10/28/13
New Guidelines for Stabilization of
Proteins by Freeze Drying
or
“Formulation and Process Heresy”!
Michael J. Pikal
School of Pharmacy
University of Connecticut
1
Historical “Rules”
•  Formulation should have “high”
glass transition temperature of the
freeze concentrate, Tg’, to facilitate
freeze drying.
•  Freeze dry below collapse
temperature for optimal stability
•  Formulation, when dried, should
have high glass transition
temperature, Tg, to facilitate elegance
and stability.
2
1
10/28/13
Process Design for Primary Drying
Key Points:
• Run at High Temperature to run fast!
• ...But, run a safe margin below the
collapse temperature!-Conventional wisdom!
• However, recent studies indicate that drying
above the collapse temperature is not
necessarily bad.
- Generally, stability is not damaged!
-”micro-collapse” may not damage any
product quality attributes.
3
Freeze Drying Above Tg’ (and Tc)
usually does not damage product quality!
Some Examples:
1.  LDH
2.  Recombinant Factor VIII
3.  Monoclonal antibody-details below
-50 mg/mL protein, 80 mg/mL sucrose(Tg’=-30°, Tc = -28°)
-Four run conditions
-Conservative, mean Tp ≈ -33 °
-Low, mean Tp ≈ -24°
-Optimal, mean Tp ≈ -20°
-High, Tp range from -10° to -24°
4
2
10/28/13
Freeze Drying with Collapse May Increase Stability
Schersch, et al., J. Pharm. Sci., 101, 2288-2306 (2012)
IgG01(4 mg/mL) in 50 mg/mL Trehalose
after 15 weeks:
1= 5°C, 2 = 40°, 3 = 50°
Soluble Aggregation
Solid symbols = no collapse
Open symbols = collapse
FD no collapse
FD with collapse
50°C
40°C
5°C
5
Collapse Temperature Measurement
•  Freeze drying microscopy (Tc)
–  1-5 µl liquid product frozen between two microscope
coverslips
–  Temperature is lowered to freeze the sample, vacuum,
temperature is raised to begin sublimation
–  Tc is visually observed
•  DSC (Tg’)
–  The glass transition of the maximally freeze concentrated
solution
–  Used as an estimate for Tc, generally 1-3oC lower than Tc
–  With protein-rich formulations, difference between Tc and
Tg’ can be much larger!
•  Note that neither Tc nor Tg’ are thermodynamic
properties.
–  Even Tc (measured by FDM) may not be representative of
collapse in vials, due to drying rate differences.
6
3
10/28/13
Accurate Prediction of Collapse
Temperature using Optical
Coherence Tomography (OCT)
Based Freeze Drying Microscopy
7
Optical Coherence
Tomography
•  Looks at collapse in a vial rather than thin film
–  More relevant for processing, manufacturing
•  OCT is the optical analog of an ultrasound imaging
–  Measures the back-reflection intensity of light instead of
sound
•  Data for 5% Sucrose:
–  DSC: Tg’ = - 34°C
–  Conventional FD Microscopy: Tc = -32°C
–  OCT: Tc = -28°C
•  Data for 1:3 BSA:Sucrose
– 
– 
– 
– 
Tg’ = -28°C
Conventional FD Microscopy: Tc =-26°C to -28°C
OCT: No Collapse! (only shrinkage)
Product freeze dried in lab freeze dryer, Tp(max) ≈ -21°C.
No collapse observed!
•  SEM and SSA suggest “micro-collapse”
8
4
10/28/13
Determining Tc by OCT-FDM
5% Sucrose
1:3 BSA:Sucrose @ -22°
Collapse
Shrinkage
En face
x-y view
En face view of vial, L - uncollapsed,
R - collapsed
y-z view
9
Freeze Drying Batch Runs – BSA/Sucrose
150
0
90
70
50
30
10
-10
5
10
15
20
25
30
35
Time (h)
Avg EV Avg CV Avg Shelf Temp.
Chamber CM Pirani
Low Temperature Run
Based on Tc by FDM
Tp(max) = -34°C
1° drying ~20hrs)
o
110
TemperatureC)(
Pressure (mT)
o
TemperatureC)(
130
60
600
40
500
20
400
Pressure (mT)
50
40
30
20
10
0
-10
-20
-30
-40
-50
0
300
-20
200
-40
100
-60
0
EV
0
1
2
Avg CV
3
4
5
6
7
8
9
Time (h)
Avg Shelf Chamber CM Pirani
High Temperature Run
Based on OCT data
Tp(max) ≈ -21°C
1° drying ~4hrs
10
5
10/28/13
Freeze Drying Batch Runs – BSA/
Sucrose
•  Freeze dried product in vials and
SEM Images (300x)
–  Left – Sample lyophilized at <
-34oC, Tp
–  Right – Sample lyophilized at ≈
-21oC, Maximum Tp
•  OCT-FDM data consistent with
batch freeze drying results – NO
COLLAPSE
11
What Formulation Factors
do Determine Stability?
