COMPUTING METHODS FOR CONTROL OF HIGH-THROUGHPUT EXPERIMENTAL PROCESSING, DIGITAL ANALYSIS, AND RE-ARRAYING COMPARATIVE SAMPLES IN COMPUTER-DESIGNED ARRAYS

Information

  • Patent Application
  • 20070021929
  • Publication Number
    20070021929
  • Date Filed
    August 24, 2006
    18 years ago
  • Date Published
    January 25, 2007
    17 years ago
Abstract
Computer-controlled automated high-throughput systems can be used to design, prepare, process, screen, and analyze a large number of samples in removable sample vials each containing a compound of interest formulated with differing component combinations and varying concentrations. The computer-controlled methods of the present invention allow for a determination of the effects of additional or inactive components, such as excipients, carriers, enhancers, adhesives, additives, and the like, on the compound of interest, such as a pharmaceutical. The invention thus encompasses the computer systems, computer methods, and computer-program products for computer-controlled automated high-throughput testing of experimental formulations in order to identify experimental formulations that can be further processed. Identified experimental formulations from multiple arrays can be removed and re-arrayed together to form a new array for further processing.
Description
BACKGROUND OF THE INVENTION

1. The Field of the Invention


The present invention relates to computer-controlled automated high-throughput devices, systems, and methods for conducting and evaluating multiple experiments on samples having different formulations, each containing and/or chemical compositions. More particularly, the present invention relates to computer systems, computer methods, and computer-program products for designing, preparing, processing, screening, and analyzing high-throughput preparation and study of a variety of formulations contained in removable vials held in computer-designed arrays.


2. The Relevant Technology


In recent years, chemical discovery has seen an explosion of new science, such as genomics, proteomic and bioinformatics, as well as high-throughput technologies for identifying and/or creating new compounds or chemical entities, such as combinational chemistry. Such technologies allow the researcher to rapidly synthesize and/or identify large numbers of compounds. At the same time, these technologies have led to the development of more compounds that are larger, greasier and more hydrophobic, and thus more challenging to develop into products.


Conducting large numbers of experiments results in the need to inspect or otherwise analyze hundreds or thousands of samples for the presence of the desired result. And, a large number of the pre-selected samples require continuing analysis. The resulting voluminous data must then be processed effectively and efficiently within a reasonable amount of time.


The physical form of a compound, particularly that of an active pharmaceutical ingredient (API), plays a role in a number of areas. For example, in order to be developed into a drug, a compound must be able to be delivered to the patient via some suitable device or formulation, and it must also pass criteria in several categories, such as safety, metabolic profile, pharmacokinetics, cost and reliability of synthetic process, stability, and bioavailability.


High-throughput technologies, when possible, enable the discovery of various physical forms of a compound, some of which may be particularly useful as pharmaceuticals, for formulating pharmaceuticals, intermediates for manufacturing drugs, foods, food additives and the like. (See, e.g. International Application Nos. WO 00/59627, WO 01/09391, and WO 01/51919). Such technologies can result in extraordinary numbers of experiments being conducted very rapidly thereby creating large amounts of data and results that must be reviewed and analyzed by the scientist in order to identify a desired form of the compound. For example, in order to discover various solid forms of a compound, often thousands of experiments, using many different conditions, solvents, additives, pH, thermal cycles, and the like, must be conducted. Dozens or even hundreds of the forms must be analyzed before a desired form of the compound can be identified and chosen for further development as a potential product.


Some devices for facilitating large numbers of experiments simultaneously are known. In addition, there are systems consisting of blocks with multiple wells for performing reactions for different applications such as combinatorial chemistry. Examples of such systems include the TITAN™ Reactor Clamp and TITAN™ PTFE MicroPlates (both available from Radleys, Shire Hill, Saffron Walden, Essex CBII 3AZ, United Kingdom). A multiple-well tray for crystallization reactions is described in U.S. Pat. No. 6,039,804. There also exist systems of block, tubes, and seals, such as the Radleys TITAN™ Glass Micro Reactor Tube System and the WebSeal System (available from Radleys, Shire Hill, Saffron Walden, Essex CBII 3AZ, United Kingdom). Many tubes or vials of different geometries also exist, including many with crimped, threaded, or snap-on caps.


Spectroscopic techniques such as infrared (IR) and Raman spectroscopy are useful for detecting changes in structure and/or order. In addition, techniques such as Nuclear Magnetic Resonance (NMR), Differential Scanning Calorimetry, ultra-violet (UV) spectroscopy, circular dichroism (CD), linear dichroism (LD), and X-ray diffraction are powerful techniques. However, each of these techniques must be coupled with data analysis and handling techniques to enable data collection and processing of hundred or thousands of samples. All of these techniques are not easily adaptable for high-throughput analysis of structural information. Indeed, high-throughput analysis still remains a challenge due to the high degree of automation desired in both physical sample handling and in analysis of the collected data.


Therefore, it would be beneficial to have computer-controlled automated systems for high-throughput processing, screening, and analyzing of a large number of samples held in individual sample vials. Additionally, it would be beneficial to have computer systems, computer methods, and computer-program products for designing, preparing, processing, screening, and analyzing formulations of active compounds held in removable sample vials in computer-designed arrays.


SUMMARY OF THE INVENTION

The present invention relates to computer-controlled automated high-throughput systems, computer-program products, and methods to design, prepare, process, screen, and analyze a large number of samples in removable sample vials each containing a compound of interest formulated with differing component combinations and/or varying concentrations. The computer-controlled methods of the present invention allow for a determination of the effects of additional or inactive components, such as excipients, carriers, enhancers, adhesives, additives, and the like, on the compound of interest, such as a pharmaceutical. The invention thus encompasses the computer systems, computer methods, and computer-program products for computer-controlled automated high-throughput testing of experimental formulations in order to identify experimental formulations that can be further processed. Identified experimental formulations from multiple arrays can be removed and re-arrayed together to form a new array for further processing.


In one embodiment, the present invention can include a computing system for controlling automated high-throughput processing of an array having removable sample vials held by an array block. The computing system can be designed to identify chemical and/or physical properties leading to optimal formulation for a given use of a compound of interest. The computing system can provide computer-aided design and processing of an experimental formulation for each sample. Each experimental formulation can have the compound of interest, and the formulations can be based on at least one experimental variable which is varied as to at least some samples. In this way, the effect in terms of changes in the chemical and/or physical properties of the compound of interest due to at least one variable can be identified across a number of comparative samples.


The computing system can implement a method of generating and analyzing data from the comparative samples, and re-array at least some of the samples based on the data. Such a method can include the following: (a) inputting into the computing system at least one compound of interest and any additional components to be included in the experimental formulations that are to be designed for a first array of samples; (b) inputting into the computing system at least one selected experimental variable of interest that is to be varied as between at least some samples of the first array; (c) the computing system thereafter determining an experimental formulation for each sample that is different as between at least some samples based on the at least one selected experimental variable of interest that is varied as between at least some of the samples of the first array; (d) the computing system thereafter controlling a process by which the experimental formulation for each sample is prepared in a removable sample vial held by an array block and tested in order to create changes in chemical and/or physical properties of the compound of interest across a number of comparative samples; (e) inputting to the computing system detected changes across the comparative samples for the at least one compound of interest; (f) the computing system thereafter automatically screening the samples of the first array by identifying those samples which contain chemical and/or physical properties most likely to lead to optimal formulation for a given use of a compound of interest, and storing as a first data set information as to the experimental formulation and the resulting chemical and/or physical properties for each of the identified samples; (g) removing from the array block sample those vials for samples not identified as part of the first data set, thereby forming a second array of samples contained by the array block by virtue of those samples not removed; and (h) the computing system thereafter controlling a process by which the identified samples remaining in the second array are further processed and/or tested in order to further identify chemical and/or physical properties leading to optimal formulation for a given use of a compound of interest.


In one embodiment, the present invention can include a computer-program product (e.g. software) for use in a computing system to control automated high-throughput processing of an array having removable sample vials held by an array block. The computer-program product can provide computer-aided design and processing of an experimental formulation for each sample. The computer-program product can include a computer-readable medium, which are well-known in the art, containing computer-executable instructions for causing the computing system to execute a method for analyzing data from the comparative samples. Such a method can include the following: (a) inputting into the computing system at least one compound of interest and any additional components to be included in the experimental formulations that are to be designed for a first array of samples; (b) inputting into the computing system at least one selected experimental variable of interest that is to be varied as between at least some samples of the first array; (c) the computing system thereafter determining an experimental formulation for each sample that is different as between at least some samples based on the at least one selected experimental variable of interest that is varied as between the at least some samples of the first array; (d) the computing system thereafter controlling a process by which the experimental formulation for each sample is prepared in a removable sample vial held by an array block and tested in order to create changes in chemical and/or physical properties of the compound of interest across a number of comparative samples; (e) inputting to the computing system detected changes across the comparative samples for the at least one compound of interest; (f) the computing system thereafter automatically screening the samples of the first array by identifying those samples which contain chemical and/or physical properties most likely to lead to optimal formulation for a given use of a compound of interest, and storing as a first data set information as to the experimental formulation and the resulting chemical and/or physical properties for each of the identified samples; (g) the computing system thereafter causing removal from the array block those sample vials for samples not identified as part of the first data set, thereby forming a second array of samples contained by the array block by virtue of those sample not removed; and (h) the computing system thereafter controlling a process by which the identified samples remaining in the second array are further processed and/or tested in order to further identify chemical and/or physical properties leading to optimal formulation for a given use of a compound of interest.


In one embodiment, the computing system can cause those sample vials removed from the array block to be placed into a different array block, and subsequently cause additional sample vials to be placed in the different array block to form a third array of removable sample vials, each having an experimental formulation including a common compound of interest. The computing system can thereafter control a process by which the samples in the third array are further processed and/or tested in order to further identify chemical and/or physical properties leading to optimal formulation for a given use of a compound of interest. Optionally, the experimental formulations in the second or third array of samples can each have a similar chemical and/or physical property.


In one embodiment, experimental data obtained from processing the experimental formulations in any of the arrays of samples can be analyzed to determine at least one optimal formulation. As such, the further processed and/or tested identified samples can be screened to further identify those samples which contain chemical and/or physical properties most likely to lead to optimal formulation for a given use of a compound of interest, and storing as a data set information as to the experimental formulation and the resulting chemical and/or physical properties for each of the further processed and/or tested identified samples. Thus, any of the data sets can be analyzed in order to identify those samples which contain chemical and/or physical properties most likely to lead to optimal formulation for a given use of a compound of interest.


These and other advantages and features of the present invention will become more fully apparent from the following description and appended claims, or may be learned by the practice of the invention as set forth hereinafter.




BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail through the use of the accompanying drawings, in which:



FIG. 1 is a schematic diagram of steps associated with an embodiment of the invention, wherein tubes are filled with a compound of interest and optional other compounds, processed, and inspected.



FIGS. 2A and 2B are views of an embodiment of a tube in its open and capped configurations, respectively.



FIGS. 3A and 3B are top and bottom perspective views of an embodiment of a block, respectively.



FIG. 3C is a top perspective view of the block filled with capped tubes.



FIG. 4A is a drawing of an embodiment of a temperature-controlled shelf assembly, or “hotel,” loaded with twelve blocks.



FIG. 4B is a drawing of an embodiment of a shelf equipped for use with a heating/cooling loop (e.g. using water, ethylene glycol, or another solvent) shown as dotted lines.



FIG. 5 shows a drawing of an embodiment of a thermal cycling system top-level assembly, including an environmental enclosure. In this arrangement, 18 hotels in a semi-circular pattern around a robotic arm are shown.



FIG. 6 shows a flowchart depicting an embodiment of the logic for addressing solid form generation using the vision station approach.



FIG. 7A is a schematic diagram of an embodiment of a vision station.



FIGS. 7B and 7C are drawings of an embodiment of a vision station in operation, showing a side view (FIG. 7B) and a perspective view (FIG. 7C) of the block wherein a lifting mechanism lifts an entire row of vials at the same time. The light source can be positioned on top of the samples (e.g. normal to the CCD camera) or opposite the samples.



FIG. 8 is a drawing that shows the difference between samples with no birefringence and samples with birefringence.



FIG. 9 is a drawing showing the scattering and diffusion of laser light pointed at a single tube and consecutive tubes.



FIG. 10 is a drawing that shows the effect of illuminating of a tube containing a colloidal suspension (a) compared to a tube containing pure water (b).



FIG. 11 is a flow diagram depicting an embodiment of a use of the vision station to detect birefringence in crystalline solid forms, or to differentiate crystalline versus amorphous solid forms by the observance of birefringence.



FIG. 12 is a flow diagram depicting an embodiment of a use of the vision station for laser light interrogation of samples. Shown are diagrams of nano-suspension compared to true solutions.



FIG. 13A is a perspective diagram of the Raman system.



FIG. 13B is a diagram showing a block of tubes being moved inside an enclosure.



FIG. 13C is a diagram showing the lifting mechanism elevating a tube to be gripped by the tube gripper.



FIG. 13D is a diagram showing the tube gripper and tube having moved in the vertical direction.



FIG. 13E is a diagram showing the tube gripper and tube having rotated.



FIG. 13F is an enlarged diagram showing the tube gripper and tube having moved in the horizontal direction to bring the tube closer to the microscope objective.



FIG. 13G is an enlarged diagram showing the tube gripper and tube having been lowered to a position near the tube holder.



FIG. 13H is an enlarged diagram showing the tube gripper having loaded the tube into the tube holder.



FIG. 13I is an enlarged diagram showing the tube gripper having retracted after loading the tube into the tube holder.



FIG. 13J is an enlarged diagram showing a tube rotator engaging the tube.



FIG. 13K is an enlarged diagram showing the tube being moved under the microscope objective.



FIG. 14A is a perspective view of an embodiment of a tube and microscope objective. The long axis of the tube is preferably at a 90 degree angle to the axis of the objective.



FIG. 14B is diagram of an embodiment of a tube and microscope objective indicating the available axes of motion for the tube.



FIG. 14C is a closer view of an embodiment of a tube with a crystal inside that is in an out-of-focus position with respect to the microscope objective. This figure also indicates the available axes of motion for the tube.



FIG. 14D is a detailed view of an embodiment of a tube with a solid, such as a crystal, inside that is located toward the narrow end of the tube and on the bottom surface with respect to the microscope objective.



FIG. 14E is a detailed view of an embodiment of a tube with a solid, such as a crystal, inside that is being moved in the horizontal direction to bring it closer to the in-focus position beneath the microscope objective.



FIG. 14F is a detailed view of an embodiment of a tube with a solid, such as a crystal inside that is being rotated to bring it closer to the in-focus position beneath the microscope objective.



FIG. 14G is a detailed view of an embodiment of a tube with a solid, such as a crystal, inside that is being moved in the vertical direction to bring it to the in-focus position beneath the microscope objective.



FIG. 15 is a flowchart depicting six stages of an embodiment of a computational binning process of one embodiment of the invention, and one optional stage in such a process.



FIG. 16A is a graph showing Raman intensity plotted as a function of Raman shift (cm−1) for an empty glass vial.



FIG. 16B is a graph showing Raman intensity plotted as a function of Raman shift (cm−1) for a fluorescent sample.



FIG. 16C is comparative graph showing Raman intensity plotted as a function of Raman shift (cm−1) for the pre-filtered sample of FIG. 17B compared to the corresponding filtered spectra after the fluorescence has been removed.



FIG. 17A is a screen shot showing the output from an embodiment of binning software captured during the binning procedure for the flufenamic acid sample set.



