TITLE
CRYO-PROTECTIVE AGENTS FOR MICROORGANISMS
FIELD OF THE INVENTION
The present invention relates to cryoprotective agents for microorganisms.
BACKGROUND OF THE INVENTION
Vaccines are often produced by growing a pathogen in a culture medium, isolating the pathogen or a portion of the pathogen or a product of the pathogen and using this material as an immunogen for formulating a vaccine. Vaccines containing whole pathogens include whole cell pertussis vaccines and measles vaccines. Vaccines containing portions of the pathogen include acellular pertussis vaccines. Vaccines containing a product of the pathogen include diphtheria and tetanus vaccines. The pathogen, portion or product may require detoxification by for example chemical treatment before it can be used as a vaccine.
An example of a pathogen from which a product is used in the production of a vaccine is Corynebacteriwn diphtheriae and the product is diphtheria toxin. Diphtheria is a life-threatening disease caused by infection with C. diphtheriae, a gram-positive, aerobic, rod-shaped bacterium. The disease is caused by local invasion of nasopharyngeal tissues by toxin-producing strains of C. diphtheriae. The organisms grow in a tough, fibrinous membrane overlying a painful, hemorrhapic, and necrotic lesion, which may be located on the tonsils or within the nasopharynx region. During typical epidemics of the past, the spread of the disease was by droplet infection. Patients who recover from diphtheria may carry toxigenic bacteria in their throats and nasopharynx for weeks or months, unless intensively treated with antibiotics.
Most of the clinical symptoms of diphtheria are due to the potent diphtheria toxin produced from corynebacterioprophage carrying the tox gene. After the prophagc infects the G diphtheriae strain and lysogenization has taken place, the strain becomes virulent. Toxin neutralizing antibodies (antitoxin) induced by active immunization with non-toxic forms (toxoids) of the diphtheria toxin can prevent diphtheria. The current immunization strategy is the utilization of diphtheria vaccines prepared by converting the diphtheria toxin into its non-toxic,

but antigenic, toxoid form by formaldehyde treatment. The diphtheria toxoid is used in various combinations with other vaccine components for mass immunization worldwide. The World Health Organization (WHO) recently estimated that about 100,000 cases worldwide and up to 8,000 deaths per year are due to decreased immunization of infants, waning immunity to diphtheria in adults and insufficient supply of vaccines.
The variant of the Parke Williams 8 (PW8) strain of Corynebacterium diphtheriae is often used to produce the exotoxin from which the toxoid is prepared by chemical modification. In general, a medium formulation with amino acids, trace vitamins, inorganic salts and a carbohydrate source such as maltose promotes excellent growth of the bacterium. Different media, such as the acid digest of casein and the enzymatic digest of beef muscle (trypsin or papain) are suitable media for toxin production. In conventional methods, the bacteria are cultivated in media containing proteinaceous material of animal origin. A commonly used medium in diphtheria production is the NZ-Amine Type A medium, which contains a casein digest. Under optimal conditions, the amount of toxin produced using NZ-Amine Type A media is 180 Lf/mL using the Limes of flocculation method.
The use of proteinaceous material of animal origin in the production of vaccines such as the exemplified diphtheria vaccine can result in the introduction of undesirable contaminants into the diphtheria toxin produced using such a medium.
Most workers have concentrated efforts on the production of growth media substantially free or devoid of animal-components for the cultivation of pathogens such as C diphtheriae. There is also a need to provide seed cultures and in particular cryoprotective agents substantially free or devoid of animal-components for microorganisms including pathogens such as C. diphtheriae.
SUMMARY OF THE INVENTION
The present invention is concerned with cryo-protective agents for microorganisms.
In one aspect of the invention, there is provided a lyophilization medium for a microorganism wherein the medium is substantiality free of animal-derived products and comprises yeast extract and monosodium glutamate. the lyophilization medium may comprise about 1-10% (w/v) monosodium glutamate and about 1-10% (w/v) yeast extract such as about 5

