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High Pressure Refolding as an Alternative Technology in Protein Manufacturing
Linda Gombos
University of Natural Resources and Life Sciences, Vienna • Boehringer Ingelheim RCV Vienna • Austria
06.08.2015
Protein Refolding
High Pressure Refolding - The Bioprocessing Summit 2015
E. coli: high-yield protein expression ↔ Drawback: dense aggregates (IB) → refold to gain biological activity
Mechanism of High Pressure Treatment
High Pressure Refolding - The Bioprocessing Summit 2015
Main charachteristics of pressure
Effects on protein stability
Densification effect High pressure treatment favors reactions that decrease the volume of a system
Protein aggregates are less dense than native proteins → aggregate dissociation and formation of native, more compact structures
Low energy High pressure only affects weak chemical bonds
Hydrogen bonding and secondary structures are not affected by elevated pressures → Solubilization and refolding without first denaturing the protein
Establishment of the High Pressure Technology at Boehringer Ingelheim RCV in Cooperation with BaroFold
High Pressure Refolding - The Bioprocessing Summit 2015
2011-2012: Feasibility study with 6 Proteins at BaroFold (Aurora, Colorado, USA)
2013-2015: Installation of a high pressure reactor at Process Science Austria Test an unlimited number of molecules at laboratory scale (1 mL-150 mL)
10 L cGMP-Reactor Production of material for toxicological and clinical studies
Large scale reactor (up to 525 L) Substantial investment (new building)
Aims
• Evaluate high pressure refolding
For our specific manufactured molecular formats on a variety of model proteins
Develop experimental approach for fast optimization of process parameters
Compare structure, stability, and activity of the protein variants refolded with high pressure vs. conventional methods
Identify potential economic benefits of high pressure compared with conventional refolding
• Evaluate high pressure for the dissolution of soluble protein aggregates
Using process-related aggregates of a relevant model protein
Show that solubilized monomers have native structure
High Pressure Refolding - The Bioprocessing Summit 2015
Experimental Approach for Optimization of Process Parameters
• Refolding yield: amount of soluble/affinity-captured protein determined
• Versatile method needed suitable for all model proteins and buffers: SDS-PAGE (percent error 13-15%)
• First challenge in protein refolding: to limit meaningful experimental space
High Pressure Refolding - The Bioprocessing Summit 2015
2-level factorial design
• Yield may tend to 0 at extremes of a factor
A
B
„Intuitive raster“
• For 3 levels of 5 factors 35=243 runs
A
B
Optimal design
• Requires the smallest number of runs
A
B
Experimental Approach for Optimization of Process Parameters
Initial screen (pH, urea, arginine, redox) to limit experimental space
Optimization of basic buffer components
Additive screen
Optimization of other parameters (e.g. Fab chain ratio, additive interactions)
Protein concentration and refolding time screen to optimize throughput
Scale-up and purification
High Pressure Refolding - The Bioprocessing Summit 2015
Example for Process Optimization Fc-Fusion Protein
Design-Expert® Software
Correlation: 0.129
1 6 11
