Stationary capillary electrophoresis system

Abstract

A system and method for capillary electrophoresis are provided for analyzing a macromolecule prepared from a complex liquid mixture. In particular applications, methods and apparatus are provided for separating and analyzing a solution containing a denatured macromolecule by employing a stationary capillary electrophoresis apparatus. An apparatus for capillary electrophoresis includes an inlet chamber and a capillary electrophoresis column. One end of the column is fixed at the interior of the inlet chamber. The column has a length of at least about 20 centimeters. Also included is a liquid source adapted for automatic control. The liquid source supplies a liquid sample through an input valve into the inlet chamber so that the sample is in fluid communication with the end of the column. A method for capillary electrophoresis includes automatically supplying the liquid sample to the apparatus.

Description

RELATED APPLICATION[0001]This application is a continuation of U.S. application Ser. No. 10 / 601,181, filed Jun. 20, 2003. The entire teachings of the above application are incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]Analysis of macromolecules in complex mixtures is challenging in many chemical and biochemical processes. For example, the analysis of a macromolecule product, e.g., a protein, typically involves first preparing a sample of a macromolecule from a complex mixture for analysis. FIG. 1 depicts an example of a macromolecule preparation process 100, which involves taking a sample from a complex liquid mixture, e.g. a biofluid in a bioreactor 102, separating a macromolecule 104 from other components in the mixture, and processing it to deliver a prepared macromolecule 104′ for analysis at analyzer 106.[0003]Effective process control generally requires accurate and frequent sampling, yet sampling of an operating bioreactor is associated with numerous probl...

AI Technical Summary

Benefits of technology

[0016]The methods and apparatus disclosed herein provide significant advantages to analyzing a macromolecule prepared from a complex liquid mixture by capillary electrophoresis. Fixing the column to the inlet chamber provides durability compared to robotic CE systems, and removes the need for calibration of the robotic system when a column is replaced. Furthermore, the fixed chamber coupled with valved input allows the system to exclude the liquid sample from the exterior environment and allows rapid sampling and high throughput compared to chip based CE systems. The longer CE column allows better separation and higher electric fields compared to chip based systems. Furthermore, the combination of high throughput with provision of automatic control allows analysis at a higher rate, enabling improved analysis and control of time-sensitive bioreactor processes.

[0017]The combination of the stationary CE system with automated macromolecule sample preparation leads to efficient, high-throughput analysis of macromolecules from complex bioreactor mixtures. Rapid, sequential sampling and analysis can be conducted without contamination or manual intervention. A sample can be automatically filtered and delivered to the CE column for analysis, without being exposed to the environment. The combination of cleaning solution reservoirs allows the system to be automatically cleaned in preparation for the next sample.

Problems solved by technology

Analysis of macromolecules in complex mixtures is challenging in many chemical and biochemical processes.

Effective process control generally requires accurate and frequent sampling, yet sampling of an operating bioreactor is associated with numerous problems, particularly contamination from sampling.

Other contaminants, e.g., chemical contaminants, can affect the growth of the process bacteria and can confound the analysis of process components in the bioreactor fluid.

Contamination can also affect the sampling and analysis apparatus.

For example, wild or process bacteria can colonize the sampling / analysis system, or the system can accumulate other components form the biofluid, e.g., as salts, nutrients, proteins, peptides, cells, cell components, biopolymers such as polysaccharides, all of which can confound analysis of the desired products.

Additionally, frequent sampling can lead to build-up of the molecule or molecules being analyzed, which can lead to inaccuracy.

In particular, the problem of “backflow”, i.e., liquid cross-contamination, is especially difficult when interfacing two fluidic systems.

Simple valve interfaces are inadequate because valves typically have crevices, joints, dead volume, and the like, where contaminants can lodge and accumulate, only to be released during another sample cycle.

Additionally, valves can fail and allow undesirable contamination to occur before much measurable fluid has leaked.

More complex valved interfaces are known, but some are costly and still suffer some of the problems of simple valve systems, while other examples are unsuitable for high pressure systems.

Needle / septa interfaces are known to avoid backflow but have issues with septa lifetime, needle contamination during transfer, and are particularly troublesome for frequent, automated sampling of larger volumes.

Furthermore, septa replacement itself opens the system for contamination.

Matrix chromatography uses expensive columns that can be prone to plugging when used with complex mixtures that include insoluble or precipitation-prone components.

Centrifugation can be effective but can cause contamination problems as there is no way to readily isolate a sample from the environment during the various sample transfers typically employed, and the size of the centrifuge limits the amount of macromolecule that can be prepared at one time.

Thus both methods are low throughput in terms of amount of macromolecule that can be prepared.

Additionally, both methods are low throughput in terms of the sampling frequency, as the time from sample extraction from a complex bioreactor mixture to analysis of the macromolecule can easily be four hours or more.

Such a slow analysis time leads to poor optimization of reactor processes, resulting in lowered yields, increased costs, increased purification demands, and increased amounts of potentially hazardous biological waste. FIG. 3 depicts a hypothetical example comparing two sampling frequencies, wherein a lower sampling frequency versus time (squares) can miss details in the level of a desired macromolecule versus time (solid line) in a reaction mixture, compared to a higher sampling frequency (circles).

Furthermore, small capillaries are physically fragile and are not suited to high-throughput separations, being easily plugged from the many macromolecules and debris in a complex mixture.

In particular, rapid separation and analysis of macromolecules from complex liquid mixtures, for example, during the analysis of proteins produced in a bioreactor, is especially challenging.

The repetitive motion can easily break the CE column.

Column replacement requires time-consuming recalibration of the robotic motion.

While this hardware is durable, the separation efficiency is limited by the length of CE channel that can be fabricated on a chip.

Attempts to extend the channel length by increasing channel density on a chip generally restrict high electric fields from use, increasing separation time.

Also, the throughput of this technique is limited.

Furthermore, sample transfer as practiced in both the robotic capillary technique and the chip technique expose the analytic solution to undesirable environmental contamination.

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