NMR webinar

1. Solid-State NMR Spectroscopy: Form
Identification, Quantitation, and
Applications to Drug Substances and
Drug Products, Including Amorphous
Solid Dispersions
Eric J. Munson
Department of Pharmaceutical Sciences
University of Kentucky

2. Disclosure
I am a partial owner of Kansas Analytical Services, a company
that provides solid-state NMR services to the pharmaceutical
industry.
The results presented here are from my academic work at the
University of Kansas and the University of Kentucky, and no
data from Kansas Analytical Services is presented here.

3. Outline
I. Introduction to Pharmaceutical Solid-State NMR
II. Form Identification/Detection
a. New polymorphs
III. Particle Size/Defects
a. Chemical stability/Particle size/Chemical purity
IV. Dynamic Nuclear Polarization
a. Sensitivity enhancement
b. Quantitation
V. Drug-Polymer Interactions
a. Miscibility
b. Hydrogen bonding
VI. Protein Stability
a. Phase miscibility
VII. Conclusions and Acknowledgements

4. q Traditional SSNMR
q
13C SSNMR
q Polymorphic form
q Crystalline/amorphous
q Quantitative – no standard
q Drug formulations
q Drug - different chemical shift
from excipient
q Host-guest interactions
q Chemical reactions
q Emerging
q
19F NMR
q “Advanced” SSNMR
q Relaxation times
q Particle size
q Miscibility
q Mobility
q Chemical stability
q Dissolution rate??
q Selective Labeling
q Probe molecules
q Peak overlap - isolation
q Enhanced sensitivity
Information Content from
Solid-State NMR

5. Non-destructive and non-
invasive
q Bulk drugs
q Drug formulations
q Drug - different chemical shift
from excipient
q Inclusion compounds
q Host-guest interactions
q Polymer matrices
q Crystalline drugs, proteins, and
peptides
q Chemical reactions
Quantitative and
Selective
q Quantitation of Forms
q Crystalline vs. amorphous
q Mixtures of forms
q Don t need standard!
q Selective Labeling
q Drug-excipient interactions
q Changes upon formulation
q Amorphous Þ crystalline
Why Use Solid-State NMR
Spectroscopy to Characterize
Pharmaceuticals?

6. Structure
q Crystalline
q Number of crystallographically
inequivalent sites
q Conformation
q Hydrogen bonding
q Packing arrangement
q Amorphous
q Degree of disorder
q Mixed phases
q Liquids in solids
Dynamics (mobility)
q Crystalline
q Determine mobility in lattice
q Amorphous
q Tg
q Plasticizers
q Formulations
q Drug
q Excipient
q Polymer
Why Use Solid-State NMR
Spectroscopy to Characterize
Pharmaceuticals?

7. Why Isn’t Solid-State NMR
Spectroscopy Used More to
Characterize Pharmaceuticals?
q Requires expertise to use properly
q Expensive
q Non-routine
q Difficult to automate
q Insensitive
q Long analysis times
q Perception of what it can do

8. Two Key Issues:
Line Broadening and Sensitivity
§ Line broadening
§ Heteronuclear dipolar
interactions
§ Chemical shift anisotropy
(CSA)
§ Sensitivity
§ Long relaxation times
§ Low natural abundance
Issue
§ Line broadening
§ High power proton
decoupling
§ Magic-angle spinning
(MAS)
§ Sensitivity
§ Cross polarization (CP)
Remedy

9. § 13C and 1H nuclei are
strongly coupled in solids
§ This coupling can cause line
widths up to 50 kHz
High Power Proton Decoupling
1H Decoupling
>50 kHz
§ This coupling can be removed
with high power RF field
§ Much narrower peaks in the
solid-state NMR spectrum

10. Chemical Shift Anisotropy (CSA)
§ In a powder, fixed orientations
of each particle relative to the
static magnetic field (Bo)
results in very broad lines
§ Continuous tumbling motion
in solution results in very
narrow lines

