High Pressure Disaggregation and Refolding of Proteins

Ted Randolph, Matt Seefeldt, Jon Webb, Rick St. John, Yongsung Kim, Ryan Crisman, Amber Haynes, John

CarpenterCenter for Pharmaceutical Biotechnology

Department of Chemical and Biological EngineeringUniversity of Colorado

Protein Therapeutics

• 125 biotechnology-based medicines on the market*

Out of more than 2700 drugs in clinical or later development*:

• 418 new biotechnology-based medicines are currently in testing*, most of which are proteins

*Pharmaceutical Research and Manufacturers of America, 2006

Therapeutic Proteins: Ripe for Engineering Progress

• Offer remarkable new treatments for cancer, AIDS/HIV, autoimmune disorders, digestive disorders, blood disorders…

• But there are number of important challenges that need to be addressed to allow more widespread use- many of which require engineering solutions.

Some Challenges of Protein Therapeutics

• Cost– Example: human growth hormone– Retail price: $50/mg (yes, that’s $50M/kg!!!)– Typical dose size: 0.3 mg/kg/week- for a 20 kg pediatric patient a

year’s treatment retails at $20,000– Cost to develop new drug: $802,000,000*– Cost to develop a new protein drug $1,240,000,000

• Safety– Immune response– Other adverse effects

• Regulatory– Regulatory environment lead to conservative approach to process

changes/improvements

*J. A. DiMasi, R. W. Hansen and H. G. Grabowski, Journal of Health Economics 22 (2003): 151-185.

Some contributors to high costs:

• Process yields are low

• Processes are inefficient

• Products are unstable

• Testing is expensive

• Regulatory burden is high

• Long development times

To become a therapeutic product…

• Protein must be produced in a form that is chemically pure

• Protein must be produced in a form that is conformationally pure (properly folded)

• Protein must be produced in the correct assembly state (monomeric, dimeric, etc.)

• Protein must remain so for duration of its labeled shelf life (typically two years)

An unfortunate start

• Proteins are synthesized within cells as linear polymers

• Polymers must “fold” to achieve correct 3D structure that imparts biological activity

• Incorrect folds typically show (greatly) reduced biological activity, and may be toxic

• Human proteins synthesized in lower organisms frequently misfold and aggregate

Protein Folding- A Bottleneck Early in the Process

(Dill et al, Proteins, 1998)

To become functionally active, proteins must fold correctly form a disordered state to the highly ordered native state

If all goes well, unfolded protein molecules fold through a biased walk as they concomitantly lower their free energy and reduce the number of available conformations- “sliding down the folding funnel”

•Radford S., “Protein folding: Progress made and promises ahead”, Trends in Biochemical Sciences, V25, 611-618, 2000.

But…

Partially folded protein intermediate states are often very “sticky”

These intermediates may assemble in “off-pathway” reactions to form aggregates

Aggregates are biologically inactive, and must be disaggregated and then folded to become active

Traditional Chaotrope-Based Refolding Methods

fermentation

Collect, wash, concentrate aggregates

• Aggregates are dissolved in large amounts of chaotropic solvents

• Chaotropes removed by diafiltration

• Low protein concentrations used to favor folding over re-aggregation

• Overall yields often 10-50%

• Multi-day process

Add Guanidinium HCl

Dissolve aggregates in chaotrope

Buffer exchange by dilution, Ultrafiltration/Diafiltration to effect refolding

Unfortunately, most of our valuable product ends up as useless aggregate

Disadvantages of Current Methods

• Low Yields• Capital cost

– Guandine incompatible with 316SS– Guanidine interferes with Ion Exchange

Chromatography- extensive dialysis required

• Product dilute• Waste handling costs• Slow

Intermediates on Folding Pathway

• Under atmospheric conditions, folding intermediates:– Exhibit attractive protein-protein interactions-

“sticky”– Self-associate to form aggregated species– Slow down folding– Reduce yields

What drives protein aggregation?

