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Kinetic Analysis of the Conformational Stability and Aggregation of mAbs Using Calorimetry
orTwo New Things To Do With Your Calorimeter
Ernesto Freire
Johns Hopkins University
Freire, E.
mAbs Denature and AggregateFreire, E.
Partial Denaturation or Complete Denaturation Can Lead to Aggregation
- Lower Concentration of Aggregating Species
Solution: Increase Tm or G
- Decrease Intrinsic Rate of Aggregation
Strategies to Slow Down Denaturation/Aggregation
Solution: Increase Activation Energy H*
Freire, E.
ki ki
Case Study: VRC07-523LS, a Broadly Neutralizing HIV-1 Antibody Currently in Phase 1 Clinical Trials
• VRC07-523LS is a VRC01 class IgG1 neutralizing antibody
• mAb VRC01 binds to CD4 binding site in envelope glycoprotein gp120. It neutralizes ∼90% of HIV-1 strains with an IC50 of less than 50 μg/ml
• VRC07 was identified through sequencing antibody-gene transcripts of VRC01 donor B cells
• Structure-based design was used to further optimize VRC07
• VRC07-523LS is 5- to 8-fold more potent than VRC01
• It neutralizes 96% of viruses tested
PDB: 4OLY, Kwon, Y.D., Kwong, P.D. 2014
Freire, E.
Temperature Denaturation of mAbs is an Irreversible ProcessDSC Usually Shows Peaks Corresponding to Fab, CH2 and CH3 Domains
Garber and Demarest BBRC 355 (2007) 751–757
Freire, E.
Irreversible Denaturation is Kinetically Controlled
ku kagg
Freire, E.
The kinetic control of irreversible protein denaturation in DSC was first discussed by Sanchez-Ruiz et al Biochemistry (1988) 27 1648.
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200
40 50 60 70 80 90
Cp
kca
l/K
*mol
Temp deg C
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Cp kca
l/K
*mol
Temp deg C
69.0°C
3days10days60days280days
1/k
Freire, E.
~1minute
The Rate of Denaturation Increases With TemperatureAt Tm the Rate of Denaturation is ~ 1 minute
Temperature Denaturation is Kinetically ControlledFreire, E.
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40 50 60 70 80 90
Cp
kca
l/K
*mo
l
Temp deg C
69.0°C1 min
1day10days60days280days
400days
800days
The Stability at Lower Temperatures Depends on the Activation Enthalpy H*.
- The Higher H*, the Higher the Stability.
- In Formulation or mAbSelection we want to Increase H* to Maximize Long Term Stability
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10-7
10-6
10-5
0.0001
0.001
0.01
0.1
1
50 55 60 65 70
k m
in-1
Temp deg C
The Temperature Dependence of the Rate of Denaturation is Determined by the Activation Energy H*
Freire, E.
The Higher ΔH* the Faster the Decrease of the Rate of Denaturation/Aggregation Upon Lowering the Temperature
ΔH* = 100 kcal/mol
ΔH* = 200 kcal/mol
ΔH* = 150 kcal/mol
𝑘−1 = 7 𝑑𝑎𝑦𝑠
𝑘−1 = 140 𝑑𝑎𝑦𝑠
𝑘−1 = 14,000 𝑑𝑎𝑦𝑠
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50 55 60 65 70 75 80
Cp
kcal/K
*mo
l
Temp deg C
ΔH Area under the curve
ΔH* Activation EnthalpyShape of the Curve
Tm
Kinetic Deconvolution of DSC for Irreversible Denaturation Freire, E.
Hi* Can be Determined from Kinetic Deconvolution
𝐶𝑝 =∆𝐻𝑖𝑑𝐹𝑑,𝑖𝑑𝑇
𝑑𝐹𝑑,𝑖
𝑑𝑇=
𝑘𝑖𝐹𝑑,𝑖
𝛼
𝑘𝑖 = exp −∆𝐺𝑖
∗
𝑅𝑇= exp(−
∆𝐻𝑖∗ −𝑇∆𝑆𝑖
∗
𝑅𝑇)
Kinetic Deconvolution of DSC for Irreversible Denaturation Using Exact Equations
Freire, E.
Hi* Can be Determined from Kinetic Deconvolution
𝑑 𝑙𝑛𝐹𝑑,𝑖
𝑑𝑇=
𝑘𝑖
𝛼=
exp(−∆𝐺𝑖
∗
𝑅𝑇)
𝛼
𝛼 =𝑑𝑇
𝑑𝑡= 𝑠𝑐𝑎𝑛 𝑟𝑎𝑡𝑒
𝑑𝐹𝑑,𝑖𝑑𝑡
= 𝑘𝑖𝐹𝑑,𝑖
-20
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Cp kca
l/K
*mol
Temp deg C
H* = 100 kcal/mol
H* = 200 kcal/mol
The Effect of the Activation Enthalpy (H*) on the Shape of the DSC CurveFreire, E.