11. CSA and Magic-Angle Spinning (MAS)
§ Observed chemical shielding is a
combination of both isotropic and
anisotropic contributions:
sobs = siso + (3cos2q - 1)saniso
§ When q = 54.74 , (3cos2q - 1) = 0,
and if we orient the sample at this
“magic” angle and spin very fast
(kHz), only the isotropic
component is observed

12. Single Pulse vs. Cross Polarization (CP)
§ In this “single pulse”
experiment we excite the
13C spins directly and
then listen to what they
have to say
§ Low natural abundance
and long relaxation times
of the 13C nucleus limit
the utility of these
experiments
§ Delay between
subsequent pulses
governed by 13C T1

13. Single Pulse vs. Cross Polarization (CP)
§ In this “CP”
experiment we
excite the 1H
spins first,
transfer that
magnetization to
13C, then acquire
§ Gain 4 times the
signal with each
pulse, and can
pulse more often
§ Delay between
subsequent
pulses governed
by 1H T1
gHB1H = gCB1C

14. § Single pulse, static, 100
scans, 240 s pulse delay,
total time = 400 min
Putting It All Together:13C
Solid-State NMR Spectra of Ibuprofen
§ Same as above, but with
1H decoupling added
§ Same as above, with MAS
added
§ CP, MAS, 1H decoupling,
100 scans, 3 s pulse delay,
total time = 5 min
§ Same as above, withTOSS
pulse sequence applied

15. Impact of Solid-State Form Changes
on Biopharmaceutical Properties
• Discovered 1992, FDA approved 1996
• Problems with dissolution observed 1998
• New polymorphic form discovered with
half the solubility
• Forced withdrawal of formulation from
market
• Eventually reformulated with both forms
Bioavailability enhancement using
amorphous vs. crystalline formulations
A – 30%
A – 20%
A – 10%
C
Hours After Dose
Plasma
Concentration
Polymorphs

16. 13C Solid-State NMR Spectra of
Crystalline Aspirin and Freeze-Dried
Aspirin
Crystalline Aspirin
Freeze Dried
Aspirin

17. Polymorphs of Proline
D-proline
DL-proline form I
DL-proline form II
DL-proline monohydrate

18. 13C Solid-State NMR
Spectra of Trehalose

19. 13C Solid-State NMR
Spectra of Trehalose

20. 1H-19F CPMAS NMR Spectra of
50%-50% Triamcinolone-HPMCAS
Amorphous Solid Dispersions
Triamcinolone (Polymorph B)
Doped Dispersion – ~100ug crystalline
(~0.2%)
Triamcinolone – HPMCAS Dispersion

21. 1H-19F CPMAS NMR Spectra of
50%-50% Triamcinolone-HPMCAS
Amorphous Solid Dispersions
Triamcinolone (Polymorph B)
Doped Dispersion – 1H T1rho filter = 120ms
Doped Dispersion – 1H T1rho filter = 80ms
Doped Dispersion – 1H T1rho filter = 40ms
Doped Dispersion – 1H T1rho filter = 10us

22. Particle Size Reduction in
Pharmaceuticals

23. Relaxation – What is Measured Using
Solid-State NMR Spectroscopy
10
-4
10
-3
10
-2
10
-1
10
0
χ''
[a.u.]
15
10
5
0
log(ω) [Hz]
α Relaxation
β-JG Relaxation
Metabasin
Barrier Crossings
Inherent State
Transitions
Β –fast
Relaxation
2D exchange
Chemical exchange
1H T1rho
1H T1
Relaxation Time
• 2D Exchange
• 10-1 – 103 Hz
• Chem. Exchange
• 100 – 103 Hz
• 1H T1rho
• 104 – 105 Hz
• 1H T1
• 108 – 109 Hz
Image Courtesy of Marc Cicerone, NIST

24. Spin Diffusion Rate
Distance α
√1H T1
n Very sensitive to distance
n 1 nm - ~1 ms
n 10 nm - ~100 ms
n 100 nm – ~10 s
n 1000 nm - ~ 1000 s
n 10,000 nm - ~100,000 s
n Crystal defects reduce
relaxation time
ñ
defect

