View the interactive recording : https://bit.ly/2JYehee Abstract: Over the last several years, the biopharmaceutical industry has had a significant focus on connected and continuous processing to improve both process economics and plant utilization. As opposed to the traditional polishing trains comprised of bind-elute chromatography operations, an all flow-through polishing train easily enables connected and continuous processing while simultaneously improving process economics, flexibility, and productivity. Leveraging commercially available and novel prototype chemistries and devices, we investigated how a properly designed flow-through polishing train can be used to meet the stringent demands expected for mAb polishing purification. A streamlined methodology will be described to investigate the performance of individual units as well as synergies between technologies. For both individual technologies and connected processes, results will be discussed on their ability to meet purity and yield targets robustly. Finally, we will show how leveraging the integrated combination of unit operations can result in improved performance over the standard batch, segregated processing paradigm. In this webinar, you will learn: • New process design for continuous flow-through polishing and its operational robustness • Economic benefits (43% savings in COGs) of implementing a robust flow-through polishing toolbox

1. Merck KGaA
Darmstadt, Germany
Process Development for
Continuous Flow Through
mAb Purification
Meghan Higson, Junyan Zhang, Jeff Barna, Sandeep Mora

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

3. 0
0
0Agenda
Flow Through Polishing:
Value Proposition1
Process Development:
The Need to Think Holistically2
A Robust Flow Through Process:
The Importance of CEX3

4. 0
0
0Agenda
Flow Through Polishing:
Value Proposition1
Process Development:
The Need to Think Holistically2
A Robust Flow Through Process:
The Importance of CEX3

5. Evolutionary Adoption
Business Drivers → Facility-of-the-Future (FoF)
5
Process Batch Intensified Connected Continuous
Format Stainless Steel Hybrid Single-Use
Single-Use
Closed/Sterile
Process Continuum
Process
Analytics
QC Methods
At-Line
On-Line
In-Line
Real Time
Release
Control Unit Op Process Facility
Predictive
Autonomous
Digital Plant Paper-Based Digital Silos Connected Plant
Fully Digital
Adaptive
PAT Continuum
Digital Continuum
Today
Facility of
Future

6. Current Process
Standard mAb Batch Process
Bioreactor Clarification Affinity
Chrom
Virus
Inactivation
CEX Bind and
Elute
Flow Through AEX Viral
Clearance
Final
Filtration
Concentration &
Diafiltration
Depth
Filtration
Seed TrainInoc Train
Batch
Production Capture VI Polishing UF/DFSeed Train
14.4%
36.9%
13.6%
2.1%
24.7%
8.2%
0
50
100
150
Thousands$
Other
Labor
Consumables
Materials
Capital
Representative Process Economics of mAb Batch Process

7. Facility-of-the-Future
Batch → Intensified → Connected → Continuous
Bioreactor Clarification Affinity
Chrom
Virus
Inactivation
CEX Bind and
Elute
Flow Through AEX Viral
Clearance
Final
Filtration
Concentration &
Diafiltration
Depth
Filtration
Seed TrainInoc Train
BatchIntensified
Intensified
Seed Train
Intensified
Production
Intensified
Capture
In-Line
VI
Flow Through
Polishing
Continuous
UF/DF
Intensified Flow Through Polishing is a critical step to move towards a continuous process

8. Properly designed flow through toolbox capable
of delivering robust impurity clearance and high
product yield
Flow Through Polishing
Robust Performance
High monomer yield
▪ 85-95%
Efficient purification
▪ Aggregates: < 1%
▪ Fragments: up to 1 LRV
▪ HCP: < 10 ppm
Integrated process
▪ All flow through
▪ 2 pH changes, no product dilutions
Methodology
▪ Adjust Pro A pool to pH 7.0
▪ Challenge AC @ 0.5 kg/L
▪ Challenge AEX @ 3 kg/L
▪ Adjust pH to ~ 5.0
▪ Challenge CEX @ 0.5-1 kg/L
Activated Carbon Flow Through
CEX
Flow Through
AEX
Move to optimization of flow through condition
Low pH pool pH Adjust to 5 Virus Filter Pool

9. Properly designed flow-through toolbox capable
of delivering robust impurity clearance and high
product yield
Developed BioSolve Model for integrated flow
through polishing
Flow Through Polishing
Process Modeling
Activated Carbon Flow Through
CEX
Flow Through
AEX
Viral
Clearance
Bioreactor: 2k L fed-batch
Titer: 5 g/L Mab