16
21
26
31
36
41
46
51
0
5
10
15
20
Run
Yie
ld (
%)
High Pressure Refolding - The Bioprocessing Summit 2015
Initial screen at 2 g/L
pH 8-10 Urea 0-2 M Tris 0.05-1 M Arg 0-0.5 M Cysteine 0-4 mM Cystine 0-4 mM
Parameter optima
pH not significant → set to 9.5 2 M Urea 1 M Tris 0.4 M Arg 3 mM Cysteine 0 mM Cystine
Reduced quadratic model R-Squared 0.6625 Adj R-Squared 0.5935 Pred R-Squared 0.4851 Adeq Precision 10.374
Design-Expert® SoftwareFactor Coding: ActualOriginal ScaleYield (%)
16.3218
0
X1 = E: CysteineX2 = F: Cystine
Actual FactorsA: pH = 9.00B: Tris = 1.00C: Urea = 2.00D: Arg = 0.40
0.0
1.0
2.0
3.0
4.0
0.0 1.0
2.0 3.0
4.0
0
5
10
15
20
25
30
Yie
ld (
%)
E: Cysteine (mM)
F: Cystine (mM)
Design-Expert® SoftwareFactor Coding: ActualOriginal ScaleYield (%)
16.3218
0
X1 = B: TrisX2 = D: Arg
Actual FactorsA: pH = 9.00C: Urea = 2.00E: Cysteine = 3.08F: Cystine = 1.06
0.00 0.10
0.20 0.30
0.40 0.50
0.05 0.15
0.24 0.34
0.43 0.53
0.62 0.71
0.81 0.90
1.00
0
5
10
15
20
25
30
Yie
ld (
%)
B: Tris (M)
D: Arg (M)
Example for Process Optimization Fc-Fusion Protein
High Pressure Refolding - The Bioprocessing Summit 2015
Additive screen at 2 g/L
At optimal conditions from initial screen
Results
Major improvement of yield by applying results of initial screen
No significant further improvement by additives ↔ candidates
A d d it iv e s
Yie
ld (
%)
Base
1 lo
w
1 h
igh
2 lo
w
2 h
igh
3 lo
w
3 h
igh 4 5 6 7 8 9
10
11
12
13
14
15 lo
w
15 h
igh
16 lo
w
16 h
igh
17 lo
w
17 h
igh 1
819
20 lo
w
20 h
igh
0
1 0
2 0
3 0
4 0
S a lts D e te rg e n ts O rg a n ic c o m p o u n d s
Example for Process Optimization Fc-Fusion Protein
High Pressure Refolding - The Bioprocessing Summit 2015
Additive screen at 8 g/L
Candidates alone and in combination
Additive and pH screen at 8 g/L
Results
Additive 15 improves yields at higher protein concentrations
Higher yields at pH 8.5
A d d itiv e s
Yie
ld (
%)
Base
13 lo
w
13 h
igh
15 lo
w
15 h
igh 4 6
12
13 lo
w +
15 lo
w
13 lo
w +
15 h
igh
13 lo
w +
4
13 lo
w +
6
13 lo
w +
12
15 lo
w +
4
15 lo
w +
6
15 lo
w +
12
15 h
igh
+ 4
15 h
igh
+ 6
15 h
igh
+ 1
2
0
5
1 0
1 5
2 0
A d d it iv e s
Yie
ld (
%)
15 h
igh
15 h
igh
+ 6
15 h
igh
+ 1
2
15 h
igh
+ 1
3 lo
w
15 lo
w +
13 lo
w
0
1 0
2 0
3 0
4 0
p H 9 .5
p H 8 .5
High Pressure Refolding Is Readily Scalable
• 1 mL caissons → 5-50 mL syringes → ca. 140 mL bioprocess bags
High Pressure Refolding - The Bioprocessing Summit 2015
scFv Affinity scaffold
200
66
55
22
14
11697
kDaRed. IB 140 mL
37
31
6
3.5
1 m
L
1 m
L
1 m
L
Std
.
200
66
55
22
14
116
97
kDa
37
31
6
1 m
L
1 m
L
1 m
L
Re
du
ce
dIB
5m
L
140 m
L
Protein Purification
High Pressure Refolding - The Bioprocessing Summit 2015
Affinity scaffold Fab scFv
Affinity (ProteinA)
CIEX
Affinity (ProteinA)
CIEX
CIEX
S E C H P L C
T im e (m in )
A2
14 (
mA
bs
)
4 8 1 2 1 6
-3 0 0
0
3 0 0
6 0 0
9 0 0
1 2 0 0
C h a o tro p e
H ig h p re s s u re
14
200
66
55
22
6
3.5
11697
kDa
37
31
Ch
ao
tro
pe
Hig
h p
ressu
reS E C H P L C
T im e (m in )
A2
14 (
mA
bs
)
4 8 1 2 1 6
-1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
C h a o tro p e
H ig h p re s s u re
14
200
66
55
22
6
116
97
kDa
3731
Ch
ao
tro
pe
Hig
h p
ressu
re
S E C H P L C
T im e (m in )
A2
14 (
mA
bs
)
4 8 1 2 1 6
-1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
C h a o tro p e
H ig h p re s s u re