25. -0.4
0.1
0.6
1.1
1.6
2.1
2.6
-2 -1 0 1 2 3
log
sqrt
(
1
H
T
1
)
log d
dicumarol
sieved salicylic acid
extrapolated theory
theory (polymers)
Correlation of 1H Solid-State NMR
Relaxation Times with Particle Size
Determined by SEM
§ The dicumarol 1H T1
times are lower than
predicted
§ The difference is
believed to be due to
the presences of crystal
defects
0.1 μm; 8.3s
§ log√(1H T1) = log d- log √6D ; with D= 0.2 nm2/ms
1 μm; 830 s
10 μm; 83000 s

26. 13C Solid-State NMR
Spectra of Gabapentin
O
OH
NH2
1
2
3
4
5
6
7
8 9
Gabapentin Lactam
Gabapentin Form II
Gabapentin Form III
Gabapentin Isomorphous
Desolvate
Gabapentin Form I

27. Correlation of 1H Solid-State
NMR Relaxation Times with
Degradation Rate of Gabapentin
y = 0.0127x - 0.1198
R² = 0.9799
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 20 40 60 80 100 120 140
-
log
Go
relaxation time
more stable
less stable

28. Correlation of 1H Solid-State NMR
Relaxation Times with Trehalose
Dihydrate Purity as Determined by HPLC
*"Purity"to"be"measured"
0"
200"
400"
600"
800"
1000"
1200"
1400"
1600"
1" 2" 3" 4" 5" 6" 7" 8" 9" 10" 11" 12" 13" 14"
H1T1$(s)$
*"
*"
*"
low
medium
high purity

29. Dynamic Nuclear Polarization (DNP)
Bruker: https://www.bruker.com/products/mr/nmr/dnp-nmr/overview.html

30. Dynamic Nuclear Polarization (DNP)
Ames Lab DNP Instrument

31. DNP – A Pharmaceutical
Scientist’s Perspective
Salicylic Acid – Time Savings
Traditional - 60s delay, 1024 scans = 17hours
DNP - 15s delay, 4 scans = 1 minute
17 Hours
1 Minute

32. DNP – A Pharmaceutical
Scientist’s Perspective
Salicylic Acid – DNP Spectra – Line Width
Anisotropic Bulk
Magnetic Susceptibility
(ABMS)
Peaks are broadened
because local environment
of molecules in particle
depends upon where the
molecules are in particle
ABMS can be reduced by
dilution with low
susceptibility material (10%
SA in lactose), reducing
line widths from ~0.7 ppm
(sheared) to ~0.4 ppm

33. 13C SSNMR Spectra of Form A and
Form C of Chlorpropamide at -40 oC
50:50
Mixture
CPA-A
CPA-C

34. Chlorpropamide 13C SSNMR Spectra
of 10% Formulations:
2% Form A and 8% Form C

35. Chlorpropamide 13C SSNMR Spectra
of 10% Formulations:
2% Form A and 8% Form C
2Ctr = 26.397
Area= 0.305
2.3%
Ctr = 24.159
Area= 1.000
7.7%

36. DNP – A Pharmaceutical Scientist’s
Perspective
Chlorpropamide – Polymorph Formulations
Traditional*SSNMR**method
DNP4enhanced*SSNMR
Form C
Form A
2% Form A and 8% Form C
Form A
DNP
Conventional CPMAS NMR -
Same sample as DNP
Form C
Form C
Form C
Form A
Form A

37. DNP – A Pharmaceutical Scientist’s
Perspective
Chlorpropamide – Polymorph Formulations
2% Form A and 8% Form C
Form A
Form C
Red – DNP Spectrum – Microwaves On
Blue – Microwaves Off
No Change in Form Upon Cooling!
Form A
Form C

38. DNP – A Pharmaceutical Scientist’s
Perspective
Dicumarol – DNP Spectra – Particle Size
Red – Ground for 5 Minutes
Blue – Unground

39. Impact of Solid-State Form Changes
on Biopharmaceutical Properties
• Discovered 1992, FDA approved 1996
• Problems with dissolution observed 1998
• New polymorphic form discovered with
half the solubility
• Forced withdrawal of formulation from
market
• Eventually reformulated with both forms
Bioavailability enhancement using
amorphous vs. crystalline formulations
A – 30%
A – 20%
A – 10%
C
Hours After Dose
Plasma
Concentration
Amorphous