10. Properly designed flow-through toolbox capable
of delivering robust impurity clearance and high
product yield
Developed BioSolve Model for integrated flow-
through polishing
Benefits:
1. COGs reduction of ~40%
Flow Through Polishing
Process Modeling
43% COGS reduction

11. Properly designed flow-through toolbox capable
of delivering robust impurity clearance and high
product yield
Developed BioSolve Model for integrated flow-
through polishing
Benefits:
1. COGs reduction of ~40%
2. Buffer reduction of ~80%
Flow Through Polishing
Process Modeling
Buffer L/g
Total Process Polishing only
Traditional 1.19 0.63
FT Polishing 0.64 0.08

12. Properly designed flow-through toolbox capable
of delivering robust impurity clearance and high
product yield
Developed BioSolve Model for integrated flow-
through polishing
Benefits:
1. COGs reduction of ~40%
2. Buffer reduction of ~80%
3. Processing time reduction of ~70%
Flow Through Polishing
Process Modeling

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

14. 1
4 Flow Through Polishing
Existing Toolbox
▪ Eshmuno® CP-FT resin
Flow through aggregate removal at high
loading
5 – 10X buffer reduction
Easy implementation
Differentiated from existing CEX media

15. 1
5 Flow Through Polishing
Existing Toolbox
▪ Eshmuno® CP-FT resin
Flow through aggregate removal at high loading
5 – 10X buffer reduction
Easy implementation
Differentiated from existing CEX media
▪ NatriFlo® HD-Q AEX membrane
Impurity clearance at high loading
High velocity operation (~1 sec RT)
▪ Eshmuno® Q resin: High viral and HCP clearance
NatriFlo® HD-Q AEX Membrane: HCP and Viral Clearance
0,0
1,0
2,0
3,0
4,0
5,0
6,0
0-150 151-300 301-450 451-600
MVMLogReductionValue(LRV)
Mass Loading ( g/L)
MVM removal using Eshmuno Q [pH 8.5, 5mS/cm]
Feed: 79g/L Post CEX SPTFF mAb02 pool
Device # 1
Device # 2Arrow denotes complete removal of MVM

16. 1
6 Flow Through Polishing
Existing Toolbox
▪ Eshmuno® CP-FT resin
Flow through aggregate removal at high loading
5 – 10X buffer reduction
Easy implementation
Differentiated from existing CEX media
▪ NatriFlo® HD-Q AEX membrane
Impurity clearance at high loading
High velocity operation (~1 sec RT)
▪ Eshmuno® Q resin: High viral and HCP clearance
▪ Millistak+® Pod CR depth filter
Activated carbon media
Binding: van der Waals interactions
Size- and charge-based selectivity
Used in sensitive applications (oral poisoning,
food and beverage, pharmaceuticals,
hemoperfusion, etc.)
Impurity (MW) Capacity
Insulin 60 g/L
Methotrexate 75 g/L
Pluronic® F68
surfactant
75 g/L
Antifoam C > 16 g/L

17. 17
Through the use of process
modeling significant improvements
identified with Flow Through
Polishing:
• COGs reduction of ~ 40% for flow-
through polishing process compared
to bind-elute template
• Significant footprint reduction
through reduced buffer volumes
(~80%) and hold tanks
• ~ 70% reduction in polishing
processing time with connected
flow through train
There is an evolution happening
in bioprocessing
• Focus on process intensification
to reduce COGs
A properly designed flow-through
polishing toolbox can robustly
remove protein A pool impurities:
• Chemistries that work
synergistically
• High capacity → small devices
Summary

18. 0
0
0Agenda
Flow Through Polishing:
Value Proposition1
Process Development:
The Need to Think Holistically2
A Robust Flow Through Process:
The Importance of CEX3

19. Experimental Plan
Two-Step Process
Step 1: Scouting Step 2: Integration
Activated Carbon
Prototype Carbon
Eshmuno® Q Resin
Prototype AEX
Eshmuno® CP-FT Resin
Prototype CEX
CEX
CEX

