1. Field of the Invention
The present invention relates to improvements in the field of systems for analyzing the crystalline physical and chemical forms of pharmaceutical compounds, and more specifically to a sample holder that is suitable for use in powder X-ray diffraction, as well as microscopy and Raman spectroscopy.
2. Description of Prior Art
As used herein, the term “pharmaceutical compound(s)” should be construed broadly to include any organic or inorganic molecule that may be crystalline. With respect to pharmaceutical research, the term includes, but is not limited to, any active pharmaceutical ingredient (API), final intermediate (AKA penultimate intermediate), pivotal intermediate, isolated intermediate, protein, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), amino acid, polypeptide, fermentation product, complex and starting material, regardless of its source (natural, unnatural or semi-synthetic). The term “pharmaceutical compound(s)” includes colloquial references, including but not limited to large molecules, small molecules, neutral molecules, semi-synthetic molecules, or biological (or fermentation) molecules. The term also includes any compound that may be used in the synthesis of a pharmaceutical.
Potential drug candidates include organic and inorganic molecules, which may crystallize in one of several different forms, or a mixture of these forms. Different physical forms (polymorphs) and chemical forms (salt forms, solvates, and complexes) of a compound can have significantly different physiochemical properties. In some instances, the different physiochemical properties (e.g., solubility, melting point, etc.) can lead to differences in bioavailability in preclinical and clinical testing.
The crystallization parameters that influence the solid state form of any compound include temperature, reaction time, pH, concentration of the compound of interest in a solvent or solvents and concentration of impurities in a solvent or solvents. Additional crystallization parameters that influence the solid state form of any compound include seeding or lack thereof, nucleation rate, precipitation rate, crystallization rate, saturation point, and solubility with respect to temperature. Additionally, the rate of adjustment of any or all of the aforementioned parameters, as well as the type and composition of the solvent used, will influence the solid state form. Henceforth, the polymorphs, solvates, complexes and salt forms of compounds will be referred to as “forms” for simplicity.
Identification of an optimal form of a compound is one of the key requirements for successful pharmaceutical development. Further, it is important to determine which polymorphic form of the compound is produced by a particular set of parameters in order to formulate the optimum protocol for producing the compound. It is therefore critical to have a means of analyzing arrays of solids for their crystalline properties.
One common diagnostic method for identifying the crystalline forms of a compound is X-ray diffraction. Because the atomic coordinates of each polymorph are different, each polymorph's crystal will diffract X-rays in a distinctive pattern. Typically, the samples screened in pharmaceutical development are a collection of randomly oriented crystallites that form a powder. For that reason, the technique is commonly referred to as “powder X-ray diffraction” (PXRD).
The X-ray diffraction from a crystalline powder sample generates a certain pattern. Typical PXRD instruments designed for laboratory use are capable of measuring a powder diffraction pattern from approximately 100-500 mgs of material. The pattern is measured by scanning a sample with an X-ray beam and at the same time scanning a detector that measures the intensity of radiation diffracted by the sample as a function of the diffracting angle. The pattern of the diffracted radiation received at the detector is characteristic of the sample. Since each crystalline structure dictates a unique pattern, it is possible to distinguish among different forms of the compound present in the sample by analyzing the diffraction pattern.
Often, a compound will exist in several different polymorphic crystalline forms. It is important to test a variety of crystallization conditions to ensure that all polymorphs that can be produced are identified, and to optimize the crystallization conditions that produce polymorphs and salts with desired physical properties.
The identification of polymorphs may require a large number of samples to be screened using high-throughput crystallization (HTC) techniques. In addition to PXRD, other techniques are used to analyze the properties of pharmaceutical compounds, including microscopy and Raman spectroscopy. Each technique has its particular advantages and commonly all three techniques are used to generate a comprehensive set of analytical data.
