The document discusses the development and application of mixed mode chromatography using mobile phase modulators to enhance protein purification processes. It highlights various modulators and their impact on protein binding characteristics, including their effects during the capture and polishing steps of biopharmaceutical manufacturing. By integrating modulators, the process aims to increase selectivity and purity while reducing levels of host cell proteins (HCPs) in the final product.

1. Leslie
S.
Wolfe,
Ph.D.
Abhinav
A.
Shukla,
Ph.D.
Process
Development
KBI
Biopharma,
Durham,
NC

2. • Background
• Mixed
Mode
Chromatography
• Mobile
Phase
Modulators
• CharacterizaGon
of
Protein
Binding
• Linear
salt
gradient
eluGon
studies
• log
k
vs.
log
salt
concentraGon
plots
• Enhancing
purificaGon
through
modulator
washes
• During
Capture
step
• During
Polishing
step
• Process
impact

3. • Takes
advantage
of
more
than
one
type
of
interacGon
» i.e.
ionic,
hydrophobic,
hydrogen
bonding
• Provides
enhanced
selecGvity,
pseudo-­‐affinity
• Can
reduce
process
steps
• Several
mixed
mode
resins
have
recently
been
developed
with:
» Increased
loading
capaci4es
» Higher
ionic
strength
tolerance
GE Healthcare, Capto MMC ligand
Ionic interactions
Hydrophobic interactions
Hydrophobic interactions
GE Healthcare, Capto Adhere ligand
Ionic interactions

4. • Mobile
phase
modulators:
addiGves
incorporated
into
process
buffers
to
alter
protein-­‐ligand
interacGons
• Modulators
can
enhance
resin
selecGvity
and
eluate
purity
when
incorporated
into
load,
wash
and/or
eluGon
process
steps
• AddiGon
of
a
combinaGon
of
modulators
can
further
improve
selecGvity
Modulator
Modulator
Effect
MgCl2,
NaSCN,
KI
Decrease
hydrophobic
interacGons
Ethanol,
Methanol,
Isopropanol
Decrease
hydrophobic
interacGons
(used
in
low
concentraGons)
Urea
Weakens
hydrogen
bonding,
denaturant
Glycerol
Weakens
hydrophobic
interacGons
Ethylene
Glycol
Weakens
hydrophobic
interacGons
and
hydrogen
bonding
Arginine
Weakens
hydrophobic
interacGons,
induces
protein
unfolding,
disrupts
electrostaGc
interacGons
Ammonium
Sulfate
Strengthens
hydrophobic
interacGons

5. • Background
• Mixed
Mode
Chromatography
• Mobile
Phase
Modulators
• Characteriza2on
of
Protein
Binding
• Linear
salt
gradient
elu2on
studies
• log
k
vs.
log
salt
concentraGon
plots
• Enhancing
purificaGon
through
modulator
washes
• During
Capture
step
• During
Polishing
step
• Process
impact

6. • Resin:
Capto
MMC
• Modulator
added
to
equilibraGon,
wash
and
eluGon
buffers
• Products
eluted
with
a
linear
NaCl
gradient
• Model
proteins:
• RNase
(pI
8.9)
• Lysozyme
(pI
9.6)
• mAb1
• mAb2
• mAb3
• mAb4
• Mobile
phase
modifiers:
• Ethylene
glycol
• Urea
• Arginine
• Sodium
Thiocyanate
• Ammonium
sulfate

7. 357 312
221 249 202
0
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
RNase
2,217 1,862 1,766 1,981
1,248
2,500
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
Lysozyme
∞
1,482 1,602
847 962 916
1,800
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
mAb4
∞
300 296
198 136
235
400
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
mAb3
∞
314 304
209
132
237
400
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
mAb2
∞
1,219 1,145 824 795 809
2,500
No
modulator
5% ethylene
glycol
50mM
arginine
50mM
sodium
thiocyanate
1M urea 1M
ammonium
sulfate
Elution[NaCl](mM)
mAb1
∞
∞ = target protein did not elute during NaCl gradient

8. • AnGbodies
behave
differently
• In
absence
of
modulator
» mAb2,
mAb3
–
low
salt
(~300mM)
» mAb1,
mAb4
–
high
salt
(~1.5M)
• Modulator
with
largest
effect
» mAb1,
mAb2,
mAb3
–
sodium
thiocyanate
» mAb4
–
arginine
• Lysozyme
requires
highest
NaCl
for
eluGon
• RNase
does
not
bind
in
the
presence
of
1M
(NH4)2SO4
• All
other
proteins
tested
irreversibly
bind
in
1M
(NH4)2SO4