20. Experimental Plan
Scouting
20
mAb Protein A elution pool
Mass Loading
(mg/mL)
pH Range
Conductivity
(mS/cm)
Column Size
(mL)
1000
4 – 8
4, 6
0.2
Carbon
200
6.5 – 8.5
4, 6
0.2
AEX
1000
4 – 6
4, 6, 10*, 15*
0.2/0.5
CEX
*Prototype CEX devices used were designed to work at higher conductivities; therefore higher ranges were also tested

21. Technology
comparison

22. Scouting Experiment – Technology Comparison
Activated Carbon vs. Prototype Carbon
Title of Presentation | DD.MM.YYYY
mAb1: Titer: 23 mg/mL HCP: 700 ppm
mAb2: Titer: 9.5 mg/mL HCP: 1600 ppm
YIELD:
mAb1:
- Results insensitive to conductivity
- Yield on Activated Carbon better than on Prototype Carbon

23. Scouting Experiment – Technology Comparison
Activated Carbon vs. Prototype Carbon
Title of Presentation | DD.MM.YYYY
mAb1: Titer: 23 mg/mL HCP: 700 ppm
mAb2: Titer: 9.5 mg/mL HCP: 1600 ppm
YIELD:
mAb1:
- Results insensitive to conductivity
- Yield on Activated Carbon better than on Prototype Carbon
mAb2:
- Insensitive to pH and conductivity for both technologies

24. Scouting Experiment – Technology Comparison
Activated Carbon vs. Prototype Carbon
Title of Presentation | DD.MM.YYYY
mAb1: Titer: 23 mg/mL HCP: 700 ppm
mAb2: Titer: 9.5 mg/mL HCP: 1600 ppm
YIELD:
mAb1:
- Results insensitive to conductivity
- Yield on Activated Carbon better than on Prototype Carbon
mAb2:
- Insensitive to pH and conductivity for both technologies
HCP Removal:
mAb1:
- pH dependent performance
- Better HCP clearance on Activated Carbon

25. Scouting Experiment – Technology Comparison
Activated Carbon vs. Prototype Carbon
Title of Presentation | DD.MM.YYYY
mAb1: Titer: 23 mg/mL HCP: 700 ppm
mAb2: Titer: 9.5 mg/mL HCP: 1600 ppm
YIELD:
mAb1:
- Results insensitive to conductivity
- Yield on Activated Carbon better than on Prototype Carbon
mAb2:
- Insensitive to pH and conductivity for both technologies
HCP Removal:
mAb1:
- pH dependent performance
- Better HCP clearance on Activated Carbon
mAb2:
- Both technologies insensitive to conductivity
- Activated Carbon more robust than Prototype Carbon

26. Scouting Experiment – Technology Comparison
Activated Carbon vs. Prototype Carbon
Title of Presentation | DD.MM.YYYY
mAb1: Titer: 23 mg/mL HCP: 700 ppm
mAb2: Titer: 9.5 mg/mL HCP: 1600 ppm
Summary:
▪ Both Carbon technologies gave high yield and
robust HCP clearance
▪ Activated Carbon performed better than Prototype
Carbon
YIELD:
mAb1:
- Results insensitive to conductivity
- Yield on Activated Carbon better than on Prototype Carbon
mAb2:
- Insensitive to pH and conductivity for both technologies
HCP Removal:
mAb1:
- pH dependent performance
- Better HCP clearance on Activated Carbon
mAb2:
- Both technologies insensitive to conductivity
- Activated Carbon more robust than Prototype Carbon

27. Scouting Experiment – Technology Comparison
Eshmuno® Q resin vs. Prototype AEX
Title of Presentation | DD.MM.YYYY
mAb1: Titer: 17 mg/mL HCP: 3300 ppm
mAb2: Titer: 10 mg/mL HCP: 650 ppm
YIELD:
mAb1:
- Eshmuno® Q resin outperformed Prototype AEX
- Prototype AEX expected to have higher yield at higher loading

28. Scouting Experiment – Technology Comparison
Eshmuno® Q resin vs. Prototype AEX
Title of Presentation | DD.MM.YYYY
mAb1: Titer: 17 mg/mL HCP: 3300 ppm
mAb2: Titer: 10 mg/mL HCP: 650 ppm
YIELD:
mAb1:
- Eshmuno® Q resin outperformed Prototype AEX
- Prototype AEX expected to have higher yield at higher loading
mAb2:
- Comparable results as seen with mAb1
- Eshmuno® Q resin had better yield with mAb2 than mAb1

