Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm

An overview of custom affinity chromatography technologies for vaccine purification.

Purification can account for majority of the manufacturing costs of most biological drugs. In the vaccine industry, purification processes are particularly complex with non-templated processes typically with low yields leading to higher than desired product costs.

We are actively working with industry partners to develop purification processing platform to address these challenges using affinity chromatography technologies. Such a purification platform could be amenable to diverse heterogeneous types of vaccines such as glycoconjugates, virus like particles and viruses.

In this webinar, you will learn:
-Custom affinity ligand discovery, characterization, and selection strategies
-Affinity ligand - base matrix immobilization strategies
-Performance evaluation techniques

1. Merck KGaA
Darmstadt, Germany
Romas Skudas
Senior Scientist, Chromatography R&D
13 September 2018

2. Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.
2

3. Non-templated processes
leading to long
timelines for Process
Development

Often have complicated
purification schemes
leading to high COGS
and low yields
Current Challenges with Vaccine Downstream Purification
Capture ClarificationBioreactor
1
st

Purification
2
nd

Purification
Final
Polishing
Final
Formulation
Sterile
Filtration
Final
Fill
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.20183

4. Current Challenges with Vaccine Downstream Purification
ClarificationBioreactor
Capture
with target
specific
custom
affinity
ligands
Final
Polishing
Final
Formulation
Sterile
Filtration
Final
Fill
Simplifying downstream
purification schemes leading
to reduced COGS and
improved yields

Potential to establish a
purification template with
target specific custom
affinity ligands
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.20184

5. Divine Project
Affinity
ligand
discovery
Functionalization
of
chromatography
resins
Full process
GMP-like
Water recycling
process
development
Vaccine
product
Industrial Process
implementation
DSP development
pilot-scale
The DiViNe consortium is a EU based commission focused on improvements to vaccine purification
processes that we are actively participating in.
Source: https://divineproject.eu/
The DiViNe project has received funding from the European Commission’s Horizon 2020 research and innovation programme under Grant Agreement n°635770.
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.20185

6. Poll Question #1

7. Ligand Immobilization and Support Synthesis
Separation media
characterization
Synthesis
upscaling
Supply
Ligand
characterization and
support choice
Small scale
immobilization
GMP Mfg & Scale-up
7 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018

8. Ligand Selection
AFFINITY
RANGE
5 to 200 nM
CIP
CONDITIONS
> 90% binding
recovery
6 hours,
0.1M
NaOH
EXPRESSION
YIELDS
beyond 500 mg/L
Expression
yield
Stability
Immunogenicity
Nanofitin
®
ligand discovery process:
In silico immunogenicity prediction
scores below characterized non immunogenic protein
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.20188

9. Nanofitin
®
Ligands
7kDa alternatives to antibodies…
Random mutations
in binding site
Discovered in Yellowstone geysers
Extremely stable to heat
and acid (85°C and pH 2).
Binding specificity redirected to
the target of choice
Specific, high affinity binders
with conserved original
features
Robinson et al., Nature (1998)
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.20189
https://www.affilogic.com

10. …Appropriate for Affinity Separation
 Simple and cost-effective manufacturing by E. coli fermentation
Extremly robust
Affordable custom ligands
 Stable to T° (>80°C) and pH (0-12)
 Highly resistant to CIP treatments
 Straightforward and regio-selective conjugation to resins
Tunable affinity ligands
100% in vitro selection process in 2 months
Capture of macromolecules, from peptides to viruses
With a level of performance comparable to Protein A
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201810
https://www.affilogic.com

11. Ligand Discovery Scheme
Ligand library
screening
Expression
of leaders
Affinity binding
Binding kinetics at
chosen conditions
(e.g. BLI assay)
Physical characterization (e.g.
caustic stability, immunogenicity)
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201811

12. Discovery
Selection Process in Ribosome Display with Affilogic Propriety
Libraries:
•4 rounds with increasing washing pressure
•Possibility of implementing desired elution parameters to orient
selection towards expected specifications
•Selection process optimized to reduce discovery time
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201812

13. Screening
Supernatants of selected Nanofitin
®

ligands were tested in ELISA, clones
were ranked according to their binding
Enzyme-Linked Immunosorbent Assay (ELISA) :
•Direct and indirect affinity screening modes
•Automated process in 96 well plate format
Ligand hits showing
specific interaction
with immobilized
target protein
13
Direct mode –
screening against
epitopes opposite to
biotinylation
Indirect mode –
screening against
random epitopes
13 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018

14. Screen ELISA:
-Biotinylated target is attached in plate bottom on streptavidin
-Clones selection based on the binding signal compared to background noise
-Binding signal at different ligand concentrations (1 µM, 0.1 µM, 0.01 µM)
Nanofitin
®
Ligand Affinity
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
Ligands
showing
strongest
interaction with
immobilized
target protein
CRM197 targets were coated on ELISA plates and binding of Nanofitin
®
ligands (1 µM) was
revealed by anti-RGS HRP antibody
Chosen Nanofitin
®
ligands
present different binding
profiles and affinities ranging
from sub micromolar to low
nanomolar
14

15. Bio-Layer Interferometry (BLI) Assay
Sensor
modification with
ligand
Blocking of
active sides
Association with
POI at defined
conditions (K
on
)
Dissociation at
defined conditions
(K
off
)
Bio-Layer Interferometry (BLI) Assay:
•Affinity constant estimation in defined conditions (e.g. pH, conductivity, additives, etc.)
•4 stage automated process
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201815