14
200
66
55
22
6
3.5
11697
kDa
37
31
Ch
ao
tro
pe
Hig
h p
ressu
re
Chaotrope: ca. 94% High pressure: ca. 96%
Chaotrope: ca. 94% High pressure: ca. 95%
Chaotrope: 41-49% High pressure: 50-56%
Comparison of Protein Stability and Structure
High Pressure Refolding - The Bioprocessing Summit 2015
Affinity scaffold Fab scFv
D S C
T e m p e r a tu r e ( ° C )
Cp
(k
ca
l/m
ol/
°C
)
0 2 0 4 0 6 0 8 0 1 0 0
-1 0
0
1 0
2 0
3 0
4 0
5 0
H ig h p re s s u re
C h a o tro p eC D s p e c tru m
(n m )
[]
(de
g c
m2 d
mo
l-1)
1 8 0 2 0 0 2 2 0 2 4 0 2 6 0
-6 .01 0 5
-4 .01 0 5
-2 .01 0 5
0
2 .01 0 5
4 .01 0 5
C h a o tro p e
H ig h p re s s u re
D S C
T e m p e r a tu r e ( ° C )
Cp
(k
ca
l/m
ol/
°C
)
0 2 0 4 0 6 0 8 0 1 0 0
-5
0
5
1 0
1 5
C h a o tro p e
H ig h p re s s u reC D s p e c tru m
(n m )
[]
(de
g c
m2 d
mo
l-1)
1 8 0 2 0 0 2 2 0 2 4 0 2 6 0
-4 .01 0 5
-2 .01 0 5
0
2 .01 0 5
4 .01 0 5
C h a o tro p e
H ig h p re s s u re
C D s p e c tru m
(n m )
[]
(de
g c
m2 d
mo
l-1)
1 8 0 2 0 0 2 2 0 2 4 0 2 6 0
-1 .51 0 6
-1 .01 0 6
-5 .01 0 5
0
5 .01 0 5
1 .01 0 6
C h a o tro p e
H ig h p re s s u re
D S C
T e m p e r a tu r e ( ° C )
Cp
(k
ca
l/m
ol/
°C
)
0 2 0 4 0 6 0 8 0 1 0 0
-2 0
0
2 0
4 0
6 0
C h a o tro p e
H ig h p re s s u re
Comparison of Activity Fc-Fusion Protein
High Pressure Refolding - The Bioprocessing Summit 2015
Strucural studies Fc-receptor binding by SPR
-50
0
50
100
150
200
250
-50 0 50 100 150 200 250 300 350 400
RU
Re
sp
on
se
Tim e s
-20
0
20
40
60
80
100
120
140
160
180
-50 0 50 100 150 200 250 300 350 400
RU
Re
sp
on
se
Tim e s
-50
0
50
100
150
200
250
-50 0 50 100 150 200 250 300 350 400
RU
Re
sp
on
se
Tim e s
Sensor surface Fc-fusion / IgG
FcRn
Fc-fusion - chaotrope
Fc-fusion - high pressure
Control - IgG
Kd = 35 11 nM
Kd = 46 8 nM
Kd = 63 3 nM
D S C
T e m p e r a tu r e ( ° C )
Cp
(k
ca
l/m
ole
/°C
)
2 0 4 0 6 0 8 0 1 0 0
-5
0
5
1 0
1 5
2 0
2 5
C h a o tro p e
H ig h p re s s u re
C D s p e c tru m
W a v e le n g th (n m )
[]
(de
g c
m2 d
mo
l-1)
2 0 0 2 2 0 2 4 0 2 6 0
-3 .01 0 5
-2 .01 0 5
-1 .01 0 5
0
1 .01 0 5
2 .01 0 5
3 .01 0 5
C h a o tro p e
H ig h p re s s u re
Low Population of Aggregate-prone Intermediates Increases Refolding Yields at High Protein Concentrations
High Pressure Refolding - The Bioprocessing Summit 2015
M axim a l re fo ld in g y ie ld s
P r o te in c o n c e n tra t io n (g /L )
Yie
ld (
%)
0 5 1 0 1 5
0
2 0
4 0
6 0
8 0
C h a o tro p e
H ig h p re s s u re
M a xim a l p ro d u ctiv ity
P r o te in c o n c e n tra t io n (g /L )P
ro
du
cti
vit
y (
g/L
/h)
0 .1 1 1 0 1 0 0
0 .0 1
0 .1
1
1 0
C h a o tro p e
H ig h p re s s u re
• Fc-fusion protein refolded conventionally and with high pressure
High Pressure Refolding Increases Productivity
Molecule class Conventional refolding
High pressure refolding
Increase in productivity
Fabs
15% at 0.8 g/L in 30 h 20% at 2 g/L in 6+24 h* 17x
15% at 0.2 g/L in 16 h 6% at 2 g/L in 6 h 11x
3 Fabs not refoldable 3 Fabs not refoldable -
scFv 80% at 0.2 g/L in 8 h 40% at 6 g/L in 16 h 8x
Other affinity scaffolds
80% at 0.9 g/L in 18 h 50% at 4 g/L in 6 h 8x
10% at 0.1 g/L in 24 h 5% at 2 g/L in 8 h 30x
50% at 1.2 g/L in 24 h 60% at 2 g/L in 2 h 24x
Fc-fusions 35% at 0.8 g/L in 3 h 20% at 15 g/L in 2 h 16x
15% at 0.7 g/L in 5 h 50% at 4 g/L in 6 h 16x
Other fusion proteins