FIG. 17B is a screen shot showing the output from the binning software captured during the binning procedure for the theophylline sample set.



FIG. 18 illustrates an implementation of an embodiment of a binning procedure.



FIG. 19A is a comparative graph of X-ray powder diffraction patterns for Form I and Form III of flufenamic acid.



FIG. 19B is a comparative graph of X-ray powder diffraction patterns for anhydrous and monohydrate forms of theophylline.



FIG. 20A is a comparative graph of DSC thermograms for Form I and Form III of flufenamic acid where heat flow (W/g) is plotted as a function of temperature (° C.).



FIG. 20B is a comparative graph of DSC thermograms for anhydrous and monohydrate forms of theophylline where heat flow (W/g) is plotted as a function of temperature (° C).



FIG. 21 is a graph showing thermograms obtained for the anhydrous and hydrous forms of theophylline. An inset graph shows an enlargement of the same thermograms between 35° C. and 150° C.



FIG. 22A is a graph showing Raman intensity (arbitrary units) plotted as a function of Raman shift (cm−1) for Form I and Form III of flufenamic acid.



FIG. 22B is a graph showing the Raman intensity (arbitrary units) plotted as a function of Raman shift (cm−1) for anhydrous and monohydrate forms of theophylline.



FIG. 23A is the output after clustering illustrating sorted cluster diagrams for the flufenamic acid sample set.



FIG. 23B is the output after clustering illustrating sorted cluster diagrams for the theophylline sample set.



FIG. 24A illustrates X-ray crystal diffraction spectra corresponding to the anhydrate and the hydrate forms of Theophylline.



FIG. 24B illustrates the binning of Raman Spectra corresponding to Hydrate distinctly from the Anhydrate form of Theophylline.




DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to computer-controlled automated high throughput systems, computer-program products, and computer-controlled methods for processing of an array having a large number of samples in order to identify at least one optimal formulation for a given use of a compound of interest. The computing system can implement a method of computer-aided design for determining an experimental formulation and experimental process for each sample. Each experimental formulation can have the compound of interest and the formulations can be based on at least one experimental variable which is varied as to at least some samples so that the effect in terms of changes in the chemical and/or physical properties of the compound of interest due to at least one experimental variable can be identified across a large number of comparative samples for a compound of interest. The computer-controlled system and methods of the present invention may be used to design, prepare, process, screen, analyze, and identify the optimal components (e.g., solvents, carriers, transport enhancers, adhesives, additives, and other excipients) for various compositions or formulations.


I. Introduction


As an alternate approach to traditional methods for discovery of new or optimal formulations and discovery of conditions relating to formation, inhibition of formation, or dissolution of solid forms, a computer-controlled automated high-throughput system and computer-program products can be used in methods to design, produce, and screen hundreds, thousands, to hundreds of thousands of samples per day. The array technology described herein is a computer-controlled high-throughput approach that can be used to generate large numbers (e.g. greater than 10, more typically greater than 50 or 100, and more preferably 1000 or greater samples) of parallel small-scale formulation experiments (e.g. crystallizations) for a given compound of interest.


Typically, each sample is designed and prepared to have less than about 1 g of the compound of interest, preferably, less than about 100 mg; more preferably, less than about 25 mg; even more preferably, less than about 1 mg; still more preferably, less than about 100 micrograms; and optimally, less than about 100 nanograms of the compound of interest. The computer-controlled systems and computer-program products are useful to optimize, select, and discover new or optimal formulations having enhanced properties. In some instances, the formulations produce novel solid forms of the compound of interest. The computer-controlled systems and computer-program products can be used in methods that are also useful to discover compositions or formulation conditions that promote formation of formulations with desirable properties. The computer-controlled systems and computer-program products are further useful to discover compositions or conditions that inhibit, prevent, or reverse formation of specific solid forms within formulations.


The computer-controlled system and computer-program products can design and prepare an array of sample sites, such as a 24-, 48- or 96-well plate, or more samples. Each sample in the array can include a mixture of a compound of interest and at least one other additional component. The array of samples can be subjected to a set of processing parameters designed and implemented by the computer-controlled system. Examples of processing parameters that can be varied to form different formulations can include adjusting the temperature; adjusting the time; adjusting the pH; adjusting the amount or the concentration of the compound of interest; adjusting the amount or the concentration of a component; component identity (e.g. adding one or more additional components); adjusting the solvent removal rate; introducing of a nucleation event; introducing of a precipitation event; controlling evaporation of the solvent (e.g., adjusting a value of pressure or adjusting the evaporative surface area); and adjusting the solvent composition.


The contents of each sample in the processed array are typically analyzed initially for physical or structural properties, for example, the likelihood of crystal formation is assessed by turbidity, using a device such as a spectrophotometer. However, a simple visual analysis can also be conducted including photographic analysis. For example, the formulation can be analyzed in order to detect a solid or crystalline or amorphous form of the compound of interest. Also, more specific properties of the solid can then be measured, such as polymorphic form, crystal habit, particle size distribution, surface-to-volume ratio, and chemical and physical stability, and the like. Samples containing active compounds can be screened to analyze properties of the formulation, such as altered bioavailability and pharmacokinetics. The active compounds can be screened in vitro for their pharmacokinetics, such as absorption through the gut (for an oral preparation), skin (for transdermal application), or mucosa (for nasal, buccal, vaginal or rectal preparations), solubility, degradation or clearance by uptake into the reticuloendothelial system (“RES”) or excretion through the liver or kidneys following administration, then tested in vivo in animals. Testing of the large number of samples can be done simultaneously or sequentially.


The computer-controlled system and methods of use are widely applicable for different types of substances (e.g. compound of interest), including pharmaceuticals, dietary supplements, alternative medicines, nutraceuticals, sensory compounds, agrochemicals, the active component of a consumer formulation, and the active component of an industrial formulation. Accordingly, optimal formulations for a variety of active compounds can be determined by using a high-throughput approach with the computer-controlled systems and methods of the present invention.


The computer-controlled system can be configured to operate with a tube and block system. The tube and block system is comprised of a block having an array of holes that are configured to receive an array of removable containers. As such, each sample in the array can be held in an individual container that can be manipulated separately from other samples in the array. That is, the individual containers can be inserted, removed, arrayed, and re-arrayed with respect to the block separately from other containers in the block and/or within other blocks. Accordingly, an array can include a block containing an array of holes for receiving individual containers and a plurality of containers, each of which contains a compound of interest and optionally one or more additional compounds.


Another embodiment of the invention encompasses a computer-controlled system and/or computer-program products that can facilitate an automated high throughput method for screening formulations containing a compound of interest. The method can include designing and preparing an array of samples, each of which comprises the compound of interest and optionally one or more additional compounds. The array can be configured to include a block containing an array of holes for receiving individual containers and a plurality of containers, each container containing a compound of interest and optionally one or more additional compounds. After the array is prepared, processed, screened and analyzed, the samples that are identified for further analysis can be re-arrayed. The processes of re-arraying the individual samples can include rearranging the individual containers in the same block or into a different block with other containers having samples that having been identified for a similar further analysis.


For example, during preparation of an array of samples, each individual sample can be formulated and held in a sealed container. The samples can then be processed by being exposed to a condition, such as heat or cold, for a particular amount of time. After processing, the samples can be screened by imaging the samples to determine, for example, whether they produced or contain a solid or liquid. The samples can be analyzed by collecting and analyzing spectroscopic data obtained from one or more of the samples. FIG. 1 provides a general illustration of this method and a system that can be used to implement it. Briefly, one or more tubes 50 are placed in an array of wells in a thermally conductive block 60. Next, chemical ingredients 52 for each experiment are dispensed into tubes 50. Optionally, a cap 54, possibly including a seal 64, is placed on tube 50 to prevent leakage, evaporation, or contamination of the contents. The tubes are then optionally processed in a temperature-controlled shelf assembly 56 using a controlled thermal cycling system to implement a thermal cycle 66. Following inspection of the contents of tubes 50 (e.g., using automated imaging equipment), experimental specimens or samples of interest are identified using data 70 obtained from the samples. The samples of interest are then optionally separated from other samples for further analysis or processing.


A. Definitions


As used herein, the term “array,” when used to refer to a plurality of objects (e.g., samples), means a plurality of objects that are organized physically or indexed in some manner (e.g., with a physical map or within the memory of a computer) that allows the ready tracking and identification of specific members of the plurality. Typical arrays of samples comprise at least 6, 12, 24, 94, 96, 380, 384, 1530, or 1536 samples.


As used herein, the term “compound of interest” refers to the substance, compound, molecule, or chemical studied, formulated, or otherwise manipulated using methods or devices of the invention. Examples of compounds of interest include, but are not limited to, pharmaceuticals, veterinary compounds, dietary supplements, alternative medicines, nutraceuticals, sensory compounds, agrochemicals, the active components of consumer products, and the active components of industrial formulations. A preferred compound of interest is the active component of a pharmaceutical, also referred to as the active pharmaceutical ingredient (API). Specific APIs are suitable for administration to humans. Specific APIs are small organic molecules that are not polypeptides, proteins, oligonucleotides, nucleic acids, or other macromolecules. Small organic molecules include, but are not limited to, molecules with molecular weights of less than about 1000, 750, or 500 grams/mol.


As used herein, the term “controlled amount” refers to an amount of a compound that is weighed, aliquotted, or otherwise dispensed in a manner that attempts to control the amount of the compound. Preferably, a controlled amount of a compound differs from a predetermined amount by less than about 10, 5, or 1 percent of the predetermined amount. For example, if one were to dispense, handle, or otherwise use 100 μg of a compound of interest, a controlled amount of that compound of interest would preferably weight from about 90 μg to about 110 μg, from about 95 μg to about 105 μg, or from about 99 μg to about 101 μg.


As used herein, the term “form” refers to the physical form of a compound or composition. Examples of forms include solid and liquid. Examples of forms of solids, or “solid forms,” include, but are not limited to, salts, solvates (e.g., hydrates), desolvates, clathrates, amorphous and crystalline forms, polymorphs, crystal habits (e.g., needles, plates, particles, and rhomboids), crystal color, crystal size, crystal size distribution, co-crystals, and complexes.


As used herein, the term “pharmaceutical” refers to a substance, compound, or composition that has a therapeutic, disease or condition preventive, disease or condition management, diagnostic, or prophylactic effect when administered to an animal or human, and includes prescription and over-the-counter pharmaceuticals. Examples of pharmaceuticals include, but are not limited to, macromolecules, oligonucleotides, oligonucleotide conjugates, polynucleotides, polynucleotide conjugates, proteins, peptides, peptidomimetics, polysaccharides, hormones, steroids, nucleotides, nucleosides, amino acids, small molecules, vaccines, contrasting agents, and the like.


As used herein, the term “sample” refers to an isolated amount of a compound or composition. A typical sample comprises a controlled amount of a compound of interest, and may also contain one or more excipients, solvents, additives (e.g. stabilizers and antioxidants), or other compounds or materials (e.g. materials that facilitate crystal growth). Specific samples comprise a compound of interest in an amount less than about 100 mg, 25 mg, 1 mg, 500 μg, 250 μg, 100 μg, 50 μg, 25 μg, 10 μg, 5 μg, 2.5 μg, 1 μg, or 0.5 μg.


II. Computer-Controlled Automated High-Throughput System


In one embodiment, the present invention is directed, in part, to computer-controlled automated high-throughput systems and/or computer-program products (e.g., software) for determining conditions that when applied to a particular compound or composition provide a particular result (e.g., a compound or composition having particular chemical and/or physical properties). The invention is further directed to computer-controlled systems and methods for the generation, synthesis, and/or identification of various forms of a compound or composition, such as, but not limited to, polymorphs, salts, hydrates, solvates, desolvates, and amorphous forms. The invention is also directed to methods and systems for the generation, synthesis, and/or identification of various forms of solids such as, but not limited to, crystal habit and particle size distribution.


The invention encompasses a complete computer-controlled system and software for planning (i.e., designing) and conducting high-throughput experiments on one or more arrays of samples. The system encompasses various computer-controlled equipment and software to implement methods that can be used to design, prepare, process, screen, and analyze samples. Additionally, the various computer-controlled equipment and software can be used to inspect, process, and screen samples. The various computer-controlled equipment and software can be used to collect spectroscopic and other data from one or more of the samples. The various computer-controlled equipment and software can be used to process, interpret, and analyze the data. The system can include robotics, computers, spectral techniques, and various mechanical devices, each designed to conduct high-throughput experiments on large or preferably small amounts of material, including materials on the milligram and microgram scales.


In particular, this invention encompasses computer-controlled systems and software for the high-throughput design, preparation, processing, screening, and/or analyzing of samples. Particular methods of the invention prepare arrays of samples, each of which comprises the compound or composition of interest in optional contact with one or more solvents or excipients. In specific embodiments of the invention, each sample is held in a container that can be manipulated separately from other samples in the array.


A. Sample and Process Design


In one embodiment, the present invention can include a computing system designed for controlling automated high-throughput preparation and processing of an array having a large number of samples. As such, the computing system can implement a method of computer-aided design for determining an experimental formulation and experimental processing for each sample. Each experimental formulation can have the compound of interest, and the formulations can be based on at least one experimental variable which is varied as to at least some samples so that the effect in terms of changes in the chemical and/or physical properties of the compound of interest due to at least one experimental variable can be identified across a large number of comparative samples for a compound of interest. Also, the sample processing can be varied to determine whether or not various processes can effect the chemical and/or physical properties of the compound of interest


The computing system can be used in implementing a method of designing an experimental formulation for each of a large number of comparative samples. Such a method of designing experimental formulations can include inputting into the computing system at least one compound of interest to be included in each of a plurality of experimental formulations that are to be designed for the array of samples. Also, the additional components to be formulated with the at least one compound of interest in the experimental formulations can be input into the computing system. Additionally, at least one experimental variable to be varied as between at least some of the samples of the array can be input into the computing system. In part, this can include identifying specific values or ranges of values in varying the variables. Accordingly, the computing system thereafter can design a plurality of unique experimental formulations that differ as between at least some samples of the array based on at least one experimental variable that is varied as between the at least some samples of the array. Each experimental formulation being designed at least in part based on at least one experimental variable and the compound of interest.


For example, the combinations of the compound of interest and various components at various concentrations and combinations can be generated using standard formulating software (e.g., Matlab software, commercially available from Mathworks, Natick, Mass.). The combinations thus generated can be downloaded into a spread sheet, such as Microsoft EXCEL. From the spread sheet, a work list can be generated for instructing the automated distribution mechanism to prepare an array of samples according to the various combinations generated by the formulating software. The work list can be generated using standard programming methods according to the automated distribution mechanism that is being used. The use of so-called work lists simply allows a file to be used as the process command rather than discrete programmed steps. The work list combines the formulation output of the formulating program with the appropriate commands in a file format directly readable by the automatic distribution mechanism. However, various computer-program products can be used for generating arrays of samples having different experimental formulations, and such computer-program products can be operated on a computer within the computing system.


In one embodiment, the experimental variable to be varied as between at least some samples of the array is varied as to at least one of concentration of the compound of interest, concentration of components in the experimental formulations, identity of the components, combination of components, additive, solvent, antisolvent composition, temperature, temperature change, heating, cooling, nucleation seeds, supersaturation, pH, pH change, or time of crystallization reaction.


In one embodiment, at least one criteria can be input into the computing system for determining the effect of at least one experimental variable for each experimental formulation that is varied as to that experimental variable. The effect of the criteria can be manifested by a change in one or more of the physical property permutations for the compound of interest between different experimental formulations. The effects can be identified by changes in microstructure, crystallinity, amorphism, polymorphism, hydrate, solvate, isomorphic desolvate, packing order, ionic crystal, interstitial space, lattice, or habit.