16. Binding Level Investigation Experiment:
-In order to evaluate the K
D
of identified clones the identified ligands were fused to sensors
-Affinity constant K
D
estimation in defined conditions (e.g. pH, conductivity, additives, etc.)
Nanofitin
®
Ligand Affinity
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201816
-0.5 M Glycin, pH 2.3
-0.1 M Glycin,
pH 2.3
-0.25 M Glycin, pH 2.3
-10 mM KH
2
PO
4
,
0.09% Tween20, pH 7.2
(binding buffer)

17. Stability
Nanofitin
®
ligands (1 µM) were incubated during 6 hours in NaOH or PBS. Affinity for
CRM197 was compared by ELISA after treatment.
Caustic Stability:
-Expected affinity ligands should be
stable enough to resist to regeneration
cycle
-Nanofitin
®
ligands were tested in
cleaning-in-place conditions used in
biopharmaceutical manufacturing
17 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018

18. Immunogenicity:
-Very low immunogenicity predicted by the calculated immunogenic score for NF A, B, C and D, and low
immunogenic score for E
Ligand Immunogenicity
The global immunogenic score
represents the trend, for each
Nanofitin® ligand and
proportionally to its sequence
length, to contain peptides with
high affinity to MHC-II molecules.
The higher scores are presumed to
have a good chance of binding,
therefore they present a potential
immunogenic risk. Detailed
profiles for the 8 HLA-DRB1 alleles
considered are available in each
Nanofitin® ligand cumulative HLA
chart.
18
Immunogenicity
scale
low
medium
high
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018

19. Ligand Immobilization and Support Synthesis
Separation media
characterization
Synthesis
upscaling
Supply
Ligand
characterization and
support choice
Small scale
immobilization
GMP Mfg & Scale-up
19 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018

20. Eshmuno
®
-type base bead:
•Available particle sizes: 50 µm, 85 µm
•Two different pore sizes
•High chemical stability (NaOH CIP)
•High mechanical stability
•Low non-specific binding
•Flexibility: Particle sizes and pore structure
could be varied, if necessary
For larger species (e.g. VLPs), other
carrier matrices might be considered (e.g.
CPG material)
Selection of Carrier Matrix
SEM images of Eshmuno
®
-type base beads
20 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018

21. Epoxy activation is a preferred method
Surface Activation/Coupling Technologies
Nf
Y
activation
X
conjugation
NfOH
21
X Activation method Y Coupling method
Epoxy Single or multistep reactionNH
2
, SH Nucleophilic substitution
Aldehyde Multistep reaction NH
2
Reductive amination
COOH Single or multistep reactionNH
2
Carbodiimide mediated condensation
Maleimide Multistep reaction SH Addition
Iodoacetyl Multistep reaction SH Nucleophilic substitution
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018

22. Immobilization:
•Ligands usually carry a unique cysteine at
their C-terminal
Improvement of reduction protocol
•pH and salt scouting for ligand
immobilization in 10 ml scale
Coupling yield up to 83% in the best
established conditions
•Comparison of binding capacities and ligand
amount coupled
Choice of the leading ligand to be
scaled up
Small Scale Ligand Immobilization
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
Ligand
reduction
step
Bead
activation
Coupling
Ligand immobilization scheme
22

23. Influence of ligand coupling
conditions on target
binding capacity:
-Optimum pH and salt
concentration window for
coupling, leading to the
highest target binding
capacity.
-A potential to increase
binding capacity through
spacer selection and/or use of
different supports.
Ligand Immobilization
Binding
capacity
(mg/ml)
23 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018

24. Support Physical Characterization
Physical resin
characterization:
•Static binding capacity
measurements using
target
•Bicinchoninic acid assay
(BCA) for the ligand
amount estimation on
surface
•SDS wash to remove non-
covalently bound ligand
BCA assay
results showing
immobilized
ligand amount
on resin
24 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018

25. Robotic Resin Lifetime Screening
Robotic resin lifetime
screening:
•TECAN runs on the
mini-chromatography
column format (e.g.
0.2-0.6 ml)
•Investigation of
different regeneration
solutions for resin
lifetime
•Binding capacity
values for numerous
cycles (e.g. depending
on the target amount
availability)
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201825

26. Robotic Resin Lifetime Screening
Robotic resin lifetime
screening:
•TECAN runs on the
mini-chromatography
column format (e.g.
0.2-0.6 ml)
•Investigation of
different regeneration
solutions for resin
lifetime
•Binding capacity
values for numerous
cycles (e.g. depending
on the target amount
availability)
26 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018

27. Poll Question #2

28. Ligand Immobilization and Support Synthesis
Separation media
characterization
Synthesis
upscaling
Supply
Ligand
characterization and
support choice
Small scale
immobilization
GMP Mfg & Scale-up
28 Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.2018
oLifetime
oOptimal bind and elution
conditions
oAffinity range: 5–200 nM
oExpression
oAbility of reuse
oFrom 10 ml scale to
1000 ml and beyond
oPhysical and function
characterization

29. Affilogic Team:
•Anne Chevrel
•Olivier Kitten
Merck KGaA, Darmstadt, Germany:
•Michael M Schulte
•Achim Schwaemmle
•Oliver Rammo
•Julia Pielok
•Dominic Zorn
Acknowledgements
Custom Affinity Chromatography for Vaccine Purification: A New PD Paradigm | 13.09.201829

30. [email protected]
The vibrant M and Eshmuno 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.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.