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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/