In one embodiment, the computing system can design a process for processing the array of samples to determine an effect on the compound of interest of at least one experimental variable for each experimental formulation. Such processing can be determined from the experimental variable input into the computing system so as to process the samples as described herein. For example, the processing of each experimental formulation can include a process consisting of at least one of mixing, agitating, heating, cooling, adjusting pressure, adding crystallization aids, adding nucleation promoters, adding nucleation inhibitors, adding acids, adding bases, stirring, milling, filtering, centrifuging, emulsifying, mechanically stimulating, introducing ultrasound energy to the experimental formulation, introducing laser energy to the experimental formulation, subjecting the experimental formulation to a temperature gradient, allowing the experimental formulation to set for a time, or heating to a first temperature then cooling to a second temperature.


In one embodiment, the present invention can include using a computer-program product having computer-modeling capabilities for determining at least one optimal formulation of a compound of interest, such as a pharmaceutical, for a desired purpose. In some instances, the formulation can include a solid form of the compound of interest. The computer-controlled system and/or computer-program product can design and screen the compound of interest. The computer-controlled system and/or computer-program product can compute an optimization algorithm in order to select a plurality of molecular descriptors and a model accepting the molecular descriptors as parameters to optimize the design and/or predictive power of the computer-modeling capabilities. The molecular descriptors and model can be used in designing and testing a large number of samples having experimental formulations to determine at least one optimal formulation for the compound of interest.


Additionally, the computer-controlled system and/or computer-program product can generate values of experimental parameters using the model to design experimental formulations and experimental processes for an array of samples. As such, high-throughput design and screening can be performed as described herein by using the values generated by the model. Also, experimental results obtained from screening the experimental formulations designed by the model can be compared with the results predicted by the model. The model and/or experimental parameters used therewith can be modulated based on the high-throughput experimental results.


The model-generated values can be used to find an extremum of an expected property of an experiment, boundaries between solid forms, regions in which desired properties of formulations change rapidly with respect to changes in experimental parameters, regions in which desired properties of formulations change slowly with respect to changes in experimental parameters, or regions of ambiguity or low confidence in classification or regression results. As such, the predictive power of the model can be determined with respect to an extremum of an expected property of an experiment, with respect to boundaries between solid forms, with respect to regions in which desired properties of formulations or solid forms change rapidly with respect to changes in experimental parameters, or with respect to one or more regions within class boundaries.


Also, a variety of optimization algorithms and models may be used in the computing system and/or computer-program product. Accordingly, an approximately maximally diverse set of values of experimental parameters for high-throughput screening can be generated using a diversification algorithm and a metric for measuring diversification. Alternatively, a set of values for experimental parameters for high-throughput screening can be generated based on a structure-activity model.


B. Sample Preparation


The computer-controlled system can include an automated distribution mechanism to add components and the compound of interest to separate sites; for example, on an array plate having sample wells or sample tubes. Preferably, the distribution mechanism is controlled by computer software, such as a computer-program product operating on the computing system, and can vary at least one variable with respect to the experimental formulation containing the compound of interest. As such, the distribution mechanism can vary the identity of the component(s), the component concentration, and the like. Also, the distribution mechanism can prepare the sample in accordance with the experimental formulation designed by the computing system. Material handling technologies and robotics can be used in the distribution mechanism and are well known to those skilled in the art. Of course, if desired, individual components can be placed at the appropriate sample site manually. This pick and place technique is also known to those skilled in the art.


Also, the computer-controlled system can include a processing mechanism to process the samples after component addition. Optionally, the processing mechanism can have a processing station that processes the samples after preparation. A processing mechanism can be any computer-controlled experimental equipment that can process the array of samples by any of the processes described herein.


Additionally, the computer-controlled system can include a screening mechanism to test each sample to detect a change in physical and/or chemical properties of the formulation and compound of interest. Preferably, the testing mechanism is automated and controlled by computer software, such as a computer-program product operating on the computing system,


A number of companies have developed array systems that can be adapted for use in the invention disclosed herein. Accordingly, array systems can be employed in a computer-controlled system as described herein. Such array systems may require modification, which is well within the range of ordinary skill in the art. Examples of companies having array systems include Gene Logic of Gaithersburg, Md. (see U.S. Pat. No. 5,843,767 to Beattie), Luminex Corp., Austin, Tex. Beckman Instruments, Fullerton, Calif. MicroFab Technologies, Plano, Tex. Nanogen, San Diego, Calif. and Hyseq, Sunnyvale, Calif. These devices test samples based on a variety of different systems. All include thousands of microscopic channels that direct components into test wells, where reactions can occur. These systems are connected to computers for analysis of the data using appropriate software and data sets. The Beckman Instruments system can deliver nanoliter samples of 96 or 384-arrays, and is particularly well suited for hybridization analysis of nucleotide molecule sequences. The MicroFab Technologies system delivers sample using inkjet printers to aliquot discrete samples into wells. These and other systems can be adapted as required for use herein.


The automated distribution mechanism delivers at least one compound of interest, such as a pharmaceutical, as well as various additional components, such as solvents and additives, to each sample well. Preferably, the automated distribution mechanism can deliver multiple amounts of each component. Automated liquid and solid distribution systems are well known and commercially available, such as the Tecan Genesis, from Tecan-US, RTP, NC. The robotic arm can collect and dispense the solutions, solvents, additives, or compound of interest from the stock plate to a sample vial or sample tube. The process is repeated until array is completed, for example, generating an array that moves from wells at left to right and from top to bottom in increasing polarity or non-polarity of solvent. The samples are then mixed. For example, the robotic arm moves up and down in each sample vial for a set number of times to ensure proper mixing.


Liquid handling devices manufactured by vendors such as Tecan, Hamilton and Advanced Chemtech are all capable of being used in the invention. A prerequisite for all liquid handling devices is the ability to dispense to a sealed or sealable reaction vessel and have chemical compatibility for a wide range of solvent properties. The liquid handling device specifically manufactured for organic syntheses are the most desirable for application to crystallization due to the chemical compatibility issues. Robbins Scientific manufactures the Flexchem reaction block which consists of a Teflon reaction block with removable gasketed top and bottom plates. This reaction block is in the standard footprint of a 96-well microtiter plate and provides for individually sealed reaction chambers for each well. The gasketing material is typically Viton, neoprene/Viton, or Teflon-coated Viton, and acts as a septum to seal each well. As a result, the pipetting tips of the liquid handling system need to have septum-piercing capability. The Flexchem reaction vessel is designed to be reusable in that the reaction block can be cleaned and reused with new gasket material.


III. Sample Containment and Preparation


The computer-controlled system and/or computer-program products operating in the computing system can be used for designing, preparing, processing, screening, and analyzing samples having experimental formulations comprising a compound of interest. After the experimental formulation for each sample has been designed by the computer-controlled system and/or computer-program products, the automated high-throughput system can prepare the array of samples. As such, compound of interest and any additional components can be delivered to a plurality of sample sites in an array, such as sample vials or sample tubes on a sample plate to give an array of unprocessed samples. The array can then be processed according to the purpose and objective of the experiment, and one of skill in the art will readily ascertain the appropriate processing conditions. Preferably, the automated distribution mechanism as described above is used to distribute or add components.


A. Tubes and Blocks


High throughput preparation and analysis of samples is aided by the assembly of arrays of samples, each of which can be the same or different from other samples in the array. In specific embodiments of this invention, arrays of samples are prepared in removable containers (e.g., vials or tubes), which fit in holes, wells, or depressions in a holder, or what is referred to herein as a “block.” This system is referred to herein as “tubes and blocks” or “tubes in blocks.”


A wide variety of containers known to those skilled in the art can be used to hold the individual samples in an array. Because preferred embodiments of the invention are directed to the high-throughput preparation, processing, and/or testing of samples that contain relatively small amounts of the compound of interest, preferred containers are sufficiently small so that many of them fit into a block. Preferred containers are also optically transparent or translucent to allow visual inspection of their contents, which are chemically inert (e.g., will not chemically react with the compounds they contain), and can withstand physical conditions (e.g., thermal processing) to which it will be exposed. Specific containers are made of glass or polypropylene. Preferred containers can be sealed or closed. For example, a septum that can be pierced by a needle or other device that can add fluids to the container or remove fluids from the container is used in a preferred embodiment. Containers may also be closed with a closure (e.g., a cap or top) that allows light to pass through into the container to illuminate its contents. Moreover, the closure may be used to imprint or otherwise provide an identifier to a single tube or a sub-block. Such an identifier may be in addition to or in lieu of an identifier associated with each block.



FIGS. 2A-2B provides a view of a tube container 50 in its open (FIG. 2A) and capped (FIG. 2B) configurations. The specific closure 54 shown in FIG. 2B can be crimped, is made of aluminum or some other suitable material, and incorporates a polymer septum 55.


The blocks that hold the containers preferably allow for the automated removal and insertion of the containers. For example, a particular block has holes with top openings large enough to accommodate containers and smaller bottom openings that allow the containers to be pushed out of their holes with a rod or pin. Such blocks can be used with particular systems of the invention that comprise a lifter mechanism capable of protruding through the access hole in the blocks to elevate one or more containers until at least partially removed from the block. Preferably, the block is thermally conductive and is made from metal (e.g., copper, steel, or aluminum), although other materials, such as plastic, may also be used. As shown in FIGS. 3A-3C, a specific block 60 is made of aluminum, and has 96 holes 61 into which containers will fit. Block 60 provides thermal transfer between the tubes and a controlled means for temperature regulation. Block 60 incorporates a set of bottom access holes 63 that provide for physical and optical access to each individual vial. Optionally, block 60 has one or more indents 65 on two opposing sides of block 60 to provide means for preventing slipping or dropping of the block 60 when automated handling, such as by a robotic arm, is used to move block 60.


The geometry, size, and materials from which a block is made can be readily adapted for use with particular containers, processing conditions, and sample and block handling devices. For example, the holes in a block may be counter-bored, counter-sunk, stepped, tapered, or more complexly shaped to fit different tube and seal shapes, although in FIGS. 3A-3B they are simply shown as illustrative straight through holes.


The tube and block system has several distinct advantages over alternative ways of performing parallel experiments. First, the use of individual containers, instead of using a plate format, allows for the individual handling of each sample, or experiment, in an array. This makes it possible to re-array containers to separate those that show desired properties from the rest, in order to perform further processing or analysis of only some of the experiments. In addition, for experiment samples or products that can exhibit different properties depending on orientation (e.g., samples that contain crystals), the containers can be precisely oriented with respect to an analysis instrument, such as a Raman spectrometer or X-ray diffractometer.


The invention also encompasses the use of various tube materials, including the use of different types of glass such as amber glass vials, which can protect their contents from degradation due to exposure to light during processing. Optical inspection of the contents of each vial is possible by illuminating the samples with a light source having a wavelength able to penetrate the vial walls, and using a detector (e.g., camera) for imaging light at that wavelength.


Second, a translucent, transparent, semitransparent, or clear container allows for optical inspection of the experiment from multiple angles, generally perpendicular to the axis of the container, but also from underneath through optional access holes in the block. Also, the use of such containers, including but not limited to, glass or clear polypropylene tubes, allows for optical inspection methods such as machine vision or microscopy. In addition, clear plastic tubes, or tubes fabricated from quartz or any other optically transparent, translucent, or clear material can be used. Chromacol Ltd. (2 Little Mundells, Wellwyn Garden City, Herts AL7 1EW, United Kingdom) offers many examples of the variety of available vial shapes and materials. The ability to visually inspect each experiment in an array, from all angles, allows analysis of the contents, such as solids or precipitates, in a number of ways, including without limitation, estimating size, color, shape, orientation and location in the container.


Third, because containers can be of any shape, the tubes and blocks system enables the testing of a wide range of experimental volumes. By selecting the shape of the containers, small volume experiments (e.g., about 2 μL) are still clearly visible in the narrow, preferably conical, tip of the preferred container, while larger volume experiments (e.g., greater than 100 μL) may also be tested due to the larger diameter top section of the tubes. Also, the container geometry in the preferred embodiment permits the use of a tightly sealing cap. An airtight seal isolates the contents of the experiments from the environment and prevents evaporation, leakage or contamination of, or changes to, the components in the containers.


Fourth, many containers can be capped. The use of a cap with an integral translucent frit or septum allows for the ability to probe, add, or remove components to/from the experiment, as well as the ability to illuminate the contents of the container through the septum. This lighting of the samples in the containers though the septa can be accomplished through the use of light sources such as fiber optic light guides or light-emitting diodes (LEDs).


Fifth, the use of thermally conductive blocks allows for quick heat transfer between a heating or cooling source and the containers, as well as a large thermal mass to maintain the containers at the desired temperature when temporarily not in contact with a heating/cooling source. As noted previously, many metals, plastics, and a variety of other materials can used to build the blocks. Although aluminum does not exhibit the best thermal conductivity or heat capacity, it is preferred in view of additional considerations such as weight, cost, corrosion-resistance, and ease of manufacturing.


Sixth, the chosen geometry of the block offers certain advantages. For example, the access holes at the bottom of each container hole allow for physical access to the containers, so that they can be partially or fully removed from the block for inspection or rearranging purposes. In addition, the holes also provide a window for optical inspection of the containers from the underside of the block that can be used alone or in conjunction with top lighting of the containers through translucent septa, to image the experiments in the containers in a block.


B. Sample Preparation


The composition of a particular sample in an array will depend on the use to which the particular method or device of the invention is put. For example, if an array is used to provide crystalline forms of a compound of interest, each sample might contain one or more solvents or solvent mixtures in addition to the compound of interest (which could be evaporated) or to which other solvents (e.g. antisolvents, reagents that affect pH, counterion concentration, or the ionic character of the solvent) or materials (e.g., nucleation promoters) could be added during the processing of the samples. The specific composition of each sample in an array might be the same (to allow redundancy) or different (to allow the simultaneous testing of numerous crystallization conditions). However, the invention also encompasses the use of arrays to attempt the crystalizations of compounds of interest from melts, in which case the samples might only contain solid compound of interest.


In another example, the array is used to determine various characteristics of a compound of interest, or how they change when exposed to particular conditions (e.g., those described below in the Sample Handling and Processing section). Examples of characteristics include, but are not limited to, form, chemical composition, solubility, physical and/or chemical stability, and hygroscopicity.


Whatever the purpose to which an embodiment of this invention is put, each container (apart from any containers used as controls, or blanks), will comprise a controlled amount of the compound of interest and, optionally, one or more additional compounds (e.g., solvents, excipients, or nucleation agents). The containers may also contain a stirbar or other device to facilitate stirring, uniform heating, or anything else that is deemed necessary for the particular use to which the invention is being put. All of these materials are preferably added to containers in an automated fashion. For example, compounds of interest and solvents can be deposited into the vials in a variety of ways, ranging from hand-pipetting to automated liquid and/or solid dispensing. Dispensing of chemicals into the vials is preferably accomplished with an automated reagent dispensing apparatus, such as Cartesian Technologies' PreSys model (available from Cartesian Technologies Inc., 17851 Sky Park Circle, Suite C, Irvine, Calif. 92614, USA), and multiple-channel liquid dispensers, such as those available from Tecan Group Ltd. (Tecan Group Ltd., Seestrasse 103, 8708 Mannendorf, Switzerland). Other models and brands of liquid dispensers can also be used. Solid compounds and compositions can also be dispensed by hand or by automated means known in the art. For example, a solution comprising a compound of interest can be dispensed into sample containers, after which the solvent can be removed to provide a controlled amount of the compound of interest (e.g., in a milligram or microgram quantity).