10. • As
pH
approaches
pI,
retenGon
decreases
• Lysozyme
does
not
elute
in
absence
of
modulator
at
pH
6.0
• AnGbodies
behave
more
similarly
as
pH
approaches
pI

11. • Background
• Mixed
Mode
Chromatography
• Mobile
Phase
Modulators
• Characteriza2on
of
Protein
Binding
• Linear
salt
gradient
eluGon
studies
• log
k
vs.
log
salt
concentra2on
plots
• Enhancing
purificaGon
through
modulator
washes
• During
Capture
step
• During
Polishing
step
• Process
impact

12. • Protein
retenGon
under
linear
loading
condiGons
is
dependent
on
the
thermodynamics
of
the
interacGon
between
the
protein
and
the
staGonary
phase
log K = (- ΔG°es / 2.3RT) + (-ΔG°hΦ / 2.3RT)
∆Ges = Gibbs free energies for retention by electrostatic interactions
∆GhΦ = Gibbs free energies for retention by hydrophobic interactions
T = the absolute temperature
R = the universal gas constant
k’ = ΦK
Retention factor (k’) relates to K by,
where Φ is the ratio of stationary and mobile phase volumes

13. • This
relaGonship
was
further
described
by
Melander
et.
al
to
describe
the
dependency
of
the
linear
retenGon
factor
on
a
mixed
mode
sorbent
as
a
funcGon
of
salt
concentraGon
as:
log k’ = A – Blog(csalt) + C(csalt)
where csalt is the mobile phase salt concentration in molar units and A, B and C are constants
The
retenGon
factor
under
isocraGc
condiGons
is
represented
by:
k’ = tr – tm /tm
tm
=
Gme
for
mobile
phase
to
pass
through
column
tr
=
target
protein
retenGon
Gme
Melander, W.; El Rassi, Z.; Horvath, Cs. Journal of Chromatography, 469, 3-27, 1989.

14. • This
relaGonship
was
further
described
by
Melander
et.
al
to
describe
the
dependency
of
the
linear
retenGon
factor
on
a
mixed
mode
sorbent
as
a
funcGon
of
salt
concentraGon
as:
log k’ = A – Blog(csalt) + C(csalt)
where csalt is the mobile phase salt concentration in molar units and A, B and C are constants
The
retenGon
factor
under
isocraGc
condiGons
is
represented
by:
k’ = tr – tm /tm
tm
=
Gme
for
mobile
phase
to
pass
through
column
tr
=
target
retenGon
Gme
Melander, W.; El Rassi, Z.; Horvath, Cs. Journal of Chromatography, 469, 3-27, 1989.
• ElectrostaGc
interacGons
predominate:
a
linear
relaGonship
is
expected
between
log
k’ vs
log[NaCl]
• Hydrophobic
interacGons
predominate:
a
linear
relaGonship
is
expected
unGl
a
minimum
is
reached
at
which
point
further
increases
in
salt
result
in
increased
retenGon

15. • RetenGon
factors
(k )
were
determined
for
» mAb1
» RNase
» Lysozyme
• Mobile
phase
modulators
tested
» No
modulator
» 1M
Urea
» 50mM
Arginine
» 5%
Ethylene
Glycol

16. • RNase
» electrostatic interactions
» No effect from urea or ethylene glycol
• Lysozyme
» hydrophobic and electrostatic interactions
» urea has largest effect
• mAb1
» driven by electrostatic interactions, hydrophobic contribution
» Urea and arginine have the largest effect
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
2.10 2.30 2.50 2.70
Logk'
Log [NaCl]
mAb1
All experiments performed at pH 7.0
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.50 2.00 2.50
Logk'
Log [NaCl]
RNase
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
2.60 3.10 3.60Logk'
Log [NaCl]
Lysozyme
¿ baseline
˜ 1M urea
¢ 5% ethylene glycol
p 50mM arginine

17. • Background
• Mixed
Mode
Chromatography
• Mobile
Phase
Modulators
• CharacterizaGon
of
Protein
Binding
• Linear
salt
gradient
eluGon
studies
• log
k
vs.
log
salt
concentraGon
plots
• Enhancing
purifica2on
through
modulator
washes
• During
Capture
step
• During
Polishing
step
• Process
impact

18. • IncorporaGon
of
modulators
into
process
can
help
increase
selecGvity
and
purity
of
product
• CombinaGons
of
modulators
can
further
enhance
process
step
• Goal:
UGlize
mobile
phase
modulators
to
decrease
HCP
levels
during
Capto
MMC
process
step
for
anGbody
purificaGon

19. • Case
Study:
» Target
molecule:
E.
coli
derived
recombinant
protein
» Process
step:
Phenyl
Sepharose
FF
» Ini4al
product
yield:
88.8%
» Result:
Individual
modulators
showed
some
selecGvity
enhancement
but
also
product
loss
A
combinaGon
of
urea,
sodium
thiocyanate
and
glycerol
in
the
wash
step
increased
product
purity
to
>95%
Shukla AA, et al., 2002. Biotechnol Prog 18: 556–564.

