This document summarizes research on using high pressure refolding as an alternative to conventional refolding methods for producing proteins. It describes how high pressure refolding can dissolve protein aggregates formed during expression more gently than chemical or thermal denaturation. The document outlines the optimization of high pressure refolding processes for several model proteins through experimental designs, and finds the refolded proteins have similar stability, structure and activity as those refolded through conventional methods.

1. 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

2. Protein Refolding
High Pressure Refolding - The Bioprocessing Summit 2015
E. coli: high-yield protein expression
↔ Drawback: dense aggregates (IB) → refold to gain biological activity

3. 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

4. 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)

5. 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

6. 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

7. 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

8. 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
Yield(%)
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 MTris
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® Software
Factor Coding: Actual
Original Scale
Yield (%)
16.3218
0
X1 = E: Cysteine
X2 = F: Cystine
Actual Factors
A: pH = 9.00
B: Tris = 1.00
C: Urea = 2.00
D: 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
Yield(%)
E: Cysteine (mM)
F: Cystine (mM)
Design-Expert® Software
Factor Coding: Actual
Original Scale
Yield (%)
16.3218
0
X1 = B: Tris
X2 = D: Arg
Actual Factors
A: pH = 9.00
C: Urea = 2.00
E: Cysteine = 3.08
F: 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
Yield(%)
B: Tris (M)
D: Arg (M)

9. 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 itiv e s
Yield(%)
B
a
s
e1
lo
w1
h
ig
h2
lo
w2
h
ig
h3
lo
w3
h
ig
h
4
5
6
7
8
9
1
0
1
1
1
2
1
3
1
4
1
5
lo
w
1
5
h
ig
h
1
6
lo
w
1
6
h
ig
h
1
7
lo
w
1
7
h
ig
h
1
8
1
9
2
0
lo
w
2
0
h
ig
h
0
1 0
2 0
3 0
4 0
S a lts D e te rg e n ts O rg a n ic co m p o u n d s

10. 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 itives
Yield(%)
B
a
s
e1
3
lo
w
1
3
h
ig
h1
5
lo
w
1
5
h
ig
h
4
6
1
2
1
3
lo
w
+
1
5
lo
w
1
3
lo
w
+
1
5
h
ig
h
1
3
lo
w
+
4
1
3
lo
w
+
6
1
3
lo
w
+
1
2
1
5
lo
w
+
4
1
5
lo
w
+
6
1
5
lo
w
+
1
2
1
5
h
ig
h
+
4
1
5
h
ig
h
+
6
1
5
h
ig
h
+
1
2
0
5
1 0
1 5
2 0
A d d itiv e s
Yield(%)
1
5
h
ig
h
1
5
h
ig
h
+
6
1
5
h
ig
h
+
1
21
5
h
ig
h
+
1
3
lo
w1
5
lo
w
+
1
3
lo
w
0
1 0
2 0
3 0
4 0
p H 9 .5
p H 8 .5

11. 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
116
97
kDa
Red. IB 140 mL
37
31
6
3.5
1mL
1mL
1mL
Std.
200
66
55
22
14
116
97
kDa
37
31
6
1mL
1mL
1mL
ReducedIB
5mL
140mL

12. Protein Purification
High Pressure Refolding - The Bioprocessing Summit 2015
Affinity scaffoldFab scFv
Affinity (ProteinA)
CIEX
Affinity (ProteinA)
CIEX
CIEX
S E C H P L C
T im e (m in )
A214(mAbs)
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 su re
14
200
66
55
22
6
3.5
116
97
kDa
37
31
Chaotrope
Highpressure
S E C H P L C
T im e (m in )
A214(mAbs)
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 su re
14
200
66
55
22
6
116
97
kDa
37
31
Chaotrope
Highpressure
S E C H P L C
T im e (m in )
A214(mAbs)
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 su re
14
200
66
55
22
6
3.5
116
97
kDa
37
31
Chaotrope
Highpressure
Chaotrope: ca. 94%
High pressure: ca. 96%
Chaotrope: ca. 94%
High pressure: ca. 95%
Chaotrope: 41-49%
High pressure: 50-56%

13. Comparison of Protein Stability and Structure
High Pressure Refolding - The Bioprocessing Summit 2015
Affinity scaffoldFab scFv
D S C
T e m p e ra tu re (°C )
Cp(kcal/mol/°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 su re
C h a o tro p e
C D s p e c tru m
 (n m )
[](degcm
2
dmol
-1
)
1 8 0 2 0 0 2 2 0 2 4 0 2 6 0
-6 .0 1 0 5
-4 .0 1 0 5
-2 .0 1 0 5
0
2 .0 1 0 5
4 .0 1 0 5
C h a o tro p e
H ig h p re s su re
D S C
T e m p e ra tu re (°C )
Cp(kcal/mol/°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 su re
C D s p e c tru m
 (n m )
[](degcm
2
dmol
-1
)
1 8 0 2 0 0 2 2 0 2 4 0 2 6 0
-4 .0 1 0 5
-2 .0 1 0 5
0
2 .0 1 0 5
4 .0 1 0 5
C h a o tro p e
H ig h p re s su re
C D s p e c tru m
 (n m )
[](degcm
2
dmol
-1
)
1 8 0 2 0 0 2 2 0 2 4 0 2 6 0
-1 .5 1 0 6
-1 .0 1 0 6
-5 .0 1 0 5
0
5 .0 1 0 5
1 .0 1 0 6
C h a o tro p e
H ig h p re s su re
D S C
T e m p e ra tu re (°C )
Cp(kcal/mol/°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 su re

