This presentation provides an introduction to the M Lab™ Collaboration Centers, an overview of chromatography theory, and highlights the benefits of next-generation chromatography. To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.merckmillipore.com/mlab

1. Merck KGaA
Darmstadt, Germany
Authors: Marine Maszelin, Emilie Egloff, Nargisse El Hajjami,
Josselyn Haas Durr, Nina Weis
Process Scale
chromatography
M Lab™ Collaboration Center Demonstration Day

2. 01
02
03
Agenda
2
Introduction
Chromatography Theory
Next Generation Chromatography

3. For Upstream,
Downstream, and
Final Fill, our non-
GMP facilities are
resource hubs for:
• Application &
Technology
Demonstrations
• Education
• Complex Process
Development,
Optimization, and
Scale-Up
• Troubleshooting and
Unit Operation
Support
• Analytical Modelling
• Customer Sizing and
Simulation Tools &
Methodologies
M Lab™ Collaboration Centers
Singapore
Molsheim,
France
Sao Paulo,
Brazil
Burlington,
USA
Mumbai,
India
Bangalore,
India
Shanghai,
China
Tokyo,
Japan
Incheon,
South Korea

4. 1904
1930
1950s
1970s
/80s
1989
1991
1992
1995
2004
2005
2008
2009
2011
2012
2010
2016
2013 2017
Aluminium
Oxide
Silica Gel
ProSep® vA HC
Fractogel® EMD SO3
(M), TMAE (M), DMAE
(M), DEAE (M)
Fractogel®
EMD Chelate
Fractogel® EMD
SE Hicap (M),
COO (M)
ProSep®
vA Ultra
Eshmuno® S
Eshmuno® Q
Eshmuno® HCX
Eshmuno® CPX
PharmPrep® P
Eshmuno® A
Eshmuno® P
anti-A &
P anti-B
Natrix® Q
Fractogel® EMD
TMAE Medcap
Fractogel® EMD
BioSEC, TMAE
Hicap (M), SO3
(S), TMAE (S)
Calcium
Carbonate LiChroprep®
ProSep®
Ultra Plus
2020
Eshmuno® CMX
A Long History of Bringing Innovative Products to Market
2018
Eshmuno® CP-FT
Eshmuno® CPS

5. 01
02
03
Agenda
5
Introduction
Chromatography Theory
Next Generation Chromatography

6. Used to separate chemical
or biological components of
a mixture for later use,
and a powerful step in the
drug purification process.
Used for the quantitation and
identification of chemical or
biological compounds to
establish relative proportions of
analytes in a mixture.
Analytical
Chromatography
Preparative
Chromatography
VS

7. The Goal of Preparative Chromatography
Isolation of the target compound from a crude
feedstock as economical as possible!
... as fast as possible
... as pure as possible
... as much as possible
... as highly concentrated as possible

8. Chromatographic Steps
Equilibrate
Column is
conditioned to
suitable pH, ionic
strength, polarity,
and hydrophobicity
for mobile phase A
Load
Sample containing
solutes is applied to
the chromatographic
bed
Wash
Bound solutes are
washed with mobile
phase A to remove
unwanted and
unbounded
molecules
Elute
Target molecule is
eluted from the
chromatographic
bed by changing
conditions to
mobile phase B
Regenerate
Re-equilibration of
the chromatographic
medium to the initial
mobile phase
conditions
Chromatography
Resin
Target Protein
Unwanted molecules

9. 9
• Enables enhanced mAb purity which are positively charged
• Removal of DNA, endotoxins, host cell protein and leached
protein A ligands
• May be efficient in reducing aggregates
• Efficient method for capturing monoclonal antibodies (mAbs)
• Removal of trace contaminants such as nucleic
acids and host cell proteins to reach the highest
level of mAb purity
• Contribute to the overall viral clearance strategy
for mammalian cell processes
How Chromatography is used in a Drug: mAb Purification Process

