High Pressure Disaggregation
and Refolding of Proteins
Ted Randolph, Matt Seefeldt, Jon Webb, Rick St. John,
Yongsung Kim, Ryan Crisman, Amber Haynes, John
Carpenter
Center for Pharmaceutical Biotechnology
Department of Chemical and Biological Engineering
University 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
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”
(Dill et al, Proteins, 1998)
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
•Radford S., “Protein folding: Progress made and promises ahead”,
Trends in Biochemical Sciences, V25, 611-618, 2000.
Traditional Chaotrope-Based
Refolding Methods
• Aggregates are
dissolved in large
amounts of chaotropic
solvents
• Chaotropes removed by
diafiltration
• Low protein
concentrations used to
favor folding over reaggregation
• Overall yields often 1050%
• Multi-day process
Collect, wash,
concentrate
aggregates
fermentation
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
Second osmotic virial coefficient
describes protein-protein interactions
B22 characterizes the overall two-body interactions between proteins
2 
B22 
(1  e U ( r ) / kT )r 2 dr
M2 0

where U(r) is the overall protein-protein interaction potential:
Hard sphere
- excluded volume
Electrostatic
- charge-charge
van der Waals
- charge-dipole, dipole-dipole, dispersion
Osmotic
- ion excluded volume
Association
- interaction to account for protein association
Solvation - hydration and hydrophobic forces
B22 > 0, repulsive interactions
B22 < 0, attractive interactions
We anticipate that systems with negative (attractive) B22 values will be more prone to
assemble into aggregates than those with positive B22 values
In the presence of ~1-2 M GuanidineHCl, B22 values for lysozyme
show a minimum, causing the protein to aggregate during refolding
Data at 1 bar: Liu, W., T. Cellmer, et al. (2005). Biotechnology
and Bioengineering 90(4): 482-490.
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
G 
 P  P0     P  P0 T  T0  
2
  T


C p T  ln  1  T0   V0  P  P0  
  T0 

S0 T  T0)   G0
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
Subunits
Multimers
P
T
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/hardcooked_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)
1700
1680
1660
1640
wavenumbers (cm-1)
1620
1600
4th derivative UV @284 nm shows native
state of rhGH is stable to >4500 bar
0.15
0.06
0.10
0.04
0.05
0.00
0.02
Y Data
d4A/d4
0.20
0.00
-0.02
-0.05
-0.04
-0.10
-0.06
270
275
-0.15
280
285
290
295
300
305
X Data
-0.20
0
1000
2000
P, bar
3000
4000
5000
rhGH Fluorescence as Function of Pressure
Shows Native State is Stable to >6500 bar
120
Fluorescence Intensity @ 340 nm
Fluorescence Intensity
100
80
60
40
110
100
90
80
70
60
0
1
2
3
4
5
6
7
Pressure, kbar
20
0
320
340
360
380
Wavelength, nm
400
420
440
Percent Recovered Soluble rhGH
Refolding of human growth hormone from agitationinduced aggregates: aggregated states destabilized
under pressure
100
90
80
70
60
50
40
30
20
10
0
0
500
1000
1500
Pressure (bar)
2000
2500
Protein refolding is independent
of protein concentration
Recovered Soluble
Protein (mg/mL)
rhGH Recovery vs. Protein
Concentration
10
8
100%
Recovery
2 kbar
6
4
2
0
0
5
10
Protein Concentration (mg/mL)
Kinetics of rhGH aggregate dissolution at 2000 bar
Normalized Absorbance (500 nm)
1.2
1
0.8
Dissolution time
constants 4.8 and
10 hours
0.6
0.4
0.2
0
0
5
10
15
time (hr)
20
25
Kinetics of rhGH refolding @ 2000 bar
Refolding time
constant = 3.2 hours
Example: Disaggregation and Folding
from Aggregates of Interferon-g IFNg
• 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
D
K ;
G   RT ln K
N
 ln K
V

P
RT
V is the difference in partial molar volumes between N and D
V  vD  vN
IFN-g Dissociation
K
Dimer  2 Monomer
Monomer 