20. • Background
• Mixed
Mode
Chromatography
• Mobile
Phase
Modulators
• CharacterizaGon
of
Protein
Binding
• Linear
salt
gradient
eluGon
studies
• log
k
vs.
log
salt
concentraGon
plots
• Enhancing
purifica2on
through
modulator
washes
• During
Capture
step
• During
Polishing
step
• Process
impact

21. 0.50
0.70
0.90
1.10
1.30
1.50
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
Normalized
HCP
Recovery
HCP
vs.
Recovery
aFer
Intermediate
Wash
for
Capto
MMC
Capture
baseline

22. • Background
• Mixed
Mode
Chromatography
• Mobile
Phase
Modulators
• CharacterizaGon
of
Protein
Binding
• Linear
salt
gradient
eluGon
studies
• log
k
vs.
log
salt
concentraGon
plots
• Enhancing
purifica2on
through
modulator
washes
• During
Capture
step
• During
Polishing
step
• Process
impact

23. 0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
Normalized
HCP
Recovery
HCP
vs.
Recovery
aFer
Intermediate
Wash
for
Capto
MMC
Polishing
baseline

24. • Background
• Mixed
Mode
Chromatography
• Mobile
Phase
Modulators
• CharacterizaGon
of
Protein
Binding
• Linear
salt
gradient
eluGon
studies
• log
k
vs.
log
salt
concentraGon
plots
• Enhancing
purifica2on
through
modulator
washes
• During
Capture
step
• During
Polishing
step
• Process
impact

25. • Inclusion
of
an
intermediate
wash
using
Tris,
0.1M
NaCl,
50mM
arginine,
5%
ethylene
glycol,
pH
7.0
resulted
in
2-­‐
fold
lower
HCP
levels
when
compared
to
process
where
a
modulator
was
not
uGlized

26. • In
depth
studies
have
afforded
us
an
understanding
of
how
modulators
affect
different
molecules
• Despite
similar
class
of
molecules,
anGbodies
behave
differently
with
Capto
MMC
ligand
• We
have
uGlized
mixed
mode
chromatography
to
improve
product
purity
and
maintain
process
step
yield
• IncorporaGon
of
a
process
step
uGlizing
a
modulator
wash
can
improve
overall
process
HCP
clearance
• At
KBI
Biopharma
we
have
developed
several
downstream
processes
and
manufactured
biopharmaceuGcal
products
where
modulator
washes
on
a
mixed
mode
resin
have
significantly
contributed
to
process
HCP
clearance
» Effec4ve
for
both
molecules
derived
from
mammalian
and
microbial
cell
culture
processes

27. Pre-Clinical Phase I Phase II Phase III
FIH Process
• Deliver clinical process
quickly
• Platform process
• Clinical Supply
Submission &
Approval
Lifecycle
management
Commercial Process
• Deliver manufacturing process for
registrational trials and market
• Design keeping large-scale manufacturing
in mind
• Improve productivity, efficiency, robustness,
manufacturability, COGs
• Analytical characterization and method
development
Process Characterization and Validation
• Develop IPC strategy through understanding of process inputs and
outputs (design space)
• Scale-down characterization and validation studies
• Large-scale process validation to demonstrate process consistency
• BLA preparation
• Supporting documents for licensure inspections
• Post-commercial process improvements (CI)
• Post-commercial process monitoring
FIH process Commercial process
Gottschalk U., Konstantinov K., Shukla A. Nature Biotechnology, 30(6), 489-491, 2012
Process
Development
Process
Validation
Process Monitoring
&
Improvement
BLA
&
PAI
Manufacturing
for
Tox
Clinical
Manufacturing
Commercial
Process
Development
Process
Characterization

28. Protein: protein interactions
Protein:resininteractions
Modulator washes can augment protein:protein interactions
and/or protein:resin interactions
• Mixed
mode
chromatography
is
advantageous
because
of
its
increased
selecGvity
by
exploiGng
mulGple
chemical
properGes
of
target
protein

29. • Abhinav
Shukla,
Ph.D.
• Sigma
Mostafa,
Ph.D.
• Cartney
Barringer
• KBI
Process
Development
Team
Work available online ahead of print in Journal of Chromatography A
Wolfe, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.02.086