The document outlines the implementation of a fully single-use, integrated monoclonal antibody biosimilars purification platform by Merck KGaA as part of a four-year project funded by the EU Horizon 2020 program. The initiative aims to create a more efficient and environmentally friendly downstream process for manufacturing biopharmaceuticals, featuring innovative techniques like continuous capture and viral inactivation. The project showcases successful technical runs at a 1000L scale, leading to significant improvements in productivity, cost reduction, and bioburden risk management.

1. Merck KGaA
Darmstadt, Germany
Implementing a fully single-use,
integrated mAb biosimilars
purification platform for next
generation manufacturing
Josselyn Haas Durr
European Biomanufacturing
Engineer Manager

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

3. 3
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0Agenda Project overview
1
Primary clarification
2
Continuous capture
3
Continuous viral inactivation
4
Full flow-through DSP
5
Implementation at 1000L scale
6
Conclusions
7

4. Project overview
10

5. Title
 Next-generation biopharmaceutical downstream process
 Acronym: nextBioPharmDSP
 Webpage: www.nextbiopharmdsp.eu
General  4 year project (March 1, 2015 – February 28, 2019)
Partners
 Industry: Lek Pharmaceuticals d.d. (Slovenia), Sandoz GmbH
(Austria), Millipore SAS (France)
 Academia: University of Natural Resources and Applied Life
Sciences (Austria), Karlsruhe Institute of Technology (Germany),
National Institute of Chemistry (Slovenia)
 SME: National Systems srl (Italy)
Objective
 Develop and implement a more efficient, cost-effective and
environmentally friendly downstream process for the
manufacture of monoclonal antibodies and biosimilars.
Horizon 2020: EU-funded Program for Research & Innovation
Snapshot
5
Budget
 Total budget: 10,6 mio €
 EU funding: 8,4 mio €
 70 % for industry
 100 % for academia

6. 1000L Fed-Batch
Bioreactor
Optimizing Biopharmaceutical Downstream Processing
Establish fully connected disposable DSP platform for biologics
production
Bioreactor Centrifuge Filtration Protein A VI CEX b/e AEX f/t VF UF/DF
Bioreactor +
pre-treatment
Filtration Continuous
VI
Continuous
capture
Carbon & AEX
f/t
CEX & VF
f/t
UF/DF
Continuous DSP - 3 days - 4kg
Capture Intermediate / Polishing VF UF/DF
Primary
Separation
6
Batch
Process
Continuous
Stainless
Steel
Format
Single-Use

7. Primary
clarification
20
Flocculation followed by depth filtration outperforms
traditional clarification strategies

8. Screening more than 70 combinations !
Screening
3 mAbs
5 CHO
harvest
techniques
High
process
window
5 pre-
treatment
agents
3 depth
filter
families
IgG1(B), IgG2(A), IgG1(C)
Fed-batch un-treated
Fed-batch centrifuge
Fed-batch pre-treated harvest
Perfusion ATF
Perfusion TFF
Caprilic Acid
Calcium Chloride
PEG
pDADMAC
Clarisolve® mPAA
Millistak+® HC depth filter (3)
Millistak+® HC Pro synthetic depth
filter (2)
Clarisolve® depth filter (3)
8
Primary clarification
Cell density: 4,9M – 10M cells/mL
Viability: 72 – 98%
Turbidity: 1040 – 2600 NTU
2
strategies
for
flocculant
addition
Batch
Continuous

9. 5.2
1.9
3.4
0
1
2
3
4
5
6
7
8
9
10
Untreated + Millistak+®
HC D0HC/F0HC depth
filter
pDADMAC + Clarisolve®
40MS synthetic depth
filter
Area(m²)
Minimum Depth Filtration Area
(Fed-Batch, 1000L, 2h)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0 100 200 300 400 500 600
FilterResistance(psi/L/m².h)
Filtrate Volume (L/m²)
Untreated - Millistak+® HC D0HC/F0HC
Caprilic Acid 0.25% - Clarisolve® 20MS
100mM CaCl2/10mM PO4 - Clarisolve® 20MS
Untreated - Clarisolve® 20MS/Millistak+® HC F0HC
Untreated - Millistak+® HC Pro D0SP/X0SP
PEG 4% - Clarisolve® 40MS
Clarisolve® mPAA 0.05% - Clarisolve® 40MS
pDADMAC 0.0375% - Clarisolve® 40MS
pDADMAC pre-treatment followed by Clarisolve® 40MS depth filter
outperformed traditional depth filtration
4,5x
9
Primary clarification

10. Surface installed 15m² 3,85m²
Clarification train
Two-step clarification:
D0HC  X0HC
One-step clarification:
pDADMAC  40 MS
Consumable cost Equivalent (including Flexware® assemblies and flocculant)
Water for flush 1500 L 385 L
Hold up volume 150 L 100 L
Operation time 1,5 day 1 day
pDADMAC pre-treatment followed by Clarisolve® 40MS depth filter
exhibit benefits compared to traditional set up
5–10M TC/mL
72–98% Viability
1040–2600 NTU
harvest turbidity
50% safety factor
€
3 mAb
1000L CHO
- 75%
- 75%
- 33%
- 33%
2:1
=
10
Primary clarification

