Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Reexamination Certificate
2000-05-19
2002-10-15
Gitomer, Ralph (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
C435S034000, C435S259000
Reexamination Certificate
active
06465201
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for rapidly reducing background adenosine triphosphate (ATP) in a mammalian cell preparation and detecting the presence or absence of microorganisms in the cell preparation using ATP bioluminescence.
BACKGROUND OF THE INVENTION
Protein-based biopharmaceutical products are often produced by cloning the desired genes into mammalian tissue culture cells and producing the protein products on a large scale in a mammalian cell fermentation facility. However, microbial contamination of mammalian cell fermentors results in cell death and leads to losses and delays in production. Proactive measures, such as monitoring cell culture production and detecting the presence of low levels of microorganism contamination, are useful to reduce loss of time and revenue.
Contaminating microorganisms are typically detected in cell culture by membrane filtration, followed by culturing on media until colonies are visible, or by sterility testing in liquid media. The culturing method typically does not produce results for several days and the standard sterility testing requires one to two weeks for results. Although other detection methods are known, including Polymerase Chain Reaction for detecting specific organisms such as Mycoplasma and methods for detecting general microbial contamination in the blood testing market such as BacT/Alert® from Organon Tecknika and Bactec™ from Becton Dickinson, these methods do not utilize ATP bioluminescence and do not have the sensitivity or speed of an ATP detection system.
SUMMARY OF THE INVENTION
It is therefore a primary object of this invention to provide a method for rapidly detecting and accurately enumerating microorganisms in mammalian cell preparations using ATP bioluminescence.
It is a further object of this invention to provide a highly sensitive method for rapidly and capably detecting and enumerating low levels of microorganisms in mammalian cell preparations.
It is a further object of this invention to provide a method of reducing ATP background in tissue culture cells for rapidly detecting and enumerating microorganisms in mammalian cell preparations using ATP bioluminescence.
It is a further object of the invention to provide a convenient method for preparing samples for detecting and enumerating microorganisms in mammalian cell preparations.
It is a further object of the invention to provide a method for detecting and enumerating microorganisms that quantifies the microbial organisms and automatically records and documents results.
It is a further object of the invention to provide a method for rapidly and capably detecting and enumerating low levels of microorganisms so that informed decisions can be made before expanding cultures or using cell lines for patient treatments.
It is a further object of the invention to provide a method for detecting and enumerating microorganisms in mammalian cell preparations in less than 24 hours.
A preferred method of the invention for rapidly detecting and enumerating microorganisms, having a level of microbial ATP, in mammalian cell preparations, comprises the steps of: providing a mammalian cell preparation comprising mammalian cells having a level of mammalian ATP; reducing the level of mammalian ATP in the cell preparation by, differentially lyzing the mammalian cells with one or more detergents to extract the mammalian ATP, treating the extracted mammalian ATP with one or more ATP hydrolyzing enzymes, such as adenosine triphosphatase (ATPase); immobilizing the microorganisms and washing away the detergent and the ATPase by filtering the mammalian cell preparation through a micropartitioned hydrophilic/hydrophobic membrane; extracting the microbial ATP using an extracting reagent; applying a bioluminescent reagent onto the membrane; and detecting and enumerating the microorganisms. The method may further comprise, after the extracting step, the step of drying the membrane at temperatures between about ambient temperature to 40° C.
The method, after the step of lyzing the mammalian cells, may further comprise the step of incubating the cell preparation for about 15 minutes at room temperature; and after the step of filtering the cell preparation, may further comprise the step of incubating the cell preparation for about 8-24 hours at 25-35° C.
The detergents preferably comprise one or more detergents selected from a group consisting of 2% Tween 80, 13% Tween 80, Milli-Q water and 0.005% Triton X-100 with or without 10-5% SDS, and the bioluminescent reagent preferably comprises a luciferin-luciferase reagent. The microbial ATP extracting reagent preferably comprises either methanol, or ethanol, mixed with 0.1 to 5% by weight of ammonium hydroxide.
Another preferred method of the invention for rapidly detecting and enumerating microorganisms, having a level of microbial adenosine triphosphate (ATP), in mammalian cell preparations, comprises the steps of: providing a mammalian cell preparation comprising mammalian cells having a level of mammalian ATP; reducing the level of mammalian ATP in the cell preparation by, differentially lyzing the mammalian cells by osmotically shocking the cells in water to extract the mammalian ATP, treating the extracted mammalian ATP with one or more ATP hydrolyzing enzymes; immobilizing the microorganisms and washing away the hydrolyzing enzyme by filtering the mammalian cell preparation through a micropartitioned hydrophilic/hydrophobic membrane; extracting the microbial ATP using an extracting reagent; drying the membrane; applying a bioluminescent reagent onto the membrane; detecting and enumerating the microorganisms.
Similar to the first described preferred method, after the step of lyzing the mammalian cells, the method may further comprise the step of incubating the cell preparation for about 15 minutes at room temperature, and after the step of filtering the cell preparation, the method may further comprising the step of incubating the cell preparation for about 8-24 hours at 25-35° C.
The bioluminescent reagent preferably comprises a luciferin/luciferase reagent and the microbial ATP extracting reagent preferably comprises methanol, or ethanol, mixed with 0.1 to 5% by weight of ammonium hydroxide.
Yet another preferred method of the invention for rapidly detecting and enumerating microorganisms, having a level of microbial adenosine triphosphate (ATP), in mammalian cell preparations, comprising the steps of: providing a mammalian cell preparation comprising mammalian cells having a level of mammalian ATP; reducing the level of mammalian ATP in the cell preparation by, differentially lyzing the mammalian cells with one or more detergents to extract the mammalian ATP, incubating the cell preparation for about 15 minutes at room temperature, treating the extracted mammalian ATP with one or more ATP hydrolyzing enzymes during and/or after the incubation step; immobilizing the microorganisms and washing away the detergent and the hydrolyzing enzyme by filtering the mammalian cell preparation through a micropartitioned hydrophilic/hydrophobic membrane; incubating the cell preparation for about 8-24 hours at 25-35° C.; extracting the microbial ATP using an extracting reagent; drying the membrane; applying a luciferin/luciferase reagent onto the membrane; and detecting and enumerating the microorganisms.
Similar to the first described preferred method, the detergents preferably comprise one or more detergents selected from a group consisting of 2% Tween 80, 13% Tween 80, Milli-Q water and 0.005% Triton X-100 plus 10
−5
% SDS; and the microbial ATP extracting reagent preferably comprises methanol, or ethanol, mixed with 0.1 to 5% by weight of ammonium hydroxide.
Other objects, features and advantages will occur to those skilled in the art from the following description of the preferred methods.
DETAILED DESCRIPTION OF THE PREFERRED METHODS
The method of the invention is based on ATP bioluminescence and utilizes the light producing enzyme, luciferase, derived from fireflies for the rapid detection of microorganisms.
Presente Esther
Upperman Susan
Young Barbara
Gitomer Ralph
Millipore Corporation
Nields & Lemack
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