However, where microscopy, Raman spectroscopy, and PXRD are all used in analyzing a pharmaceutical compound, the preparation of PXRD samples typically creates a bottleneck. Samples for microscopy and Raman spectroscopy may suitably be held in a 96-well plate, and the same plate may be used for both techniques. The 96 wells in the sample plate are arranged in a rectangular matrix, and an automated system using two-dimensional X-Y or Cartesian coordinates may be incorporated to allow detection and analysis of the samples contained in each well in the matrix.
Prior art 96-well plates, however, have been found to be unsuitable for use in PXRD. Thus, samples held in a 96-well plate must be transferred onto a suitable PXRD sample holder or, alternatively, fresh samples of the pharmaceutical compound must be prepared for use in PXRD. In addition to increasing the amount of time required to analyze a new pharmaceutical, transferring samples or preparing additional samples increases the risk of human error. Also, new pharmaceuticals are typically prepared in very small quantities, and it is therefore highly desirable to use as little of the pharmaceutical as possible for testing purposes.
To address such issues and others, an aspect of the invention provides a sample plate for use in PXRD and other testing techniques. The plate comprises a quartz body having substantially planar top and bottom surfaces that are parallel with each other. The quartz body includes a matrix of cylindrical apertures that are substantially perpendicular to the top and bottom surfaces. Each aperture has an upper opening at the top surface and a lower opening at the bottom surface of the plate body. A synthetic quartz bottom sheet is fused to the bottom surface of the quartz body. The quartz bottom sheet includes respective portions that close the lower opening of each aperture in the quartz body, such that each aperture and respective portion of the bottom sheet together form a well for receiving a sample. Each well is suitably dimensioned to allow a powder X-ray diffraction technique to be performed on a sample contained therein.
Additional features and advantages of the present invention will become apparent by reference to the following detailed description and accompanying drawings.
An aspect of the present invention provides a 96-well quartz plate for use in high throughput crystallization (HTC) workflow. As discussed in detail below, a plate according to the present invention is suitable for collecting powder X-ray diffraction (PXRD) patterns. The collected PXRD data exceeds acceptable minimums for resolution and intensity. Although multi-well quartz sample plates have been used in other applications, they have been considered to be unsuitable for direct use in PXRD because of the physical properties of quartz and because of the requirements for preparing a suitable PXRD sample.
Using a plate according to the present invention, it is possible to perform screening experiments on samples loaded into wells in the plate and then collect critical analytical data, including birefringence, Raman spectra, and PXRD, which can identify solids in the wells as leads for active pharmaceutical ingredient (API) final form and polymorph studies. The present invention eliminates sample filtration and transfer steps. Also, a plate according to the present invention may be used to conduct slurry studies including relative stability study of forms and transition point study of solvates.
Thus, overall productivity is increased by minimizing sample preparation time and minimizing the amount of sample needed to gain the maximum knowledge of solids, including birefringence, chemical identification and composition of salts, and polymorphism of crystalline samples (hits) obtained through HTC. This solid characterization capability enables an investigator to make more data-driven decision on hits for scale up studies. Currently, Raman spectroscopy is used for chemical identification of HTC hits, for example, by comparing API vs. counter ions, and other such techniques. Using Raman spectra data, polymorphs often show only subtle differences, which can make polymorph screening in form selection a challenging process. Typically, PXRD pattern data can be grouped more effectively than Raman spectra.
Collecting PXRD pattern data at early stages of drug development can also utilize the simulated patterns obtained from single crystal work completed at the discovery stage to make the HTC product identification easier. In a competitive industry, it is desirable to be able to collect all these analytical data from a single sample plate at a very early stage of API form screening.
