Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-09-12
2004-08-24
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S004000, C435S007100, C435S007200, C435S239000, C436S063000, C436S169000, C436S518000, C436S523000, C436S546000
Reexamination Certificate
active
06780581
ABSTRACT:
FIELD
The present invention relates to a method for the preparation of products containing a quantum of bioparticles, preferably viable bioparticles, and to said products produced thereby. More particularly, the invention relates to a method for the preparation of products containing a defined quantum of bioparticles, and to said products produced thereby.
BACKGROUND
There are many procedures performed in life sciences that involve the manipulation of small bioparticles such as cells, bacteria, viruses, protozoa, sperm, eggs, embryos and larvae. Generally the manipulation of these small bioparticles is inherently difficult because the bioparticles are too small to be visualised with the naked eye.
Where one is performing an experiment or procedure that involves adding bioparticles to a vessel (for example, a test tube) there is currently no simple technology available which allows one to know exactly, or at least with a minimal degree of error, how many bioparticles have been added. Typically one would prepare a suspension of the bioparticles and then perform an analysis (for example, enumeration by microscopy or culture on an agar plate), to estimate the number of bioparticles per volume of fluid. An aliquot of this suspension, containing an estimated number of bioparticles, would then be used for a desired purpose; the exact number of bioparticles in the aliquot not being known.
In addition to problems associated with estimation of the numbers of bioparticles by sampling, further problems may result during manipulation of the bioparticles in a particular procedure. For example, an unknown amount of the bioparticles are inevitably lost due to factors such as adhesion to surfaces of vessels or pipettes used, or to denaturation or death of some of the bioparticles. Further, bioparticles can lose their viability over time and accordingly products containing such particles may suffer from a short shelf life. Combined with the above problems, these factors may create gross inaccuracies in experimental data.
A number of products are known which attempt to provide a standardised product having a defined number of bioparticles. However, these products unfortunately fail to address all of the problems above mentioned, and accordingly, may be considered to fall short of providing a desirable product. For example, the degree of error in respect of the number of bioparticles present from one sample of a product to another sample of the same product is likely to be in the order of greater than 50%, in many cases the number of bioparticles present may vary 10 to 100 fold or more.
One example of such known products is Cultiloops® (Oxoid, Australia). Cultiloops® are disposable bacteriological culture loops that contain a loopful of freeze-dried culture of a specific microorganism and are generally used for quality control purposes in microbiology laboratories. While Cultiloops® save time in the preparation of cultures for quality control they unfortunately do not contain accurately defined numbers of cells per loopful. Further, it is possible that a number of the cells present may not be in a viable state.
Several companies supply vials containing an approximate number of microorganisms in a freeze-dried form. Typically, these are manufactured to an accuracy of 1 order of magnitude; for example, a vial will contain between 1000 and 10000 bacteria. To use these products one generally adds water to the vial to resuspend the freeze-dried microorganisms, subsequently using a pipette to transfer the microorganisms to a sample. Due to the nature of this product, and the means by which it is used, it may not be considered to adequately address the issue of providing accurate and consistent numbers of bacteria, or the issue of the loss of unknown quantities of bacteria during manipulation as a result of adhesion to the side of the vial or the pipette.
BTF Pty Ltd (Australia) market a product known as EasySeed C&G that provides an accurately defined number of inactivated Cryptosporidium and Giardia in fluid in a test tube. While it may be considered that this product overcomes many of the issues associated with providing accurate numbers of microorganisms, during use of the product an unknown number of the Cryptosporidium and Giardia are generally lost due to adhesion to the side of the test tube or pipette. Further, the cells are not provided in a viable state.
A further example of a presently available product are lenticles, freeze-dried quality control samples prepared by the UK Public Health Laboratory Service (PHLS). Lenticles are prepared by pipetting drops of a viscous bacterial culture onto a cold surface, freezing the drops, and then freeze-drying the drops to form a lens-shaped freeze-dried pellet. While the lenticules may be considered to overcome the inaccuracies associated with handling liquid quality control samples, they unfortunately do not contain accurately defined numbers of bacteria.
A further product, known as TrueCount® (Becton Dickinson, San Jose, USA), is used in conjunction with flow cytometry to allow one to determine the number of specific cells per milliliter of blood, for example. The product consists of dried balls of approximately 1 mm diameter that contain approximately 50,000 fluorescent beads of approximately 5 &mgr;m diameter. While this product may overcome problems associated with the loss of particles during manipulation of a liquid sample, it does not contain an accurately defined number of beads within the dried ball. Further, as the beads do not represent biological material, the product, and the procedure of producing the same, is not concerned with, and therefor may not adequately address, the issue of maintenance of viability of the particles.
U.S. Pat. No. 3,932,943 describes a process for the production of a homogeneous, lyophilised particulate product containing at least one biologically active component. The process involves spraying a solution or colloidal suspension containing the biologically active component into a moving bath of fluorocarbon refrigerant, subsequently lyophilising the resultant frozen droplets. The inventors report that the product has a spherical shape, free-flowing properties, and rapid dissolution times. However, the process does not address the issue of preparing a product that contains accurately defined numbers of bioparticles. In addition, it may be considered that this process does not adequately address the issue of maintenance of bioparticle viability, especially where such bioparticle is a cell. U.S. Pat. No. 6,106,836 describes a process for the production of a vaccine product comprising a container with freeze-dried vaccine components therein. The process involves the formation of spheres containing biological components of estimated numbers utilising the steps of freezing droplets of a suspension containing the biological components in a cryogenic liquid and subjecting them to freeze-drying. The process of this patent does not immediately address the issue of preparing a freeze-dried product that contains accurate numbers of bioparticles. By contrast, products containing estimated numbers of components are made via the above mentioned process, their titre measured, and then a number of products combined, or used to supplement another product, to obtain a desired quantity of components. In addition, the process of U.S. Pat. No. 6,106,836 may not be considered to adequately address the issue of maintenance of the viability of bioparticles during processing; rather, the process centres on the loss of viability of the bioparticles followed by supplementation of the resultant product with additional viable particles.
Further, U.S. Pat. No. 3,655,838 describes a method for the preparation of pelletised reagents purportedly in a stable, accurate form. In this method a suspension containing predetermined concentrations of desired reagents is formed into calibrated droplets which are allowed to fall into a liquid having certain characteristics, one of which is a temperature gradient suitable to freeze the droplets. Subsequentl
Gauci Mark
Vesey Graham
BTF Pty Ltd
Morgan & Lewis & Bockius, LLP
Srivastava Kailash C.
Weber Jon P.
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