Dsc Presentation (Aocs)
Transcript of Dsc Presentation (Aocs)
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Method for Predicting Enzyme Storage Stability in Liquid Surfactant Systems
Debbie Winetzky, Louise Wallace & Douglas Dale
99th Annual AOCS Meeting, Seattle, WA19 May 2008
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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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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=16http://upload.wikimedia.org/wikipedia/en/c/c5/Protein_folding_schematic.png
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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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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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Adiabatic-type DSC
From MicroCal MC-2 User’s Manual
How much more heat needs to be supplied to the sample cell to keep it at the same temp as the reference cell?
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Pan-type DSC
http://hekabe.kt.dtu.dk/~vigild/2005_04_melitek/dsc.htm
How much more heat needs to be supplied to the sample pan to keep it at the same temp as the reference pan?
Thermoelectric Disc
Measurement Thermocouples
Cylindricalfurnace
Reference PanBase HDL
Sample PanHDL w/ enzyme
Cylindricalfurnace
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Ideal DSC Curve
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Thermal Measurements Have Little Dependence on Protein Concentration
Effect of Protein Concentration on Tm
0.0
20.0
40.0
60.0
80.0
0.0 2.0 4.0 6.0
[Protein], mg/mL
T m, °
C
71.0629271.6646671.73301
71.5644
72.01371
72.33306
40 50 60 70 80 90
-0.00015
-0.00010
-0.00005
0.00000
0.00005
0.00010
0.00015
0.00020
0.00025
0.00030
0.00035
0.00040
0.00045
0.00050
buffer 0.1 mg/ml 0.2 mg/ml 0.5 mg/ml 1 mg/ml 2 mg/ml 4 mg/ml
Cp(
cal/o C
)
Temperature (oC)
Protein concentration dependence 10 mM HEPES, pH 8.0; 0.1 - 4 mg/ml200 oC/hr
Note: A decreasing Tm with increasing protein concentration would indicate aggregation
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DSC Will Rapidly Distinguish Whether A Protein Is Stabilized Or Destabilized Relative To A Control
74.533279.3462569.20858
40 50 60 70 80 90-0.00010
-0.00005
0.00000
0.00005
0.00010
0.00015
0.00020
0.00025
0.00030
Wild-type Protein A Stable variant of Protein A Destabilized variant of Protein A
Cp(
cal/o C
)
Temperature (oC)
IncreasedStability
DecreasedStability
• Loss in enzyme stability = unfolding
• Some factors that may influence enzyme stability:
• pH• Ionic strength• Chemical interactions• Oxidation
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Enzymes Stored in HDL Are Less ThermostableThan Enzymes Stored in Buffer
Effect of detergent on protein stability compared with buffer system (pH 8.0)
temperature (oC)
40 50 60 70 80
Cp
(cal
/o C)
-0.0002
0.0000
0.0002
0.0004
0.0006
0.0008
Protein in detergent 1hr incubationProtein in detergent overnight incubationProtein control in buffer
50.3 oC 69.2 oC
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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
Enzyme in buffer
HDL #2 HDL #1
Protease Y Protease X
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Whole Detergent Effects
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
Sample Tm inBuffer, °C
Tm inHDL #1, °C
Tm inHDL #2, °C
Protease X 77.7 72.3 68.7Alt. Form. A 75.4 71.9 66.9Alt. Form. B 76.5 71.8 67.3Alt. Form. C 75.3 71.3 64.7
Protease Y 72.5 66.7 63.2
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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:
Protease YProtease X
Increasing [hydrotrope]
Increasing [hydrotrope]
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Component Effects
Results show that changing the surfactant type or increasing the hydrotrope concentration cause a decrease in stability
pH is not a factor
-7.762.0-7.769.68.0% Hydrotrope-4.665.1-3.973.44.0% Hydrotrope-2.367.4-1.575.81.6% Hydrotrope
-69.7-77.3Buffer-1.264.0-1.467.9Alt. Surfactant-0.165.1069.3HDL #1, pH 8.4
-65.2-69.3HDL #1, pH 9Δ TmProt Y TmΔ TmProt X TmComponent
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Effect of Known Stabilizing Components on Protease Stability in HDL
-1.0-0.50.00.51.01.52.02.53.03.54.04.5
Base H
DL0.05
% Ca
0.1% Ca
0.05% Ca;
0.7% Borate
0.05% Ca,
0.7% Form
ate
0.05% Ca,
1.4% PG
0.05% Ca,
1.4% G
lycerol
0.05% Ca,
0.7% Borate, 1
.4% PG
0.05% Ca,
0.7% Borate, G
lycD
elta
Tm
(°C
)
Protease #1Protease #2Protease #3Protease #4
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Effect of pH and Ions on Thermostability
64.066.068.070.072.074.076.078.080.0
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
pH
T m (°
C)
No Ca 2+w/ Ca 2+
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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 72.32 0.0 0.0 2.0 98.0 5.74 73.43 7.5 7.5 1.5 83.5 5.96 76.84 10.0 0.0 1.0 89.0 5.47 75.05 2.5 2.5 0.5 94.5 5.59 73.76 5.0 5.0 1.0 89.0 5.53 75.67 2.5 7.5 1.5 88.5 5.85 76.08 0.0 10.0 0.0 90.0 5.70 69.99 5.0 5.0 1.0 89.0 5.54 75.610 10.0 10.0 2.0 78.0 5.92 79.011 7.5 2.5 0.5 89.5 5.49 74.612 0.0 0.0 2.0 98.0 5.55 73.413 0.0 10.0 0.0 90.0 5.86 69.914 10.0 10.0 1.0 79.0 5.72 78.5
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Evaluation of the Stability of a Protease in Mixtures of Known Stabilizers: Correlation Between Tm and Storage Stability
-20.0%
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
120.0%
68 70 72 74 76 78 80
Tm (°C)
% A
ctiv
ity R
emai
ning
1 wk @ 40°C
2 wk @ 40°C
5 wk @ 40°C
Correlation Coefficients:1 wk r = 0.95692 wks r = 0.95975 wks r = 0.9228
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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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More Information
Microcal: http://www.microcal.com/
TA Instruments: http://www.tainstruments.com/
Setaram: http://www.setaram.com/
General info: http://www.ThermalCal.com/