11. Primary clarification
Large scale implementation
11
Bioreactor Flocculation
in the
bioreactor
pDADMAC
10 % stock
solution
Water
Buffer
Bioburden
reduction
Clarisolve®
40 MS depth filter
Clarified
harvest

12. Continuous
capture
30
Continuous Protein-A capture enhances productivity and
provides a constant quality eluate

13. Continuous Multi-Column (CMC) Capture
Batch Operation
10% BT
Batch loading
(~70% resin utilization)
Loading time
Proteinconcentration
• Load
• Wash
• Elute
• Clean
• Regen
1
Protein conc.
Continuous Operation
60% BT
Continuous loading
(~90% resin utilization)
Loading time
Proteinconcentration
1 2
3
• Wash
• Elute
• Clean
• Regen
• Load
Speed and Cost benefits
•Process Intensification
•Reduced COGs
•Higher binding capacity
•Maintained performances
and robustness
13
Load Wash-
Regen
STEP 1
3(ProtA)
2(ProtA)
1(ProtA)
Optimizes resin utilization and enhances productivity

14. Small beads offer better performances for continuous capture
Particle size: 35 – 49 μm
Particle size: ~ 85 μm
Particle size: 35 – 49 μm
Particle size: ~ 85 μm
Batch chromatography
Continuous chromatography
10 Protein A resins evaluated
• DBC for several residence times (RT)
• BT curve shape & BT point
• Removal of HCP, aggregates and step
yield  no significant differences
• DNA removal  some differences - not
critical
14
Protein A resins screening
®
®
Resin
Resin

15. Continuous Chromatography
Continuous capture on 3 columns: a robust and consistent operation
15
Processes developed on several resins – optimized step lengths
50 – 60 cycles performed – consistent performance
Method duration: 65 – 75 min / cycle
RT 1.5 min, high loadings (60 – 70 mg/mL)
High productivity
and resin utilization

16. Development of a disposable 3-columns system
• Fully disposable flowpaths (incl. flowmeters, pump
heads and UV)
• 8 to 120 l/h per line
• 30 days of continuous operation
• Fit for columns from 7 to 25 cm i.d.
• 3 inlets product line, 6 inlets buffer line
16
Continuous Multi-Column (CMC) Capture

17. From concept to reality
17
Continuous Multi-Column (CMC) Capture

18. Continuous viral
inactivation
40
Fulfill a double challenge: make it single use and
continuous!

19. Viral inactivation is dependant on pH, buffer type and temperature, but NOT
on mAB concentration
19
 Virus inactivation at low pH is rapid and requires less than
5 minutes.
 The kinetics depend on pH, temperature, and buffer
conditions, and are not dependent on protein
concentration.
 The incubation chamber that can achieve a target
inactivation time with an appropriate safety factor must
be designed appropriately.

20. Semi-continuous virus inactivation : Concept
20
Ensure homogeneity and correct incubation time
Continuous feed
20

21. From 3D concept to reality
21
Semi-continuous virus inactivation
Pumps for pH
regulation
C2
WYE inlet Flexware®
and F1/F2 valve
C1 Acid/Base bags holder
C1 to C1B
pump
C1B

22. Full flow-through
DSP polishing
50
New technologies

23. pH 5
Protein A elution material after VI
AEX FT-CEXCarbon
AEX
FT-CEX
FT-CEX
pH 7
MAb
acidic pI basic
LowMWhigh
Larger acidic
HCP, DNA, and viruses
Anion Exchangers
HCP
Cation Exchangers
Smaller HCP’s
and cell culture components
Activated Carbon Functionality Media
23
Toolbox Approach
Flow-Through Polishing

24. Flow-Through Polishing
Technologies investigated
24
Virus filtration
Eshmuno® Q resin, (2) AEX resin prototypes
Natriflo® HD-Q Membrane Adsorber, Sartobind® Q, Sartobind® STIC,
Mustang® Q, QyuSpeed® D
(1) AEX fiber prototype
Eshmuno® CP-FT resin, Eshmuno® CPX resin
Sartobind® S, Mustang® S, Natriflo® HD-Sb hydrogel membrane
Millistak+® Carbon Filter
Viresolve® Pro solution
Activated Carbon
Cation Exchange
Anion Exchange

25. Millistak+® CR40 depth filter is the main contributor to HCP reduction
level
25
Activated Carbon
Binding: van der Waals
interactions
Size-based selectivity
Protects AEX
CAP.E. AFTER AC AFTER AEC AFTER CECCAP.E. AFTER AC AFTER AEC AFTER CEC

26. Eshmuno® CP-FT resin outperforms CEX membrane adsorbers for
aggregates removal in Frontal chromatography mode
26
FT cation exchange

