Differential scanning calorimetry (DSC) can be used to quickly predict the storage stability of enzymes in liquid detergents. DSC measures the thermal stability of enzymes by determining the temperature at which they denature (Tm). Higher Tm values indicate better thermal stability. DSC studies found that enzymes are less stable in liquid detergents than buffers, and stability decreases with increasing hydrotrope concentration or changing surfactants. Adding known stabilizers like glycerol increased Tm and correlated with improved long-term stability. Thus DSC provides a fast way to screen enzyme and detergent formulations for thermal stability.

1. Method for Predicting Enzyme Storage
Stability in Liquid Surfactant Systems
Debbie Winetzky,
Louise Wallace &
Douglas Dale
99th Annual AOCS
Meeting,
Seattle, WA
19 May 2008
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2. Background
Need a quick method for predicting the storage stability
of enzymes in liquid detergents
• Screen a higher number variations faster
• Faster feedback than storage stability study
• Reduction in time
Under utilized Differential Scanning Calorimeter in-house
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3. Definitions
DSC: Differential Scanning Calorimetry
• Measures the heat changes that occur during the controlled increase
(or decrease) in temperature
• It is ideal for evaluating the effects of formulation changes on enzyme
stability
• pH
• Surfactant systems
• Builder or buffering systems
• Minor ingredients
Tm: Thermal transition temperature
• The temperature in which 50% of the molecules are in their native, folded
state and 50% of the molecules are in a denatured, unfolded state
• Changes that increase the Tm lead to improved thermal stability of the
enzyme in the formulation
http://www.microcal.com/index.php?id=16
http://upload.wikimedia.org/wikipedia/en/c/c5/Protein_folding_schematic.png
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4. DSC is Used in the Pharmaceutical Industry to
Develop Formulations for Protein Therapeutics
DSC has been shown to be a valuable predictor of liquid formulation stability
for proteins and other biological macromolecules (Remmele and Gombotz,
Biopharm, June 2000, pp 36-46; Remmele et al, 1998).
• Excipients, preservatives, and other additives in the formulation can stabilize or
destabilize proteins.
• Stabilizing additives increase the Tm of proteins while destabilizing materials have
the opposite effect.
• DSC is used to determine the stabilizing effects of different solution conditions
and additives.
Heat capacity changes associated with protein unfolding are primarily due to
changes in hydration of side chains that were buried in the native state, but
become solvent exposed in the denatured state.
http://www.microcal.com/index.php?id=16
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5. Why Use DSC Instead of Other
Instruments?
Pros
• In-solution method
• No purification, separation, or special preparation needed
• Useful with turbid, colored or viscous solutions
• Not dependent on optical measurements
Cons
• Low through-put due to the difficultly in loading neat detergents
into the cells
• Does not measure changes due to other degradation mechanisms
• Proteolytic cleavage
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6. Adiabatic-type DSC
How much more heat needs to be supplied to the sample
cell to keep it at the same temp as the reference cell?
From MicroCal MC-2 User’s Manual
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7. Pan-type DSC
Cylindrical How much more heat needs to be supplied to the sample Cylindrical
furnace pan to keep it at the same temp as the reference pan? furnace
Reference Pan Sample Pan
Base HDL HDL w/ enzyme
Thermoelectric Disc
Measurement Thermocouples
http://hekabe.kt.dtu.dk/~vigild/2005_04_melitek/dsc.htm 7

8. Ideal DSC Curve
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9. Thermal Measurements Have Little
Dependence on Protein Concentration
Effect of Protein
0.00050
Protein concentration dependence
72.33306
Concentration on Tm
0.00045 10 mM HEPES, pH 8.0; 0.1 - 4 mg/ml
o
200 C/hr
0.00040
0.00035
80.0
0.00030
Cp(cal/ C)
buffer 72.01371
0.00025
o
0.1 mg/ml
0.00020
0.2 mg/ml
0.5 mg/ml
60.0
1 mg/ml
Tm, °C
0.00015 2 mg/ml 71.5644
0.00010 4 mg/ml
0.00005
71.73301
71.66466
40.0
71.06292
0.00000
-0.00005 20.0
-0.00010
-0.00015
40 50 60 70 80 90
0.0
Temperature ( C)
o
0.0 2.0 4.0 6.0
[Protein], mg/mL
Note: A decreasing Tm with increasing protein
concentration would indicate aggregation
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10. DSC Will Rapidly Distinguish Whether A Protein Is
Stabilized Or Destabilized Relative To A Control
0.00030 • Loss in enzyme stability =
74.5332 unfolding
69.20858 79.34625
0.00025
• Some factors that may
