Multi-sample fermentor and method of using same

Details Multi-sample fermentor and method of using same Multi-sample fermentor and method of using same

2002-02-08

2004-04-20

Redding, David A. (Department: 1744)

Chemistry: molecular biology and microbiology

Apparatus

Bioreactor

C435S296100, C435S813000, C435S818000, C099S276000, C099S323100

Reexamination Certificate

active

06723555

ABSTRACT:

COPYRIGHT NOTIFICATION
Pursuant to 37 C.F.R. 1.71(e), a portion of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
Fermentation is a key technology in many fields and industries and is performed both on a mass production scale and on an experimental, bench top scale. For example, fermentation systems are used for the production of a large number of products such as antibiotics, vaccines, synthetic biopolymers, synthetic amino acids, and proteins. Fermentation technology is integral in the production of recombinant proteins using biological organisms, such as
E. coli
, and many other cell cultures. For example, production of commercial pharmaceuticals such as recombinant insulin (Eli Lilly), erythropoietin (Amgen), and interferon (Roche) all involve fermentation as an essential step.
In addition, the recent identification of the tens of thousands of genes comprising the human genome highlight an important use of fermentation, namely the production of the proteins encoded by those genes. The determination of each gene's function is of paramount importance and therefore, the proteins encoded by those genes must be produced, e.g., by fermentation methods. Because each gene encodes at least one protein, tens of thousands of proteins must be produced and isolated. However, fermentation and isolation of the resulting protein products typically requires several labor intensive and time-consuming procedures. Fermentation systems that can produce tens of thousands of different proteins, e.g., in amounts sufficient for analysis are therefore needed. An additional advantage would be fermentation systems that are amenable to high throughput processes and the microtiter plate format used in many biotechnolgy applications.
Although, rapid advances in biotechnology have enabled the development of high throughput alternatives to traditional laboratory bench top processes, fermentation methods have not been amenable to automation. For example, limits in current fermentation technology prevent the uninterrupted processing flow that characterizes automated high throughput systems. Existing fermentation systems typically involve multiple handling steps by either a batch processing method or a continuous processing method.
Fermentations are typically carried out in batch mode or continuous mode. Batch mode processes are those in which a fermentor is filled with a medium in which cells are grown and the fermentation is allowed to proceed with the entire contents removed from the fermentor at the end for downstream or post-processing. The fermentor is then cleaned, re-filled, and inoculated for the fermentation process to be performed again. For example, current production scale batch processes involve first fermenting in large scale, bulk fermentation vessels, then processing the fermentation medium to isolate the desired fermentation product, followed by transferring this product into the production stream for further processing, and finally cleaning the fermentation apparatus for the next batch. In a large scale batch culture, it is generally necessary to provide a high initial concentration of nutrients in order to sustain cell growth over an extended time. As a result, substrate inhibition may occur in the early stages of cell growth and then may be followed by a nutrient deficiency in the late stages of fermentation. These disadvantages result in sub-optimal cell growth rates and fermentation yields. Another disadvantage of this method lies in the need to individually dispense the fermentation products from the bulk fermentation apparatus into separate sample vessels for further processing. Thus, by producing the fermentation product on a bulk scale, the fermentation product is not immediately available for automated processing. Further disadvantages include the decreased efficiency of both transferring the material to another sample vessel, as well as cleaning and sterilizing the fermentation apparatus for the next batch. These disadvantages result in increased production costs, inefficient production times and decreased yields.
Continuous batch processes involve siphoning off the fermentation product from the bulk fermentation vessel and continuously adding nutrients to the fermentation medium according to a calculated exponential growth curve. This curve, however, is merely an approximation that does not accurately predict cell growth in large, industrial scale quantities of fermentation medium. Consequently, due to the unpredictable nature of large scale fermentation environments, experienced personnel are required to monitor the feeding rate very closely. Changes in the fermentation environment may result in either poisoned fermentation products being siphoned off into the production stream or sub-optimal production yields due to starved fermentation mediums. As a further disadvantage, unpredictable fermentation product yields affect the accuracy of subsequent processing steps. For example, when the fermentation yield decreases, the amount of aspirating, the amount of reagent dispensed, or the centrifuge time is no longer optimized, or even predictable. Frequent or continuous monitoring of the fermentation process and adjustment of the fermentation conditions is often not practicable or efficient in a production scale process.
Neither of the current processes provides an efficient, automated production scale fermentation. However, fermentation remains a key processing step in a number of industries, particularly in biotechnology industries, and thus a need exists for incorporating fermentation processes into automated high throughput systems. A process that produces a precise, known, and repeatable amount of untainted fermentation product with limited human interaction or sample vessel transfer is essential to integrating fermentation into modern production processes. The present invention meets these as well as other needs that will be apparent upon review of the following detailed description and figures.
SUMMARY OF THE INVENTION
The present invention provides methods and apparatuses for simultaneously fermenting a plurality of samples, e.g., small samples in an 8 by 12 array. For example, the present invention provides a fermentation apparatus comprising a container frame configured to contain a plurality of sample vessels and a gas distribution arrangement coupled to the container frame. The fermentor provides for fermentation of large numbers of samples, e.g., to produce a large number of proteins. Alternatively, the fermentors of the invention provide a more efficient route for production scale fermentations.
In one aspect the invention provides a container frame configured to contain a plurality of sample vessels, e.g., in an array; and, a gas distribution arrangement configured to provide gas to a plurality of sample vessels, e.g., when the sample vessels are positioned in the container frame. The container frame is typically configured to contain an array of sample vessels, e.g., an 8 by 12 array, e.g., holding at least about 96, 384, or 1536 samples. The gas distribution arrangement typically comprises a gas inlet configured to deliver gas to a plurality of cannulas, which are configured to provide gas to the sample vessels.
In one embodiment, the container frame is a transportable container frame, e.g., configured for transport to a post-fermentation processing station. In addition, the container frame is optionally configured for placement within a temperature controlled area, e.g., water bath or a temperature controlled room, wherein a temperature controller is coupled to the container frame and/or to one or more sample vessels within the container frame.
In other embodiments, the container frame is autoclavable. For example, the container frame is autoclavabl

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