45% at 1 g/L in 24 h 30% at 30 g/L in 1+8 h* 480x
20% at 1.2 g/L in 12 h 35% at 10 g/L in 1+20 h* 175x
45% at 2 g/L in 14 h 50% at 15 g/L in 1+10 h* 117x
High Pressure Refolding - The Bioprocessing Summit 2015
* Time in refolding vessel used for the calculation of productivity
Large-scale Manufacturing Feasibility for 2 kg Cleaved and Purified Fusion Protein
High Pressure Refolding - The Bioprocessing Summit 2015
Protein mass: 48 kg Titer: 12 g/L Volume: 3,700 L
3 columns Overall yield: 50% Target protein: 2 kg
Step yield: 90% Protein mass: 44 kg
Step yield: 90% Protein mass: 27 kg
Target protein: 4 kg Fusion protein: 12 kg 45% yield at 1 g/L in 24 h 27 kg protein in 27,000 L Max. capacity at BI: 35,000 L 2 cycles at full capacity
Target protein: 4 kg Fusion protein: 12 kg 30% yield at 30 g/L in 1+10 h 40 kg protein in 1,300 L Max. capacity : 525 L 3 cycles within 1 day
Fermentation
IB recovery
Purification
Fill/finish
Solubilization
Refolding by dilution
Refolding by pressure
treatment
Step yield: 90% Protein mass: 30 kg
Protein mass: 30 kg Titer: 12 g/L
Volume: ca. 2,500 L
Dissolution of Soluble Protein Aggregates
High Pressure Refolding - The Bioprocessing Summit 2015
• Origin of soluble aggregates:
Mammalian cell expression
Protein processing and purification
• Fields of application:
Early in downstream processing dissolve large amounts of soluble aggregates formed during expression
Polishing step solubilize trace amounts of aggregates to meet quality criteria
• Model protein:
Expressed in CHO cells
Capture pool contains ca. 12% aggregates
S E C H P L C c h ro m a to g ra m
T im e (m in )
A2
14 (
mA
U)
2 4
-1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
4 6 8 1 0 1 2
A g g re g a te s
M o d if ie d m o n o m e rs
N a tiv e m o n o m e rs
S E C H P L C c h ro m a to g ra m
T im e (m in )
A2
14 (
mA
U)
2 4
-1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
4 6 8 1 0 1 2
Effects of Pressure and pH
High Pressure Refolding - The Bioprocessing Summit 2015
p HA
gg
re
ga
te c
on
ten
t (%
)
6 .0 7 .0 8 .0 9 .00
5
1 0
1 5 A m b ie n t p re s s u re
2 5 0 0 b a r
P re s s u re
Ag
gre
ga
te c
on
ten
t (%
)
2 0 0 0 b a r 3 0 0 0 b a r0
5
1 0
1 5 A m b ie n t p re s s u re
H ig h p re s s u re
Additives Do Not Improve the Solubilization of Aggregates
High Pressure Refolding - The Bioprocessing Summit 2015
A d d it iv e
% a
gg
re
ga
tes
HP
/% a
gg
re
ga
tes
AP
base 1 2 3 4 5 6 7 8
9 lo
w
9 h
igh
10 lo
w
10 h
igh
11 lo
w
11 h
igh
12 lo
w
12 h
igh
13 lo
w
13 h
igh
14 lo
w
14 h
igh
15
16 lo
w
16 h
igh
0 .0 0
0 .2 5
0 .5 0
0 .7 5
1 .0 0
1 .2 5
1 .5 0D e te rg e n ts O rg a n ic c o m p o u n d s
Effects of Pressurization Time and Protein Concentration
High Pressure Refolding - The Bioprocessing Summit 2015
P ro te in c o n c e n tra t io n (g /L )Fo
ld r
ed
uc
tio
n o
f a
gg
re
ga
te c
on
ten
t
0 .5 1 2 40
1
2
3
41 6 h
1 h
T im e (m in )
A2
14 (
mA
U)
2 4
-1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
4 6 8 1 0 1 2
a g g re g a te s
S E C c h ro m a to g ra m s
A m b ie n t p re s s u re
3 0 0 0 b a r
P ro te in c o n c e n tra t io n (g /L )
Ag
gre
ga
te c
on
ten
t (%
)
0 .5 1 2 4 60
5
1 0
1 5A m b ie n t p re s s u re
1 h a t 3 0 0 0 b a r
Are the monomers which were dissolved from aggregates identical to the monomers which have always been monomers?