27. Unit Operation Technology Selection
Flow-Through Polishing – Implementation Strategy for 1000L Reactor
Protein A
Eluate after
VI
Millistak+®
CR40
Activated
Carbon Depth
Filter
(1.2 m2)
Eshmuno®
Q
AEX FT Resin
(2 x 1.1L)
Eshmuno®
CP-FT
CEX FT Resin
(2 x 1.1L)
Viresolve®
Pro
Virus Filtration
(0.22 m2)
adjust pH=5
pH = 7
Conductivity < 5 ms/cm
27

28. Implementation
at 1000L scale
60

29. Technical runs
1000L Scale Setup
CAP VIN
AC-AEX
CEX-VF
29

30. DSP continuous process control: system handshaking
Bioreactor +
pre-treatment
Clarification
Continuous
Virus
Inactivation
Continuous
capture
f/t Carbon
f/t AEX
f/t CEX
& VF
UF/DF
pH
adjustment
Switch of VIN
collection tank
Full VIN tanks stop CAP
VIN ready starts
Carbon/AEX
pH adj. starts
CEX/VF
Breakthrough
detection
Peak
detection
Flow
VPE
VIN = Viral Inactivation30

31. Continuous capture during the 4 runs
Data Run 1 Run 2 Run 3 Run 4
Number
of cycles
Total 90 92 94 139
col 1 30 30 32 46
col 2 30 31 31 46
col 3 30 31 31 47
Loading speed
(cm)
180 240 210 210
Elution speed
(cm)
180 280 280 280
31

32. 32
Capture – CMC skid
Trends (run 4) demonstrate the consistency of the capture process
32

33. 4 technical runs
at 1000L scale
2,5 kg mAb purified in 2,5 days
> 80% yield all runs and steps considered
9–12 g/l mAb concentration after Viresolve® Pro solution
CONSISTENT purity and quality over runs
• No bioburden
• RPC and CZE profiles comparable to reference33

34. 34
HCP removal consistently < 40 ppm (Target < 100 ppm)
0
50
100
150
200
250
300
350
400
450
500
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 50 52 54 56 58 60 62
HCP(ppm)
Time (h)
Run 3
AC
AEX.E
CEX.E
VIN
0
50
100
150
200
250
300
350
400
450
500
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58
HCP(ppm)
Time (h)
Run 4
AC
AEX.E
CEX.E
VIN
Technical runs detailed results

35. 35
Aggregates removal < 0.2% (target < 1%)
0
1
2
3
4
5
6
7
8
9
10
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 52 54 56 58
Aggregatecontent(%)
Time (h)
Run 4
AC
AEX.E
CEX.E
VIN
0
1
2
3
4
5
6
7
8
9
10
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 50 52 54 56 58 60 62Aggregatescontent(%)
Time (h)
Run 3
AC
AEX.E
CEX.E
VIN
Technical runs detailed results
Aggregates content in pool
Run 2: < LOQ
Run 3: 0.2%
Run 4: 0.1 %

36. 36
Protein A removal below LOD (target < 5 ppm)
0
5
10
15
20
25
30
35
40
45
50
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 50 52 54 56 58 60 62
ProteinA(ppm)
Time (h)
Run 3
AC
AEX.E
CEX.E
VIN
0
5
10
15
20
25
30
35
40
45
50
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58
ProteinA(ppm)
Time (h)
Run 4
AC
AEX.E
CEX.E
VIN
Technical runs detailed results
Activated carbon
and
Eshmuno® CP-FT resin
are the main contributors

37. Conclusions
70

38. Main benefits
38
€
• Facility investment: 35%
• Running costs: 30%
• Cost of materials: 50%
• CO2 emissions: 25%
• Reduced water, buffer, resins consumption (water 10x)
Productivity
Footprint
Economics
Single-Use
Environmental
• Reduce bioburden risk
• No carry-over issues
• No cleaning, regeneration, steaming
• Rapid change-over
• Complete DSP in 30 m2 (10-15x smaller)
• Elimination of large intermediate hold tanks
• Flexibility (mobile equipment)
• Capable of processing up to 3000L harvest in 24 hours (up 12 kg of mAb)
nextBioPharmDSP project helps to make highly efficient
biopharmaceuticals more accessible to patient

39. You are invited!
Next Generation Bioprocessing
Forum
October 8-9, 2019
M Lab™ Collaboration Center
Molsheim, France

40. Trademarks and logos used in this presentation are the property of companies
collaborating in the nextBioPharmDSP project.
Thank you!
www.nextbiopharmdsp.eu
This project has received funding from the European
Union’s Horizon 2020 research and innovation
program under grant agreement No 635557

41. josselyn.haas@milliporesigma.com
Josselyn Haas
The vibrant M, Eshmuno, Viresolve, Millistak+, Flexware, NatriFlo and BioContinuum 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.
© 2019 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.