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Invited Papers
GREGORY A. STEPHENSON
Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, Indiana 46285. E-mail: gas@lilly.com
The objective of the following article is to present an overview of the application of X-ray
powder diffraction in the pharmaceutical industry, with discussion covering the stages of dis-
covery, development, and post product launch. Such applications range from lead optimization
where many molecules are examined for their solid-state characteristic, that is developability
assessment, in parallel with studies assessing toxicological effects and pharmacological activ-
ity. Other applications relate to development stages where the technique is used primarily for
form identification and monitoring of form during pharmaceutical processing and later quan-
tification activities aimed at product control. The role of additional applications such as struc-
ture solution using the powder method is discussed.
3000
LY333531(HCl)
2500 LY333531(M esylate)
ng/mL Plasma
LY338522 (HCl)
2000
LY338522 (M esylate)
1500
1000
500
0
0 8 16 24 32 40 48 56 64 72 80 88 96
Hours
Fig. 1. Mean plasma concentrations in male beagle dogs orally administered. The AUC val-
ues in each dog were approximately 2.6 times higher following administration of mesylate
salts compared to HCl.
Fig. 2. High throughput salt crystallization and characterization. The design of the chemistry
to be conducted is dispensed and crystallized by the three most common industrial proce-
dures, cooling, evaporation, and antisolvent addition.
The combination of the large number of mole- makes this a problem ideally suited for applica-
cules to evaluate, the large array of potential tion of a combinatorial chemistry approach. The
crystallization conditions and methods to be ex- chemistry is conducted in an arrayed fashion,
amined, and the need for assessment of the typically a 96-well titer plate format, see Figure
physical properties of the crystallization hits 2, so that the samples may be collectively trans-
ferred from where the chemistry occurs to tion geometry may afford one the ability of in-
where the samples are characterized. In the salt troducing the sample into the diffraction beam
screen, the amount of sample available for without transferring the sample from the crys-
analysis in our process is between one half to tallizer to the diffractometer, pharmaceuticals
two milligrams per well. As for characterization typically have large unit cells having diffraction
of the results from the screen we use birefrin- peaks that fall within four and forty degrees
gence microscopy, Raman microscopy, X-ray two-theta, using a Copper Ka radiation source.
powder diffraction and solubility analysis by In order to collect data at low angles, the foot-
high performance liquid chromatography print of the incident beam impinging upon the
(HPLC). X-ray diffraction remains the gold stan- sample is elongated and is elliptical in shape
dard technique for distinguishing crystals forms when using a pin-hole collimator. This large
of a drug substance, as shown in Figure 3, how- footprint limits how close the samples may be
ever there are numerous challenges one faces positioned with respect to one another if the
in designing a system capable of conducting samples are introduced in an arrayed format.
diffraction analysis from such a sample format. Consideration must be made as to the over-
Moreover, the sample deviates from the ideal- spray into an adjacent sample. The ideal
ized perfectly flat, infinitely thick, compact of geometry for diffraction by high throughput
fine particles, having dimensions of less than screening (HTS) would most likely be transmis-
ten microns. In such instances, an area detec- sion geometry. In such a scenario, the incident
tor is best suited for a variety of reasons. The beam passes through the entire organic sam-
speed of data collection is considerably faster, ple. Unfortunately, the sample must be crystal-
the collection of a significant portion of the dif- lized or moved to a highly localized position or
fraction cone provides better averaged intensity spot. This requires transfer or manipulation of
than would be obtained using a conventional the sample so that it is presented to the incident
point detector. When one characterizes a large beam. This is not easily automated and many
number of samples using very small sample crystallization attempts result in gels or oils
sizes of particles of variable size, one is provid- such that transfer becomes problematic. In our
ing sub-optimal data for the task of phase iden- laboratory we have found both reflection and
tification. Poor particle statistics and preferred transmission approaches yield suitable diffrac-
orientation effects are at their worst. The task of tion data.
sorting through thousands of patterns collected One additional benefit of the HTS is that
on a material presents itself as a new challenge many times the crystals obtained are large
to the diffractionist and is greatly facilitated by enough to isolate and determine their crystallo-
application of chemometric approaches such as graphic structure. Figure 4 provides photomi-
hierarchical cluster or principle component crographs and unit cell packing diagrams of the
analysis [3]. single crystal structures determined on five dif-
There is a choice to be made between trans- ferent polymorphs that were isolated from a
mission and reflection geometry. While reflec- single plate during a HTS run. Thanks in part to
2000
Free Base
Acid
0
5 10 20
2-Theta - Scale
Fig. 5. The X-ray powder diffraction pattern showing that disproportionation of the salt oc-
curred during recrystallization.
of the diffraction pattern it is easy to establish Fig. 6. X-ray powder diffraction patterns of erythro-
that the salt was not formed and different crys- mycin A dihydrate before and after dehydration. The
tallization conditions should be explored to pro- shifting of unit cell parameters toward higher angle indi-
cated a reduction of unit cell volume during relaxation of
vide the desired salt. The sample shown in Fig-
the desolvated lattice.
ure 5 was prepared for toxicological testing and
was analyzed by diffraction prior to testing. If it
using an environmental chamber where the
had been tested, one might anticipate the sam-
drug, erythromycin A dihydrate, was dehy-
ple to have lower bioavailability compared to
drated at low humidity and allowed to exist in a
the desired salt form and potentially hinder es-
moisture free environment for an extended pe-
tablishment of appropriate dosing levels for
riod of time. Though the crystal form does not
clinical investigations.