After samples have been prepared, the containers that hold them are preferably sealed to prevent leakage, contamination, and evaporation (unless otherwise desired), as well as to prevent outside factors (e.g., humidity changes) from affecting the samples. Preferred containers are vials which can be sealed using crimpable metal caps or compliant gaskets, such as a silicon frits or septa. Other means of sealing containers include, but are not limited to, wax plugs, threaded caps, caps that snap over the vial opening, and compression or adhesive seals. Preferred septa allow for the illumination of the contents of a container from the top, and also allow for the addition or withdrawal of materials or components to/from the tube. The capping or sealing of the containers is preferably accomplished using an automated means, such as a Wheaton Crimpmaster Crimping Station (Wheaton Science Products, 1501 No. 10th Street, Millville, N.J. 08332, USA) pneumatically powered crimper. Alternatively, hand powered crimper tools (also Wheaton Science Products) may be used.


The invention encompasses the labeling of either the vial itself or the crimped seal cap that allows the ready identification of individual samples. Both crimp caps and glass vials may be labeled, for instance, through laser and inkjet marking, by using human-readable, alphanumeric codes, as well as using machine-readable codes such as DataMatrix 2-D codes. Such codes may advantageously be scanned and tracked with optical readers. Similarly, other types of barcodes and marking technologies may be used without limitations.


IV. Sample Handling and Processing


Particular embodiments of the invention encompass exposing the samples in an array to one or more conditions such as, but not limited to, pH, ion concentration, solvent, temperature, and light for a particular amount of time. A typical condition is temperature, and one embodiment of the invention encompasses a thermal cycling system capable of processing many blocks simultaneously. This system comprises one or more shelves, preferably thermally conductive, onto which blocks can be placed, and heating and/or cooling means such as, but not limited to, chillers, baths (e.g., water), dry baths, hot plates, temperature-controlled rooms, ovens, thermoelectric devices, such as devices employing Peltier-effect cooling and/or joule-heating, and environmental chambers. The temperature of the samples can be controlled by heating or cooling the thermally conductive shelves.


The thermal cycling system can be used to simply incubate an array of samples at a specific temperature for a particular time (isothermal incubation), or can be used to cycle their temperatures to vary their temperature as a function of time. When employed, thermal processing comprises varying the temperature of the contents of each vial in a controlled cycle, usually a heating period is followed by a cooling period. Heat transfer through the blocks that hold the arrays of containers changes the temperature of the containers. Thus, when thermal processing is used to process the samples, the blocks used should allow heat transfer between a heating/cooling source (e.g., thermally controlled shelves) and the sample containers (e.g., vials).



FIGS. 4A-4B illustrate a specific example of a thermal cycling system, which comprises temperature-controlled shelf assemblies 56, which are also referred to herein as “hotels.” A number of hotels 56 can be arranged to include shelves 58 containing a number of different blocks 60.



FIG. 5 provides an illustration of one example of a thermal cycling system, which comprises 18 hotels 56 in a semi-circular arrangement around a robotic arm 55. In a preferred embodiment, a hotel comprises twelve shelves 58 arranged in a vertical fashion as shown in FIG. 4A, held in place with supporting members and incorporating locating features for securing the assembly in the desired position. In a preferred embodiment, each individual shelf 58 contains an internal loop through which a liquid, such as water, is circulated to control the shelf temperature. The loops are piped to a bath acting as the cooling/heating source. Finally, the thermal cycling system can optionally include an environmental-control enclosure 57 that regulates the humidity and/or ambient temperature of the air surrounding the blocks, preventing condensation on the containers and other components. One embodiment of an environmental control system 72 is shown in FIG. 5. Alternatively, the thermal cycling system can be located in an environmentally-controlled room.


In a specific embodiment of the invention, different water baths (which may also employ various other fluids for conducting heat or cold to the samples) allow for the processing of multiple blocks at different temperatures. The blocks are located in hotels that are connected to the baths, the temperatures of which are computer controlled. In this embodiment, computers also record the heating/cooling time and temperature for each assembly of shelves, or “hotel.” Because each block contains a plurality of sample containers, each of which is identifiable by is location in the block and/or the use of a bar code or other identifier, the conditions to which each sample in a given hotel is exposed is recorded and tracked by computer.


The processing of samples or arrays of samples can involve more than simply subjecting the samples to a particular temperature or range of temperatures. For example, the samples can be exposed to other environmental conditions, such as humidity, using an environment-controlled room. As shown in FIG. 5, environment control is achieved in one embodiment of the invention using an enclosure 57 that surrounds the shelf assemblies 56, and is connected to a supply of air that has been treated to provide the desired humidity level inside the enclosure.


Samples in an array can be processed in any number of ways. For example, samples in an array can all be subjected to the same temperature for the same amount of time, or can be processed individually using, for example, robotic techniques. For example, a solvent or antisolvent can be added to just one or a few of the containers held in a block with the aid of automated dispensing devices and robotic arms, such as that shown in FIG. 5.


Samples can also be subjected to a combination of different processes. For example, in what is referred to as a “mixed-mode” crystallization process, more than one processing mode is applied to samples in an array either serially or in parallel. For instance, thermal processing (described above), followed by antisolvent addition to the container(s) and/or partial or complete evaporation of the volatile contents of the container(s) can be used to facilitate crystallization of a compound of interest. Here, the term “antisolvent” refers to a solvent in which the compound to be crystallized has very low solubility. An evaporation process entails allowing the sample solvent systems to evaporate and may involve flowing a dry, inert gas over the samples and/or heating the samples to an extent and for a time sufficient to effect concentration of the compound of interest in the sample. In a specific example of mixed-mode processing, a thermal process is followed by an evaporative step in which the sample vessels are opened (uncrimped) and dry nitrogen is blown over the surface of the samples to promote evaporation of the solvent to an extent and for a time sufficient to allow crystallization. In another example of mixed-mode processing, a thermal process is followed by addition of an antisolvent to the sample vessels in an amount sufficient to allow crystallization. In still another example of mixed-mode processing, a thermal process is performed on duplicate sets of sample formulations followed by an evaporative step on one set and antisolvent addition to the other set. A mixed-mode crystallization process may conclude with an incubation step, where the samples are incubated at a temperature and for a time sufficient to allow crystallization. Any combination of individual process steps (e.g., thermal, antisolvent addition, and evaporation) may be used in serially or sample arrays may be split to allow different process modes to be used in parallel.


Visual inspection of the samples is preferably done at least once during their processing (i.e., their exposure to one or more chemical or environmental conditions). Such inspection can occur at any time before, during, or after the processing of the samples, and is preferably done using automated means. For example, a robotic arm 55 as shown in FIG. 5 can be used to removed the blocks 60 from the shelves 58 and transfer them to an imaging or vision station. Depending on the result of the imaging, the block 60 can then be replaced onto a shelf in the thermal cycling system, or its containers can be separated, rearranged into new blocks, or removed entirely for more detailed (e.g., spectroscopic) analysis. As mentioned above, the location and processing history of each sample is preferably tracked and recorded, so that it can be located, analyzed, and reproduced at any time. Because each container can be imaged separately from others in the array to which it belongs, this invention allows the rapid identification of samples that can be further processed or removed for detailed analysis even when such samples are just a few of hundreds or even thousands of samples being processed.


In one embodiment of the invention, the processing of one or more samples in an array is stopped at a specific time using what is referred to herein as a “quenching station.” It is at such a station that the condition(s) to which a sample is exposed are removed. For example, if the condition to which a compound of interest has been exposed involves contact with a particular solvent, the samples can be quenched by extracting any fluid component that remains in each container. This can be accomplished by puncturing the seal of the container, or tube, with a needle that can extract the liquid from the tube and provide a relief path through which air can flow into the tube, so as not to create a vacuum. In addition, samples can be air-dried after removal of the liquids in a vial by using a similar needle assembly to punch through the septum and inject dry air into the vial for a specific amount of time. The dry air (or other gases) removes remaining liquids from the sample through evaporation, and vents them outside the vial. As with sample preparation, quenching can be automated, and can be triggered by a human operator or by computer.


V. Sample Imaging


A result of conducting a large number of small scale experiments using various processing methods creates the need to interrogate or inspect each of the samples in the containers for the presence (or absence) of solid forms or other products of interest. Although visual inspection can be done manually, preferred embodiments of the invention utilize what is referred to herein as a “vision station,” which is an automated system that allows for the rapid and efficient imaging and screening of samples. Preferred vision stations are designed for the analysis of samples contained in tubes and blocks-type arrangements.


In one embodiment of the invention, the vision station comprises a device for capturing an image of small particles, such as a microscope/camera system with a highly magnifying lens to capture images of small (down to sub-micron) particles onto a CCD such as the Canty Particle Size Vertical Imaging Microscope J M Canty Inc., Buffalo, N.Y. USA). Another example is the published report from December 2000 on image analysis of protein crystals: An optical system for studying the effects of microgravity on protein crystallization, Alexander McPherson et al., application note from American Biotechnology Laboratory, December 2000 issue, which is incorporated herein in its entirety by reference.


Depending on the use to which the invention is put, sample imaging can be used to determine the presence of a solid form in a sample or container. Alternatively, the absence of solids can be also be detected. Consequently, vision stations of the invention can be used to determine the stability of liquid formulations (e.g., drug formulations for intravenous administration to patients) and the stability of a formulation in a simulated body (e.g., gastric) fluid.


Samples can be imaged at any time after their preparation. Consequently, imaging information can be used to determine whether or not a sample should be processed, how it should be processed, and whether or not it should be subjected to more detailed, (e.g., spectroscopic) analysis.


A typical vision station of the invention comprises a light source and a camera. A suitable camera can be any unit capable of yielding photographic images of the contents of containers, e.g., the presence or absence of solids or solid forms, but is preferably capable of digital capture. In a preferred embodiment of this invention, a charge coupled device (CCD) camera provides adequate sensitivity, but other digital capture devices may also be used. The light source is selected based on the types of containers being used and the design of the experiment. Examples of light sources include, but are not limited to, visible light, laser light of varying wavelengths, monochromatic laser, plane-polarized, or circularly polarized light. In an example embodiment, the light source is white light from one or more tungsten lamps. Depending on the mode of application of the vision station, light can be brought in from the top of the array, the bottom, or from the side. Blocks containing removable containers allow improved access by light to the sample due to the ability to elevate the containers from the block, either by hand, or using an automated means.


In one embodiment, the vision station system is adapted for use with the tubes and blocks system. In this embodiment, the vision station system comprises a camera, a light source, and, optionally, a mechanism to elevate containers (e.g., tubes) from a block, thus presenting the containers to the camera. The mechanism to elevate containers from a block can lift containers out of a block individually, or in groups, including without limitation, lifting all the containers in one or more rows or columns of a block at the same time, and preferably, lifting all the containers in one row or column at the same time. Additionally, the system can employ software to capture, store, and analyze images and digitally flag or select tubes containing contents of interest. Furthermore, the vision station system may optionally comprise a database for warehousing of the results and collation of information on the identity, composition and history of samples in order to allow further detailed analysis of the combined data.


Ultimately, the vision station system enables the automatic selection of specific samples (or containers containing samples) from an array based on their appearance. Advantages of the vision station system include, but are not limited to, speed of acquisition coupled with the details of the solid form, such as gross crystal habit, color, form, and location of solids (e.g., crystals) in a container. Such information about where solid formation occurs (such as where a crystal nucleates), and shape of the crystals or precipitate is useful in studying and controlling crystallization. The vision station also provides many automation opportunities (both in hardware as well as in software analysis of images) and the ability to capture a variety of data regarding the detailed physical form of the compound of interest (e.g., its crystallinity, amorphous character, physical stability, and size range information). In terms of speed, embodiments of the vision station system can observe 96 sample tubes in less than one minute, and the image capture is rapid (e.g., on the order of 30 milliseconds with current digital camera technology).


In a specific embodiment of the invention, the vision station system can accept different arrays or blocks of containers for analysis in rapid succession. Using the vision station system, the information obtained can include: (1) detection of solids based on illumination (e.g., white light) and image capture; (2) observation of birefringence (backlit crystalline samples seen with the help of cross-polarized light); (3) observation of nano-particle presence (using laser beams at various angles to the camera lens); and (4) temporal information (nucleation kinetics and kinetic stability of colloidal suspensions toward growth and phase separation are two examples). In addition, automated exemplary and example machine vision algorithms further enhance the utility of the system by obviating the need for a user to manually select tubes that are of interest.


In another specific embodiment, the vision station system is adapted to process blocks that contain about 96 containers in an arrangement of 8 rows of 12 columns. FIG. 6 illustrates the logic used in one embodiment to address solid form generation using the vision station approach. The embodiment in the flowchart 80 involves tubes in blocks, but the process can be adapted for use with other container systems. This embodiment comprises a vision station analysis 82, which includes optical inspection of vials holding samples. If no solids are detected 84 in the vial, a determination is made as to whether or not that particular reaction is of further interest 88. If it is not of further interest, the experiment may be stopped 92 for that particular vial. If a sample in a vial is still of interest, it may be returned to the block 94 and sent back for further processing, such as in the thermal cycling system. The process may then be repeated as to that vial. Alternatively, it may be removed without further processing. If the experiment is designed to detect solids, vials that contain solids are sent to a re-arraying process 86 whereby multiple vials with solids present are grouped together in the same output block. At every step, the address of each vial is tracked and updated if necessary. Such tracking can be done using various methods known to the skilled artisan, including without limitation using bar codes. Optionally, the entire output block can then be sent for detailed (e.g., spectral) analysis 90. The output block is preferably entirely filled, but it need not be.



FIG. 7A shows a schematic diagram 100 of a vision station. A block 60 containing an array of vials 50 is placed before an imaging device 104. While it is recognized that a camera can be oriented underneath the block so as to be capable of viewing the contents of the vials through the access holes in the bottom of the block, the preferred embodiment contemplates raising the vials at least partially out of the blocks into view of the imaging device. Thus, FIGS. 7B and 7C are drawings of a vision station in operation, showing a side view 111 (FIG. 7B) and a perspective view 113 (FIG. 7C) of the block 60, wherein a lifting mechanism 115 lifts an entire row of vials 50 at the same time. Alternatively, vials 50 can be lifted one by one.


As shown in FIGS. 7A-7C, vial 50 is illuminated by a light 102 that can be placed in a variety of locations to light up different portions of the vial 50, depending on where in the vial 50 illumination is desired. The level of illumination is determined by inspecting the resulting images for the desired contrast and is controlled by the operator adjusting the level or voltage until the desired contrast is obtained. Alternatively, the level of illumination can be automatically adjusted using appropriate sensors and/or algorithms. The resulting illumination provides sufficient contrast for an image or picture to be captured. Various software 108 can be used to capture the image, such as Component Works IMAQ Vision (National Instruments). In a preferred embodiment, the image capture software is integrated into a custom VB software. The camera 104 then takes a picture of the vial 50. The picture is then stored on a computer 106. A hardware card, such as National Instruments Image Capture Card, model number PCI-1422, is used to capture the image in conjunction with image capture software 108. A custom software application then displays the picture of the vial and the vial can then be designated as containing various results, such as, but not limited to, solids, lack of solids, sediment, phase separation. The pictures and the vial 50 designations can then be stored in a database 110. The process is repeated for all of the vials 50 in a block 60, and for all the blocks in a given experiment run or design. The vials can all be processed at one time, or it can be done in smaller groups.