40. 13C Solid-State NMR Spectra of
Melt-Quenched Nifedipine-PVP
Solid Dispersions
175 150 125 100 75 50 25 0
200
ppm
Amorphous
nifedipine
95:5
90:10
75:25
60:40
PVP
50:50
N
CH
O
H2C
n
PVP
Nifedipine

41. Relaxation – What is Measured Using
Solid-State NMR Spectroscopy
10
-4
10
-3
10
-2
10
-1
10
0
χ''
[a.u.]
15
10
5
0
log(ω) [Hz]
α Relaxation
β-JG Relaxation
Metabasin
Barrier Crossings
Inherent State
Transitions
Β –fast
Relaxation
2D exchange
Chemical exchange
1H T1rho
1H T1
Relaxation Time
• 2D Exchange
• 10-1 – 103 Hz
• Chem. Exchange
• 100 – 103 Hz
• 1H T1rho
• 104 – 105 Hz
• 1H T1
• 108 – 109 Hz
Image Courtesy of Marc Cicerone, NIST

42. Miscibility Determination Using
Solid-State NMR Spectroscopy
T1 values T1ρ values Number of Phases
Same Same
1
(domain size < 2-5nm)
Same Different
2
(domain size 5-20 nm)
Different Different
2
(domain size > 20-50 nm)
2-5
nm
20-50
nm

43. 1H T1 values were the same for both Nifedipine and PVP,
indicating that they are intimately mixed at < 50 nm, but 1H T1ρ
values were different for varying polymer weight fractions
Difference in 1H T1ρ Relaxation Times
in ASDs of Nifedipine and PVP
0
5
10
15
20
25
30
0 0.1 0.2 0.3 0.4 0.5 0.6
ΔT1ρ
(ms)
PVP weight fraction
T1 values T1ρ values Number of Phases
Same Same
1
(domain size < 2-5nm)
Same Different
2
(domain size 5-20 nm)
Different Different
2
(domain size > 20-50 nm)

44. Solid-State Forms of Indomethacin
γ-IMC
α-IMC
amorphous IMC

45. Carboxylic acid
carbon (HB)
Solid-State Forms of Indomethacin
γ
α
Amide carbon
(No HB)
Amorphous
A
B C
19
10
19
10
19 19
19
10
10
10
19 10
?

46. Hydrogen Bonding of Amorphous
Indomethacin
carboxylic acid
carbon
Amorphous IMC
(13C labeled)
Amorphous IMC
(natural abundance)

47. Hydrogen Bonding of Amorphous
Indomethacin
179.4 ppm 170.4 ppm
IMC-Polystyrene
1% IMC
0.2% IMC
2% IMC
5% IMC Amorphous IMC
171.9 ppm
179.4 ppm
polystyrene
free
dimer

48. Hydrogen Bonding of Amorphous
Indomethacin
n 179 ppm = cyclic dimer
n 176 ppm = disordered chains/rings
n 172 ppm = carboxylic acid-amide complex
n 170 ppm = free

49. Hydrogen Bonding of Amorphous
Indomethacin
IMC-Polystyrene
0
0.5
1
1.5
2
2.5
20 70 120 170
Dimer/Free
T (°C)
1% IMC
0.2% IMC
1% IMC in polystyrene as a
function of temperature
2% IMC
5% IMC
Summary: ratio of dimer to free
changes as a function of
temperature, and is different above
and below the glass transition.
170.4 ppm
free
179.4 ppm
dimer

50. Hydrogen-Bonding Interactions of
IMC Amorphous Solid Dispersions
PVP
H-bond acceptor
Indomethacin
H-bond donor and
acceptor
Model System
PVP/VA
H-bond acceptor