29. Scouting Experiment – Technology Comparison
Eshmuno® Q resin vs. Prototype AEX
Title of Presentation | DD.MM.YYYY
mAb1: Titer: 17 mg/mL HCP: 3300 ppm
mAb2: Titer: 10 mg/mL HCP: 650 ppm
YIELD:
mAb1:
- Eshmuno® Q resin outperformed Prototype AEX
- Prototype AEX expected to have higher yield at higher loading
mAb2:
- Comparable results as seen with mAb1
- Eshmuno® Q resin had better yield with mAb2 than mAb1
HCP Removal:
mAb1:
- Both technologies resulted in ~0.5 LRV HCP removal

30. Scouting Experiment – Technology Comparison
Eshmuno® Q resin vs. Prototype AEX
Title of Presentation | DD.MM.YYYY
mAb1: Titer: 17 mg/mL HCP: 3300 ppm
mAb2: Titer: 10 mg/mL HCP: 650 ppm
YIELD:
mAb1:
- Eshmuno® Q resin outperformed Prototype AEX
- Prototype AEX expected to have higher yield at higher loading
mAb2:
- Comparable results as seen with mAb1
- Eshmuno® Q resin had better yield with mAb2 than mAb1
HCP Removal:
mAb1:
- Both technologies resulted in ~0.5 LRV HCP removal
mAb2:
- More robust HCP clearance
- HCP removal independent of conductivity

31. Scouting Experiment – Technology Comparison
Eshmuno® Q resin vs. Prototype AEX
Title of Presentation | DD.MM.YYYY
mAb1: Titer: 17 mg/mL HCP: 3300 ppm
mAb2: Titer: 10 mg/mL HCP: 650 ppm
Summary:
▪ Higher yield on Eshmuno® Q resin
▪ HCP clearance strong function of pH but
essentially independent of conductivity
YIELD:
mAb1:
- Eshmuno® Q resin outperformed Prototype AEX
- Prototype AEX expected to have higher yield at higher loading
mAb2:
- Comparable results as seen with mAb1
- Eshmuno® Q resin had better yield with mAb2 than mAb1
HCP Removal:
mAb1:
- Both technologies resulted in ~0.5 LRV HCP removal
mAb2:
- More robust HCP clearance
- HCP removal independent of conductivity

32. Scouting Experiment – Technology Comparison
Eshmuno® CP-FT resin vs. Prototype CEX
Monomer Yield:
mAb1: Titer: 17 mg/mL HCP: 650 ppm
Aggregates: 3%
mAb2: Titer: 10 mg/mL HCP: 3700 ppm
Aggregates: 3%
mAb1:
- Better performance with Eshmuno® CP-FT resin

33. Scouting Experiment – Technology Comparison
Eshmuno® CP-FT resin vs. Prototype CEX
Monomer Yield:
mAb2:
- Eshmuno® CP-FT resin has higher monomer yield at higher pH
- Prototype CEX able to achieve monomer yield >90% at
higher conductivities
mAb1: Titer: 17 mg/mL HCP: 650 ppm
Aggregates: 3%
mAb2: Titer: 10 mg/mL HCP: 3700 ppm
Aggregates: 3%
mAb1:
- Better performance with Eshmuno® CP-FT resin

34. Scouting Experiment – Technology Comparison
Eshmuno® CP-FT resin vs. Prototype CEX
Monomer Yield:
HCP Removal:
mAb2:
- Eshmuno® CP-FT resin has higher monomer yield at higher pH
- Prototype CEX able to achieve monomer yield >90% at
higher conductivities
mAb1:
- Robust HCP clearance over entire pH/conductivity window
mAb1: Titer: 17 mg/mL HCP: 650 ppm
Aggregates: 3%
mAb2: Titer: 10 mg/mL HCP: 3700 ppm
Aggregates: 3%
mAb1:
- Better performance with Eshmuno® CP-FT resin