12
6
10/28/13
Storage Stability is Very Sensitive to Formulation
Human Growth Hormone Formulations
Rate Constants, k(√t), at 40°C (40°C << Tg)
Storage Stability of hGH Formulations at 40°C
STD
NEW
*
1.00
*
0.10
kRP,%/√lmo
kSEC,%/√mo
6)
se
(
3)
se
(
se
(
1)
6)
se
(
3)
se
(
1)
se
(
1)
n(
os
e(
5)
)
ES
(1
on
e
0.01
cr
o
Su
cr
o
Su
cr
o
Su
lo
Tr
eh
a
lo
Tr
eh
a
lo
Tr
eh
a
hy
St
ac
G
ly
(1
)
:M
H
an
* residual water in range 0.7% to 2.5%; stability not correlated with %H2O
* all formulations except Gly:Mann are glassy
** Trend is same for both chemical degradation and aggregation!
N
Rate Constant, %/√mo
10.00
Stabilizer Why?
System
13
Significance of Results
•  Standard, “Current most used”
commercial formulation
– ≈ 3% aggregation in 2 yrs at Refrigerated
Storage (estimated from literature data)
•  New, “best Formulation” (currently
known)
–  ≈ 3000 years to form 3% aggregate at 25°C!
(estimated from literature data)
14
7
10/28/13
Pharmaceutical Stability in Solids is
Not Driven by Thermodynamics
•  Thermal Denaturation of proteins in solids is very high,
≈ 130°C-190°C
–  addition of disaccharides stabilizes against degradation, but
lowers thermal denaturation temperature.
•  Molecular mobility in glasses is very slow---> system is
Not in equilibrium!
–  thermodynamics does not apply?
•  Some limited correlation between secondary structure
and pharmaceutical stability
–  thermodynamics “could” be critical in determining structure
formed in the freeze drying process (where mobility is high),…
or not!
15
Key Factors in Stabilization
• Generally Accepted Concepts#
•  Stabilizer must remain amorphous and in same phase
as the drug!
–  i.e., physical mixture does not stabilize; want to dilute the drug
in the stabilizer matrix#
•  Stabilizer must be “inert”!
–  i.e., reducing sugars react with proteins#
•  Formulation must not allow selective buffer
crystallization and pH shift (for pH sensitive
molecules)#
•  Low Water Content#
16
8
10/28/13
Key Factors in Stabilization
• Factors of Probable Importance
•  Dynamics (Mobility) is Important: The Formulation
Should be in the Glassy “Solid State”#
–  i.e., a protein should be diluted in an inert glassy matrix to
minimize mobility! ( F. Franks, H. Levine & L. Slade, ...)#
•  Structure is Important for Proteins: The Formulation
Should Provide a “Non-Reactive” Solid State Conformation
After Freeze Drying#
–  i.e., presumably the “native” conformation (J. Carpenter, S.
Prestrelski, ...)#
However, glassy does not mean perfect stability,
and a “native” conformation is not always stable
during storage (i.e., otherwise, why add stabilizers & freeze dry?)
17
Free Volume, Mobility, and Stability
A consideration of the effect of “free volume” on dynamics leads to the
WLF Equation (M. H. Cohen and D. Turnbull, J. Chem. Phys., 31, 1164-1169 (1959))
The Concept of Free Volume
“structure in a snapshot in time”
volume of one molelcule.
“van der Waals volume”,
v0, is volume/molecule
in “closest packing”
(i.e., crystal, more than
volume of one molecule)
free volume per
molecule, vf
volume of amorphous system, v
vf = v - v 0
18
9
10/28/13
Volume Change on Mixing HES and
Disaccharides from Densities
Volume of Mixing of HES and Amorphous Disaccharides
0.010
Volume of Mixing, cc/g
HES & Trehalose
HES & Sucrose
0.005
0.000
-0.005
-0.010
0.00
0.20
0.40
0.60
0.80
1.00
1.20
Wt fraction Disaccharide
19
Density and Free Volume:
Analysis of Density Data: Volume Decrease on Mixing
Amorphous Polyols with HES:Trehalose
Note: Compare with total free volume in sucrose of 0.034 cc/g
Using amorphous densities of components
0.015
Sorbitol Systems
Glycerol Systems
0.010
Series1
0.005
ro
l
Tr
e
h
:g
ly
ce
:3
%
5
%
H
ES
H
5
%
ES
:1
%
5
%
H
Tr
e
h
ES
:g
:g
ly
ce
ro
l
ro
l
ly
ce
it
ol
or
b
:S
h
Tr
e
:3
%
ES
H
5
%
H
ES
:1
%
H
Tr
e
h
ES
:S
:S
or
b
or
b
it
ol
it
ol
0.000
5
%
volume decrease on mixing, cc/g
Volume Decrease on Mixing 1% Polyol with HES:Trehalose
20
System
10
10/28/13
Density Comparison for Disaccharide:hGH
Comparison of Normalized Densities
Densities Normalized to Density of hGH (w buffer)
1.14
sucrose experimental
trehalose experimental
van der Waals density
Relative Normalized density
1.12
1.10
1.08
1.06
1.04
1.02
1.00
0
10
20
30
40
50
60
70
80
% Disaccharide
• Density increases greater than atomic density increase
• Slightly greater density for sucrose:hGH systems
21
-consistent with less free volume & better stability
Implications of Density
Analysis
•  If loss of volume indicates loss of free volume,
and stability correlates with free volume
–  Addition of sucrose to HES should improve
stability
•  As observed!