• Non-native conformations of proteins such as partially unfolded molecules more reactive

• Hydrophobic effect causes protein-protein interactions to be attractive

To understand this, we’ll have to dive into thermodynamics

But it’s really not that unfriendly

B22 characterizes the overall two-body interactions between proteins

where U(r) is the overall protein-protein interaction potential:

Hard sphere - excluded volumeElectrostatic - charge-charge van der Waals - charge-dipole, dipole-dipole, dispersionOsmotic - ion excluded volumeAssociation - interaction to account for protein associationSolvation - hydration and hydrophobic forces

Second osmotic virial coefficient describes protein-protein interactions

B22 > 0, repulsive interactionsB22 < 0, attractive interactions

0

2/)(222 )1(

2drre

MB kTrU

We anticipate that systems with negative (attractive) B22 values will be more prone to assemble into aggregates than those with positive B22 values

Data at 1 bar: Liu, W., T. Cellmer, et al. (2005). Biotechnology and Bioengineering 90(4): 482-490.

In the presence of ~1-2 M GuanidineHCl, B22 values for lysozyme show a minimum, causing the protein to aggregate during refolding

Is there a way around this?

• One way of influencing hydrophobic effects is by manipulating the system pressure

• Studies dating back nearly 100 years have shown that high pressures can so drastically alter hydrophobic effects as to cause proteins to unfold

P-T Stability Boundaries

Integration of the relation d(G)=-SdT +VdP

2

0 0 0

0 0 00

0 0) 0

2

ln 1p

G P P P P T T

TC T T V P P

T

S T T G

Protein Unfolding in P-T space

Hawley, 1971, Biochemistry 10, 2436-2442

(chymotrypsinogen)

Protein Folding “Pressure Window”

• Multimeric proteins dissociate @1-3 kbar• Monomeric proteins unfold @ >5 kbar• Aggregates may be thought of as ill-defined

“multimers”

• In “window” between ca. 1-5 kbar, pressure should dissociate aggregated state, while still favoring native conformation for monomers

Pressure Window

P

T

Subunits Multimers

A new process for folding proteins

• Take aggregated protein, pressurize to dissolve aggregates

• Reduce pressure to point where native conformation is favored, but aggregation is disfavored

• Allow to refold, then reduce pressure

The experiment

• Boil egg for 14 minutes• Remove aliquots of polymerized egg

white• Refold under pressure

– Aggregated protein at 2 mg/ml– Add Disulfide-Shuffling Agents: 4mM

glutathione, 2 mM dithiothreitol– Pressurize at 400 MPa, 25°C– Depressurize– Test for Lysozyme Activity, measure

soluble protein (size exclusion chromatography)

• Compare with “conventional” refolding– Solubilize 2 mg/ml protein in 6M

guanidine, 4mM glutathione, 2 mM dithiothreitol

– Dilute to 0.5M guanidine– Test for Lysozyme Activity, measure

soluble protein (size exclusion chromatography)

http://www.aeb.org/recipes/basics/hard-cooked_eggs.htm

The result- an egg unboiled!

• High-Pressure Process:– 25 % of starting protein recovered as soluble

protein– Lysozyme activity recovered

• Conventional process:– Negligible protein soluble– Negligible lysozyme activity recovered

Example I Human Growth Hormone

• Monomeric protein

• Aggregates easily, especially at surfaces

• High thermodynamic stability of native conformation

• Strategy: Single high pressure step for aggregate dissolution, protein refolding

Agitation-Induced Aggregation of rhGH

• rhGH aggregates nearly quantitatively after 24 hours of mild agitation

• Aggregates are irreversible at 1 atm, 25 C

• Aggregates formed by agitation in citrate buffer or citrate buffer with 0.75 M guanidine

Structure of agitation-induced rhGH aggregates (FTIR)

160016201640166016801700

wavenumbers (cm-1)

4th derivative UV @284 nm shows native state of rhGH is stable to >4500 bar

X Data

270 275 280 285 290 295 300 305

Y D

ata

-0.06

-0.04

-0.02

0.00

0.02

0.04

0.06

P, bar

0 1000 2000 3000 4000 5000

d4 A/d

4

-0.20

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

0.20

rhGH Fluorescence as Function of Pressure Shows Native State is Stable to >6500 bar