collapse to an amorphous state nor does it
2.2.2. Monitoring Drying of Active Pharmaceu- transform to a crystallographically distinct
tical Ingredient phase, it shows relaxation behavior over time
After the drug is recrystallized and is filtered that relates to a reduction in unit cell volume.
from its mother liquor, it is typically dried by The lattice energy is expressed as a function of
one of a variety of processes. If the drug is ini- the van der Waals, coulombic, and hydrogen
tially isolated as a hydrated form, it can un- bonding energies, see equation 1. While the van
dergo dehydration and conversion to an anhy- der Waals contribution is not as well described
drous form or perhaps remain in the same crys- as hydrogen bonding interactions for organic
tallographic form, but with altered physical molecules, the relatively large number of van
properties. Figure 6 shows a study conducted der Waals contacts make its contribution to the
Fig. 10. Characterization of changes occurring during drying of a wet granulated formula-
tion. The top trace is of lactose monohydrate. The middle three are of the formulation com-
posed of a mixture of hydrated and anhydrous lactose, as well as the drug (D) at 15, 60, and
120 minutes (top to bottom). The lower trace shows a diffraction pattern of anhydrous lactose.
binder is typically added in the dry mix state or method is usually satisfactory. It is important to
dissolved in the fluid used for granulating. The understand the phase conversions that occur
granulating solution or suspension is added to during processing. By profiling the process, one
the dry powders in the mixer and mixed until can determine which phases dissolve and then
the desired characteristics are achieved. This recrystallized upon drying. If the API dissolves,
usually produces a granule that is of suitable it is important to understand whether it recrys-
characteristics for producing tablets with ade- tallizes or remains amorphous, or if it forms a
quate hardness, dissolution, content uniformity, hydrate or different polymorph. Certainly if it
and other physical characteristics. After the wet dissolves and recrystallizes, its particle size will
granulation step, the product is most often differ from that of the API introduced into the
dried and then milled after drying to get a major dry blend of the formulation. All of these factors
percentage of the product within a desired size can impact the chemical stability of the drug
range. The dry granulation is then processed to product, and potentially its bioavailability or
get an acceptable size range by first screening tablet disintegration. Figure 10 shows a profil-
with a sieving device, and then milling the over- ing of a formulation that is predominantly com-
sized particles. For normal compressed tablets, posed of lactose monohydrate, a smaller
the broad particle size range produced by this amount of anhydrous lactose and a small con-
3.2. Structures Solution from Powder Diffrac- and when similar fragments are found to have
tion Data consistent conformations in different structures
While single crystal X-ray diffraction will con- that are reported in the Cambridge Structural
tinue to be the sole provider of absolute config- Database (CSD) [27], or by searching ones own
uration data for organic molecules [21], X-ray library of molecules of a particular therapeutic
powder diffraction continues to advance to a category. Alternatively, conformations can be
state where it can provide reasonably accurate calculated using molecular mechanics, semi-
information about molecular conformation, empirical, or ab initio methods. As a result,
three dimensional packing arrangement, and seemingly complex molecules can be reduced
hydrogen bonding patterns in organic molecu- to merely a few torsion variables describing the
lar solids. With the increasing performance and molecules internal degrees of freedom, three
availability of programs that apply Monte Cartesian coordinates describing the position of
Carlo/Simulated Annealing (MC/SA) or molecu- the molecule in the unit cell, and three Eulerian
lar searching methods to powder diffraction angles describing molecular orientation.
data, the solution of structures that were previ- As described in section 2.2.5, a previously un-
ously inaccessible to single crystal methods are known hydrated crystalline phase was identified
now possible [19, 2224]. The use of powder using low temperature X-ray powder diffrac-
diffraction for the solution of structures has re- tion. That crystalline phase had been implicated
cently been reviewed [25]. Certain types of crys- in the common vial breakage problem of
talline substances are difficult or beyond the ca- freeze-drying mannitol containing parenteral
pability of single crystal techniques, even when formulations. Despite the sample consisting of
performing micro-crystal diffraction using a a mixture of crystalline phases of mannitol, the
synchrotron radiation source. In particular, structure was solved on the phase-mixture pat-
metastable polymorphic forms that are isolated tern, having the peaks associated with the
by rapid crystallization from the melt or that phases whose structures had previously been
rapidly grow from solution are typically ex- determined subtracted from the pattern [28]. The
tremely small or highly flawed crystals. Desol- intensity and resolution afforded by the synchro-
vation processes commonly result in crystals tron radiation source resulted in the reasonably
that appear to have the same particle size as the low R factor. The information gained from the
crystals from which they were formed; how- studies of the freeze drying process using low
ever, upon examination by polarized light mi- temperature X-ray diffraction combined with
croscopy they are usually composed of micro- the subsequent structure solution by the pow-
crystalline aggregates [26]. As in single crystal der method highlights the important role that X-
diffraction, the first step, indexing, presents per- ray diffraction can play in the pharmaceutical
haps the biggest obstacle. Typically if one can industry as it helped understand a problem that
successfully index a crystal, its structure can be has plagued parenteral product manufacturing
determined. This is apparently true for the pow- for more than one half of a century.
der method also. The number of variables of
the problem can be limited by restraining bond 4. Conclusions
distances and angles of the molecule to known X-ray powder diffraction continues to be the
values. Portions of molecules may be consid- gold standard technique for identification of
ered rigid bodies based on chemical intuition different crystalline forms of a given substance