www.clipartbest.com
Enrichment of Monomers Dissolved from Aggregates
High Pressure Refolding - The Bioprocessing Summit 2015
HIC regeneration pool
SEC (S200) purification of aggregates
High pressure dissolution of aggregates
SEC (S200) purification of monomers
S E C H P L C c h ro m a to g ra m s
T im e (m in )
A2
14 (
mA
U)
2 4
-1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
4 6 8 1 0 1 2
H IC re g e n e ra tio n p e a k
S E C p u rif ie d a g g re g a te s
H P tre a te d a g g re g a te s
S E C p u rif ie d m o n o m e rs
R e fe re n c e
4 1 % A g g re g a te
2 0 % M o d if ie d
3 9 % M o n o m e r
8 8 % A g g re g a te
8 % M o d if ie d
4 % M o n o m e r
2 0 % A g g re g a te
1 1 % M o d if ie d
6 9 % M o n o m e r
2 % A g g re g a te
9 % M o d if ie d
8 9 % M o n o m e r
Monomers Dissolved from Aggregates Are Highly Similar to the Reference Material
High Pressure Refolding - The Bioprocessing Summit 2015
S e c o n d a ry s tru c tu re b y C D s p e c tro s c o p y
V a w e le n g th (n m )
[ ]
(de
g c
m2 d
mo
l-1)
2 0 0 2 2 0 2 4 0 2 6 0
-3 0 0 0 0 0
-2 0 0 0 0 0
-1 0 0 0 0 0
0
1 0 0 0 0 0
2 0 0 0 0 0R e fe re n c e
le s s s tr in g e n t c o n d it io n s
H P d is s o lv e d
le s s s tr in g e n t c o n d it io n s
R e fe re n c e
m o re s tr in g e n t c o n d it io n s
H P d is s o lv e d
m o re s tr in g e n t c o n d it io n s
Monomers Dissolved from Aggregates Are Highly Similar to the Reference Material
High Pressure Refolding - The Bioprocessing Summit 2015
T h e rm a l s ta b ility b y D S C
T e m p e r a tu re (° C )
Cp
(k
ca
l/m
ol/
°C
)
2 0 4 0 6 0 8 0 1 0 0
-1 0
0
1 0
2 0
3 0R e fe re n c e
le s s s tr in g e n t c o n d it io n s
H P d is s o lv e d
le s s s tr in g e n t c o n d it io n s
R e fe re n c e
m o re s tr in g e n t c o n d it io n s
H P d is s o lv e d
m o re s tr in g e n t c o n d it io n s
T h e rm a l s ta b ility b y in tr in s ic f lu o re s c e n c e
T e m p e r a tu re (° C )
BC
M w
av
ele
ng
th (
nm
)
2 0 4 0 6 0 8 0 1 0 0
3 3 0
3 4 0
3 5 0
3 6 0R e fe re n c e
le s s s tr in g e n t c o n d it io n s
H P d is s o lv e d
le s s s tr in g e n t c o n d it io n s
R e fe re n c e
m o re s tr in g e n t c o n d it io n s
H P d is s o lv e d
m o re s tr in g e n t c o n d it io n s
A g g re g a tio n b y lig h t s c a tte r in g
T e m p e r a tu re (° C )
SL
S2
66 (
co
un
ts.n
m)
2 0 4 0 6 0 8 0 1 0 0
0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0R e fe re n c e
le s s s tr in g e n t c o n d it io n s
H P d is s o lv e d
le s s s tr in g e n t c o n d it io n s
R e fe re n c e
m o re s tr in g e n t c o n d it io n s
H P d is s o lv e d
m o re s tr in g e n t c o n d it io n s
Summary
Evaluated high pressure for
Refolding a variety of BI‘s model proteins
Dissolution of soluble protein aggregates
Inclusion body refolding:
High pressure increases productivity compared with conventional chaotrope-based techniques
Structure, stability, and activity of proteins refolded with high pressure vs. conventionally are highly comparable
High pressure refolding is readily scalable
High pressure can enable large-scale manufacturing
Aggregate solubilization:
High pressure treatment converts soluble aggregates into native-like monomers
High Pressure Refolding - The Bioprocessing Summit 2015
Acknowledgement
High Pressure Refolding - The Bioprocessing Summit 2015
• Boehringer-Ingelheim RCV Process Science
Susanne Schweiger
Gerald Bieder
Martin Kellner
Srjib Banerjee
Cornelia Walther
Dean Harde
Robert Wandl
Matthias Berkemeyer
Jan Schöning
Wolfgang Buchinger
• University of Natural Resources and Life Sciences, Vienna
Alois Jungbauer
• BaroFold
Matt Seefeldt