35. Scouting Experiment – Technology Comparison
Eshmuno® CP-FT resin vs. Prototype CEX
Monomer Yield:
HCP Removal:
mAb2:
- Eshmuno® CP-FT resin has higher monomer yield at higher pH
- Prototype CEX able to achieve monomer yield >90% at
higher conductivities
mAb1:
- Robust HCP clearance over entire pH/conductivity window
mAb2:
- HCP clearance strong function of pH
- HCP clearance independent of conductivity
mAb1: Titer: 17 mg/mL HCP: 650 ppm
Aggregates: 3%
mAb2: Titer: 10 mg/mL HCP: 3700 ppm
Aggregates: 3%
mAb1:
- Better performance with Eshmuno® CP-FT resin

36. Scouting Experiment – Technology Comparison
Eshmuno® CP-FT resin vs. Prototype CEX
Monomer Yield:
HCP Removal:
mAb2:
- Eshmuno® CP-FT resin has higher monomer yield at higher pH
- Prototype CEX able to achieve monomer yield >90% at
higher conductivities
mAb1:
- Robust HCP clearance over entire pH/conductivity window
mAb2:
- HCP clearance strong function of pH
- HCP clearance independent of conductivity
Aggregate Removal:
mAb1:
- Higher aggregate removal at lower pH for Eshmuno® CP-FT resin
- Aggregate clearance optimized at higher conductivity for
Prototype CEX
mAb1: Titer: 17 mg/mL HCP: 650 ppm
Aggregates: 3%
mAb2: Titer: 10 mg/mL HCP: 3700 ppm
Aggregates: 3%
mAb1:
- Better performance with Eshmuno® CP-FT resin

37. Scouting Experiment – Technology Comparison
Eshmuno® CP-FT resin vs. Prototype CEX
Monomer Yield:
HCP Removal:
mAb2:
- Eshmuno® CP-FT resin has higher monomer yield at higher pH
- Prototype CEX able to achieve monomer yield >90% at
higher conductivities
mAb1:
- Robust HCP clearance over entire pH/conductivity window
mAb2:
- HCP clearance strong function of pH
- HCP clearance independent of conductivity
Aggregate Removal:
mAb1:
- Higher aggregate removal at lower pH for Eshmuno® CP-FT resin
- Aggregate clearance optimized at higher conductivity for
Prototype CEX
mAb2:
- Eshmuno® CP-FT resin performance similar to that of mAb1
- Broad operating window with Prototype CEX
mAb1: Titer: 17 mg/mL HCP: 650 ppm
Aggregates: 3%
mAb2: Titer: 10 mg/mL HCP: 3700 ppm
Aggregates: 3%
mAb1:
- Better performance with Eshmuno® CP-FT resin

38. Scouting Experiment – Technology Comparison
Eshmuno® CP-FT resin vs. Prototype CEX
Monomer Yield:
HCP Removal:
mAb2:
- Eshmuno® CP-FT resin has higher monomer yield at higher pH
- Prototype CEX able to achieve monomer yield >90% at
higher conductivities
mAb1:
- Robust HCP clearance over entire pH/conductivity window
mAb2:
- HCP clearance strong function of pH
- HCP clearance independent of conductivity
Aggregate Removal:
mAb1:
- Higher aggregate removal at lower pH for Eshmuno® CP-FT resin
- Aggregate clearance optimized at higher conductivity for
Prototype CEX
mAb2:
- Eshmuno® CP-FT resin performance similar to that of mAb1
- Broad operating window with Prototype CEX
mAb1: Titer: 17 mg/mL HCP: 650 ppm
Aggregates: 3%
mAb2: Titer: 10 mg/mL HCP: 3700 ppm
Aggregates: 3%
mAb1:
- Better performance with Eshmuno® CP-FT resin
Summary:
▪ Eshmuno® CP-FT resin achieved high yield and
robust HCP and aggregate removal
▪ Prototype CEX performs better at higher
conductivities

39. Molecule
comparison

40. Scouting Experiment – Molecule Comparison
mAb1 vs. mAb2
Title of Presentation | DD.MM.YYYY
mAb1 Starting HCP: 700 ppm
mAb2 Starting HCP: 650 ppm
Prototype Carbon – HCP Removal:
mAb1 vs. mAb2:
- Prototype Carbon has much better HCP removal for mAb1

