Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Preserving or maintaining micro-organism
Reexamination Certificate
2002-03-08
2003-12-16
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Preserving or maintaining micro-organism
C435S235100, C435S870000, C435S948000, C424S488000, C424S211100, C424S212100, C424S214100, C424S217100, C424S218100, C424S219100, C424S264100
Reexamination Certificate
active
06664099
ABSTRACT:
The present invention relates to the preservation of viruses and mycoplasma. In particular, it relates to an ultra-rapid method by which such materials can be preserved using the disaccharide, trehalose. By this method, a long term preservation of viruses and/or mycoplasma can be achieved and, especially, living attenuated vaccines can be prepared.
The preservation of biodegradable materials by dehydration and osmoconcentration is a familiar and ancient technology. When the task of preserving sensitive biomolecules became necessary, simple drying by dehydration failed, as structural water was removed, causing subsequent denaturation and loss of vital activity. Cryopreservation in liquid nitrogen and lyophilisation have become the accepted methods for the long term preservation of sensitive biomolecules, the latter method being used extensively for the preservation of live attenuated vaccines.
Improved thermotolerance of freeze dried Rinderpest vaccine has been achieved by extending the secondary drying cycle, in order to reduce residual moisture (RM) levels to around 1%-2%. This entails long and high energy consuming operational cycles of up to 72 hours as described by Mariner, J. C. et al., Vet. Microbiol., 1990, 21, 195-209. Vaccines produced by this method are known and are distinguished from the standard vaccine by the name “THERMOVAX”.
As mentioned above, these currently used processes are time consuming and involve high energy input. Furthermore, lyophilisation confers only a modest level of thermotolerance in the final product and refrigeration is still required to reduce deterioration during storage. This is a particular problem for live vaccines to be used in tropical climates since these lose potency with the unfortunate result that vaccination programs carried out in the field in tropical countries, where monitoring the “cold chain” is difficult, ultimately lead to vaccination of patients with substandard or, in some cases, useless vaccine.
During evolutionary natural selection, certain species of plants and animals acquired the remarkable and elegant ability to tolerate extreme dehydration, remaining dormant in hostile environments for very long periods of time and yet able to assume complete vital activity on rehydration. Examples include the resurrection plant
Selaginella lepidophya
, the brine shrimp
Artemia salina
(Clegg, J., J.Comp.Biochem.Physiol, 1967, 20, 801-809), the yeast
Saccharomvces cerevisiae
(Coutinho, E., Journal of Biotechnology, 1988, 7, 23-32) and the tardigrade
Macrobiotus hufelandi
(Kinchin, I. M., Biologist, 1995, 42, 4). Such organisms are termed cryptobiotic and the process by which they survive is known as anhydrobiosis. All species of animals and plants which display this ability contain the disaccharide trehalose (&agr;-D-glucopyranosyl-&agr;-D-glucopyranoside). Its presence generally in the order of 0.2 g/g dry cell weight in most cryptobionts enables them to resist extreme dehydration, high temperatures, X-rays and also in some species of tardigrades, pressures as high as 600 Mpa.
Colaco et al., Biotechnology, 1992, 10, 1007-1011, describe the benefit of rapid drying of biological materials using trehalose. This method mostly refers to the drying of restriction enzymes and immunoglobulins onto preformed solid matrices, such as cellulose fibres or onto the surfaces of plastic plates for diagnostic purposes such as ELISA or similar diagnostic applications in the laboratory. Problems arise, however, with these techniques when scaling up to industrial applications, such as large scale commercial vaccine production where much larger unit numbers and volumes have to be handled using mandatory aseptic techniques in partially sealed vials. To meet the operational requirements of large scale vaccine production, where unit volumes from 1.0 ml upwards and production batches of 20 litres are typical, a different strategy is required to remove that volume of water in an economically acceptable time. Drying at atmospheric pressure even at the highest physiologically tolerated temperatures would require an unacceptably long time to remove water quickly enough from partially stoppered vaccine vials and would inevitably result in denaturation and loss of potency.
The present invention is concerned with a method of preservation of viruses or mycoplasma using trehalose under conditions which cause water to be removed while, at the same time, allew the biological integrity of the material to be maintained.
Accordingly, the present invention provides a method of preserving a biologically-active material comprising a live virus or mycoplasma which method comprises the steps:
(i) mixing an aqueous suspension of the biologically-active material with a sterile aqueous solution of trehalose to give a trehalose concentration in the mixture in the range of from 0.2 to 10% w/v;
(ii) subjecting the mixture to primary drying, for 30 to 60 minutes, at a pressure of less than atmospheric and at a temperature initially no greater than 37° C., and which is controlled not to fall to 0° C. or below and which finally is no greater than 40° C. to form a glassy porous matrix comprising glassy trehalose having a residual moisture content of not greater than 10% and containing, within the matrix, desiccated biologically-active material; and
(iii) subjecting the glassy porous matrix of step (ii) to secondary drying for 10 to 30 hours at a pressure not greater than 0.1 mbar and at a temperature which finally is in the range of from 40 to 45° C. to form a trehalose matrix having a residual moisture content of not greater than 2% containing, within the matrix, desiccated biologically-active material.
By using the method of the invention it is possible to produce a live vaccine with, compared to prior art methods, enhanced biological characteristics and distinct commercial advantages. Vaccines prepared using the method of the invention are dried much more quickly than those using conventional freeze drying procedures. For instance, the method of the invention can be used to dry trehalose/biologically-active material mixtures to a moisture content of about 10% in less than one hour. Further dehydration to a residual moisture content of about 1-2% can be achieved in less than 30 hours, for instance about 20 hours, compared to a period of 50 hours by conventional freeze drying procedures. Furthermore, damage caused by solute concentration is minimised according to the present invention and particularly damaging ice crystallisation is avoided. The thermostability of the biologically-active material preserved in the trehalose glassy matrix is greater than that of materials preserved by prior art methods and, thus, the necessity of the “cold chain”, which is a serious constraint with conventional freeze-dried vaccine, is minimised. The product of the present invention can be exposed to high ambient temperatures, e.g., up to about 45° C., for prolonged periods without any substantial loss of biological activity. In addition to these and other advantages of the present invention the product of the method exhibits instantaneous “flash solubility” upon rehydration.
The method of the present invention is suitable for achieving the long term preservation of viruses and mycoplasma. In particular, it can be used to preserve highly labile live attenuated viral components and mycoplasma components that can be rehydrated to form vaccines. Examples of such biologically-active materials that can be preserved according to the method of the invention include:
Family: Paramyxoviridiae
Subfamily: Paramyxovirinae
Genera: Parainfluenza virus group Measles, Rinderpest, canine
distemper, Peste des Petits Ruminants (PPR)
Paramyxovirus: mumps virus (Mumps)
Genus: Rubivirus, Rubella (German Measles)
Genus: Flavivirus, Yellow fever virus (Yellow Fever)
Genus: Rhabdoviruses, Lyssaviridiae (Rabies virus)
Picoma viruses (Polio virus)
Newcastle Disease virus
Mycoplasma:
Mycoplasma mycoides
(Contagious Bovine Pleuropneumonia)
Brucella abortus: Strain 19 vaccine
Chlamydia:
Chlamydia psittaci
(Enzootic abor
Anhydro Limited
Bozicevic, Field & Francis
Field Bret E.
Stucker Jeffrey
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