A preferred embodiment of the vision station system comprises a camera 104, preferably a CCD camera, for example, a CCD camera manufactured by Roper Scientific (model MegaPlus ES: 1.0) (now Redlake MASD, Inc., 11633 Sorrento Valley Road, San Diego, Calif. 92121 USA) with an 9×9 mm image array with a total of 1008×1008 pixels. Another suitable source of imaging cameras is Spectral Instruments, Inc., Tucson Ariz. that provides a CCD camera that can be cooled to −50° C. Alternatively, image plate technology based on CMOS can be used for obtaining images, but CCD is the preferred capture mechanism.


In one implementation, an area of the width of roughly 72 mm is observed when 8 tubes (a row at a time) are pushed out of a block for vision analysis, although, for instance, tubes may be viewed in groups of fewer than 8 such as single tubes or two tubes per captured image. This observed area leads to a pixel resolution of about 70 microns/pixel. A resolution range from about 5 to 1000 microns is useful in the many embodiments of this invention, since most organic crystalline materials in a powdery state range in particle size from a few microns to hundreds of microns. Single crystals are often a few hundred microns on the shortest edges, while on the other hand extreme colloidal particles, such as titania (TiO2) and silica (SiO2) can be stably prepared in the nanometer size range.


The vision station can be used to identify amorphous, as well as crystalline, solids. The amorphous form can be of significant interest with regard to certain compounds of interest, such as, but not limited to, increased solubility relative to crystalline forms. Generally, amorphous forms of a given compound are thermodynamically unstable compared with crystalline forms, but can be rendered kinetically stable toward physical form change. Amorphous particles are typically irregular in size, and the material lacks the property of optical birefringence. This is defined as the ability of most crystalline materials to interact with polarized light by changing the direction of the polarization as it passes through the crystals. Plane-polarized light is generally rotated upon traveling through a crystalline material. If the light is subsequently sent through an analyzing filter (this is another plane-polarized filter where the polarization direction is 90 degrees perpendicular to the first filter) at a right angle to the plane-polarizing filter on the light source, the rotated light escapes the analyzer. Therefore, true crystals appear as bright spots on a dark background. Conversely, amorphous disordered materials generally do not rotate plane-polarized light such that minimal light (equal to background) escapes the filter resulting in a dark image. It may be advantageous to look for the presence or absence of crystallinity in this way, and by comparison of birefringence image with the plain image rather than simply looking for the presence of solids.


The lighting used to capture white light images of elevated tubes is flexible, in that it can be brought in (a) from the top of the tubes (if the top is either open or any seal is transparent), and (b) from the side of the tubes, behind the camera. The latter is referred to as backlighting and this approach is required when one wants to capture birefringence information. In principle, the light can be brought in at a number of angles, but the preferred orientations are either vertical or horizontal. The lighting can be provided by fiber optics (for example, NT39-366 from Edmund Industrial Optics, 101 East Gloucester Pike, Barrington, N.J. 08007), although white light strips (for example, Stocker Yale, Imagelite brand) can also be used. Various polarizing filters can be obtained from a number of commercial sources, such as NT45-669 available from Edmund Industrial Optics.



FIG. 8 illustrates an embodiment of the vision station adapted for the detection of birefringence. On the left side, a pair of tubes with water 112 and another pair of tubes with varying amounts of glycine crystals 114 are shown with backlighting without a polarizing filter on the camera lens. On the right are the same samples 116 and 118 with a polarizer in place. With use of color images, one can capture polychromism (i.e., multi-color crystals) information from the experiment with a suitable camera, or simply run the analysis with black and white images and look for bright pixels. In addition, a quarter-wave retarder filter can be used to confirm the presence of crystals by causing a color shift when the filter is applied.



FIG. 9 shows the use of a combination of white light and laser scattering. A laser beam 124 (which can be of any color, such as red, green, or blue) can be brought into proximity with a tube or vial 50. Single tube analyses 120 are typically preferred, due to some scattering and diffusion of the laser beam in cases where one attempts to send the beam 124 through several tubes consecutively 122. The laser beam, which can be generated by any number of laser devices such as with a He—Ne Class II laser pointer at <1 mW power, will interact with sub-micron particles inside the tubes and the radiation is scattered, resulting in a contiguous trail of laser light through the tube. If no significant colloidal component is present (the sample is a true solution) no such trail of laser light will be observed in the image. Using this application, the vision station system with the laser beam can be used to obtain kinetic information regarding colloidal stability (i.e., how long it takes a suspension to settle or ripen to microcrystals), solution physical stability (how stable is a solution toward nucleation), or phase segregation.


Another embodiment of the invention utilizes laser light at an angle different from 90 degrees (e.g., at a 45 degree angle) relative to the camera lens. This is shown in the example of FIG. 10, where the image 126 in panel (a) clearly shows a contiguous path of laser light due to the presence of the colloids. In contrast, the image 125 in panel (b) shows a single point of scatter on the right side of the tube (where the laser beam hits the tube). This effect is due to partial scattering of the laser light by the glass, and becomes more pronounced in the image as the angle between the camera lens and the laser beam is decreased.



FIGS. 11 and 12 show flow charts for the logic used in specific vision station systems for the detection of birefringence 130 and laser light interrogation 132, respectively. The funnel widths roughly represent the number of samples at a given stage of the experimental workflow. The charts illustrate how a vision station system can facilitate analysis of crystallinity or lack thereof in a set of solid forms (FIG. 11), and also allows analysis of nano-particulate and true solutions along with the stability of each (FIG. 12).


In a preferred embodiment, the analysis of images obtained by the vision station is automated. For example, software (e.g., National Instruments IMAQ VISION software) is employed in image acquisition and analysis. When image analysis is performed manually, an operator flags the samples that satisfy the criteria used in the particular experiment (e.g., which ones contain a solid) using a software interface. Such software can perform a variety of function, such as, but not limited to, automated capture and storage of images, creating and storing logic for each sample (e.g., which ones contain solid, was a sample in solution at the start of the experiment), and ultimately containing algorithms for time-based measurements as well as automated isolation of containers that satisfy given criteria. Such software can also inform the user which samples are of interest, and facilitates the re-array of hit tubes from the source block into a destination block for further off-line processing or characterization. Preferred software provides an actual image of vials that allows a user to observe and manually select vials of interest for further processing.


In another specific embodiment of the invention, the vision station system further comprises a means of determining the optimal laser light configuration relative to the tubes for interrogation of colloidal suspensions (e.g., as to the size of the particles they contain). In another embodiment, the vision station system comprises a means of optimizing the capture of birefringence information, including the investigation using a quarter wave plate and other filters in concert with plane or other polarizers to ensure that light scattering is not interfering with image analysis and interpretation.


In a specific embodiment of the invention, once a number of blocks have been processed through the vision station system, there will be one or more output blocks holding vials containing solids. Optionally, in a preferred embodiment, these blocks are then processed further (e.g., moved to a quenching station).


VI. Spectroscopic Data Collection and Analysis


In a typical embodiment of the invention, one or more samples in an array are analyzed using spectroscopic techniques. In preferred embodiments of the invention, the sample(s) that are analyzed have been screened or selected from an original array of samples. For example, the vision station can be used to identify samples that contain solids, and the contents of those samples are then analyzed further using spectroscopic techniques.


The specific analysis done will depend on the purpose to which a particular embodiment of the invention is put. For example, if the invention is used to prepare solid forms of a compound of interest, the solids that have been identified in samples can be analyzed to determine their chemical and physical form, such as whether they are salts or solvates (e.g., hydrates) of the compound of interest, whether or not they are crystalline, and, if they are crystalline, the nature of their crystal form (e.g., their crystal structures). Spectroscopic analysis can also be used to determine if any of the compounds in a sample (e.g., the compound of interest) decomposed or reacted with other compounds in that sample.


Spectroscopic techniques can also be used to identify samples that share one or more characteristics. For example, if a solid compound of interest can exist in more than one solid form, and each of a plurality of samples comprises a solid compound of interest, it may be desirable to identify which samples contain the compound of interest and in which form. The grouping of samples as a function of a particular characteristic (e.g., a spectral characteristic unique to a particular solid form) is referred to herein as “binning.” Such binning provides a means of avoiding unnecessary duplication of further experiments. For example, if a group of samples are binned based on a particular spectral characteristic which corresponds to a previously unknown solid form of the compound of interest, further analysis of that solid form need not require a detailed analysis of each sample in the group.


Examples of spectroscopic techniques that can be used to bin or analyze samples are numerous, and will be readily apparent to those skilled in the art. Some specific examples include, but are not limited to, optical absorption (e.g. UV, visible, or IR absorption), optical emission (e.g., fluorescence or phosphorescence), Raman spectroscopy (including resonance Raman spectroscopy), nuclear magnetic resonance spectroscopy (e.g., single and multi-dimensional 1H and 13C), X-ray diffraction (e.g., powder X-ray diffraction), neutron diffraction, and mass spectroscopy. For the sake of convenience, other methods of analysis are encompassed by the term “spectroscopic technique,” as it is used herein, include, but are not limited to, microscopy (e.g., light and electron microscopy), second harmonic generation, circular dichroism, linear dichroism, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGS), and melting point. Preferred embodiments of the invention utilize Raman spectroscopy.


A. Raman Spectroscopy


The use of Raman spectroscopy for the high-throughput screening and/or analysis of multiple samples is believed to be novel, particularly in view of the relatively low intensity of Raman scattering as compared to other spectroscopic techniques. When coupled with the devices and techniques disclosed herein, however, Raman spectroscopy has been found to be particularly useful in the high-throughput screening and analysis of samples.


The Raman spectrum of a compound can provide information both about its chemical nature as well as its physical state. For example, Raman spectra can provide information about intra- and inter-molecular interactions, inclusions, salts forms, crystalline forms, and hydration states (or solvation states) of samples to identify suitable or desirable samples, or to classify a large number of samples. With regard to the hydration states of molecules, methods and devices of this invention, particularly the binning methods discussed in more detail below, allow their determination in situ.


Raman spectroscopy can also be used in this invention to examine kinetics of changes in the hydration-state of a sample or compound of interest. Moreover, the ability of Raman spectroscopy to distinguish, in certain situations, forms with different hydration states is comparable to X-ray diffraction, thus promising specificity and sensitivity. The lack of a strong Raman signal from water, a common solvent or component in preparations allows collection of Raman data in-situ in a manner relevant to many applications.


This invention also encompasses the use of Raman spectroscopy to determine the amount of a compound of interest that is dissolved in a particular sample. Advantageously, it has been discovered that for many compounds of interest and solvents, a correlation between the amount of compound of interest dissolved in a liquid sample and certain characteristics of its Raman spectrum can be obtained using one solvent, yet can be applied to the high-throughput analysis of samples prepared using a variety of other solvents.


These and other aspects of the invention are made possible by the utilization of several devices and methods described herein, which overcome problems inherent to Raman spectroscopy that would otherwise limit its usefulness as a high-throughput analytical technique. Examples of such problems include, but are not limited to, weak signals, background (e.g., solvent) emissions, and signals due to other solids or liquids in a sample, as well as the sample container itself.


Improvements in reproducibly obtaining Raman spectra for samples of interest include rapid and sensitive spectra acquisition and rejection of background noise. The strength of Raman emissions is improved by the use of lasers to excite the target substance. Use of a carefully selected wavelength also results in resonance Raman spectra. Sample preparation techniques resulting in adsorbing of a target to a surface further increase Raman signals, although such preparation is not always possible desirable in the case of in-situ data collection. Since the strength of the Raman signal can vary depending on many factors, it is important to use on-line data analysis in order to determine when a sufficient quality and quantity of data have been collected to meet the goals of the measurement (e.g. a prescribed signal-to-noise threshold). Of course, optical amplifiers further improve sensitivity and specificity. Each of these techniques or process steps may be used alone or in combination.


Filtering techniques encompassed by the invention that can be used to reject noise include, but are not limited to, temporal, spatial, and frequency domain filtering. Spatial filtering requires collecting emissions from a small area to reject noise from surrounding sources. Such confocal techniques, for instance with the target in the focus of an objective and/or using a pinhole arrangement, allow scanning of a target to reduce unwanted noise due to emissions from the material surrounding the target area.


The invention also encompasses temporal filtering, which rejects or accepts signals received in a particular time window. In the case of Raman spectra, temporal filtering relies on the different times taken for emission of Raman spectra and the background fluorescence spectra. Notably, Raman emissions, although weak, can be detected much earlier than fluorescence following excitation. Furthermore, fluorescent radiation continues over a significantly longer period, thus making possible selection of time windows for collecting Raman signal with a higher S/N ratio than otherwise. An example of such filtering is provided by Matsousek et al. in “Fluorescence suppression in resonance Raman spectroscopy using a high-performance Picosecond Kerr Gate,” in J. Raman Spectroscopy, vol. 32, pages 983-988 (2001). The Kerr gate realized by Matousek et al. exhibits a response time of about 4 picoseconds, thus allowing collection of Raman emissions during a window of 4 picoseconds following an exciting ilaser pulse. This example should be regarded as illustrative and not limiting as to temporal considerations in collecting and filtering spectra in possible embodiments since other gates, including virtual gating techniques are also intended to be within the scope of the claimed invention. Such filtering techniques, which can be used separately or together, can be augmented with mathematical filtering (e.g., convolution with the characteristic shape of a Raman line to further reduce the noise and reject unwanted frequencies and emissions).


In another aspect, the invention encompasses the use of polarized excitation and detection. Raman scattering emissions are sensitive to the orientation of the polarization of the exciting light relative to the molecules being examined. If the exciting light (typically from a laser) is polarized and the molecules in a crystal have fixed orientations, the Raman signal varies as a function of the orientation of the crystal. This property, while useful for detecting and evaluating crystalline samples, presents challenges in collecting representative Raman spectra due to the change in the amplitude of individual lines. The use of spectral binning, which is discussed elsewhere herein in more detail, can be used to overcome such challenges. Following the collection of a plurality of spectra that are, optionally, preprocessed to remove contaminating signals, as described more fully below, it is possible to identify peaks in each of the spectra. Optionally, from these identified peaks of the spectra it is possible to generate, for instance, a peak height or binary spectra reflecting the peak positions. The use of binary spectra reduces the computational overhead in binning and otherwise interpreting the data while taking into account variations due to orientation and the like. Filtered raw spectra, peak height spectra generated from identified peaks of filtered raw spectra, or binary spectra may be used to calculate similarity scores using any suitable metric, and the similarity scores allow binning of the spectra in accordance with various clustering techniques.


B. Data Collection


Spectroscopic data can be obtained for one or more samples by manually removing the containers that contain them from the block holding them, and presenting the containers to the particular analytical device being used (e.g., Raman spectrometer). Preferably, a mechanical system (such as an automated robotic arm) is used to select, or “cherry-pick,” particular containers (e.g., those identified as satisfying certain criteria by the vision station) from the block(s) that contain them.


In a specific embodiment of the invention used to detect and/or characterize solid forms of compounds of interest, a container is presented to a Raman spectrometer, and is imaged down the centerline at predetermined x, y positions. At each x, y position, two predetermined z positions are selected in order to focus imaging on the upper and lower inside face of the container (e.g., the upper and lower inside glass faces of a glass tube). Preferably, at least one position is used to focus imaging. This image acquisition step is repeated for different angles of rotation of the container until the entire inside surface of the container (e.g., glass tube) is imaged. After each image capture, an analysis is performed to determine where the “areas of interest” in a container are, where “areas of interest” can include solids or solid forms (e.g., crystals), and in some instances, any remaining droplets of solution or solvent.


