Polymorph Quantitation

Investigations in Pharmaceutical Polymorph

Quantification using PXRD, ATR‒FTIR,

and FT‒Raman at Roche Palo Alto

Richard E. YoungResearch Scientist II

Analytical Research

Roche Palo Alto LLC

12/30/2009 Richard E. Young 2

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


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 CompoundJ. 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

AmorphousCrystalline

Single Entity

Polymorphs

Molecular Adduct

Stoichiometric

Solvates (hydrates)

Nonstoichiometric

Inclusion compounds

Channel Layer Cage (clathrate)

The Crystallinity Continuum

The “crystallinity continuum” is expressed by the relative amount

of order of contiguous unit cells


Large single crystal

Powdered crystals

Small crystallites(more or less ordered)

Semi-crystalline (short range order)

Amorphous(lacks a distinct crystal form )

Low

High

Cry

sta

lline o

rder

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


• 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

Tube

Divergence slit Antiscatter-

slit

Mono-

chromater

Detector

slit

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

Bragg Diffraction

• A crystal modeled as a series of parallel planes with distance

“d” between planes (d-spacing)

• Constructive interference of reflections creates a Bragg peak


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)

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


2-Theta

3 10 20 30 40

100% Amorphous

70% Amorphous

50% Amorphous

30% Amorphous

0% Amorphous

N N

N

N

O

O O

O

F

F

F

F

O

O

O

O

RS-104253-146

PXRD: Example of Polymorph Transitions by

Temperature


2-Theta

2 10 20 30

30 C

110 C

155 C

175 C

200 C

RO0130830-000

Cl

O

O

S

O

O

NO

O

30 °C

110 °C

155 °C

175 °C

200 °C

Multi-bounce Attenuated Total Reflectance -

FTIR

• 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


Sample

Sample clamp

Multi-bounce

ATR crystal

IR Radiation IR DetectorMirror Mirror

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

Rayle

igh s

catt

erin

g

Sto

kes

Anti-s

tokes

Excitation energy

(laser radiation)

Excitatio

n e

nerg

y

1

2

3

4

Vib

ratio

nal

energ

y s

tate

s

0

Sample

Rayleigh scatter

(same wavelength as excitation energy))

Raman scatter

(stokes & anti-stokes - different

wavelengths than excitation energy)

Exited e

nerg

y

sta

tes

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


2-Theta

2 10 20 30 40

Ganciclovir Phase I

Ganciclovir Phase II

Ganciclovir Phase III

Phase I

Phase II

Phase III

2-Theta

Equations for Calculating Ganciclovir Phase

Percents using PXRD Analysis


% Phase II100%

Peak I

Peak II+ 1( )

=

Slope 1

% Phase I % Phase II100% -=

% Phase II100%

Peak III

Peak II+ 1( )

=

Slope 3

% Phase III % Phase II100% -=

% Phase I100%

Peak III

Peak I+ 1( )

=

Slope 2

% Phase III % Phase I100% -=

% Phase I100%

Peak III

Peak I+ 1( )Peak II

Peak ISlope 1( )+ Slope 3

=

% Phase III =Peak III

Peak I( )Slope 3% Phase I

% Phase II % Phase I % Phase III100% - -=

If Only Phases I & II

If Only Phases II & III

If Only Phases I & III

If All Three Phases (I, II, & II)

Phase I Phase II Phase III

Quantifation of Standards of Ganciclovir

Polymorph Mixtures by PXRD (Set A)


Ratios 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

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

Phase I Phase II Phase III

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

• 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


Int J of Pharm 163, 157-166 (1998)

ATR-FTIR Ganciclovir Polymorph Calibrations

Using Partial Least Squares


Phase I r2 = 0.962

Phase II r2 = 0.964

Phase III r2 = 0.972

Int J of Pharm 163, 157-166 (1998)

FT-Raman Spectra of Ganciclovir Polymorphs


Phase I

Phase II

Phase III


Phase I Polymorph by ATR & FT-Raman


Actual

wt%

Mean Stnd Dev Difference

ATR RAM ATR RAM ATR RAM

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

ATR Raman

Int J of Pharm 163, 157-166 (1998)


Phase II Polymorph by ATR & FT-Raman

12/30/2009 Richard E. Young

Actual

wt%



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

ATR Raman

29

Int J of Pharm 163, 157-166 (1998)


Phase III Polymorph by ATR & FT-Raman


Actual

wt%



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

ATR Raman

Int J of Pharm 163, 157-166 (1998)

Ganciclovir Polymorph Validation Mixtures


Validation

Mix ID

Phase I

(wt%)

Phase II

(wt%)

Phase III

(wt%)

Phase I 100 0.0 0.0

Phase II 0.0 100 0.0

Phase III 0.0 0.0 100

Mix 1 5.6 21.2 73.2

Mix 2 7.3 9.8 82.8

Mix 3 14.2 43.1 42.7

Mix 4 14.1 78.6 7.4

Mix 5 26.8 56.0 17.2

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

• One of Each Pure Phase

– 100 % Phase I

– 100% Phase II

– 100% Phase III

• Ternary Phase Mixtures

– Ten mixtures of all three

phases in varying

amounts

Int J of Pharm 163, 157-166 (1998)

Quantifation of Validation Mixes of Ganciclovir

Phase I Polymorph by ATR & FT-Raman


Validation

Mix ID

Actual

wt%

Calc wt% Difference

ATR RAM ATR RAM

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

ATR Raman

Int J of Pharm 163, 157-166 (1998)


Phase II Polymorph by ATR & FT-Raman


Validation

Mix ID

Actual

wt%

Calc wt% Difference

ATR RAM ATR RAM

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

ATR Raman

Int J of Pharm 163, 157-166 (1998)


Phase III Polymorph by ATR & FT-Raman


Validation

Mix ID

Actual

wt%

Calc wt% Difference

ATR RAM ATR RAM

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

ATR Raman

Int J of Pharm 163, 157-166 (1998)

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

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


Identification and quantification of two phases of a Roche

research compound in the presence of excipients

PXRD Patterns of Phase A, Phase B,

and Placebo


PXRD Patterns of Polymorph Standards

(% Phase B in Phase A & Placebo)


Phase A

9.0 2θ

Phase B

13.1 2θ

PXRD Reference Intensity Ratio (RIR)

Technique for Two Polymorph Mixtures

• 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


wt% B = 100 %

1 + (m × IA÷ IB)

PXRD RIR Slope Determination for Weight%

Ratio Versus Peak Intensity Ratio


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

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Weig

ht

% R

ati

o (

XBX

A)

2θ Peak-Height Ratio (IB/IA )

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


ATR-FTIR r2 = 0.989

FT-Raman r2 = 0.970

Calculated vs Actual Plot - Phase B

Corr. Coeff.: 0.99445 RM SEC: 1.54

Calibration

Validation

Correction

-2 51Actual

-25

1C

alc

ula

ted

Calculated vs Actual Plot - Phase B

Corr. Coeff.: 0.98488 RM SEC: 2.53

Calibration

Validation

Correction

-2 51Actual

-25

1C

alc

ula

ted

Determined Weight% of Phase B by

PXRD, ATR-FTIR, and FT-Raman


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

Lot 1: 0 wt% Phase B

Determined Weight% of Phase B by

PXRD, ATR-FTIR, and FT-Raman


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

Lot 2: 10 wt% Phase B

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


Documents

Polymorph Quantitation