Polymorph Quantitation

Investigations in Pharmaceutical Polymorph
Quantification using PXRD, ATR‒FTIR,
and FT‒Raman at Roche Palo Alto

Richard E. Young
Research Scientist II
Analytical Research
Roche Palo Alto LLC

Agenda

 Basic Principles of Polymorphism
 Polymorphism’s Importance to Pharmaceuticals
 Polymorph Discovery Techniques and the Allied
Analytical Tools
 Case Study 1: Three Phases of Gancilclovir
 Case Study 2: Two Phases of a Roche Research
Compound
 Conclusion

12/30/2009 Richard E. Young 2

What is a Chemical Polymorph?

A compound with a single molecular structure that possess more
than one crystal form. A polymorph form is often termed a
“phase.”

http://en.wikipedia.org/wiki/Bravais_lattice


Differentiating Habit and Crystal Chemistry of
a Compound
J. K. Haleblian, “Characterization of habits and crystalline modification of solids and their pharmaceutical applications,” J. Pharm. Sci., 64(8), 1270 (1975).

Chemical Compound (Solid)

Habit Internal Structure

Crystalline Amorphous

Single Entity Molecular Adduct

Polymorphs Nonstoichiometric Stoichiometric
Inclusion compounds Solvates (hydrates)

Channel Layer Cage (clathrate)


The Crystallinity Continuum

The “crystallinity continuum” is expressed by the relative amount
of order of contiguous unit cells

High Large single crystal
Crystalline order

Powdered crystals
Small crystallites
(more or less ordered)

Semi-crystalline
(short range order)

Low Amorphous
(lacks a distinct crystal form )


The Importance of Polymorphism in
Pharmaceuticals
• Pharmaceutical performance
Each polymorph may have different physical properties (melting point,
solubility, dissolution rate, processibility, etc.), which may affect:
– Stability
– Formulation
– Potency
– Bioavailability
– Storage
• Intellectual Property
– Each polymorph can be patented if it shows better properties than
any previously patented polymorph
– A rival company may legally sell the same drug substance in a
different crystal form if the new polymorph is not patented


Polymorph Discovery Techniques

No method can predict polymorphs.
Discovery must be done by empirical techniques.

• Stability studies with varying temperature, humidity, and time
• Re-crystallization with different solvents
• Melt re-crystallization
• Anti-solvent addition
• Annealing


Polymorph Analytical Tools

• Powder X-Ra Diffraction (PXRD or XRPD)
• Thermal techniques
– Differential scanning calorimetry (DSC)
– Differential thermal analysis (DTA)
– hermal gravimetric analysis (TGA)
– Melting point
– Thermal microscopy
• Solid State NMR
• Vibrational Spectroscopy
– FTIR (ATR, microscopy, etc.)
– FT-Raman
– NIR


The Polymorph Analytical Tools to be
Examined

• Powder X-Ray Diffraction
• AT-FTIR
• FT-Raman


PXRD Bragg-Brentano Theta-Theta
Configuration

Mono-
chromater
Divergence slit Antiscatter-
slit

Detector
slit
Tube

http://www.thermo.com/eThermo/CMA/PDFs/Product/productPDF_11602.pdf http://www.smcr.fisica.unam.mx/8temasutiles/articulosutiles/Bas-XRD.pdf

• A crystalline solid will produce a distinctive “fingerprint” pattern of sharp peaks;
amorphous materials will produce a broad “hump”
• Phase characterization and identification - not optimum for structure
determinations
• Transmission or reflection configuration

Bragg Diffraction

Bragg’s Law:
nλ = 2d sin θ
θ is the scattering angle
λ is the X-ray wavelength
d is the distance between planes

http://www.smcr.fisica.unam.mx/8temasutiles/articulosutiles/Bas-XRD.pdf)

• A crystal modeled as a series of parallel planes with distance
“d” between planes (d-spacing)

• Constructive interference of reflections creates a Bragg peak


Powder X-Ray Diffraction Advantages and
Disadvantages
• Advantages
– Well established and accepted (the gold standard)
– Rapid and simple sample analysis
– Can readily differentiate polymorphs
– Can analyze mixed polymorphs
– Quantitative and qualitative

• Disadvantages
– Sensitive to sample preparation technique
– Requires radiation license and fees to operate
– X-ray hazard
– Very expensive