41. Scouting Experiment – Molecule Comparison
mAb1 vs. mAb2
Title of Presentation | DD.MM.YYYY
mAb1 Starting HCP: 3300 ppm
mAb2 Starting HCP: 650 ppm
Prototype Carbon – HCP Removal:
mAb1 vs. mAb2:
- Prototype Carbon has much better HCP removal for mAb1
Prototype AEX – HCP Removal:
mAb1 vs. mAb2:
- Prototype AEX has broader operating window for mAb2

42. Scouting Experiment – Molecule Comparison
mAb1 vs. mAb2
Title of Presentation | DD.MM.YYYY
Prototype Carbon – HCP Removal:
mAb1 vs. mAb2:
- Prototype Carbon has much better HCP removal for mAb1
Prototype AEX – HCP Removal:
mAb1 vs. mAb2:
- Prototype AEX has broader operating window for mAb2
Prototype CEX – Aggregate Removal:
mAb1 vs. mAb2:
- Prototype CEX has a broader operating window for mAb2
mAb1 Starting Aggregate Level: 3%
mAb2 Starting Aggregate Level: 3%

43. Scouting Experiment – Molecule Comparison
mAb1 vs. mAb2
Title of Presentation | DD.MM.YYYY
Prototype Carbon – HCP Removal:
mAb1 vs. mAb2:
- Prototype Carbon has much better HCP removal for mAb1
Prototype AEX – HCP Removal:
mAb1 vs. mAb2:
- Prototype AEX has broader operating window for mAb2
Prototype CEX – Aggregate Removal:
mAb1 vs. mAb2:
- Prototype CEX has a broader operating window for mAb2
Summary:
▪ Optimal conditions are molecule dependent
mAb1 Starting Aggregate Level: 3%
mAb2 Starting Aggregate Level: 3%

44. Summary
Scouting is important
• Every molecule is different
• Need to determine optimal operating window to maximize product yield
and impurity
Select technologies with broad operating windows
▪ Activated Carbon
▪ Eshmuno® Q resin
▪ Eshmuno® CP-FT resin
Select technologies that work synergistically
▪ Different mechanisms of removal
▪ Work under comparable solutions
4444

45. 0
0
0Agenda
Flow Through Polishing:
Value Proposition1
Process Development:
The Need to Think Holistically2
A Robust Flow Through Process:
The Importance of CEX3

46. Linked Process
Selection Criteria
Title of Presentation | DD.MM.YYYY
1: Technology Selection – Robust window and work synergistically
• Activated Carbon
• Eshmuno® Q resin
• Eshmuno® CP-FT resin
2: Process Conditions (pH and Conductivity)
• Conductivity Aggregate clearance optimized at lower conductivity for Eshmuno® CP-FT resin
3: Process Unit order
• pH Carbon: Results independent of pH/conductivity; to be linked with AEX; conditions to match AEX conditions
AEX: pH chosen from scouting for highest HCP removal; higher pH chosen to aid in virus removal
CEX: pH chosen from scouting with highest HCP removal and lowest aggregate level in the pool
mAb1
pH 6.5 - 4 mS/cm 42%
pH 6.5 - 6 mS/cm 40%
pH 7 - 4 mS/cm 50%
pH 7 - 6 mS/cm 51%
pH 7.5 - 4 mS/cm 41%
pH 7.5 - 6 mS/cm 42%
pH 8 - 4 mS/cm 44%
pH 8 - 6 mS/cm 39%
pH 8.5 - 4 mS/cm 45%
pH 8.5 - 6 mS/cm 47%
Eshmuno® Q resin
HCP Removal (Pool) @ 200 mg/ml Loading
• Eshmuno® Q resin Remove impurities that increase the robustness/loading capacity of Eshmuno® CP-FT resin
• Eshmuno® CP-FT resin Protect Viresolve® Pro Solution to increase loading capacity
Agg in Pool HCP Removal
pH 4 - 4 mS/cm 0.2% 90%
pH 4 - 6 mS/cm 0.2% 87%
pH 5 - 4 mS/cm 0.3% 93%
pH 5 - 6 mS/cm 0.2% 93%
pH 6 - 4 mS/cm 1.2% 90%
pH 6 - 6 mS/cm 1.9% 82%
Eshmuno® CP-FT resin @ 1000 mg/mL Loading
mAb1• Activated Carbon Increase loading on Eshmuno® Q resin