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pH 6.8 datapH 6.8 fitpH 6.5 datapH 6.5 fitpH 6.2 datapH 6.2 fitpH 6.0 datapH 6.0 fitpH 5.85 datapH 5.85 fitpH 5.7 datapH 5.7 fitpH 5.5 datapH 5.5 fit
Cp ca
l/K
*mol
Temp deg C
DSC Data and Kinetic Deconvolution as a Function of pH for mAb VRC07
50mM Histidine, 100mM NaCl
Freire, E.
Freire, E.
Kinetic Deconvolution of Cp Function of VRC07 pH 6.8
SumCp1Cp2Cp3
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Cp
kcal/K
*mol
Temp deg C
pH Dependence of Tm and Activation Enthalpy Freire, E.
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200
5.5 6 6.5 7 7.5
dHa1dHa2dHa3
H
a kcal/m
ol
pH
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70
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78
5 5.5 6 6.5 7 7.5 8
Tm1 deg CTm2 deg CTm3 deg C
Tm
d
eg C
pH
Combination of Tm and Activation Enthalpy Indicate that pH 6.8 Offers Best Stability Profile
Freire, E.
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100
150
200
5.5 6 6.5 7 7.5
dHa1dHa2dHa3
H
a kcal/m
ol
pH
62
64
66
68
70
72
74
76
78
5 5.5 6 6.5 7 7.5 8
Tm1 deg CTm2 deg CTm3 deg C
Tm
d
eg C
pH
Cp2 and Cp3 are the first to start denaturation. Even though they have higher Tm’s they have lower H*
Low Temperature Region Provides Clues to Long Term Stability.Kinetic Deconvolution Allows Extrapolation of Cp Components
Freire, E.
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1
2
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8
56 58 60 62 64
Cp kca
l/K
*mol
Temp deg C
SumCp1Cp2Cp3
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50 55 60 65 70 75 80 85 90
Cp
kcal/K
*mol
Temp deg C
pH 7.5
pH 6.8
At Low Temperatures Cp3 is the Least Stable Domain.pH 6.8 Appears to be the Most Appropriate pH
Freire, E.
SumCp1Cp2Cp3
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buffer 1 databuffer 1 fit
Cp
(kca
l/K
/mo
l)
Temperature (°C)
0
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150
50 60 70 80 90
buffer 2 databuffer 2 fit
Cp (
kcal/K
/mol)
Temperature (°C)
0
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150
50 60 70 80 90
buffer 3 databuffer 3 fit
Cp
(kcal/K
/mo
l)
Temperature (°C)
0
50
100
150
50 60 70 80 90
buffer 4 databuffer 4 fit
Cp
(kca
l/K
/mo
l)
Temperature (°C)
Freire, E.
Clarkson et al (2018) Analytical Biochemistry
Further Formulation Optimization at pH 6.8. Addition of Excipients: DSC of VRC07 in Buffers 1 - 4
Buffer 1 - 50mM Histidine, 100mM NaCl, pH 6.8
Buffer 2 - 50mM Histidine, 100mM NaCl, 5% w/v sucrose, pH 6.8
Buffer 3 - 50mM Histidine, 100mM NaCl, 5% w/v sorbitol, pH 6.8
Buffer 4 - 50mM Histidine, 50mM NaCl, 5% w/v sucrose, 2.5% w/v sorbitol, pH 6.8
Effect of Excipients in Tm’s and H*’s of VRC07 at pH 6.8 Freire, E.
Tm’s show an increase for all transitions. Activation enthalpy is fairly constant for transitions 1 and 3. Buffer 4 is the best overall.
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1 2 3 4
Tm1 deg CTm2 deg CTm3 deg C
Tm
deg C
buffer
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200
1 2 3 4
dHa1dHa2dHa3
H
* kca
l/m
ol
buffer
Buffer 1 Buffer 4 SumCp1Cp2Cp3
Notice that Formulation Optimization is a Combination of Tm and Activation Enthalpy
Improvement of Low Temperature Stability by Excipients
Freire, E.
Isothermal CalorimetryFreire, E.
ΔHu Qagg
Denaturation and Aggregation Involve the Absorption or Release of Heat and Therefore Can Be Measured Calorimetrically at Constant Temperature
TAM Isothermal CalorimeterFreire, E.
VP DSC. Little Known Isothermal ModeFreire, E.