PXRD: Example of an Amorphous and
Crystalline Drug Substance Mixture
O
O O

N N
O
N
O
O
F F N
O O
F
0% Amorphous
RS-104253-146
F

30% Amorphous

50% Amorphous

70% Amorphous

100% Amorphous

3 10 20 30 40

2-Theta


PXRD: Example of Polymorph Transitions by
Temperature
200 °C
200 C

O
O
Cl
N S
O
O O
O
RO0130830-000
175 °C
175 C

155 °C
155 C

110 °C
110 C

30 °C
30 C

2 10 20 30

2-Theta


Multi-bounce Attenuated Total Reflectance -
FTIR

Sample clamp

Sample
Multi-bounce
ATR crystal

IR Radiation IR Detector
Mirror Mirror

• Solid & liquid samples may be analyzed without preparation

• Solid samples are firmly clamped against crystal to provide intimate contact reducing the
distorting effect of trapped air

• ATR crystals: germanium, KRS-5, zinc selenide, silicon, diamond


ATR-FTIR (with diamond) Advantages and
Disadvantages
• Advantages
– Sample holder (diamond) scratch & abrasion resistant
– Rapid sample analysis
– Minimal sample preparation required

• Disadvantages
– Diamond absorbs in the 2300 to 1800 cm-1 region
– Polymorph distinction is compound dependent
– Not as well established for polymorph analysis
– Expensive (diamond)

Raman Scattering

Scattered radiation
Sample

Excitation energy
(laser radiation)
Exited energy
states

Rayleigh scatter
Rayleigh scattering

(same wavelength as excitation energy))
Excitation energy

Anti-stokes
Stokes

4 Raman scatter
energy states

3
Vibrational

2 (stokes & anti-stokes - different
1 wavelengths than excitation energy)
0


FT-Raman Advantages and Disadvantages

• Advantages
– Rapid sample analysis
– Minimal sample preparation required

• Disadvantages
– Polymorph distinction is compound dependent
– Not as well established for polymorph analysis


Case Study 1: Ganciclovir
9-(1,3-dihydroxy-2-propoxymethyl) guanine

• Antiviral drug for the treatment for cytomegalovirus (CMV)
infections
• Four known polymorphs: Phases I, II, & III and Phase I Hydrate
• PXRD quantifiation method for Phases I, II, and III

PXRD Patterns of Three Polymorphs of
Ganciclovir
Ganciclovir Phase I

Phase I

Ganciclovir Phase II

Phase II

Ganciclovir Phase III

Phase III

2 10 20 30 40

2-Theta
2-Theta


Equations for Calculating Ganciclovir Phase
Percents using PXRD Analysis
If Only Phases I & II If Only Phases I & III
100% 100%
% Phase II = % Phase I =
Slope 1 ( Peak I
Peak II ) +1 (
Slope 2
Peak III
Peak I ) +1

% Phase I = 100% - % Phase II % Phase III = 100% - % Phase I

If Only Phases II & III If All Three Phases (I, II, & II)
100% 100%
% Phase I =
( ) ( )
% Phase II =
( )
Peak III Peak II Peak III
Slope 3 +1 Slope 1 + Slope 3 +1
Peak II Peak I Peak I

% Phase III = 100% - % Phase II % Phase III = % Phase I (Peak III
Peak I ) Slope 3

% Phase II = 100% - % Phase I - % Phase III


Quantifation of Standards of Ganciclovir
Polymorph Mixtures by PXRD (Set A)
Ratios Phase I Phase II Phase III Phase I Phase II Phase III
05/95/00 -3.39 3.39 0.00
10/00/90 -1.16 0.00 1.16
45/50/05 -3.64 3.59 0.06
20/75/05 -5.49 6.23 -0.74
60/35/05 -3.78 3.40 0.38
70/05/25 -1.38 -0.48 1.86
80/10/10 -2.21 0.05 2.16
00/95/05 0.00 0.65 -0.65
95/05/00 0.06 -0.06 0.00
95/00/05 -0.45 0.00 0.45


