banking
Characterization testing
Lot Release testing
We are your process development and manufacturing partner
Formulation

21. From Cell line development to
GMP Manufacturing
Three Biodevelopment Centers
EU
NA
Successful process
development lab and GMP
Facility since 2012
Expansion to a Process
development lab in
Burlington (MA) since 2017
Expansion to a lab and a GMP
facility in Shanghai since
2017
CN
21 Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins

22. Fast-track implementation of innovative early downstream process solutions to address challenges with Host Cell Proteins22
Thanks

23. The vibrant M, BioReliance, CHOZN, Millistak and Clarisolve are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the
property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.
© 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.