𝑄 = ∆𝐻𝑢 1 − 𝑒−𝑘𝑢𝑡 + 𝑄𝑎𝑔𝑔 1 − 𝑒−𝑘𝑢𝑡 (1 − 𝑒−𝑘𝑎𝑔𝑔𝑡)
Quantity Measured by Calorimeter is Heat Flow: dQ/dt
Isothermal Calorimetry
𝑑𝑄/𝑑𝑡 = 𝑘𝑢∆𝐻𝑢𝑒−𝑘𝑢𝑡 + 𝑘𝑎𝑔𝑔𝑄𝑎𝑔𝑔𝑒
−𝑘𝑎𝑔𝑔𝑡 + 𝑘𝑢𝑄𝑎𝑔𝑔𝑒−𝑘𝑢𝑡 − (𝑘𝑢 + 𝑘𝑎𝑔𝑔)𝑄𝑎𝑔𝑔𝑒
−(𝑘𝑢+ 𝑘𝑎𝑔𝑔)𝑡
Freire, E.
ΔHu Qagg
𝑄 =𝐻𝑒𝑎𝑡
𝑣[𝑃𝑇]= ∆𝐻𝑢𝐹𝑢 + 𝑄𝑎𝑔𝑔𝐹𝑢𝐹𝑎𝑔𝑔
Two Different Situations for Protein Denaturation/AggregationFreire, E.
ku kagg
ku >> kagg
ku << kagg
k
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0 2 4 6 8 10
q expq fit
He
at flo
w (k
ca
l/da
y/m
ol)
time days
Denaturation Precedes Aggregation
Denaturation, Aggregation Occur Simultaneously
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0 0.5 1 1.5 2 2.5 3 3.5 4
expfit
dQ
/dt (k
cal/d
ay/m
ol)
Time (days)
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Cp
kca
l/K
*mol
Temp deg C
-100
0
100
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300
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500
0 2 4 6 8 10
q expq fit
Heat flow
(k
cal/day/m
ol)
time days
k = 1.82 days-1 (1/k = 14h)
Isothermal Calorimetry at 60°C, 100 mg/mL
Temperature Denaturation and Aggregation of mAb VRC07
DSC of VRC07
Freire, E.
1 2 3 4 5 6 7 8
q 1-100
qfit 1-100
q 2-100
qfit 2-100
q 3-100
qfit 3-100
q 4-100
qfit 4-100
time days
Clarkson et al (2018) Analytical Biochemistry, In Press
Denaturation/Aggregation Measured by Isothermal Calorimetry (100mg/mL)
Freire, E.
Kinetics of Denaturation/Aggregation Buffers 1 and 4 at 60C(Isothermal Calorimetry Data)
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0 2 4 6 8 10
Heat F
low
k
cal/d
ay*m
ol
time days
k = 1.82 days-1 (1/k = 0.55 days) buffer 1
k = 0.37 days-1 (1/k = 2.7 days) buffer 4
Freire, E.
Clarkson et al (2018) Analytical Biochemistry, In Press
VRC07 Denaturation/Aggregation Rates in Different Formulations at 60C
Discrimination Between Different Formulations
Freire, E.
0
0.5
1
1.5
2
A B C D
TA
M d
enat/agg r
ate
s days
-1
Formulation1 2 3 4
Formulation
SEC chromatograms of VRC07 in the four formulation buffers
after 12 weeks storage at 25°C:
red: buffer 1
blue: buffer 2
green: buffer 3
black: buffer 4
Freire, E.
90
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96
97
0 5 10 15
Buffer 1Buffer 2Buffer 3Buffer 4
y = 95.808 - 0.42633x R= 0.99687
y = 95.775 - 0.3389x R= 0.98572
y = 95.755 - 0.2465x R= 0.98028
y = 95.941 - 0.23289x R= 0.9873
%M
on
om
er
Time (weeks)
SEC Data for Four Different Formulations at 25°CFreire, E.
Correlation Between SEC and Isothermal Calorimetry Rates
Freire, E.
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6
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9
10
0 0.5 1 1.5 2
y = 5.6946 + 1.4968x R= 0.92814
SE
C %
Agg
regate
s
10 w
eeks incubation 2
5oC
TAM rate days-1
Conclusions
- Kinetic Deconvolution of DSC: Activation enthalpies for each denaturation peak. Temperature dependence of denaturation rates. Combined analysis of H* and Tm allows selection of best formulation.
- Isothermal Calorimetry: Rates of denaturation/aggregation at constant temperature can be determined in one week. Correlates well with SEC data and can be used to assess quality of antibody or formulation.
Freire, E.
For additional information or to discuss a collaboration please contact E. Freire at [email protected]