Wavelength, nm

320 340 360 380 400 420 440

Flu

ores

cenc

e In

tens

ity

0

20

40

60

80

100

120

Pressure, kbar

0 1 2 3 4 5 6 7Flu

ores

cenc

e In

tens

ity @

340

nm

60

70

80

90

100

110

Refolding of human growth hormone from agitation-induced aggregates: aggregated states destabilized

under pressure

010203040

5060708090

100

0 500 1000 1500 2000 2500

Pressure (bar)

Per

cent

Rec

over

ed S

olub

le r

hGH

Protein refolding is independent of protein concentration

rhGH Recovery vs. Protein Concentration

0

2

4

6

8

10

0 5 10

Protein Concentration (mg/mL)

Re

cove

red

So

lub

le

Pro

tein

(m

g/m

L)

100%Recovery

2 kbar

Kinetics of rhGH aggregate dissolution at 2000 bar

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25

time (hr)

Nor

mal

ized

Abs

orba

nce

(500

nm

)

-4

-3

-2

-1

00 10 20

time (hr)

Dissolution timeconstants 4.8 and 10 hours

Kinetics of rhGH refolding @ 2000 bar

283.5

284

284.5

285

285.5

286

0 2 4 6 8 10 12

Time (hr)

ma

x (n

m) Refolding time

constant = 3.2 hours

-2.5

-2

-1.5

-1

-0.5

00 2 4 6

time (hr)

Example: Disaggregation and Folding from Aggregates of Interferon-

IFN• Protein is dimeric in its native state

• Aggregates easily

• Strategy: High pressure to dissolve aggregates; moderate pressure to refold

→ Choose operating points based on equilibrium unfolding as f(P)

Pressure Effects on Equilibria

Assume a two state transition N D

; ln

ln

is the difference in partial molar volumes between N and D

D N

DK G RT K

N

K V

P RT

V

V v v

IFN- Dissociation

2

2

0

2

[ ]

fraction native protein

1K=4N

K

N

N

N

Dimer Monomer

MonomerK

Dimer

f

f

f

IFN- UV Spectra as f(P)

Wavelength, nm

260 270 280 290 300 310

Abso

rba

nce

0.0

0.2

0.4

0.6

0.8

1.0

4th Derivative UV Spectra of IFN- as f(Pressure)

Wavelength, nm278 280 282 284 286 288 290 292 294 296

d4A

/dl4

-0.20

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

0.20

P

Convert UV data to fraction protein folded as f(P)

Calculate folding equilibrium constants

Calculate ln K

P

Partial Molar Volume Change of IFN- Dissociation, 40oC

Pressure, Bar

400 600 800 1000 1200 1400 1600 1800 2000 2200

G (

ml b

ar

mol

-1)

-2e+5

-2e+5

-1e+5

-5e+4

0

5e+4

1e+5

V= -176 ml/mol

IFN-Elliptical stability diagram generated from pressure-induced dissociated data used

to choose process operating points

0

50

100

150

200

250

300

-20 -10 0 10 20 30 40 50 60 70

Temperature (oC)

Pre

ssur

e (M

Pa)

AggregateDissolution Conditions

Refolding Conditions

Aggregate Dissolution at 2500 bar

0.0

0.2

0.4

0.6

0.8

1.0

0 1 2 3 4 5 6 7

Time (min)

Ab

sorb

ance

at

310

nm

(A

U/c

m)

Refolding Rate at 100 MPa

0 20 40 60 80 100

Time (min)

Arb

itra

ry H

eig

ht

B

Monomer-Dimer Equilibrium Re-established from γ-Interferon Aggregates

0

10

20

30

40

3 3.5 4 4.5 5 5.5 6 6.5 7

EM Diameter (nm)

Mas

s P

erce

nt

A

0

1

2

3

4

5

6

7

7 12 17 22 27

EM Diameter (nm)M

ass

Per

cent

B Aggregates ~650kDaDimers

Monomers

Red – Size distribution before pressure

Black – after high pressure treatment

At high pressures, aggregation is suppressed- why?