–  Sucrose should be better stabilizer than trehalose
•  ???
–  Addition of glycerol or sorbitol should stabilized
protein:disaccharide system
•  As observed!
•  High precision density measurements may be a
useful stability prediction tool??
22
11
10/28/13
Formulation and Stability: First
Rule
•  First Approximation
–  Maximize the Tg!
23
Stability and “T-Tg” for MoAb:Vinca Conjugate
Roy, et. al., Develop. biol. Standard., 74, 323-340 (1991)!
KS1/4 HD Conjugate Decomposition
25°C and 40°C; 1.4%, 3%, and 4.7% Water
5
lnR/Rg
4
3
2
Dimer
Free Vinca
Vinca Loss
1
calculated
0
0
5
10
15
T-Tg
20
25
30
• Good Correlation of Stability with T-Tg (above Tg),
as in WLF equation!#
24
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10/28/13
Addition of Sucrose to Proteins
Decreases Tg but increases Stability
Tg in Sucrose: IgG1 System
130
120
100
90
80
70
0
0.2
0.4
0.6
0.8
1
1.2
Wt Fraction Sucrose
25
Correlation of Stability and “T - Tg”
“Dry Solids” Stored Well Below Tg
Correlation of hGH Degradation Rate with Tg
Freeze Dried Samples
8
Rate Constant(√t) for Degradation
Tg, °C
110
Chemical deg./40°
Aggregation/40°
Chemical deg./50°
Aggregation/50°
6
4
2
0
-80
-70
-60
-50
T-Tg
• No Obvious Sensible Correlation!
• Tg is not the whole story, at least well
below Tg!!!
26
13
10/28/13
Formulation and Stability
•  First Approximation
–  Maximize the Tg!
•  Second Approximation
–  The first approximation is frequently a
very poor approximation!
27
Stabilization and Molecular Mobility
•  Mobility in a glass depends on more than “T-Tg”
•  Does molecular mobility determine
pharmaceutical degradation in the solid state?
–  Or, at least is it a critical factor?
•  Degradation Rate = (Mobility)C, C = coupling coefficient, = 1
for diffusion controlled Rx with Stokes-Einstein where
Diffusion ∝ 1/η
•  What kind of molecular mobility is most
relevant?
–  All?
–  None?
28
14
10/28/13
Classes of Dynamics in Glasses
•  Global Dynamics (α relaxation)
–  Directly related to viscosity
•  Enthalpy relaxation
•  Dielectric relaxation
–  Long time scale, long length scale
–  Tg marks division between “solid” and “liquid” behavior
•  “Fast” Dynamics (example: β relaxation)
–  “local” motion,
–  small length scale, short time scale
–  Various measures
•  β-relaxation via Dielectric,
•  amplitude of nanosec time scale motion via neutron scattering.
•  NMR Relaxation Times
29
Stability,Global Dynamics, and “Structure”
Correlations Between Protein Structure, Glass Dynamics,
and Stability for an IgG1 Antibody
3
0.8
Glass Dynamics,
ln(τβ)
2.5
2
0.75
Structure,
0.7
FTIR fraction Native
("marker" band area)
1.5
0.65
1
Stability,
Agg. Rate constant
(sq root of time)
ln (td^b)
0.5
0.6
log(k)+1, SEC(sqrt of time)
fraction of native structure
0
0.55
0
0.2
0.4
0.6
0.8
1
Fraction of Sucrose
• Sucrose addition decreases Tg (previous slide)
• Correlation with Global Mobility (relaxation time)
30
is far from perfect!!