A vision algorithm designed to automatically detect areas of interest (e.g., solid forms) in a container carries out the following: 1) locates or recognizes the presence or absence of a container; 2) locates the meniscus, if any, of the sample in a container; and 3) searches the area between the meniscus and the bottom of the container for particles, solids, solid forms, or other areas of interest.


After identifying areas of interest in a container, the Raman stage is moved to the center of the excitation source (e.g., laser) on to each of areas of interest in a container, and the Raman detection apparatus is focused using manual or automated means.


In one embodiment, auto-focusing of the Raman spectrometer can be performed. One way in which auto-focusing can be performed is by taking a series of Raman spectra at various z positions (to change the focus), for each x, y position representing an area of interest in a container. The one with the “best” Raman signal is marked, wherein the “best” Raman signal is defined by predetermined criteria, including, for example, by filtering each spectrum for a location and taking the maximum peak other than the normal peak associated with the effects of the container (e.g., glass tube). The resulting series of “best” Raman spectra for various areas of interest in a container can then be sorted based on similarities, and clustered into bins with spectra from other containers in an experiment. Automated focusing of a Raman spectrometer can result in a series of “best” Raman spectra for various areas of interest. These spectra can be sorted to distinguish droplets of solution or solvent from solids and clustered with data (spectra) from the other containers in the experiment.


When multiple spectra are obtained, one or more of the following can be also done: (1) find the one “best” spectra of a set of spectra for an area of interest or a solid form, with best being defined in a predefined way, including without limitation, highest peak signal, highest average signal, best S/N ratio, most peaks, and the like; (2) construct an average spectrum of all the spectra for an area of interest or a solid form, and use this spectrum in further processing; (3) construct an “agglomerated spectrum” that contains the highest peak of the set for every peak window, wherein a peak window is defined as a region in which peaks are considered to be the same; and/or (4) keep all of the spectra and perform downstream analysis on all of the spectra.


In processing (e.g., sorting and clustering) spectral data, the knowledge that several spectra come from each sample can be used to score the clustering results, or the labeled spectra can be used to influence the clustering run. For example, a k-means clustering run can be altered in the following manner: for each step of the k-means run, cluster assignments are made in the traditional sense, such that each point is assigned to the cluster with the nearest centroid, resulting in precluster assignments that are not the final assignments for the step; the precluster assignments for all points coming from the area of interest or solid form are then compared, and the most popular cluster assignment is assigned to all of the points in the group as the final assignment; and new centroids are determined from these final cluster assignments.


C. Data Analysis


In particular embodiments of the invention, spectroscopic data is processed using what is referred to herein as a “spectra binning system,” which allows the rapid analysis and identification of samples in an array by creating, for example, a family or similarity map. Preferred embodiments of the spectra binning system comprise a hardware-based instrumentation platform and a software-based suite of algorithms. The computer software is used to analyze, identify and categorize groups of samples having similar physical forms, thus identifying a group from which the operator, or scientist, can then select a few samples for further analysis. This selection can be performed independently by the scientist or using an automated means, such as software designed to automatically select samples of interest. Although, many applications made possible by the spectral binning system will be apparent to those skilled in the art, preferred systems of this invention is used to identify and characterize samples or compounds of interest. Particular binning and analytical methods useful in the invention are disclosed in U.S. patent application Ser. No. 10/142,812, filed May 10, 2002, the entirety of which is incorporated herein by reference.


The spectral binning system is generally used in this invention to detect similarities in the properties of a plurality of samples by observing their binning behavior. Thus, the number of forms of a substance can be estimated by binning spectra. The plurality of samples are examined with a device for generating a corresponding spectrum of acceptable quality (i.e., sufficient S/N ratio). Spectral peaks or other features are next identified to obtain a binary fingerprint. Advantageously, the spectra are compared pairwise in accordance with a metric to generate a similarity score. Other comparisons that use more than two spectra concurrently are also acceptable, although possibly complex.


One or more clustering techniques can be used to generate bins that are preferably well defined, although this is not an absolute requirement since it is acceptable to generate a reduced list of candidate forms for a given substance as an estimate of the heterogeneity of the structure of the substance. Advantageously, the generation of bins facilitates the ready evaluation of structure heterogeneity among samples. For instance, frequency, frequency shift, amplitude, and other similar measurements based on Raman spectra are often limited by the lack of suitable standards. However, the number of bins generated from evaluation of Raman spectra obtained by sampling a substance of interest is a measure that does not directly depend on having a good standard.


The invention also encompasses the use of hierarchical clustering to represent the data in the form of a similarity matrix having similar spectra/samples listed close together. Such a similarity matrix may be sorted to generate similarity regions along a diagonal. The resulting sorted similarity matrix may be used as a basis for setting the number of clusters for k-means clustering or other clustering techniques based on a specified number of clusters such as Gaussian Mixture Modelling.


Advantageously, although the clusters are actually in higher dimensional space, they can be projected into 2 or 3 dimensional space and visualized. Therefore, the binning procedure allows for both steady state and kinetic evaluation of states (e.g., hydration states, crystalline states, and other states, or forms, that can vary over time). This method is well suited for such measurements since individual Raman spectra can be collected rapidly (e.g., in a few seconds). Preferably, the turn-around time for generating a spectrum and assigning the spectrum to a bin is less than about two minutes, one minute, ten seconds, or one second. Moreover, limited real time processing is often possible if an acquired spectrum is to be assigned to existing bins, or, in a preferred embodiment of the invention, a library of binned spectra is updated with newly acquired spectra. In a preferred embodiment, newly acquired spectra from a single sample may all be binned into a single bin based on a majority of them being more related to the single bin in accordance with a metric, such as those discussed below and elsewhere herein.


Once the spectra from all of the samples to be analyzed have been collected, they are processed by a series of algorithms. These algorithms facilitate the binning of sample spectra according to one or more spectral features. Examples of such features include, but are not limited to, the locations of peaks, peak shoulders, peak heights, and peak areas. In a preferred embodiment, the spectral binning process bins spectra based on the locations of their scattering peaks and peak shoulders, expressed as wavelength or Raman shift (cm−1).


In the spectra binning system, the collected spectra can be binned using the raw or filtered spectra, peak height spectra generated using peaks selected from the raw or filtered spectra, and binary spectra generated using the raw or filtered spectra.



FIG. 15 represents the computational process applied by a specific embodiment of the spectra binning system. As shown in the flow chart 270, the process can be divided into preprocessing 271, peak finding 275, binary spectra generation 279, similarity matrix calculation 281, spectral clustering 283, visualization 285 stages, and storing 287. The binary spectra generation stage 279 can be optional in some instances. Each of these stages, which are applicable to the analysis of data obtained using a variety of spectroscopic techniques. For the sake of convenience, however, each is discussed in more detail below in reference to Raman spectroscopy.


1. Preprocessing


The purpose of the preprocessing step is to eliminate artifacts of the Raman spectra that are not caused by Raman scattering and to make the Raman scattering peaks as sharp as possible. Raman spectra often contain large fluorescence peaks spread over a broad spectral range and much smaller, narrower peaks caused by measurement, glass background, and instrument noise. Several different filtering techniques can be used in order to eliminate these deleterious features: Fourier filtering, wavelet filtering, matched filtering, and the like. The preferred embodiment uses a matched filter approach where the filter kernel is a zero-mean, symmetric product of sinusoids matched approximately to an average Raman peak width.


Preferably, the bandwidth of the main kernel peak is set to be equal to or slightly smaller than the bandwidth of an average Raman peak. When matched filters of this type are viewed in the Fourier domain, they may be seen to perform as bandpass filters, almost completely attenuating low- and high-frequency spectral components. Furthermore, with the bandwidth of the filter kernel chosen to be equal to or slightly smaller than the average Raman peak bandwidth, this filter detects peaks that are very close to each other. A raw, unfiltered spectrum will often display two close peaks as a main peak with a “shoulder” on one of its sides. After a matched filtering step, though, the shoulder will often be distinguished as a separate peak. This separation is useful for the peak picking procedure described below.


An example of the effect of such filtering means is provided in FIGS. 16A-16C. Specifically, FIG. 16A shows Raman intensity plotted as a function of Raman shift (cm−1) for an empty glass vial. The resulting waveform shows the pattern of absorbance present. FIG. 16B shows a Raman intensity of a fluorescent sample as a function of Raman shift. FIG. 16C shows the same pre-filtered plot as that of FIG. 16B, but also shows the corresponding filtered spectra after the fluorescence has been removed.


2. Peak Finding


The process of finding peaks in a spectrum is an important aspect of many spectral processing techniques, and there are many commercially available programs for performing this task. Many variations of peak finding algorithms can be found in the literature. An example of a simple algorithm is to find the zero-crossings of the first derivative of a smoothed or unsmoothed spectrum, and then to select the concave down zero-crossings that meets certain height and separation criteria. For the preferred embodiment, the peak finding function available in the software provided with the Almega dispersive Raman spectrometer (Thermo Nicolet, OMNIC software) was used. This function allows the threshold and sensitivity values to be set by the user. The threshold sets the lowest peak height that will be counted as a peak, and the sensitivity controls how far apart each peak must be to count as a separate peak. (See FIGS. 17A and 17B for an illustration of a graphical user interface of a peak-finding computer-program product.)


3. Binary Spectra Representations


Once the peaks have been found for all of the spectra, binary spectral representations are preferably created for all of the spectra. These binary spectra representations comprise vectors of ones and zeros. Each zero represents the absence of a peak feature and each one represents the presence of a peak feature. A peak feature is simply a peak that occurs within a certain spectral range, preferably a few wave numbers. The vectors for all of the spectra are preferably the same length and corresponding elements of these vectors correspond to the same peak feature.


In order to create these binary spectra, the peaks are clustered into ranges of peak features. The process used to perform this peak clustering is a modified form of a 1-dimensional iterative k-means clustering algorithm. The process begins with the picked peaks from a single spectrum. These peak positions are used to define the centers of peak feature ranges. The peak feature bins cover a range of wave numbers that can be specified by a user (the default is 5 wave numbers). The rest of the spectra are then iteratively added to the peak feature representation. At each step any peak that fits into a pre-existing peak feature range is added to that range. For any peak that does not fit into a range, a new range is created. Centers are not permitted to move so that peak feature ranges overlap. Then, the centers of all of the ranges are re-calculated and the peak feature ranges are re-defined relative to the new centers. This process can leave some peaks outside of an existing peak feature range. In this case, a new range is created for these peaks. This process creates a matrix with each row of the matrix corresponding to a binary spectrum specified in terms of range to which its peaks correspond.


4. Similarity Matrix Calculation


From either the spectra themselves, floating point or integer vectors representing the spectra, or from binary spectra representations such as those generated using the process described above, a similarity measure between pairs of spectra is calculated. Preferably, the similarity measure is calculated between each distinct pair of spectra. This similarity measurement is used to determine one or more clusters of similar spectra. Example similarity measurements include metric distances such as Hamming, Lp, or Euclidean distance, or non-metric similarity indices such a the Tversky similarity index (or its derivatives such as the Tanimoto or Dice coefficients) or functions thereof The selected similarity measure is preferably calculated for each distinct pair of spectra.



FIG. 18 illustrates an implementation of the binning procedure. At step 1800 the filter spectra are obtained. In one branch of the possible procedure, the peaks are located and corresponding binary spectra constructed in step 1805. The binary spectra are used to create the similarity matrix during step 1810. Next, hierarchical clustering results from sorting the similarity matrix to place similar spectra close to each other during step 1815. This matrix is suitable for visualization during step 1830. In one of the many alternative ways of processing the raw spectra of step 1800, the peaks are located and instead of binary vectors of step 2405, peak height vectors are generated during step 1820. Control can flow to step 1810 for the construction of a similarity matrix or directly point based clustering may be performed during step 1825 followed by visualization of the results in step 1830. Other alternative embodiments include control flowing from step 1805 following generation of binary vectors to point based clustering in step 1825 and then onto visualization in step 1830.


5. Spectral Clustering


Using the similarity measure calculated between spectra, a clustering algorithm is applied to determine one or more clusters of similar spectra. A variety of different clustering algorithms may be used.


Hierarchical clustering, including agglomerative and stepwise-optimal hierarchical clustering, k-means clustering, Gaussian mixture model clustering, or self-organizing-map (SOM) -based clustering, clustering using the Chameleon, DBScan, CURE, or Rock clustering algorithms are some of the clustering methods that may be used.


In a preferred embodiment, hierarchical clustering is used as a first-pass method of spectral data processing. Using the information from the hierarchical clustering run, a step of k-means clustering is then performed with user-defined cluster numbers and initial centroid positions.


In another embodiment, the number of clusters can be automatically selected in order to minimize some metric, such as the sum-of-squared error or the trace or determinant of the within cluster scatter matrix.


6. Visualization


Hierarchical clustering produces a dendrogram-sorted list of spectra, so that similar spectra are very close to each other. This dendrogram-sorted list is used to rearrange both axes of the original similarity matrix and then present the “sorted similarity” matrix in a coded manner wherein similarity indicia are used for each similarity region, including without limitation different symbols (such as cross-hatching), shades of color, or different colors. In a preferred embodiment, the “sorted similarity” matrix is presented in a color-coded manner, with regions of high similarity in warm colors and regions of low similarity in cool colors. Using this preferred three-dimensional (two spatial dimensions plus color) visualization, many clusters become apparent as warm-colored square regions of similarity along the matrix diagonal. These square regions represent a high degree of similarity between all of the spectral (i,j) pairs in those regions.


It should be noted that the failure of the similarity matrix to present a diagonal form is to be expected with some types of samples, although the matrix is still useful in representing more complex similarity relationships. Furthermore, in some cases there can be similarity regions along more than one possible diagonal that correspond to different rearrangements. Such rearrangements result in off-diagonal similarity square regions becoming part of the diagonal similarity square regions.


Along with the matrix representation of the cluster data, it is also useful to show where all of the spectra and the cluster boundaries lie in a dimensionally reduced space (usually 2-dimensions). There are several ways to perform this dimensionality reduction. In a preferred embodiment, a linear projection is made of a binary spectra matrix onto its first two principal components. Alternatively, the chosen similarity matrix could be used in order to create a map of the data using multidimensional scaling.


An example Raman clustering application is written in Visual Basic (VB). This VB program allows a user to select a group of spectra and set processing parameters. Preprocessing is performed within the VB application and then the filtered spectra are sent to OMNIC for peak finding through the Macros/Pro DDE communication layer provided by OMNIC. Once peaks are found, binary spectrum and distance matrix generation is performed in the main VB application. Then, the distance matrix is sent to MATLAB through a socket communication layer. In MATLAB, clusters are generated and visualizations are created. These visualizations are made available to the main VB application through a web server present on the same machine as the MATLAB instance. The resulting visualization allows for the easy identification of groups of samples that all have similar physical structure.


After clusters have been calculated, it is desirable to correlate clusters with corresponding solid forms. This is preferably accomplished by selecting one sample, or preferably, a plurality of samples from each cluster and characterizing the selected sample or samples with additional experimental techniques, such as powder X-Ray diffraction and/or differential calorimetry. In a preferred embodiment, the clustering and techniques result in clusters of experimental results all of which produced the same solid form. Based on the additional experimental characterization, solid-form labels reflecting the solid form produced by the experiments of the cluster are associated with the experimental result sets by the computational informatics subsystem. These labels are preferably used in combination with the experimental result sets and the corresponding values of experimental parameters to generate one or more regression models and/or classifiers for use in planning and assessing further experiments, or estimating properties for conditions that have not been experimentally verified. For example, regression models may be used to estimate properties over a continuous range reflecting an infinite number of different conditions.