51. IMC Carboxylic
Acid in Amorphous
Solid Dispersions
51
IMC-PVP IMC-PVP/VA
160
165
170
175
180
185
190
13
C Chemical Shift (ppm)
Amorphous IMC
Chemical Shift
(ppm)
Species
Peak Area
(%)
Linewidth
(Hz)
179.3 ± 0.006 cyclic dimer 58.5 ± 0.5 216 ± 0.8
176.3 ± 0.02 carboxylic acid chain 15.2 ± 0.4 303 ± 5
172.4 ± 0.004 carboxylic acid-amide 18.9 ± 0.4 212 ± 0.6
170.4 ± 0.05 free carboxylic acid 7.5 ± 0.3 225 ± 5
90% IMC
80% IMC
70% IMC
60% IMC
50% IMC

52. Hydrogen-Bonding Interactions in
IMC Amorphous Solid Dispersions
Summary:
• PVP disrupted IMC cyclic
dimers; with 40% (wt) of PVP
present, no cyclic dimers could
be detected.
• PVP/VA also disrupted the
IMC self interactions in a
similar fashion as PVP, but
less effectively.
IMC-PVP
IMC-PVP/VA

53. How does H-Bonding Influence
Miscibility?
Indomethacin
methyl ester
H-bond acceptor
Indomethacin
H-bond donor and
acceptor
Differences of SSNMR 1H T1ρ Relaxation Times
-10
-5
0
5
10
15
20
25
30
35
40
45
50
0 10 20 30 40 50 60
Δ
1
H
T
1ρ
(ms)
PVP (w/w %)
IMC-PVP
IMC methyl ester-
PVP

54. H-Bonding Interactions of 80-20 IMC-
PVP ASD - Function of Water Content
1.2 % (wt) water
0.2% (wt) water
Chain
(21%)
Dimer
(4%)
IMC-amide
(75%)
Chain
(13%)
Dimer
(5%)
IMC-amide
(54%)
IMC-water
(28%)
Chain
(9%)
Dimer
(3%)
IMC-amide
(52%)
IMC-water
(36%)
1.6 % (wt) water
0
10
20
30
40
50
60
70
80
90
100
0 0.5 1 1.5 2 2.5
HB
Fraction
of
IMC
Carboxyl
Water Content (wt %)
carboxyl-amide
carboxyl-carboxyl
IMC-water
Free

55. Physical Stability of
70:30 IMC : PVP K25
PVP
Indomethacin
+
50 C/ 0% RH
40 C/57% RH
40 C/75% RH
Storage Conditions
Tg = 72 C amorphous
Tg = 52 C amorphous
Tg = 41 C crystallized
after 1 month
API:Polymer = 7:3
70:30 IMC : PVP K25
50 °C dry 40 °C 57%RH 40 °C 75%RH
Crystallize
?
Tg
(°C)
Tstorage-Tg
(°C)
Crystallize
?
Tg (°C)
Tstorage-Tg
(°C)
Crystallize
?
Tg
(°C)
Tstorage-Tg
(°C)
Time 0 No 62.4 -12.4 No 62.4 -22.4 No 62.4 -22.4
1 wk No 71.7 -21.7 No 52.7 -12.7 No 41.4 -1.4
2 wks No 71.4 -21.4 No 52.8 -12.8 No 41.1 -1.1
1 mnth No 70.7 -20.7 No 51.8 -11.8 Yes 41.3 -1.3
2 mths No 73.0 -23.0 No 50.4 -10.4 Yes 39.9 0.1
6 mths No 74.3 -24.3 No 52.0 -12.0 Yes 43.7 -3.7
• 70:30 IMC:PVP K25 only crystallized at 40 C and 75% RH
• Is the temperature (above Tg), the water, or both the cause
for the crystallization?