14. 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
Response
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
Response
Tim e s
-50
0
50
100
150
200
250
-50 0 50 100 150 200 250 300 350 400
RU
Response
Tim e s
Sensorsurface 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 ra tu re (°C )
Cp(kcal/mole/°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 )
[](degcm
2
dmol
-1
)
2 0 0 2 2 0 2 4 0 2 6 0
-3 .0 1 0 5
-2 .0 1 0 5
-1 .0 1 0 5
0
1 .0 1 0 5
2 .0 1 0 5
3 .0 1 0 5
C h a o tro p e
H ig h p re s s u re

15. Low Population of Aggregate-prone Intermediates
Increases Refolding Yields at High Protein Concentrations
High Pressure Refolding - The Bioprocessing Summit 2015
M axim al refolding yields
P ro te in c o n c e n tra tio n (g /L )
Yield(%)
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 su re
M axim al produ ctivity
P ro te in c o n c e n tra tio n (g /L )
Productivity(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 su re
• Fc-fusion protein refolded conventionally and with high pressure

16. 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

17. 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: 35,000 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

18. 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 )
A214(mAU)
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 ifie 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 )
A214(mAU)
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

19. Effects of Pressure and pH
High Pressure Refolding - The Bioprocessing Summit 2015
p HAggregatecontent(%)
6 .0 7 .0 8 .0 9 .0
0
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
Aggregatecontent(%)
2 0 0 0 b a r 3 0 0 0 b a r
0
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

20. Additives Do Not Improve the Solubilization of Aggregates
High Pressure Refolding - The Bioprocessing Summit 2015
A d d itiv e
%aggregatesHP/%aggregatesAP
b
a
s
e
1
2
3
4
5
6
7
89
lo
w9
h
ig
h
1
0
lo
w
1
0
h
ig
h
1
1
lo
w
1
1
h
ig
h
1
2
lo
w
1
2
h
ig
h
1
3
lo
w
1
3
h
ig
h
1
4
lo
w
1
4
h
ig
h
1
5
1
6
lo
w
1
6
h
ig
h
0 .0 0
0 .2 5
0 .5 0
0 .7 5
1 .0 0
1 .2 5
1 .5 0
D e te rg e n ts O rg a n ic c o m p o u n d s

21. 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 tio n (g /L )
Foldreductionofaggregatecontent
0 .5 1 2 4
0
1
2
3
4
1 6 h
1 h
T im e (m in )
A214(mAU)
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 ssu re
3 0 0 0 b a r
P ro te in c o n c e n tra tio n (g /L )
Aggregatecontent(%)
0 .5 1 2 4 6
0
5
1 0
1 5
A 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

22. 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 )
A214(mAU)
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 rifie 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 rifie d m o n o m e rs
R e fe re n ce
4 1 % A g g re g a te
2 0 % M o d ifie d
3 9 % M o n o m e r
8 8 % A g g re g a te
8 % M o d ifie d
4 % M o n o m e r
2 0 % A g g re g a te
1 1 % M o d ifie d
6 9 % M o n o m e r
2 % A g g re g a te
9 % M o d ifie d
8 9 % M o n o m e r

23. 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 )
[](degcm
2
dmol
-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 0
R e fe re n c e
le s s s trin g e n t c o n d itio n s
H P d is s o lv e d
le s s s trin g e n t c o n d itio n s
R e fe re n c e
m o re s trin g e n t c o n d itio n s
H P d is s o lv e d
m o re s trin g e n t c o n d itio n s

24. 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 ra tu re (°C )
Cp(kcal/mol/°C)
2 0 4 0 6 0 8 0 1 0 0
-1 0
0
1 0
2 0
3 0
R e fe re n c e
le s s s trin g e n t c o n d itio n s
H P d is s o lv e d
le s s s trin g e n t c o n d itio n s
R e fe re n c e
m o re s trin g e n t c o n d itio n s
H P d is s o lv e d
m o re s trin g e n t c o n d itio n s
T h e rm a l s ta b ility b y in trin s ic flu o re s c e n c e
T e m p e ra tu re (°C )
BCMwavelength(nm)
2 0 4 0 6 0 8 0 1 0 0
3 3 0
3 4 0
3 5 0
3 6 0
R e fe re n c e
le s s s trin g e n t c o n d itio n s
H P d is s o lv e d
le s s s trin g e n t c o n d itio n s
R e fe re n c e
m o re s trin g e n t c o n d itio n s
H P d is s o lv e d
m o re s trin g e n t c o n d itio n s
A g g re g a tio n b y lig h t s c a tte rin g
T e m p e ra tu re (°C )
SLS266(counts.nm)
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 0
R e fe re n c e
le s s s trin g e n t c o n d itio n s
H P d is s o lv e d
le s s s trin g e n t c o n d itio n s
R e fe re n c e
m o re s trin g e n t c o n d itio n s
H P d is s o lv e d
m o re s trin g e n t c o n d itio n s

25. 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

26. 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