10. Types of Chromatography and How They Are Used
Mode Affinity Ion
Exchange
Reverse
Phase
Membrane Hydrophobic
Interaction
Size
Exclusion
Capture
Purification
Polishing
Capture: step used to isolate and
concentrate the target molecule
Moderate to High Purity | High Yield
Purification: step used to remove
bulk impurities
Very High Purity | Balanced Yield
Polishing: step used to remove final
contaminants
Highest Purity | Moderate Yield

11. Which Impurities are Removed by Chromatography Type
Host Cell
Proteins
DNA Virus Leached
Protein A
Aggregates
Protein A
Cation
Exchange
Anion
Exchange
Somewhat
Effective
Moderately
Effective
Extremely
Effective
Marginally
Effective
Very
Effective

12. Products to Suit a Diversified Landscape of Molecules
Type of molecule Chromatography Mode
Monoclonal antibody
• Affinity
• Ion exchange
• Mixed-mode
Other recombinant proteins
• Ion exchange
• Mixed-mode
• Reversed phase
• Affinity
• Size exclusion
Plasma protein
• Ion exchange
• Size exclusion
• Affinity
Vaccine
• Ion exchange
• Size exclusion
Small molecule, peptide
• Reversed phase
• Ion exchange
Removal of impurties
For example:
• Aggregates
• Fragments
• Charge variants
• HCPs (Host Cell
Proteins)
• DNA
• Virus
• Endotoxin
• Proteases
Impurities depend on
expression system,
product, and process.

13. Theoretical Plates Theory
Assumption:
Column consists of a
large number of
imaginary layers
Separation Action:
analytes equilibrate
between the mobile
and stationary phase
in each layer
Column Efficiency:
measured by
calculating the
height equivalent of
a theoretical plate
(HETP)
Theoretical Plate
Column

14. Retention Time
DetectorResponse
How to read a Chromatogram
Injection
Retention Time = time between injection and detection of analyte
(Capacity factor)
Obtaining the
optimum resolution
(Rs) in the minimum
time is the goal when
optimizing
chromatographic
steps
Rs= Retention x
Selectivity x Efficiency
Efficiency
Selectivity (Separation Factor)
Peak maximum
ability of the chromatographic system
to ‘chemically’ distinguish between
sample components

15. Elution of unbound
molecules
Elution of unwanted
material
Elution of target
molecule
Elution of tightly
bound molecules
Retention Time
DetectorResponse
10-20 Column Volumes
2-4 Column Volumes
2-4 Column Volumes
4 Column VolumesHow to read a Chromatogram

16. Base Bead
A
A
A
A
A
A
A
AAA
A
A
A
A
A
A
A
A
A
A A A
A
Variable Region
Fc Fragment
Fab Fragment
F(ab)₂ Fragment
Heavy Chains Light Chains
Specific and selective interaction
between target molecule and resin
Protein A Affinity Chromatography: Separation Principle

17. Ion Chromatography: Separation Principle
Base Bead
Negatively Charged
Bound Protein
Positively Charged Ligand
Unbound Protein
Bind: proteins attach as they are
loaded onto the column at low
ionic strength
Elute: performed by increasing salt
concentration or changing pH to
retrieve attached target protein

18. Architecture of Chromatographic Resins
O
O
H3N+-
Si
O H
H O
H
CH3 CH3
R R R R
R R R OH
OH
Base Material
porous, spherical or
irregular shaped
particles in the µm-
range
Linker
linear or branched
chains or tentacles to
present the ligands in
an optimal way.
Ligand
Point of Interaction
on molecular level:
(ionic, hydrophobic,
H-bridges, π-π-
interaction)
Three main parts: Base Material, Linker and Ligand

19. Hydraulic Properties
Spherical or Irregular
Average size and
distribution
Functionality
Pore diameter and
volume
1 Shape
2 Size
3 Porosity
4 Rigidity
5 Chemistry
Chromatography Resin – Heart of the Chromatography Unit Operation
+ More Efficient
+ Good mass transfer
- Higher pressure drop
- More expensive
- Less Efficient
- Poorer Mass Transfer
+ Lower pressure drop
+ Less Expensive
vs.
+ Larger surface area
→ larger capacity
+ Stronger
- Poorer mass transfer
- Less surface area
→ less capacity
- weaker
+ Better mass transfer
vs.