56. Physical Stability of 70:30 IMC:PVP
K12 and 70:30 IMC:PVP/VA at 70 oC
PVP
Indomethacin
+
50 C/ 0% RH
40 C/57% RH
40 C/75% RH
Storage Conditions
Tg = 72 C amorphous
Tg = 52 C amorphous
Tg = 41 C crystallized
after 1 month
API:Polymer = 7:3
IMC : PVP K12 -- Oven at 70 °C IMC : PVP/VA -- Oven at 70 °C
Ratio Tstorage-Tg 0 wk 1 wk 20 wks Ratio Tstorage-Tg 0 wk 1 wk 28 wks
50-50 -12.0 °C No No No 50-50 - 4.5 °C No No No
60-40 - 6.0 °C No No No 60-40 + 1.5 °C No No No
70-30 -0.5 °C No No No 70-30 + 7.0 °C No No No
80-20 + 8.5 °C No No No 80-20 + 12.5 °C No Yes Yes
90-10 + 15.5 °C No Yes Yes 90-10 + 18.0 °C No Yes Yes
• IMC crystallizes into different polymorph based on polymer (PVP/VA: Alpha,
PVP k12: Gamma)
• Crystallization only occurs at both high temperatures (> 10 oC above
Tg) and at high drug concentrations
• Which is the bigger cause for the crystallization, Tg or polymer
concentration?

57. 57
PVP
Indomethacin
+
API:Polymer = 7:3
IMC : PVP K12 -- Oven at 80 °C IMC : PVP K12 -- Oven at 70 °C
Ratio Tstorage-Tg 0 wk 1 wk 6 wks Ratio Tstorage-Tg 0 wk 1 wk 6 wks
50-50 - 0.0 °C No No No 50-50 - 10.0 °C No No No
60-40 + 6.5 °C No No No 60-40 - 3.6 °C No No No
70-30 + 13.6 °C No No No 70-30 + 3.6 °C No No No
80-20 + 18.2 °C No No YES 80-20 + 8.2 °C No No No
90-10 + 28.2 °C No YES YES 90-10 + 18.2 °C No YES YES
IMC : PVP K12 -- Oven at 60 °C
Ratio Tstorage-Tg 0 wk 1 wk 6 wks
50-50 - 20.0 °C No No No
60-40 - 13.6 °C No No No
70-30 - 6.4°C No No No
80-20 - 1.9 °C No No No
90-10 + 8.2 °C No No YES
Physical Stability of 70:30 IMC:PVP
K12 at 60 oC, 70 oC, and 80 oC
• Crystallization occurs at high drug concentrations, but lower
drug loading can retard crystallization at high temperatures (>
10 oC above Tg)
• Which is the bigger cause for the inhibition of crystallization,
Tg or polymer concentration? Polymer concentration!

58. Challenges Associated with
Protein Formulations
• Chemical degradation
• Maintain structure
• Maintain activity
• Aggregation
• Sterile
Human IgG1
1. Rouet, et al. Stability engineering of the human antibody repertoire, In FEBS Letters, Volume 588, Issue 2, 2014,
Pages 269-277, ISSN 0014-5793

59. What is Lyophilization?
• Steps in Freeze-Drying • Freeze-Drying Process
Freezing
• Lock API and
excipient in
place
Primary
Drying
• Removal of
95% of water
• Sublimation
Secondary
Drying
•Removal of
leftover bound
water
•Desorption

60. Stabilization of Proteins
Water Replacement
Theory:
• In solution, hydrogen
bonds between water and
protein keep protein
folded
• As temperature
decreases, ice
crystallizes first and a
freeze-concentrate forms
• The sugar hydrogen
bonds in place of
removed water
Vitrification Theory:
• Tg’ – glass transition of
maximally freeze-concentrated
solution
• At temperature below Tg’ -
disaccharide forms sugar glass
and immobilizes drug
mAb and
sugar in
solution
Freezing
sugar
Freeze-
concentrate forms

61. q Microenvironmental pH
q Phase miscibility
q Liquid vs. solid
q Crystalline/amorphous
q Stability prediction
Other techniques
~30 nm (DSC)
Challenging (Raman)
Possible (PXRD)
Neutron, H-D EX
In Situ Study of Lyophilization
SSNMR
2-20 nm
Easy
Easy
Relaxation

62. Protein Phase Separation
• Looked at two proteins in six different sugars to
determine phase separation after lyophilization was
performed.
• Proteins: IgG and LDH (20% protein)
• Excipients:
• Trehalose
• Inulin (2 kDa, 5 kDa)
• Dextran (2 kDa, 5 kDa, 70 kDa)
• Systems were one of the three cases based on protein
and excipient:
• Intimately mixed (Same 1H T1 and 1H T1rho)
• Partially miscible (Common 1H T1, different 1H T1rho)
• Phase separated (Different 1H T1 and 1H T1rho)
Mike Pikal and Maartin Mensink, UConn