0.00020 Wild-type Protein A influence enzyme
Stable variant of Protein A
Destabilized variant of Protein A stability:
Cp(cal/ C)
0.00015
• pH
o
• Ionic strength
0.00010
• Chemical interactions
0.00005 • Oxidation
0.00000
-0.00005 Decreased Increased
Stability Stability
-0.00010
40 50 60 70 80 90
o
Temperature ( C)
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11. Enzymes Stored in HDL Are Less Thermostable
Than Enzymes Stored in Buffer
Effect of detergent on protein stability compared with buffer system (pH 8.0)
0.0008
50.3 C
o
69.2 oC
0.0006
Cp (cal/ C)
0.0004
o
0.0002
0.0000
-0.0002
40 50 60 70 80
temperature (oC)
Protein in detergent 1hr incubation
Protein in detergent overnight incubation
Protein control in buffer
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12. DSC: Whole Detergent Effects
The Tm of two different enzymes were measured in two detergent bases and
buffer to determine if there were stability differences
Both enzymes have poorer stability in HDL #2 than in HDL #1
HDL #2 HDL #1
Protease Y Protease X
Enzyme in buffer
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13. Whole Detergent Effects
Sample Tm in Tm in Tm in
Buffer, °C HDL #1, °C HDL #2, °C
Protease X 77.7 72.3 68.7
Alt. Form. A 75.4 71.9 66.9
Alt. Form. B 76.5 71.8 67.3
Alt. Form. C 75.3 71.3 64.7
Protease Y 72.5 66.7 63.2
There are slight differences between the enzyme formulations within
a given HDL base
The more dramatic effect for all formulations is the difference
between the two HDL bases
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14. DSC: Component Effects
Single component changes were made in HDL #2 to help determine
which may be causing instability
Increasing the hydrotrope concentration decreases stability:
Increasing Increasing
[hydrotrope] [hydrotrope]
Protease X Protease Y
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15. Component Effects
Component Prot X Tm Δ Tm Prot Y Tm Δ Tm
HDL #1, pH 9 69.3 - 65.2 -
HDL #1, pH 8.4 69.3 0 65.1 -0.1
Alt. Surfactant 67.9 -1.4 64.0 -1.2
Buffer 77.3 - 69.7 -
1.6% Hydrotrope 75.8 -1.5 67.4 -2.3
4.0% Hydrotrope 73.4 -3.9 65.1 -4.6
8.0% Hydrotrope 69.6 -7.7 62.0 -7.7
Results show that changing the surfactant type or
increasing the hydrotrope concentration cause a
decrease in stability
pH is not a factor
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16. Delta Tm (°C)
B
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
as 4.5
e
HD
L
0.
05
%
C
a
0.
05 0.
% 1%
C
Protease #4
Protease #3
Protease #2
Protease #1
a; Ca
0. 0 .7
05 %
% B
C or
a, at
e
on Protease Stability in HDL
0.
7%
Fo
0. rm
05
% at
e
0. C
05 a,
0. % 1.
4%
05 C
% a, PG
C 1.
4%
a,
Effect of Known Stabilizing Components
0. G
7% ly
B c er
0. or ol
05
% at
e,
C 1.
a, 4%
0.
7% PG
B
or
at
e,
G
ly
c
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17. Effect of pH and Ions on
Thermostability
80.0
78.0 No Ca 2+
76.0 w/ Ca 2+
74.0
Tm (°C)
72.0
70.0
68.0
66.0
64.0
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
pH
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18. Evaluation of the Thermal Stability of a Protease in
Mixtures of Known Stabilizers: Formulations
% % % %
Run # Glycerol Betaine Formate Water pH Tm (°C)
1 10.0 10.0 0.0 80.0 5.72 72.3
2 0.0 0.0 2.0 98.0 5.74 73.4
3 7.5 7.5 1.5 83.5 5.96 76.8
4 10.0 0.0 1.0 89.0 5.47 75.0
5 2.5 2.5 0.5 94.5 5.59 73.7
6 5.0 5.0 1.0 89.0 5.53 75.6
7 2.5 7.5 1.5 88.5 5.85 76.0
8 0.0 10.0 0.0 90.0 5.70 69.9
9 5.0 5.0 1.0 89.0 5.54 75.6
10 10.0 10.0 2.0 78.0 5.92 79.0
11 7.5 2.5 0.5 89.5 5.49 74.6
12 0.0 0.0 2.0 98.0 5.55 73.4
13 0.0 10.0 0.0 90.0 5.86 69.9
14 10.0 10.0 1.0 79.0 5.72 78.5
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19. Evaluation of the Stability of a Protease in Mixtures of
Known Stabilizers: Correlation Between Tm and
Storage Stability
Correlation Coefficients:
1 wk r = 0.9569
120.0% 2 wks r = 0.9597
1 wk @ 40°C 5 wks r = 0.9228
100.0%
% Activity Remaining
2 wk @ 40°C
80.0% 5 wk @ 40°C
60.0%
40.0%
20.0%
0.0%
68 70 72 74 76 78 80
-20.0%
Tm (°C)
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20. Conclusion
DSC provides a rapid method for assessing
• The influence of formulation changes on the thermal stability of an
enzyme
• The influence of enzyme stabilizers on the thermal stability of an
enzyme
• The optimum conditions for storage stability
Good correlation between Tm and stability measurements
Useful method for screening enzyme formulations and
detergent formulations for thermal effects that may be
indicative of long-term stability
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21. More Information
Microcal: http://www.microcal.com/
TA Instruments: http://www.tainstruments.com/
Setaram: http://www.setaram.com/
General info: http://www.ThermalCal.com/
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