47. Experimental Plan
Linked Experiment
mAb Protein A elution pool – Aggregates 4-5%
Eshmuno®
CP-FT Resin
Evaluate
Eshmuno® CP-FT resin
Load: 1000 g/L
pH 5, 4 mS/cm
Eshmuno®
Q Resin
Eshmuno®
CP-FT Resin
Activated
Carbon
Eshmuno®
Q Resin
Eshmuno®
CP-FT Resin
Eshmuno® Q resin
Load: 300 g/L
pH 7, 4 mS/cm
Evaluate
Eshmuno® Q → Eshmuno® CP-FT resin
Load: 2000 g/L
pH 5, 4 mS/cm
Activated Carbon
Load: 1000 g/L
pH 7, 4 mS/cm
Evaluate
Activated Carbon → Eshmuno® Q resin
Load: 2000 g/L
pH 7, 4 mS/cm
Evaluate
Activated Carbon → Eshmuno® Q resin →
Eshmuno® CP-FT resin
Load: 2000 g/L
pH 5, 4 mS/cm

48. Linked Process Experiment
mAb1
Titer: 18 mg/mL
HCP: 3100 ppm
Leached ProA: 9 ppm
Aggregates: 4.5%
Eshmuno® CP-FT resin:
Yield: ≥90%
Leached ProA Removal: ≥96% (0.32 ppm)
HCP Removal: ≥97% (96 ppm)
Aggregate Removal: ≥92% (0.31% in pool)
Leached ProA Removal: ≥92% (0.68 ppm)
HCP Removal: ≥98% (73 ppm)
Aggregate Removal: ≥79% (0.95% in pool)
Leached ProA Removal: ≥50% (4.2 ppm)
HCP Removal: ≥98% (73 ppm)
Aggregate Removal: <1% removal (4.27% in pool)
Leached ProA Removal: ≥94% (0.45 ppm)
HCP Removal: ≥99% (6 ppm)
Aggregate Removal: ≥82% (0.80% in pool)
Eshmuno® Q resin → Eshmuno® CP-FT resin:
Yield: ≥94%
Activated Carbon → Eshmuno® Q resin:
Yield: ≥96%
Activated Carbon → Eshmuno® Q resin → Eshmuno® CP-FT resin:
Yield: ≥94%
CEX
(1000 mg/mL)
CEX
(1000 mg/mL)
AEX → CEX
(2000 mg/mL)
CEX
(1000 mg/mL)
AEX → CEX
(2000 mg/mL)
AC → AEX
(2000 mg/mL)
CEX
(1000 mg/mL)
AEX → CEX
(2000 mg/mL)
AC → AEX → CEX
(2000 mg/mL)
CEX
(1000 mg/mL)
CP-FT
(1000 mg/mL)
Q → CP-FT
(2000 mg/mL)
CEX
(1000 mg/mL)
AEX → CEX
(2000 mg/mL)
AC → AEX
(2000 mg/mL)
CEX
(1000 mg/mL)
AC → AEX → CEX
(2000 mg/mL)
AEX → CEX
(2000 mg/mL)
CEX
(1000 mg/mL)
CEX
(1000 mg/mL)
AEX → CEX
(2000 mg/mL)
CP-FT
(1000 mg/mL)
AEX → CEX
(2000 mg/mL)
AC → AEX → CEX
(2000 mg/mL) CEX
(1000 mg/mL)
CEX
(1000 mg/mL)
AEX → CEX
(2000 mg/mL)
CEX
(1000 mg/mL)
AEX → CEX
(2000 mg/mL)
AC → AEX
(2000 mg/mL)
CEX
(1000 mg/mL)
AEX → CEX
(2000 mg/mL)
AC → AEX → CEX
(2000 mg/mL)

49. 49
A designed-for-purpose cation-
exchange resin (such as Eshmuno®
CP-FT resin) can be a central
component of a polishing toolbox
• Robust aggregate removal at
product loadings > 1 kg/L
• Major contributor to HCP and
leached protein A removal
• Key enabler for connected and
continuous processing
A robust mAb flow through
polishing toolbox can offer
significant economic and facility-
fit benefits without sacrificing
performance
• COGs, buffer volume, process
footprint, process time
Optimization of a flow through
process requires an holistic
perspective
• Maximize performance while
minimizing number and volumes
of buffers
Summary

50. The vibrant M, Eshmuno, Millistak+, NatriFlo, Viresolve 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.
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