Polymorph Mixtures by PXRD (Set B)
Ratios Phase I Phase II Phase III Phase I Phase II Phase III
05/95/00 -5.75 5.75 0.00
10/00/90 0.04 0.00 1.76
45/50/05 -11.04 9.38 1.66
20/75/05 -8.21 7.35 0.87
60/35/05 -5.92 4.86 1.06
70/05/25 -5.25 -0.08 5.32
80/10/10 -2.06 0.55 1.51
00/95/05 0.00 -0.41 0.41
95/05/00 0.20 -0.20 0.00
95/00/05 0.23 0.00 -0.23


ATR-FTIR Spectra of Three Polymorphs of
Ganciclovir Salari, A. and R. Young, "Application of Attenuated Total Reflectance FTIR Spectroscopy to the Analysis of
Mixtures of Pharmaceutical Polymorphs," International Journal of Pharmaceutics, 163, 157-166 (1998).

Phase I

Phase II

Phase III


Ganciclovir Polymorph Calibration Standards
Int J of Pharm 163, 157-166 (1998)

• Single Phase Standards
I, II, & III

• Binary Phase Standards
I & II I & III II & III
Three sets: 5, 10, 25, 50, 75, 90, 95 wt% each

• Ternary Phase Standards
I, II, & III
– Six mixtures consisting of 10, 30, 60 wt% each
– One mixture of 33.3 wt% each


ATR-FTIR Ganciclovir Polymorph Calibrations
Using Partial Least Squares
Int J of Pharm 163, 157-166 (1998)

Phase I r2 = 0.962

Phase II r2 = 0.964

Phase III r2 = 0.972


FT-Raman Spectra of Ganciclovir Polymorphs

Phase I

Phase II

Phase III


Phase I Polymorph by ATR & FT-Raman Int J of Pharm 163, 157-166 (1998)

Actual Mean Stnd Dev Difference
wt% ATR RAM ATR RAM ATR RAM
ATR Raman
0.0 2.8 -1.2 4.0 2.0 -2.8 1.2
5.0 7.6 6.6 5.7 2.2 -2.6 -1.6
10.0 8.3 12.2 1.8 2.4 1.7 -2.2
25.0 21.8 26.9 3.5 1.5 3.2 -1.9
30.0 19.7 32.2 1.8 3.3 10.3 -2.2
33.3 25.2 33.5 0.8 3.8 8.1 -0.2
50.0 50.9 54.2 5.2 4.3 -0.9 -4.2
60.0 56.3 65.2 3.2 2.8 3.7 -5.2
75.0 71.6 75.9 9.2 5.2 3.4 -0.9
90.0 92.4 88.8 8.8 3.3 -2.4 1.2
95.0 95.0 93.1 1.2 3.7 0.0 1.9
100 106 99.3 3.2 3.2 -6.0 0.7


Phase II Polymorph by ATR & FT-Raman Int J of Pharm 163, 157-166 (1998)

ATR Raman
0.0 -0.9 0.0 4.3 1.6 0.9 0.0
5.0 4.0 4.5 1.3 1.1 1.0 0.5
10.0 12.5 10.0 2.8 0.7 -2.5 0.0
25.0 29.2 26.6 3.0 2.6 -4.2 -1.6
30.0 32.6 28.4 3.8 2.0 -2.6 1.6
33.3 39.8 33.0 3.0 2.7 -6.5 0.3
50.0 47.0 47.0 5.4 4.6 3.0 3.0
60.0 66.8 56.2 2.7 2.9 -6.8 3.8
75.0 73.0 76.8 8.8 2.2 2.0 -1.8
90.0 87.1 89.3 8.0 1.3 2.9 0.7
95.0 89.1 94.2 10.5 1.5 5.9 0.8
100 94.0 102 9.7 1.7 6.0 -2.0


Phase III Polymorph by ATR & FT-Raman Int J of Pharm 163, 157-166 (1998)

ATR Raman
0.0 2.9 1.30 5.6 2.3 -2.9 -1.3
5.0 4.8 6.1 1.3 2.5 0.2 -1.1
10.0 12.6 9.50 5.8 1.7 -2.6 0.5
25.0 22.2 21.8 6.9 1.9 2.8 3.2
30.0 27.3 27.4 0.6 5.3 2.7 2.6
33.3 33.9 33.5 3.2 1.5 -0.6 -0.2
50.0 48.1 47.1 6.6 3.5 1.9 2.9
60.0 62.8 60.1 7.3 2.2 -2.8 -0.1
75.0 75.4 73.4 6.4 2.9 -0.4 1.6
90.0 89.1 88.8 7.6 3.5 0.9 1.2
95.0 95.9 92.9 2.0 3.4 -0.9 2.1
100 102 107 7.7 0.4 -2.0 -7.0


