Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
1994-11-03
2002-02-05
Le, Long V. (Department: 1641)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S081000, C422S105000, C210S656000, C210S198200
Reexamination Certificate
active
06344172
ABSTRACT:
BACKGROUND OF THE INVENTION
Chromatographic techniques are well known in the art as means for separating components (solutes) present in a mixture. These techniques are particularly useful in the chemical and biotechnological arts. True chromatography describes the separation of solutes according to their different partitioning between two (or three) phases. The phases generally are solid and liquid, and solute partitioning results in their differing mobilities through a layer of solid, typically particulate, matrix in the presence of a flowing phase. Solute transfer through the layer may be along a pressure gradient, generally referred to as “liquid chromatography”. Typically, the sample to be separated is applied to a column filled with pellets or grains of a chromatography separation medium, and a solvent flow is maintained through the column at a steady rate. Components of the mixture are carried along by the solvent flow until each substance exits the column as a “peak” in the output, different peaks being more or less broad and overlapping.
Chromatographic matrices can separate components by any of a number of criteria, including size, electrical charge, hydrophobic interaction, and/or specific affinity for the matrix or binding sites thereon. Because the components in the mixture will vary in their affinity for the matrix, their partitioning as they pass through the matrix separates the components so that they exit the matrix sequentially, separated temporally and spatially. Determination of the location of the various separated components, or of a given component of interest within the sequence, generally is achieved by collecting the fluid phase exiting the matrix (i.e., the effluent stream) as a series of fractions and sampling these fractions to identify their contents by any of a number of means known in the art.
Resolution of the various components in the mixture depends on several considerations, chief among them being the partitioning ability of the matrix and the system's theoretical plate height and plate number (see infra). In general, a large surface area-to-volume ratio is desired. Matrices for liquid chromatography systems typically are housed in cylindrical chromatography systems known as columns. In electrophoresis systems, high resolution also demands efficient removal of the heat generated by the applied electric field. Capillary electrophoresis, or other electrophoretic modules which provide a large surface area-to-volume ratio dissipate Joule heat well, allowing rapid analysis without significant loss of resolution.
Techniques are known for treating the chromatography medium to enhance the affinity of the column generally for cationic or anionic substances, or to cause a reversible bonding to particular chemical groups or biologically active materials, so that samples containing these groups or materials may be releasably bound to the column and subsequently eluted.
To achieve a particular separation, the general practice of chromatographic separation involves identifying or selecting a particular medium or coated medium, and an optimum solvent, solvent flow rate, pH, ionic concentrations and other environmental conditions, such that the starting mixture will separate into a number of relatively narrow bands and such that at least the substance of interest passes, or may be made to pass, as a distinct output.
The determination of an appropriate set of separation conditions for a particular substance, which may have an as yet undetermined chemical structure and conformation and unknown chromatographic affinities, is a task that involves experience, experimentation, intuition and luck. Because of the complex dependence of the transport and adsorption mechanisms of biomolecules on multiple different conditions, further experimentation is usually necessary even when it is desired only to scale up a known process to operate at greater speed or to utilize a larger column. In order to meet the separation objectives of high purity, high speed and/or high volume separation, a very large number of separation conditions must be experimentally analyzed to determine one suitable set of operating conditions.
In general, the transport of material in a separation column proceeds on a macroscopic level by flow past and between the grains or pellets of the chromatography medium, while the degree of separation and column capacity are governed more by the rates at which the particular components diffuse along branching paths into and out of pores in the medium, and are repeatedly adsorbed and released along the diffusion path. By increasing the flow rate to increase process output, one generally broadens the eluted peak width of each component, thus sacrificing the resolution and hence the purity of the separated components; above a certain flow rate threshold, premature solute breakthrough may occur.
The need to monitor a product's status during its synthesis or purification is well known in the art. Status monitoring is particularly important in multistep preparation protocols. Frequently, the identity and, often, the quality of a product in a mixture must be determined at each step. Product monitoring also may be used as part of a feedback system to adjust process parameters. Generally, identification is determined using a previously established criterion for identification, for example, a characteristic absorbance measured at a given wavelength. If the product of interest is a protein, identification also may be by molecular weight, activity, and/or immunoaffinity.
It is an object of the invention to provide a rapid, adaptable, and repeatable system and apparatus for identifying the presence and/or location of a molecule of interest during any preparative or analytic protocol. The ultimate goal is to separate one or more components of a protein mixture by exploiting the benefits of high speed chromatographic techniques. Objects of the invention include two dimensional analysis to enhance resolving power of a chromatographic system, real time monitoring of solute concentration in a process mixture, detection of trace solute contaminants in a solution that contains a major amount of a dissolved product, rapid determination of the presence and location of a solute in a chromatography effluent during, e.g., any step of a preparative procedure, production of a profile of a mixture representative of the nature and relative concentration of structured variants of a given solute, and the rapid assessment of the success of a purification or separation protocol.
SUMMARY OF THE INVENTION
The invention features an apparatus and methods for the rapid and efficient separation of proteins and other biological macromolecules. The apparatus includes sample input means, a first liquid chromatography column, a multiport injection valve connecting the sample input means to the column, a second chromatography column in communication with the multiport injection valve, the second column being operative successively with or alternatively to the first column, pump means for providing variable pressure delivery of a solution to the column via the multiport valve, and program means for specifying a sequence of system control programs.
In preferred embodiments, the apparatus further includes control means in communication with the pump means for controlling the pressure of delivery of the solution; and solution input means including plural solution reservoirs, and a mixing valve, connecting the solution input means to the sample input means, operative to mix solution from the reservoir, wherein the program means specifies the mixing of solution by the mixing valve, and the delivery of the mixed solution to the column via the multiport injection valve; and detector means for detecting and recording column output; and matching means for identifying a pattern of detected output data, the template matching means being operatively keyed to means for developing a control program for liquid chromatography separation.
In another embodiment, the invention features an apparatus for the separ
Afeyan Noubar
Gordon Neal F.
Counts Gary W.
Le Long V.
PerSeptive Biosystems Inc.
Testa Hurwitz & Thibeault LLP
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