K
2
[ Dimer ]
f N  fraction native protein
K=4N 0
1  f N 
fN
2
IFN-g UV Spectra as f(P)
1.0
Absorbance
0.8
0.6
0.4
0.2
0.0
260
270
280
290
Wavelength, nm
300
310
4th Derivative UV Spectra of IFN-g as
f(Pressure)
0.20
0.15
Convert UV data to
fraction protein
folded as f(P)
d4A/dl 4
0.10
0.05
Calculate folding
equilibrium
constants
0.00
-0.05
-0.10
-0.15
Calculate
P
-0.20
278 280 282 284 286 288 290 292 294 296
Wavelength, nm
 ln K
P
Partial Molar Volume Change of IFN-g
Dissociation, 40oC
1e+5
G (ml bar mol-1)
5e+4
V= -176 ml/mol
0
-5e+4
-1e+5
-2e+5
-2e+5
400
600
800
1000 1200 1400 1600 1800 2000 2200
Pressure, Bar
IFN-g: Elliptical stability diagram generated
from pressure-induced dissociated data used
to choose process operating points
300
Aggregate
Dissolution
Conditions
Pressure (MPa)
250
200
150
100
Refolding
Conditions
50
0
-20
-10
0
10
20
30
40
Temperature (o C)
50
60
70
Aggregate Dissolution at 2500 bar
Absorbance at 310 nm (AU/cm)
1.0
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
Time (min)
5
6
7
Refolding Rate at 100 MPa
Arbitrary Height
B
0
20
40
60
Time (min)
80
100
Monomer-Dimer Equilibrium
Re-established from γ-Interferon Aggregates
7
A
B
Aggregates
~650kDa
6
Dimers
30
Mass Percent
Mass Percent
40
Monomers
20
5
4
3
2
10
1
0
0
3
3.5
4
4.5
5
5.5
6
EM Diameter (nm)
6.5
77
12
17
22
27
EM Diameter (nm)
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”?
Subunits
Multimers
P
T
Hydrophilic Surface, Low P
Hydrophilic Surface, 2 kbar
Hydrophobic Surface, Low P
Hydrophobic Surface, 2 kbar
Giovambattista, Debenedetti1, and Rossky, J. Phys. Chem. B.
At 1 kbar, protein-protein interactions for HEW lysozyme are repulsive during
folding- in contrast to folding at atmospheric pressure!
4
3
1000 bar
2
Liu et al.
(atmospheric)
3
2
B22 *10 (ml mol/g )
5
1
0
-1
0
2
4
GdnHCl Concentration [M]
Data at 1 bar: Liu, W., T. Cellmer, et al. (2005). Biotechnology
and Bioengineering 90(4): 482-490.
6
WT
L99A
L99A/A130S
1
0.8
3
 Gunf [kcal/mol]
Fraction Unfolded
1.2
0.6
0.4
2
1
0
-1
-2
-3
-4
0.2
0
1
2
3
GdnHCl [M]
0
0
2
4
6
GdnHCl [M]
Model system: T4 lysozyme variants exhibit widely varying
conformational stabilities, but nearly identical folds (see Matthews et al.,
Sathish, et al.)
Gunf (kcal/mol)
Wild Type
11.1 (1.8)
L99A
6.4 (0.5)
L99A/A130S
4.8 (0.6)
B22*10 4 (ml mol/g2)
Pressure makes intermolecular interactions more
repulsive
9
8
7
6
5
4
3
2
1
0
-1
1kbar
atm
0
2
4
6
8
GdnHCl [M]
T4 Lysozyme L99A/A130S at 1 bar (solid
symbols) and 1kbar (open symbols)
B22*10 4 (ml mol/g2)
At atmospheric pressure, B22 values show a minimum
near 1M guanidine HCl, independent of
conformational stability
9
8
7
6
5
4
3
2
1
0
-1
WT
L99A
L99A/A130S
0
2
4
GdnHCl [M]
6
8
B22*10 4 (ml mol/g2)
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!
9
8
7
6
5
4
3
2
1
0
-1
WT
L99A
L99A/A130S
0
2
4
GdnHCl [M]
6
8
Collapse of water around protein surface is
reflected in refractive index increment- and is
independent of conformational stability
0.24
dn/dc [ml/g]
0.22
0.2
0.18
0.16
0.14
WT
0.12
L99A/A130S
L99A
0.1
0
1000
2000
Pressure [bar]
3000
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