Ganciclovir Polymorph Validation Mixtures
Int J of Pharm 163, 157-166 (1998)

• One of Each Pure Phase Validation Phase I Phase II Phase III
Mix ID (wt%) (wt%) (wt%)
– 100 % Phase I
Phase I 100 0.0 0.0
– 100% Phase II
Phase II 0.0 100 0.0
– 100% Phase III Phase III 0.0 0.0 100
Mix 1 5.6 21.2 73.2
Mix 2 7.3 9.8 82.8
• Ternary Phase Mixtures
Mix 3 14.2 43.1 42.7
– Ten mixtures of all three Mix 4 14.1 78.6 7.4
phases in varying
Mix 5 26.8 56.0 17.2
amounts
Mix 6 33.6 11.4 55.0
Mix 7 40.8 18.2 41.0
Mix 8 46.4 32.9 20.7
Mix 9 46.5 28.2 25.4
Mix 10 58.2 18.2 23.5

Quantifation of Validation Mixes of Ganciclovir
Phase I Polymorph by ATR & FT-Raman Int J of Pharm 163, 157-166 (1998)

Validation Actual Calc wt% Difference
Mix ID wt% ATR RAM ATR RAM
ATR Raman
Phase I 100 102 99.3 -2.0 0.7
Phase II 0.0 3.2 -2.7 -3.2 2.7
Phase III 0.0 5.6 -4.6 -5.6 4.6
Mix 1 5.6 9.0 6.9 -3.4 -1.3
Mix 2 7.3 9.0 9.7 -1.7 -2.4
Mix 3 14.2 21.4 10.2 -7.2 4.0
Mix 4 14.1 12.5 14.3 1.6 -0.2
Mix 5 26.8 23.6 27.4 3.2 -0.6
Mix 6 33.6 39.5 44.8 -5.9 -11.2
Mix 7 40.8 45.6 42.2 -4.8 -1.4
Mix 8 46.4 47.7 45.9 -1.3 0.5
Mix 9 46.5 47.2 49.9 -0.7 -3.4
Mix 10 58.2 61.5 59.7 -3.3 -1.5

Phase II Polymorph by ATR & FT-Raman Int J of Pharm 163, 157-166 (1998)

ATR Raman
Phase I 0.0 -2.1 -0.1 2.1 0.1
Phase II 100 104 102 -4.0 -2.0
Phase III 0.0 1.2 -2.7 -1.2 2.7
Mix 1 21.2 22.0 21.7 -0.8 -0.5
Mix 2 9.8 11.1 10.0 -1.3 -0.2
Mix 3 43.1 46.0 47.8 -2.9 -4.7
Mix 4 78.6 86.8 82.4 -8.2 -3.8
Mix 5 56.0 65.9 58.4 -9.9 -2.4
Mix 6 11.4 7.2 4.9 4.2 6.5
Mix 7 18.2 22.7 17.7 -4.5 0.5
Mix 8 32.9 37.5 34.5 -4.6 -1.6
Mix 9 28.2 35.4 28.1 -7.2 0.1
Mix 10 18.2 21.1 18.1 -2.9 0.1

Phase III Polymorph by ATR & FT-Raman Int J of Pharm 163, 157-166 (1998)

ATR Raman
Phase I 0.0 -1.1 0.8 1.1 -0.8
Phase II 0.0 -6.7 0.3 6.7 -0.3
Phase III 100 94.1 107 5.9 -7.0
Mix 1 73.2 68.5 71.4 4.7 1.8
Mix 2 82.8 80.3 80.3 2.5 2.5
Mix 3 42.7 32.2 42.0 10.5 0.7
Mix 4 7.4 0.7 3.2 6.7 4.2
Mix 5 17.2 10.2 14.1 7.0 3.1
Mix 6 55.0 54.7 50.3 0.3 4.7
Mix 7 41.0 31.6 40.0 9.4 1.0
Mix 8 20.7 14.3 19.6 6.4 1.1
Mix 9 25.4 17.0 22.0 8.4 3.4
Mix 10 23.5 17.6 22.2 5.9 1.3