As shown in
Specifically, it has been found that an ultrathin bottom sheet 30 produces acceptable results. As discussed below, where the height H of each well 26 is 6 mm, suitable thicknesses B for the bottom sheet 30 have been found to include 0.18 mm, 0.25 mm, and 0.37 mm. Different well diameters D are used for each bottom thickness. For the 0.18 mm bottom, 3 mm was found to be a suitable well diameter. For the 0.25 mm bottom, 4 mm was found to be a suitable well diameter. For the 0.37 mm bottom, 6 mm was found to be a suitable well diameter. The minimum bottom thickness of each plate is governed by the well diameter to ease the tension at the bottom. It should be noted that these dimensions are exemplary, and it is not intended to limit the invention to these dimensions. For the purposes of the following discussion, the three plates are respectively referred to as the “0.18 mm plate,” the “0.25 mm plate,” and the “0.37 mm plate.” The respective volumes of the individual wells in each plate are 41 μL, 72 μL and 159 μL.
As illustrated in
As part of the fabrication process, the plate 10 is polished to a suitable optical standard.
Initial tests were performed on the 0.18 mm, 0.25 mm, and 0.37 mm plates using as samples the N1, H0, and N4 forms of Aripiprazole.
The three plates were then tested using a powder X-ray diffractometer, the Bruker AXS CST system. Initial evaluation of the plates determined that only a 15 degree, two theta range data, half of that expected, was offered by the 0.18 mm plate due to shading. As a corrective measure, the Bruker instrument was used in an inverted configuration, with the X-ray tube on top and the detector below the sample library to avoid shading by the sample plate. The sample-detector distance was 15 cm. An incident beam graphite monochromator was used at 40 kV, 50 mA.
A first series of tests was conducted to determine an optimum source accessory for the Bruker instrument. No angle opening problem was observed. Three source accessories were subjected to comparison testing to determine which accessory achieved the best combination of intensity (signal to noise ratio) and resolution: a 500 μm collimator, a 500 μm monocap, and a 300 μm monocap. The results of these tests are shown in
In order to determine an optimal sample amount, a study was conducted using the 0.18 mm plate to test the samples in wells A1, B2, B3, A4 and B5. These wells contained the following respective amounts of the N1 form of Aripiprazole: 2 mg, 1 mg, 0.5 mg, 0.25 mg, and 0.1 mg. The results of the study are shown in
One issue encountered during the comparison testing was a difference in background counts in the samples. It was found that the total count rate over the whole range decreased with increased sample amounts. The decrease in total count rate is illustrated in
As shown in
A composition study was also performed using the 0.18 mm plate. Samples with different compositions of N1 and H0 were studied.
To summarize the above analyses, the optimal configuration of the Bruker diffractometer was attained by inverting the radiation source so that the source was from the top of the instrument in order to eliminate shading. The best combination of intensity of resolution was achieved with the 500 μm monocap and sample-to-detector distance of 15 cm. Acceptable PXRD patterns were collected with both the 0.18 mm and 0.25 mm plates, with as little as 0.25 mg of sample in each well. Although about 19% intensity was lost going from the 0.18 mm to the 0.25 mm plate, the 0.25 mm plate may be desirable for certain processes because of its larger volume and well opening. To increase the well volume for compounds with poor solubility, it may be useful to modify the 0.25 mm plate to have a different height, such as 8 mm or 9 mm. The plate also could be used to detect mixtures of two polymorphs with acceptable resolution and intensity.
A further aspect of the invention provides an improved technique for analyzing slurries produced during crystallization. In prior art techniques, it was typically necessary at some point to transfer slurry samples between different holders in order to be able to perform microscopy, Raman spectroscopy, and PXRD on those samples. As described below, the present aspect of the invention eliminates this need to transfer samples.
According to the present aspect of the invention, HTC screening is performed directly on a 96-well plate, such as the plate 10 shown in
While the foregoing description includes details which will enable those skilled in the art to practice the invention, it should be recognized that the description is illustrative in nature and that many modifications and variations thereof will be apparent to those skilled in the art having the benefit of these teachings. It is accordingly intended that the invention herein be defined solely by the claims appended hereto and that the claims be interpreted as broadly as permitted by the prior art.
This application claims priority from U.S. Provisional Application No. 60/637,165, filed Dec. 17, 2004, incorporated herein in its entirety by reference.
Number | Date | Country | |
---|---|---|---|
60637165 | Dec 2004 | US |