High Pressure Refolding BPS 2015 LG

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


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


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


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


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


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 (

%)


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)



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



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


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


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


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


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


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


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


D S 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


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


D S 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


Comparison of Activity Fc-Fusion Protein


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


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


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


Low Population of Aggregate-prone Intermediates Increases Refolding Yields at High Protein Concentrations


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


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


• 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




Fc-fusions 35% at 0.8 g/L in 3 h 20% at 15 g/L in 2 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


* Time in refolding vessel used for the calculation of productivity

Large-scale Manufacturing Feasibility for 2 kg Cleaved and Purified Fusion Protein


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


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


Protein mass: 30 kg Titer: 12 g/L

Volume: ca. 2,500 L

Dissolution of Soluble Protein Aggregates


• 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


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


Additives Do Not Improve the Solubilization of Aggregates


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


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?

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Enrichment of Monomers Dissolved from Aggregates


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 % M o d if ie d

4 % M o n o m e r


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


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


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


Monomers Dissolved from Aggregates Are Highly Similar to the Reference Material


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


H P d is s o lv e d


R e fe re n c e


H P d is s o lv e d


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


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


H P d is s o lv e d


R e fe re n c e


H P d is s o lv e d


A g g re g a tio n b y lig h t s c a tte r in g


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


H P d is s o lv e d


R e fe re n c e


H P d is s o lv e d


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


Acknowledgement


• 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

High Pressure Refolding BPS 2015 LG

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