20. Different Column Dimensions
© Copyright Merck KGaA, Darmstadt Germany
Lab-scale, analytical HPLC-instruments
Semi-prep
4 mm
10 mm
25 mm
50 mm
100 mm
400 mm
Process-scale
800 mm

21. Chromatography Resin Properties and the Factors They Influence
Resolution, product
purity, removal of
impurities, pressure
drop, dynamic
capacity
Throughput,
potential scale of
manufacturing,
maximum operating
velocity
1
Binding capacity,
selectivity, and
recovery
2
Lifespan, ease and
efficacy of
sanitization, and
effectiveness of
cleaning in place
5
4
Dynamic binding
capacity, flow rate
3
Product recovery,
and effectiveness of
cleaning in place
6
Mechanical
Properties
Ligand Density
& Distribution
Pore Size &
Distribution
Particle Size &
Distribution
Chemical
Stability
Hydrophilicity &
Hydrophobicity
Source: Handbook of Process Chromatography – Development, Manufacturing, Validation and Economics. 2nd Edition. 2008,
Hagel, Gunter, Sofer. <https://www.sciencedirect.com/book/9780123740236/handbook-of-process-chromatography>

22. • Increase Diameter
• Constant Bed Height &
Linear Flow Rate
Scale-Up
22
Preserve the quality of separation achieved at small scale
in order to produce comparable results at large scale
Economic Scale-Up
Challenges
1) Column Size
2) Reuse & Cycles
3) Throughput
4) Costs
Technical Scale-Up
Challenges
1) Quality of the
Separation
2) Comparable Results
Impacts
1) Capacity
2) Column Loading
3) Product Titer
4) Lot Size
Impacts
1) Process Parameters
2) Packing Efficiency
3) Pressure Drop
Constraints
4) Flow Distribution
Conventional Scale-Up1
Residence Time Scale-Up2
• Linear increase of Bed
Height & Flow Rate
• Pressure-Flow Limitations
Due to Compressibility
Combined Scale-Up2
• Modern resin maintain
low pressure drop
• Permits scale-up in any
dimension at constant
residence time
• Greater equipment
flexibility

23. 01
02
03
Agenda
23
Introduction
Chromatography Theory
Next Generation Chromatography

24. 2018
2018-2020+
>2025
Biomanufacturing moves to the next generation of biotherapeutic
production with the BioContinuum™ Platform
Smaller
Footprint
Less
Capital Intense
Reduction in
Construction Time
Reduction in
OPEX
90%
90%
70%
90%

25. Intensified Upstream
& harvest
Intensified
Capture
In-Line VI
In-Line Buffer
Flow Through
Polishing
Continuous
UFDF
The next generation of chromatography will be important for
intensified biomanufacturing and continuous processing
✓ Reduced Footprint
✓ Enhanced Utilization of Protein A Resin
✓ Enables Single-Use and Closed Operations
✓ Increased Productivity
✓ Reduced Footprint & Capital Expenditure
✓ Reduces Consumables Needed Including
Buffer Requirements
✓ Enables Single-Use and Closed Operations
✓ Reduced Set-Up and Switching Time

26. 26
Bioreactor Virus Filtration UF/DFProtein A f/t CEX f/t AEXFiltration
Eshmuno® CP-FT resin: a novel cation exchange resin used for flow
through polishing
Tentacular Ligand
Chemistry AggregatesMonomer

27. Eshmuno® CP-FT resin: Reduces the volume of columns required to
purify 1 kg of mAb
Volume for 80 g/L
Loading
Volume for 1000 g/L
Loading
Traditional CEX Media
Bind/Elute Chromatography
Eshmuno® CP-FT resin
Flow-Through Chromatography
20cmbedheight
20cmbedheight
12.5 L 1 Lvs.

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Follow Your
Curiosity

29. Schedule an in-person or remote
visit today.
MerckMillipore.com/mlab

30. The vibrant M, M Lab, LiChroprep, Fractogel, Eshmuno, Natrix, ProSep, PharmPrep, 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.
© 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.