15
10/28/13
Stability and “Fast Dynamics”
Relationship between the normalized aggregation rate constant and fast local
mobility (1/<u2>) at 50 oC for five different proteins
5.50
A
B
C
D
E
3.50
Ln (k/Xp) +2
Linear (
A)
Linear (
E)
Linear (
D)
1.50
-0.50
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
2
1/<u >
Increasing Sucrose level
• Excellent correlation between stability & “Fast Dynamics”.
31
KGF Degradation Tracks with <u2>-1
Carpenter Lab
32
16
10/28/13
Other Systems:
Degradation Tracks with <u2>-1
150 kD
19 kD
Cicerone & Douglas,33 Soft
Matter 8 2983 (’12)
Aggregation in IgG1:Disaccharide Systems
Effect of Small Additions of Sorbitol
Effect of Sorbitol on Aggregation in IgG1:Disaccharide Systems at
40°C. All systems are 1:1 weight ratio of disaccharide:protein
1.2
Trehalose Systems
Sucrose Systems
Relative Rate Constant
1
0.8
0.6
0.4
0.2
0
0
0.1
0.2
0.3
0.4
0.5
Sorbitol Level (sorbitol:disaccharide)
0.6
• Small amounts of sorbitol stabilize!
WHY???#
34
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10/28/13
Addition of Sorbitol Also
Improves Hydrolytic Stability
in Small Molecules
“Fast Dynamics” Important for Small
Molecules Too?
35
Sorbitol is not Unique:
Stabilization by Addition of Methionine
Aggregation During 3 months at 50°C
rHSA:Sucrose:Methione in 1:11:X
2
Increase in % Aggregation
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1
Level of Methionine, X
• Tg is decreased
• Role of “Fast Dynamics”
-small amount “anti-plasticizes
36
18
10/28/13
Small Amounts of Salts Also Stabilze
Effect of Salts on Aggregation Stability in 1:1 BSA:Sucrose
Hiroshika Goshima
9
%P = P0 + k √t
8
7
k at 65
6
5
Is this also a
“Fast Dynamics”
Effect???
4
3
2
1
0
0
5
10
15
20
Salt (w/w)%
37
Could Water Also Antiplasticize Fast Dynamics?
•  Some protein systems are more
stable at intermediate water content
–  Is this an Anti-plasticization effect??
38
19
10/28/13
The Effect of Residual Water on Storage
Stability of an IgG1 MAB at 50°C
Protein:Sucrose 1:1
Pure Protein
lnτβ
kagg
kchem
Note: error bars are standard errors estimated from the fit, and are unusually
large in some cases due to marginal quality of the fit. The same trends are
obtained using % degradation at a given time point.
• Seems to be minimum in degradation rate at
“intermediate” water content,… anti-plasticization?
39
Fast Dynamics Seems to Best
Predict Stability: Why??
•  Why is it that dynamics on a nanosecond
timescale tracks with stability on a timescale
of months?
–  Relevant Observations?
•  Stability in viscous systems, and glasses, are likely
diffusion controlled
–  Diffusional motion of groups of atoms
•  Structural relaxation times (i.e., τβ) track with viscosity
•  Glasses are heterogeneous in local structure and
dynamics-consist of independent relaxing regions
•  Relaxation times “average” linearly but diffusion
coefficients average as reciprocal diffusional times.
•  Viscosity and diffusion “decouple” as the glassy state
is entered
–  Stokes-Einstein Equation become invalid
•  Diffusion behavior follows “fast track”
40
20
10/28/13
A Partial Explanation: Maybe?
Averaging behavior of molecules
Two State Example:
for τ s  τ f
τ α = 0.5(τ s + τ f ) ≈ 0.5τ s
DT = 0.5(
Fast Route, Diffusion below Tg
Slow Route, below Tg
Many Small Jumps, Small E*
Dominates well below Tg
E
1 1
+ ) ≈ 0.5τ −1
f
τs τ f
One large highly cooperative jump
Becomes very slow well below Tg
E
Reaction Coordinate
Reaction Coordinate
41
Complications
•  Argument is Qualitative- need
quantitative model
•  Specific Effects do seem to be
present
–  Different proteins behave differently
•  Sucrose stabilizes hGH better than
trehalose, but with KGF, no real difference
–  Likely that “coupling” between matrix
and protein varies between stabilizers
42
21
10/28/13
Conclusions
•  You can Freeze Dry above the Collapse
Temperature and not damage product.
–  Sometimes get better stability
•  Decreasing Tg is not necessarily bad! It can
STABILIZE!
–  As long as normal storage is still well below Tg
•  Decrease Tg in disaccharide formulations by addition of
“small molecules”
•  Decrease Tg’ by addition of NaCl in small amounts.
•  “deal with” lower Tg’
•  “Fast Dynamics” often drives stability
differences, not Tg or even global dynamics.
43
22