EXAMPLES

Some specific, non-limiting examples of particular features of the invention are provided below.


Example 1
Raman Data Acquisition System

An automated robotic mechanism has been constructed and integrated with a microscope to facilitate selecting the sample containers (e.g., tubes) from the blocks and positioning the containers under the microscope objective for spectral acquisition. The spectral data collection system comprises a dispersive Raman microscope (Almega dispersive Raman by Thermo Nicolet, 5225 Verona Road, Madison, Wis. 53711, USA), which is a research grade dispersive Raman instrument, combining a confocal Raman microscope and a versatile macro sampling Raman spectrometer. The highly automatable and versatile system offers multiple laser options under software automation, for optimized sensitivity, spatial resolution, and confocal operation. The Almega dispersive Raman spectrometer is capable of housing up to two lasers. Selection of the lasers and control of laser power is accomplished through software. In addition, the appropriate Rayleigh rejection filters, apertures and gratings are automatically selected when the laser excitation wavelength is changed. The high-resolution setting provides better than 2 cm−1 resolution for all laser wavelengths. The spectral range of operation for CCD based detection is 400-1050 nm, allowing collection of Raman spectra over the full range for laser wavelengths. The example system is equipped with a 785 nm laser, a 256×1024 k CCD detector, and a NTSC video camera to monitor samples in the microscope with a spectral range, when using the 785 nm laser, of 100-3200 cm−1.



FIG. 13A shows a schematic of an entire automated spectra collection in Raman system 150. Material handling automation has been designed around the microscope to allow automated sample handling in and out of Raman system 150. Operationally, block 60 containing samples, in tubes 50, is placed into block nest 25. Block nest 25 is attached to XY stage 27 (Parker) to allow individual addressing of tubes within the block. XY stage 27 is attached to linear actuator 16 (Parker) that moves in the X direction. When block 60 is placed in block nest 25, a sensor (commercially available from Keyence) is activated, causing linear actuator 16 to move block 60 into light-tight enclosure 18 surrounding the Raman microscope, shown in FIG. 13B. Once inside enclosure 18, bar code reader 22 (commercially available from Keyence) reads the bar code on the sample block in order to track the contents of block 60. Referring to FIG. 13C, lift mechanism 24 presents individual tube 50 to tube gripper 26 by pushing tube 50 up through the access hole in the bottom of block 60. In FIG. 13D, tube gripper 26 and tube 50 are raised vertically from block 60. Tube gripper 26 is attached to linear actuator 32 and rotary actuator 30. FIG. 13E shows rotary actuator 30 rotating 90 degrees counter-clockwise to position tube 50 in a horizontal direction. Tube gripper 26 is then ready to travel horizontally along linear actuator 32 to move tube 50 near tube holder 36 in input stage 40 of microscope 20 as shown in FIG. 13F. FIG. 13G shows tube gripper 26 lowering tube 50 to tube holder 36. Tube 50 is then placed in tube holder 36, as shown in FIG. 13H, and tube gripper 26 is retracted vertically, as shown in FIG. 13I. Tube rotator 46 then engages tube 50 in tube holder 36, shown in FIG. 13J. Finally, in FIG. 13K, microscope input stage 40, under computer control, then actuates tube holder 36 under objective 38 for Raman analysis.


FIGS. 14A-G shows a procedure for focusing the Raman spectrometer on a solid form inside tube 50. The solid form is typically found attached to an inside surface of the tube. Therefore, the Raman spectrometer is preset to first look at that position and depth. This focusing also reduces the noise due to out of focus fluorescent emissions. Although confocal techniques are not used in this example implementation, in alternative embodiments of the invention they provide greater reduction in the noise since only the radiation through a pinhole is used at any time with integration over time to reconstruct the entire image. Naturally, data collection is over longer periods of time.


Returning to the described embodiment, it becomes necessary to properly position the tube beneath objective 38 of the microscope so that the solid form is at the right depth. As shown in FIG. 14B, this is accomplished by moving the entire microscope stage (not shown) supporting the tube holder (tube holder 36, as shown in FIG. 14K) in the X, Y and Z directions, as indicated by arrows 152, as well as rotating tube 50, as shown by arrow 154, to present the solid form at the depth expected by the spectrometer. In a preferred embodiment, tube 50 has the geometry as shown in FIG. 2, where the top half of tube 50 is cylindrical, but the bottom half of tube 50 is tapered. If the solid form is located in the tapered portion of the tube, then moving the tube in the XY plane to position it under the objective will also result in changing the Z-height of the sample with respect to the spectrometer, thus the need for controlling the Z-height of the stage as well.



FIG. 14C shows a detailed perspective view of solid 160 inside tube 50 in an out-of-focus position and indicates the available axes of motion 152 and 154. FIG. 14D shows a solid 160 that is out of focus because it is attached to the inside bottom (with respect to objective 38 located above) wall of tube 50 and is also located closer to the end of tube 50 than where the objective is focused. FIG. 14E shows how the microscope (not shown) stage is moved in the horizontal direction to bring solid 160 closer to the focal position. FIG. 14F then shows how tube 50 is rotated 154 to bring solid 160 closer to the focal position. However, after this rotational movement, solid 160 is now closer to the focal point and needs to be lowered. FIG. 14G shows how tube 50 is moved in vertical direction 152 to complete the process of bringing solid 160 into focus just below the inner surface of tube 50.


Using spectral signal intensity feedback from the Raman CCD, focal distance is “auto-focused” by computer controlling the XY position and the Z-height between the tube and the microscope objective. This auto-focus capability allows for the automated collection of Raman spectra once the tube is in place under the objective. Additionally, the NTSC video camera on the Raman allows for video capture and frame grabbing of the sample as it is being analyzed. This feature further allows for a spatial “history” to be created whereby the exact location of laser on the tube can be associated with a specific Raman spectrum. In order to implement the previously mentioned auto-focus capability, the tube holder has a computer controlled, motorized rotation axis. This controllable rotation allows the system, again under feedback control, to rotate the tube under the microscope objective in order to scan the entire inside surface of the tube.


When this feature is used, it is often not quite as important to pre-align the samples in the tube so that the sample is in the field of view as discussed above. Moreover, this feature allows for rotation during collection of a Raman spectrum. This is important to minimize so-called orientation effects that are sometimes observed in Raman spectra from anisotropic crystalline samples. Orientation effects exist when a sample has two or more unequivocal crystallographic “faces” that can be targeted by the laser source. Depending on the analyzed face, different spectra are generated, although the sample is physically unchanged. These different spectra might cause one to draw the conclusion that two or more different samples were present.


Once the sample in the tube is analyzed, the tube gripper removes the tube from the tube holder and returns it to the original location in the tube block followed by the XY stage indexing to the next tube to be analyzed.


Example 2
Data Collection and Binning

The effectiveness of binning was demonstrated using two test sets that included the Raman spectra of a polymorphic material and a material with two hydration states. First, the authenticity of the samples was validated. Next, Raman spectra for each sample under varying acquisition conditions were collected. The spectra were then filtered and binned using the previously described algorithms and method. Finally, the results were cross-checked by comparison of the known sample identification to the bin/cluster assignment. Each of these steps is outlined below.


Authentic polymorphic forms (polymorphs) and anhydrate/hydrate forms for a given material each exhibit a unique X-ray powder diffraction pattern and melting transition. Such criteria were deemed sufficient evidence to verify authenticity of each sample. Representatives of each of the forms of sample sets 1 and 2 were therefore characterized using X-ray diffraction (XRD) and differential scanning calorimetry (DSC), generating X-ray powder diffraction patterns and thermal transition data to determine sample uniqueness. Aliquots of samples from set 2 were further characterized using thermo-gravimetric analysis (TGA) to confirm the hydration state (i.e., water content) of the samples.


Two test sets were used to demonstrate the binning procedure for Raman spectra. Set 1 had two polymorphic forms of Flufenamic acid (2-[[3-(Trifluoromethyl)phenyl]-amino]benzoic acid), and set 2 had the anhydrate and monohydrate of theophylline (3,7-Dihydro-1,3-dimethyl-1-H-purine-2,6-dione). Anhydrous theophylline was obtained from Fluka Biochemica (Lot & Filling Code 403967/1 13700). The monohydrate was prepared by suspending 4.0 g of anhydrous theophylline in 20 ml of methyl alcohol. While stirring, 20 ml of de-ionized water was added to the suspension and the as-diluted suspension was warmed to approximately 40° C. to promote conversion to the hydrated form. The resulting suspension was continuously stirred and allowed to cool to 25° C. under ambient conditions. An aliquot of the suspension was collected by filtration after 6 hours and allowed to air dry. The solid obtained was characterized as described below to verify its hydration state.


All X-ray powder diffraction patterns were obtained using the D/Max Rapid X-ray Diffractometer (Rigaku/MSC, The Woodlands, Tex. U.S.A.), which uses as its control software RINT Rapid Control Software, Rigaku Rapid/XRD, version 1.0.0 (©01999 Rigaku Co.), equipped with a copper source (Cu/K 1.5406), manual x-y stage and 0.3 mm collimator. Samples were loaded in to 0.3 mm quartz capillary tubes supplied by Charles Supper Company by tapping the open end of the capillary into a bed of the powdered sample. The loaded capillary was mounted in a holder that was placed into the x-y stage. Diffractograms were acquired under ambient conditions at a power setting of 46 kV at 40 mA in transmission mode, while oscillating about the omega-axis from 0-5 degrees at 1 degree/s and spinning about the phi-axis at 2 degrees/s. Exposure times were 30 minutes unless otherwise specified. The diffractograms obtained were integrated over 2-theta from 2-60 degrees and chi (1 segment) from 0-40 degrees at a step size of 0.02 degrees using the cyllnt utility in the RINT Rapid display software version 1.18 provided by Rigaku with the instrument. No normalization or omega, chi or phi offsets were used for the integration.


The resultant X-ray powder patterns, plotted as intensity (arbitrary units) as a function of 2-theta (degrees), are shown in FIGS. 19A and 19B for the flufenamic acid 176 and theophylline 178 samples, respectively. Comparison of the X-ray powder patterns within each set clearly shows unique reflections (e.g., shifted peaks) in each pattern, indicating structural differences between the samples within each set and hence, validating the authenticity of the samples. Note, comparable X-ray powder patterns for the anhydrous and monohydrate forms of theophylline have been reported by Zhu et al. (International Journal of Pharmaceutics, 135:151-160 (1996)).


Further confirmation of the authenticity of the test sets was provided by DSC thermal analysis. An aliquot of each sample was weighed into an aluminum sample pan obtained from TA Instruments (pan number 90078.609, lid number 900779.901). Pans containing flufenamic acid samples were crimped closed, whereas pans containing theophylline samples were fit pressed to avoid pressure build up due to potential water vaporization. Sample pans were loaded into the apparatus and thermograms were obtained by individually heating the samples at a rate of 10° C./min from 20° C. to 350° C. using an empty crimped aluminum pan as a reference.


The DSC thermograms for the flufenamic acid 176 and theophylline 178 sample sets are shown in FIGS. 20A and 20B, respectively, where heat flow (W/g) is plotted as a function of temperature (° C.). The melt transition (peak temperature) of flufenamic acid samples was observed at 134.4° C. for Form I showing it to be pure Form I, whereas Form III exhibited a melt at 126.1° C., followed by re-crystallization and another melt at 133.9° C., indicating the conversion of Form III (melting point=126.1° C.) to Form I upon heating. The DSC thermogram for anhydrous theophylline shows a single sharp endotherm at 273.1 ° C. corresponding to the melting transition of the sample. The DSC curve for the hydrated sample exhibits two endotherms, the first occurring at a peak temperature of approximately 77.4° C. where dehydration of the sample is expected. This is followed by an endotherm at 273.1° C., where the anhydrous form melts.


Thermo-gravimetric analysis (TGA) was performed on samples from set 2 to verify water content. An aliquot of each sample was transferred into a platinum sample holder obtained from TA Instruments (#952019.9061) and loaded into the apparatus. Thermograms were obtained by individually heating the samples at 10° C./min from 25° C. to 300° C. under flowing dry nitrogen (balance purge 40 ml/min; sample purge 60 ml/min).


The thermograms obtained for the anhydrous and hydrous forms of theophylline are shown in FIG. 21, where the weight change (%) is plotted 182 as a function of temperature (° C.). As illustrated in FIG. 21, the hydrated sample undergoes a two-step weight loss. The first weight loss 184 of 9.2% begins at approximately 25° C. and continues until approximately 70° C. This weight change is associated with loss of loosely bound water from the hydrate structure and corresponds to a water mole fraction of 0.50, indicating the sample is a monohydrate of theophylline. For comparison, the theoretical water content for the monohydrate of theophylline is 9.09%. The small deviation in the measured sample is attributed to surface absorbed water, typically ranging from 0.0-0.3%. At approximately 172° C., the second weight loss 186 indicative of decomposition of the compound is observed. Note, the anhydrous theophylline sample exhibits only one weight loss 187 corresponding to decomposition beginning at approximately 172° C.


For reference, Raman spectra were collected for each of the samples in sets 1 and 2. An aliquot of the sample was transferred to a glass slide that was positioned in the sample chamber. The measurement was made using the Almega™ Dispersive Raman system fitted with a 785 nm laser source. The sample was manually brought into focus using the microscope portion of the apparatus with a 10× power objective, thus directing the laser onto the surface of the powdered sample atop a glass slide.


The unfiltered Raman spectra generated for each sample are shown in FIGS. 22A and 22B, where the Raman intensity (arbitrary units) is plotted as a function of Raman shift (cm−1). Note that the appearance or disappearance of peaks and/or shifts in peak position between the samples within a set was observed. For example, the spectra shown in FIG. 22A for flufenamic acid polymorphs of sample set 1 show a doublet 190 centered around 450 cm−1 for form I and a singlet 192 for form III at that position, as well as significant shifting of the three peaks 193 in the 1150-1250 cm−1 range. Such peak appearance/disappearance and/or shifts in peak position indicate a unique crystal packing configuration, thus differentiating the forms and showing that the Raman spectra can be used as a unique signature for a given form.


Evaluation of the filtering and binning algorithms was carried out by acquiring at least 20 spectra for each of the samples from sets 1 and 2, filtering the spectra to remove background signals, and binning the spectra. To collect the Raman spectra, an aliquot of each sample was transferred onto a glass slide or into a glass vial. Measurements were made by directing the laser onto the surface of the powdered sample atop the glass slide (theophylline) or through the glass vial (flufenamic acid). Half of the spectra collected for each polymorph of set 1 (flufenamic acid) were collected using the 50× microscope objective rather than the 10× objective. The sampling location was varied either by moving the glass slide or rotating the glass vial.


All spectra were filtered to remove background signals, including glass contributions and sample fluorescence. This is particularly important as large background signals or fluorescence limit the ability to accurately pick and assign peak positions in the subsequent steps of the binning process. Such background contributions to the Raman spectra are shown in FIGS. 16A and 16B for representative glass and fluorescent samples, respectively. Spectra from all samples of test sets 1 and 2 were filtered using a matched filter of feature size 25. An example of the original and filtered spectra for a fluorescent sample is shown in FIG. 16C.


Filtered spectra were binned using the algorithm described above under the peak picking and binning parameters and screen shots showing the output from the binning software captured during the binning procedure are provided in FIGS. 17A and 17B for the flufenamic acid and theophylline sample sets, respectively.


The sorted cluster diagrams 194 and 196 showing the output for each sample set are illustrated in FIGS. 23A and 23B and the corresponding cluster assignments for each spectral file are provided in Tables 1 and 2, respectively.