• High pressures generally conformationally destabilize proteins, leading to higher populations of molecules with non-native conformations. Why doesn’t this accelerate aggregation?

• How does pressure affect protein-protein interactions?

Aim: Explore the interplay between conformational and

colloidal stability as a function of pressure- what

causes the “pressure window”?

P

T

Subunits Multimers

Hydrophilic Surface, Low P

Hydrophilic Surface, 2 kbar

Hydrophobic Surface, Low P

Hydrophobic Surface, 2 kbar

Giovambattista, Debenedetti1, and Rossky, J. Phys. Chem. B.

Data at 1 bar: Liu, W., T. Cellmer, et al. (2005). Biotechnology and Bioengineering 90(4): 482-490.

At 1 kbar, protein-protein interactions for HEW lysozyme are repulsive during folding- in contrast to folding at atmospheric pressure!

-1

0

1

2

3

4

5

0 2 4 6

GdnHCl Concentration [M]

B22

*103

(m

l m

ol/

g2)

1000 bar

Liu et al.(atmospheric)

Gunf (kcal/mol)

Wild Type 11.1 (1.8)

L99A 6.4 (0.5)

L99A/A130S 4.8 (0.6)

Model system: T4 lysozyme variants exhibit widely varying conformational stabilities, but nearly identical folds (see Matthews et al., Sathish, et al.)

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6

GdnHCl [M]

Fra

ctio

n U

nfo

lded

WT

L99A

L99A/A130S

-4

-3

-2

-1

0

1

2

3

0 1 2 3

GdnHCl [M]

G

unf

[kca

l/mol

]

T4 Lysozyme L99A/A130S at 1 bar (solid symbols) and 1kbar (open symbols)

Pressure makes intermolecular interactions more repulsive

-10123456789

0 2 4 6 8

GdnHCl [M]

B22

*10

4 (m

l mo

l/g2 )

1kbar

atm

At atmospheric pressure, B22 values show a minimum near 1M guanidine HCl, independent of conformational stability

-1

0

1

2

3

4

5

6

7

8

9

0 2 4 6 8

GdnHCl [M]

B22

*10

4 (m

l mo

l/g2 )

WT

L99A

L99A/A130S

In contrast, at 1 kbar pressure, no minimum in B22 values is seen as a function of guanidine HCl concentration, also independent of conformational stability!

-1

0

1

2

3

4

5

6

7

8

9

0 2 4 6 8

GdnHCl [M]

B22

*10

4 (m

l mo

l/g2 )

WT

L99A

L99A/A130S

Collapse of water around protein surface is reflected in refractive index increment- and is

independent of conformational stability

0.1

0.12

0.14

0.16

0.18

0.2

0.22

0.24

0 1000 2000 3000

Pressure [bar]

dn

/dc

[m

l/g]

WT

L99A/A130S

L99A

Result:

• Hydrophobic effect less pronounced at high P• Aggregated protein molecules, which are typically

held together through hydrophobic interactions, become less “sticky”

• Results in rapid dissolution of aggregates• Dissolution occurs under conditions where native

secondary structures are thermodynamically favored

• Results in folding of protein under conditions where aggregation is blocked

Is it scaleable?

•Used in food industry:

•guacamole

•self-shucking oysters

•orange juice

In the Pharmaceutical Industry…

• Commercialized by BaroFold, Inc.

• Over 200 proteins successfully refolded

• In commercial operation under GMP conditions at European partner

• Scaled to match refolding requirements for protein production in 10,000 L commercial fermentors.

Conclusions

•Pressure can be used to aid refolding from a variety of protein aggregates•Pressure can dissolve aggregates even under conditions where native protein is favored•Pressure changes the interactions between folding intermediates, allowing refolding to occur preferentially over aggregation•High Pressure refolding combines high concentrations with high yields

Funding

• BaroFold, Inc.

• NIH

• NSF