Summary of Case Study 1
Three Polymorphs of Ganciclovir

• ATR and Raman have the capability to quantify complex
mixtures of polymorphs

• ATR and Raman produced quantitative results comparable to
the PXRD

• The Raman, in general, gave smaller differences than ATR or
PXRD


Case Study 2: Anhydrate and Hydrate Phases
of a Roche Research Compound in Tablets
Identification and quantification of two phases of a Roche
research compound in the presence of excipients

Research Compound & excipients (50:50) in 150-mg
tablets
• Anhydrate (“Phase A”)

• Hydrate (“Phase B”)

• Excipients (“Placebo”): Pharmatose 350M, Povidone K30,
Ac-Di-Sol, Avicel PH102, magnesium stearate


PXRD Patterns of Phase A, Phase B,
and Placebo


PXRD Patterns of Polymorph Standards
(% Phase B in Phase A & Placebo)
Phase A Phase B
9.0 2θ 13.1 2θ


PXRD Reference Intensity Ratio (RIR)
Technique for Two Polymorph Mixtures

100 %
wt% B = 
1 + (m × IA ÷ IB)

• wt% B is the weight percent of Phase B in the sample

• m is the slope of the line of the (XB / XA) vs (IB / IA) linear regression

• IA is the peak height of the Phase A peak at 9.0 2θ in the sample

• IB is the peak height of the Phase B peak 13.1 2θ in the sample


PXRD RIR Slope Determination for Weight%
Ratio Versus Peak Intensity Ratio
2
1.8
Weight % Ratio (XBXA)

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
2θ Peak-Height Ratio (IB/IA )

(XBXA) = 1.976 × (IB/IA) – 0.0013 r2 = 0.991

ATR-FTIR Spectra of Phase A, Phase B,
and Placebo

Placebo

Phase A

Phase B


FT-Raman Spectra of Phase A, Phase B,
and Placebo
Placebo

Phase A

Phase B


ATR-FTIR and FT-Raman Polymorph
Calibrations
Calculated vs Ac tual Plot - Phas e B
51

Corr. Coeff.: 0.99445 RM SEC: 1.54

ATR-FTIR r2 = 0.989
Ca lcula te d

Calibration
Validation
Correction
-2

-2 Actual 51

C alculated vs Ac tual Plot - Phas e B
51

C orr. C oeff.: 0.98488 R M SEC : 2.53

FT-Raman r2 = 0.970
Ca lcula te d

C alibration
Validation
C orrection
-2

-2 Actual 51


Determined Weight% of Phase B by
PXRD, ATR-FTIR, and FT-Raman
Lot 1: 0 wt% Phase B
Sample ID PXRD ATR-FTIR FT-Raman
Rep-1 0.0 1.7 2.2
Rep-2 0.0 0.0 -0.10
Rep-3 0.0 1.0 0.7
Rep-4 0.0 2.0 1.8
Rep-5 0.0 2.0 2.4
Mean 0.0 1.3 1.4
Stnd. Dev. 0.000 0.853 1.07


Determined Weight% of Phase B by
PXRD, ATR-FTIR, and FT-Raman
Lot 2: 10 wt% Phase B
Sample ID PXRD ATR-FTIR FT-Raman
Rep-1 12 12 16
Rep-2 12 12 10
Rep-3 11 14 15
Rep-4 11 15 16
Rep-5 12 14 17
Mean 12 13 15
Stnd. Dev. 0.548 1.34 2.77


Summary of Case Study 2
Two Polymorphs of a Research Compound

• PXRD produced quantitative results superior to either ATR or
Raman.

• ATR produced quantitative results superior to Raman

• Another chemometric calibration method might work improve the
vibrational spectrocopic results


Conclusions

• All of the Techniques (PXRD, ATR, Raman) are Non-destructive

• All of the Techniques can be Applied to APIs as well as Drug Products

• The Vibrational Techniques (ATR and FT-Raman) have Demonstrated
Capabilities for Quantification of Complex Polymorph Mixtures

• The Superiority of a Particular Technique for Quantification is on a
Case by Case Basis


Acknowledgements

• Amid Salari
• Kewei Xu
• Fujun Li
• Tobin Koppelmaa
• Lourdes Javier
• Lilia Limon


Questions


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