TABLE 1Cluster Assignments for Each SpectralFile for Flufenamic Acid Sample SetClusterOriginalSortedFile NameNumberNumberNumberFiltered flufenamic I 10×111Filtered flufenamic I 10× 10.SPA125Filtered flufenamic I 10× 2.SPA136Filtered flufenamic I 10× 3.SPA149Filtered flufenamic I 10× 4.SPA154Filtered flufenamic I 10× 5.SPA167Filtered flufenamic I 10× 6.SPA178Filtered flufenamic I 10× 7.SPA1815Filtered flufenamic I 10× 8.SPA192Filtered flufenamic I 10× 9.SPA1103Filtered flufenamic I 50× 1.SPA11116Filtered flufenamic I 50× 10.SPA11211Filtered flufenamic I 50× 2.SPA11317Filtered flufenamic I 50× 3.SPA11418Filtered flufenamic I 50× 4.SPA11520Filtered flufenamic I 50× 5.SPA11612Filtered flufenamic I 50× 6.SPA11719Filtered flufenamic I 50× 7.SPA11813Filtered flufenamic I 50× 8.SPA11914Filtered flufenamic I 50× 9.SPA12010Filtered flufenamic III 10× 1.SPA22121Filtered flufenamic III 10× 10.SPA22228Filtered flufenamic III 10× 11.SPA22329Filtered flufenamic III 10× 2.SPA22426Filtered flufenamic III 10× 3.SPA22522Filtered flufenamic III 10× 4.SPA22623Filtered flufenamic III 10× 5.SPA22731Filtered flufenamic III 10× 6.SPA22830Filtered flufenamic III 10× 7.SPA22927Filtered flufenamic III 10× 8.SPA23024Filtered flufenamic III 10× 9.SPA23125Filtered flufenamic III 50× 1.SPA23233Filtered flufenamic III 50× 10.SPA23334Filtered flufenamic III 50× 2.SPA23436Filtered flufenamic III 50× 3.SPA23535Filtered flufenamic III 50× 4.SPA23632Filtered flufenamic III 50× 5.SPA23737Filtered flufenamic III 50× 7.SPA23839Filtered flufenamic III 50× 8′.SPA23938Filtered flufenamic III 50× 9.SPA24040









TABLE 2










Cluster Assignments for Each Spectral


File for Theophylline Sample Set











Cluster
Original
Sorted


File Name
Number
Number
Number













Filtered Theophylline Hydrate1.SPA
1
1
1


Filtered Theophylline Hydrate10.SPA
1
2
14


Filtered Theophylline Hydrate11.SPA
1
3
7


Filtered Theophylline Hydrate12.SPA
1
4
8


Filtered Theophylline Hydrate13.SPA
1
5
9


Filtered Theophylline Hydrate14.SPA
1
6
15


Filtered Theophylline Hydrate15.SPA
1
7
10


Filtered Theophylline Hydrate16.SPA
1
8
11


Filtered Theophylline Hydrate17.SPA
1
9
16


Filtered Theophylline Hydrate18.SPA
1
10
12


Filtered Theophylline Hydrate19.SPA
1
11
17


Filtered Theophylline Hydrate2.SPA
1
12
19


Filtered Theophylline Hydrate20.SPA
1
13
2


Filtered Theophylline Hydrate3.SPA
1
14
3


Filtered Theophylline Hydrate4.SPA
1
15
4


Filtered Theophylline Hydrate5.SPA
1
16
18


Filtered Theophylline Hydrate6.SPA
1
17
13


Filtered Theophylline Hydrate7.SPA
1
18
5


Filtered Theophylline Hydrate8.SPA
1
19
6


Filtered Theophylline Hydrate9.SPA
1
20
20


Filtered Theophylline1.SPA
2
21
21


Filtered Theophylline10.SPA
2
22
27


Filtered Theophylline11.SPA
2
23
33


Filtered Theophylline12.SPA
2
24
28


Filtered Theophylline13.SPA
2
25
34


Filtered Theophylline14.SPA
2
26
29


Filtered Theophylline15.SPA
2
27
30


Filtered Theophylline16.SPA
2
28
22


Filtered Theophylline17.SPA
2
29
31


Filtered Theophylline18.SPA
2
30
23


Filtered Theophylline19.SPA
2
31
40


Filtered Theophylline2.SPA
2
32
36


Filtered Theophylline20.SPA
2
33
24


Filtered Theophylline3.SPA
2
34
38


Filtered Theophylline4.SPA
2
35
25


Filtered Theophylline5.SPA
2
36
26


Filtered Theophylline6.SPA
2
37
39


Filtered Theophylline7.SPA
2
38
37


Filtered Theophylline8.SPA
2
39
32


Filtered Theophylline9.SPA
2
40
35









In each sample set, two distinct clusters are observed as represented by sorted spectra numbers 1-20 and 21-40 that correspond to the file names and sample identifications provided in Tables 1 and 2. In comparing the cluster assignments to the sample identification (by file number), 100% binning accuracy is observed for each test set. For example, all form I samples are binned in cluster 1 and all form III samples are binned together in cluster 2 for flufenamic acid test set 1.


While the invention has been described in connection with what is presently considered to be the practical and preferred embodiments, the invention is not limited to the disclosed embodiments. In particular, it will be clear to those skilled in the art that this invention may be embodied in other specific forms, structures, and arrangements, and with other elements, and components, without departing from the spirit or essential characteristics thereof One skilled in the art will appreciate that the invention may be used with many modifications of structure, arrangement, and components and otherwise, used in the practice of the invention, which are particularly adapted to specific environments and operative requirements without departing from the principles of this invention. The presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims.


The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims
  • 1. In a computing system for controlling automated high-throughput processing of an array having removable sample vials held by an array block, wherein the computing system is designed to identify chemical and/or physical properties leading to optimal formulation for a given use of a compound of interest, and wherein the computing system provides computer-aided design and processing of an experimental formulation for each sample, each experimental formulation having the compound of interest and being based on at least one experimental variable which is varied as to at least some samples so that the effect in terms of changes in the chemical and/or physical properties of the compound of interest due to at least one variable can be identified across a number of comparative samples, a method of analyzing data from the comparative samples comprising steps for: inputting into the computing system at least one compound of interest and any additional components to be included in the experimental formulations that are to be designed for a first array of samples; inputting into the computing system at least one selected experimental variable of interest that is to be varied as between at least some samples of the first array; the computing system thereafter determining an experimental formulation for each sample that is different as between at least some samples based on the at least one selected experimental variable of interest that is varied as between the at least some samples of the first array; the computing system thereafter controlling a process by which the experimental formulation for each sample is prepared in a removable sample vial held by an array block and tested in order to create changes in chemical and/or physical properties of the compound of interest across a number of comparative samples; inputting to the computing system detected changes across the comparative samples for the at least one compound of interest; the computing system thereafter automatically screening the samples of the first array by identifying those samples which contain chemical and/or physical properties most likely to lead to optimal formulation for a given use of a compound of interest, and storing as a first data set information as to the experimental formulation and the resulting chemical and/or physical properties for each of the identified samples; removing from the array block sample those vials for samples not identified as part of the first data set, thereby forming a second array of samples contained by the array block by virtue of those sample not removed; and the computing system thereafter controlling a process by which the identified samples remaining in the second array are further processed and/or tested in order to further identify chemical and/or physical properties leading to optimal formulation for a given use of a compound of interest.
  • 2. In a computing system for controlling automated high-throughput processing of an array having removable sample vials held by an array block, wherein the computing system is designed to identify chemical and/or physical properties leading to optimal formulation for a given use of a compound of interest, and wherein the computing system provides computer-aided design and processing of an experimental formulation for each sample, each experimental formulation having the compound of interest and being based on at least one variable which is varied as to at least some samples so that the effect in terms of changes in the chemical and/or physical properties of the compound of interest due to at least one experimental variable can be identified across a number of comparative samples, a computer-program product for implementing a method of analyzing data from the comparative samples, the computer-program product comprising a computer-readable medium containing computer-executable instructions for causing the computing system to execute the method, and wherein the method is comprised of steps for: inputting into the computing system at least one compound of interest and any additional components to be included in the experimental formulations that are to be designed for a first array of samples; inputting into the computing system at least one selected experimental variable of interest that is to be varied as between at least some samples of the first array; the computing system thereafter determining an experimental formulation for each sample that is different as between at least some samples based on the at least one selected experimental variable of interest that is varied as between the at least some samples of the first array; the computing system thereafter controlling a process by which the experimental formulation for each sample is prepared in a removable sample vial held by an array block and tested in order to create changes in chemical and/or physical properties of the compound of interest across a number of comparative samples; inputting to the computing system detected changes across the comparative samples for the at least one compound of interest; the computing system thereafter automatically screening the samples of the first array by identifying those samples which contain chemical and/or physical properties most likely to lead to optimal formulation for a given use of a compound of interest, and storing as a first data set information as to the experimental formulation and the resulting chemical and/or physical properties for each of the identified samples; the computing system thereafter causing removal from the array block those sample vials for samples not identified as part of the first data set, thereby forming a second array of samples contained by the array block by virtue of those sample not removed; and the computing system thereafter controlling a process by which the identified samples remaining in the second array are further processed and/or tested in order to further identify chemical and/or physical properties leading to optimal formulation for a given use of a compound of interest.
  • 3. A method as in claims 1 or 2, further comprising: the computing system causing those sample vials removed from the array block to be placed into a different array block; the computer system causing additional sample vials to be placed in the different array block to form a third array of removable sample vials each having an experimental formulation including a common compound of interest; and the computing system thereafter controlling a process by which the samples in the third array are further processed and/or tested in order to further identify chemical and/or physical properties leading to optimal formulation for a given use of a compound of interest.
  • 4. A method as in claims 1 or 2 wherein the at least one selected experimental variable to be varied as between at least some samples of the first array is varied as to at least one of the following: concentrations of the compound of interest, concentrations of components in the experimental formulations, identity of the components, combination of components, additives, solvents, antisolvent compositions, temperatures, temperature changes, heating, cooling, nucleation seeds, supersaturation, pH, pH change, time of crystallization reaction, and combinations thereof.
  • 5. A method as in claims 1 or 2, further comprising inputting into the computing system at least one criteria for determining the effect of at least one experimental variable for each experimental formulation that is varied as to that experimental variable, where said effect is manifested by a change in one or more of the following for the compound of interest between different experimental formulations: microstructure, crystallinity, amorphism, polymorphism, hydrate, solvate, isomorphic desolvate, packing order, ionic crystal, interstitial space, lattice, or habit.
  • 6. A method as in claims 1 or 2, wherein the computing system further designs a process for processing each of the experimental formulations in the first or second array of samples to determine an effect on the compound of interest of at least one experimental variable for each experimental formulation having a value for the experimental variable.
  • 7. A method as in claims 1 or 2, wherein the processing of each experimental formulation in the first or second array includes a process consisting of at least one of the following: mixing, agitating, heating, cooling, adjusting pressure, adding crystallization aids, adding nucleation promoters, adding nucleation inhibitors, adding acids, adding bases, stirring, milling, filtering, centrifuging, emulsifying, mechanically stimulating, introducing ultrasound energy to the experimental formulation, introducing laser energy to the experimental formulation, subjecting the experimental formulation to a temperature gradient, allowing the experimental formulation to set for a time, heating to a first temperature then cooling to a second temperature, and combinations thereof.
  • 8. A method as in claims 1 or 2, wherein the effect in terms of changes in the chemical and/or physical properties of the compound of interest is at least one of causing crystallization, inhibiting crystallization, or formation of a solid form.
  • 9. A method as in claims 1 or 2, wherein each identified sample in the first or second array is selected based on a desired property.
  • 10. A method as in claims 1 or 2, further comprising: analyzing data regarding the processing of experimental formulations in the first or second array of samples to obtain a data set having the experimental data for each sample; and analyzing the data set to determine at least one optimal formulation.
  • 11. A method as in claims 1 or 2, wherein experimental formulations in the second array of samples each have a similar chemical and/or physical property.
  • 12. A method as in claims 1 or 2, further comprising the computing system automatically screening the further processed and/or tested identified samples remaining in the second array by further identifying those samples which contain chemical and/or physical properties most likely to lead to optimal formulation for a given use of a compound of interest, and storing as a second data set information as to the experimental formulation and the resulting chemical and/or physical properties for each of the further processed and/or tested identified samples.
  • 13. A method as in claim 12, the computing system thereafter selecting from the first and second data sets those samples which contain chemical and/or physical properties most likely to lead to optimal formulation for a given use of a compound of interest.
CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 11/447,592, filed Jun. 6, 2006, which is a continuation of U.S. patent application Ser. No. 11/051,517, filed Jan. 31, 2005, now U.S. Pat. No. 7,061,605, which is a continuation of U.S. patent application Ser. No. 10/235, 922, filed Sep. 9, 2002, now U.S. Pat. No. 6,977,723 (which claims the benefit of U.S. Provisional Patent Applications Nos. 60/318,152, 60/318,157, and 60/318,138, each of which was filed on Sep. 7, 2001), which is a continuation-in-part of U.S. patent application Ser. No. 10/142,812, filed Jun. 10, 2002 (which claims the benefit of U.S. Provisional Application No. 60/290,320, filed Jun. 11, 2001), which is a continuation-in-part of U.S. patent application Ser. No. 10/103,983, filed Mar. 22, 2002 (which claims the benefit of U.S. Provisional Application No. 60/278,401, filed Mar. 23, 2001), which is a continuation-in-part of U.S. patent application Ser. No. 09/756,092, filed Jan. 8, 2001 (which claims the benefit of U.S. Provisional Application No. 60/175,047, filed Jan. 7, 2000, U.S. Provisional Application No. 60/196,821, filed Apr. 13, 2000, and U.S. Provisional Application No. 60/221,539, filed Jul. 28, 2000), which is a continuation-in-part of U.S. patent application Ser. No. 09/628,667, filed Jul. 28, 2000, which is a continuation-in-part of U.S. patent application Ser. No. 09/540,462, filed Mar. 31, 2000 (which claims the benefit of U.S. Provisional Application No. 60/121,755, filed Apr. 5, 1999), and U.S. patent application Ser. No. 10/103,983 is also a continuation-in-part of U.S. patent application Ser. No. 09/994,585, filed Nov. 27, 2001 (which claims the benefit of U.S. Provisional Application No. 60/253,629, filed Nov. 28, 2000). All the foregoing patents and applications are incorporated herein by reference.

Provisional Applications (9)
Number Date Country
60318152 Sep 2001 US
60318157 Sep 2001 US
60318138 Sep 2001 US
60290320 May 2001 US
60278401 Mar 2001 US
60175047 Jan 2000 US
60196821 Apr 2000 US
60221539 Jul 2000 US
60253629 Nov 2000 US
Continuations (2)
Number Date Country
Parent 11051517 Jan 2005 US
Child 11447592 Jun 2006 US
Parent 10235922 Sep 2002 US
Child 11051517 Jan 2005 US
Continuation in Parts (7)
Number Date Country
Parent 11447592 Jun 2006 US
Child 11467096 Aug 2006 US
Parent 10142812 May 2002 US
Child 11051517 Jan 2005 US
Parent 10103983 Mar 2002 US
Child 11051517 Jan 2005 US
Parent 09756092 Jan 2001 US
Child 11051517 Jan 2005 US
Parent 09628667 Jul 2000 US
Child 11051517 Jan 2005 US
Parent 09540462 Mar 2000 US
Child 09628667 Jul 2000 US
Parent 09994585 Nov 2001 US
Child 10103983 US