Liquid purification or separation – With means to add treating material – Chromatography
Reexamination Certificate
2000-05-11
2002-10-01
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
With means to add treating material
Chromatography
C210S656000, C210S659000
Reexamination Certificate
active
06458273
ABSTRACT:
TECHNICAL FIELD
The present invention is directed to apparatus and methods usable for sample purification, and more particularly, to sample separation apparatus and methods usable for high throughput purification of samples.
BACKGROUND OF THE INVENTION
The relationship between structure and functions of molecules is a fundamental issue in the study of biological and other chemistry-based systems. Structure-function relationships are important in understanding, for example, the function of enzymes, cellular communication, cellular control and feedback mechanisms. Certain macromolecules are known to interact and bind to other molecules having a specific 3-dimensional spatial and electronic distribution. Any macromolecule having such specificity can be considered a receptor, whether the macromolecule is an enzyme, a protein, a glycoprotein, anantibody, or an oglionucleotide sequence of DNA, RNA, or the like. The various molecules which bind to receptors are known as ligands.
A common way to generate ligands is to synthesize molecules in a stepwise fashion in a liquid phase or on solid phase resins. Since the introduction of liquid phase and solid phase synthesis methods for peptides, oglionucleotides, and small organic molecules, new methods of employing liquid or solid phase strategies have been developed that are capable of generating thousands, and in some cases even millions of individual compounds using automated or manual techniques. A collection of compounds is generally referred to as a chemical library. In the pharmaceutical industry, chemical libraries of compounds are typically formatted into 96-well microtiter plates. This 96-well formatting has essentially become a standard and it allows for convenient methods for screening these compounds to identify novel ligands for biological receptors.
Recently developed synthesis techniques are capable of generating large chemical libraries in a relatively short period of time as compared to previous synthesis techniques. As an example, automated synthesis techniques for sample generation allows for the generation of up to 4,000 compounds per week. The samples, which contain the compounds, however, typically include 20%-60% impurities in addition to the desired compound. When sample impurities are screened against selected targets, such as a novel ligand or biological receptors, the impurities can produce erroneous screening results. As a result, samples that receive a positive result from initial screening must be further analyzed and screened to verify the accuracy of the initial screening result. This is verification process requires that additional samples be available. The verification process also increases the cost and time required to accurately verify that the targeted compound has been located.
Samples can be purified in an effort to achieve an 85% purity or better. Screening of the purified samples provides more accurate and meaningful biological results. Conventional purification techniques, however, are very slow and expensive. As an example, conventional purification techniques using high-pressure liquid chromatography (HPLC) take approximately 30 minutes to purify each sample. Therefore, purification of the 4,000 samples generated in one week would take at least 2000 hours (i.e. 83.3 days or 2.77 months).
Conventional purification techniques, such as HPLC, also require large volumes of solvents and result in large volumes of waste solvent. Disposal of the solvents, particularly halogenated solvents, must be carefully controlled for legal and environmental reasons, so the disposal process can be laborious and very costly. Disposal of non-halogenated solvents is less rigorous. Accordingly, when halogenated and non-halogenated solvents are used, the waste solvents are separated. The separation process of large volumes of solvents, however, can be a difficult process to perform efficiently and inexpensively. Accordingly, purification of large chemical libraries can be economically prohibitive. Therefore, there is a need for a faster and more economical manner of purifying samples of large chemical libraries,
Supercritical fluid chromatography (SFC) provides faster purification techniques than HPLC. SFC utilizes a multiphase flow stream that includes a gas, such as carbon dioxide, in a supercritical state, a carrier solvent and a selected sample. The flow stream passes through a chromatography column, and is then analyzed in an effort to locate target compounds. SFC is beneficial because the solvent and sample are carried by the gas and the amount of solvent needed during a purification run is substantially less than the volume used in HPLC. Also, the amount of waste solvent at the end of a run is substantially less, so less waste solvent needs to be handled. SFC, however, requires pressure and temperature regulation that is difficult to control accurately and reliably long term.
There are many different configurations of the purification instruments. They typically share commonality in the concept wherein that samples are delivered to a chromatography instrument where compounds are separated in time, and a fraction collector collects the target compound. In order for these instruments to maintain the high throughput process, the instruments must be able to handle large sample numbers, as well as large samples in terms of mass weight and solvent volume. Tradition would specify the use of a semiprep or prep scale chromatography system for a typical milligram synthesis. While this is achievable, it has a low feasibility in a high throughput environment because several issues become apparent in such practice: large solvent usage, generation of large amounts of solvent waste, expensive large-bore columns, and relatively large collection volumes of target compounds. If the proper flow rate or column size is not used, sufficient chromatographic purity will not be achieved.
A variety of column configurations have been developed in an effort to improve the chromatographic results. U.S. Pat. No. 4,554,071 discloses a pre-column for high pressure pre-concentration of material to be chromatographed when the substances are provided in trace amounts. The pre-column is a vessel-shaped body that narrows internally at both ends and that is packed with a selected carrier material. The pre-column is connectable to a conventional chromatography column. Liquid sample is added at high pressure into the narrowed top end, and the selected components are absorbed by the carrier material. The non-absorbed fluid is drained from the pre-column through a separate outlet tube not connected to the chromatography column. The concentrated material is eluted with a solvent or solvent mixture and the concentrated sample and solvent are then loaded into the chromatographic column. This concentration process and subsequent separation process through the column can utilize a large amount of solvent to achieve a desired separation of lo the sample.
U.S. Pat. No. 4,719,011 discloses a modular, high pressure liquid chromatography column. The column includes segments with flanged sections that can be combined to increase or decrease the column length. Segments having different inner diameters can also be combined to provide an inner diameter deemed necessary to provide the type of chromatography for the mobile phase being treated. Accordingly, the same modular components are usable in different combinations for different chromatographic runs. The mass sample and solvent volume, however, dictate the diameter and length of the column to be constructed with these modular segments.
Columns used for high throughput processes must be able to handle large sample numbers and large samples in terms of mass weight and solvent volume. Conventional chromatography for large samples typically uses large-bore columns and large volumes of solvent. If the proper flow rate or column size is not used, the desired chromatographic purity will not be achieved. As a result, chromatography of large samples results in large solvent usage, generation of large amounts of solvent wast
Krakover Jonathan D.
Maiefski Romaine
Ontogen Corporation
Perkins Coie LLP
Therkorn Ernest G.
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