63. Protein – Sugar
Sample
Protein 1H
T1 (s)
Sugar 1H T1
(s)
Protein 1H T1rho
(ms)
Sugar 1H T1rho
(ms)
IgG – Trehalose 4.6+0.5 4.3+0.5 9.0+0.7 10.4+0.5
IgG – Inulin 2 kDa 2.2+0.3 2.1+0.3 7.8+0.5 6.8+0.3
IgG – Inulin 5 kDa 1.7+0.2 1.8+0.3 9.3+0.6 6.3+0.3
IgG – Dextran 1.5 kDa 3.7+0.5 3.5+0. 4 17.0+1.0 21.9+0.6
IgG – Dextran 5 kDa 1.5+0.3 2.2+0.3 12.3+0.9 22.8+0.5
IgG – Dextran 70 kDa 1.5+0.2 2.9+0.4 10.0+0.6 17.4+0.5
LDH – Trehalose 1.7+0.2 2.0+0.2 10.1+0.7 11.3+0.3
LDH – Inulin 2 kDa 1.6+0.2 1.9+0.2 9.7+0.7 7.2+0.3
LDH – Inulin 5 kDa 0.90+0.10 1.4+0.2 10.5+1.0 7.6+0.4
LDH – Dextran 1.5 kDa 2.4+0.3 2.4+0.2 15.1+1.6 22.7+0.7
LDH – Dextran 5 kDa 1.9+0.2 1.8+0.2 14.3+0.7 23.5+0.8
LDH – Dextran 70 kDa 1.9+0.2 1.8+0.2 15.0+1.6 26.0+1.1
Protein Phase Separation

64. Mike Pikal and Maartin Mensink, UConn
Protein Phase Separation
and Stability
Storage: SEC
0"
1"
2"
3"
4"
5"
6"
Trehalose" Dex"
1.5kDa"
Inulin"
1.8kDa"
Inulin"
4kDa"
Dex"5kDa" Dex"
70kDa"
T1#(s)#
IgG##20,50nm#phase#separa5on#
IgG"
Sugar"

65. Overall Conclusions
q Polymorphic forms of drugs are easily detected, even in
formulations
q
1H T1 relaxation times can be used to predict drug stability,
measure particle size, and determine chemical purity
q Dynamic Nuclear Polarization (DNP) is a promising technique
for pharmaceutical analysis, with challenges
q Amorphous solid dispersions can be analyzed for phase
separation and hydrogen bonding, including dynamics and
equilibria
q Protein stability in lyophilized formulations can be determined
based upon relaxation times and phase separation
q Solid-State NMR spectroscopy is a powerful technique for
the analysis of pharmaceutical solids

66. Acknowledgments
q Current and Former Students
q Joe Lubach Loren Schieber Diana Sperger Robert Berendt
q Eric Gorman Dr. Dewey Barich Robert Berendt Elodie Dempah
q Donia Arthur Xioada Yuan Nick Winquist Sarah Pyszczynski
q Kanika Sarpal Ashley Lay Travis Jarrells Dr. Matthew Nethercott
q Julie CalahanDr. Sean Delaney Dr. Steve Rheiner Ben Nelson
q National Institute for Pharmaceutical Technology and Innovation
q Iowa, Minnesota (Stability team)
q Michigan, Puerto Rico, Minnesota, FDA (Quantitation team)
q Center for Pharmaceutical Development – Industrial Advisory Board
q Ken Qian and Marc Cicerone, NIST (and collaborators)
q Aaron Rossini and Michael Hanrahan, Iowa State University
q University of Copenhagen – Jacco van de Streek
q Mike Pikal and Maarten Mensink - UConn
q Funding
q NSF (CHE 0416214, 0750467, 1710453)
q University of Kansas Madison and Lila Self Fellowships
q University of Kentucky
q NSF Center for Pharmaceutical Development (CPD) (IIP 1063879,
1540011)