% (w/v) monosodiuin glutamate and about 10% (w/v) yeast extract. The microorganism may be a strain of bacteria including Corynebacterium diphtherias
In a second aspect of the invention, there is provided a method for preparing a fireeze-dried cultnre of a microorganism comprising the steps of providing a quantity of the microorganism, mixing said quanity with a lyophilization medium wherein the medium is substantiality free of animal-derived products and comprises yeast extract and monosodium glutamate to provide a mixture and fteeze-drying said mixture. The lyophilization medium may comprise about 5 % (w/v) monosodium ghitamate and about 10% (w/v) yeast extract such as about 5 % (w/v) monosodium glutamate and about 10% (w/v) yeast extract. Hie freeze-drying of said mixture may comprise steps of achieving a first temperature of about -30 °C for said mixture to provide a cooled mixture and maintaining said cooled mixture in a vacuum for a time until said cooled mixture is substantially dry to provide a dried mixture. Suitable vacuums are about 120 mT and suitable times are between about 10 and about 12 hours. The step of maintaining the cooled mixture in a vacuum for a time until said cooled mixture is substantially dry to provide a dried mixture may comprise maintaining said cooled mixture in a vacuum for a time of between about 10 and about 12 hours and increasing said temperature of about -30 °C to a second temperature of about +20 °C. Suitable vacuums are about 120 mT. The microorganism may be a strain of bacteria including Corynebacterium diphtheriae.
There is also provided a freeze-dried lyophile comprising cells of a microorganism and a lyophilization medium wherein the medium is substantiality free of animal-derived products and comprises yeast extract and monosodium glutamate. The lyophilization medium may comprise about 1-10% (w/v) monosodium glutamate and about 1-10% (w/v) yeast extract such as about 5 % (w/v) monosodium glutamate and about 10% (w/v) yeast extract The microorganism may be a strain of bacteria including Corynebacterium diphtheriae.
BRIEF DESCRIPTION OF DRAWINGS
The present invention will be father understood from the following description with reference to the drawing, in which:
Figure 1 shows a flow diagram outlining the preparation and lyophilization of a C. diphtheriae culture.

DETAILED DESCRIPTION OF THE INVENTION
A flow diagram outlining the preparation and lyophilization of C. diphtheriae culture is shown in Figure 1. A lyophile of C diphtheriae strain 1M1514N3S was inoculated onto an agar plate containing Phytone™ peptone agar and incubated at 36°C for 43-48 hours. The composition of Phytone™ peptone medium is described in Tables 1-2 below.
Table 1. Composition of the Phvtone™ peptone medium containing 15 g/L of Fhvtone™


Table 3. A typical analysis of Phytone™ Peptone as provided by the manufacturer Difco Laboratories is provided below:
Nitrogen Content/Physical Characteristics
Total Nitrogen (TN) (%) 9.0
Amino Nitrogen (AN) (%) 2.4
AN/TN 0.27
Ash (%) 12.4
Loss on Drying (%) 1.5
NaCl (%) 4.0
pH (2% solution) 7.1
Elemental Analysis
Calcium (ug/g) 1001
Magnesium (ng/g) 2435
Potassium (µg/g) 31547
Sodium (µg/g) 34037
Chloride (%) 0.76
Sulfate (%) 0.67
Phosphate (%) 0.64
Amino Acid Analysis
Free Total
Alanine (%) 0.3 2.6
Aspartic Acid (%) 0.3 3.9
Glutamic Acid (%) 0.3 5.9
Histidine (%) 0.2 0.8
Leucine(%) 0.8 2.3
Metbionine (%) 0.2 0.2
Proline(%) 0.1 1.8
Threonine (%) 0.1 0.5
Tyrosine (%) 0.2 0.8
Argkine(%) 0.6 2.1
Cystine (%) 0.4 Destroyed by hydrolysis
Glycine (%) 0.2 1.5
Isoleucine (%) 0.2 1.3
Lysine (%) 1.2 2.4
Phenylalanine (%) 0.2 1.4
Serine (%) 0.4 0.5
Tryptophan (%) Below level of detection Destroyed by hydrolysis
Valine(%) 0.1 1.5

The culture was resuspended in 5 mL of Phytone™ peptone medium and 1.5 mL of the culture transferred to a primary shake flask containing 90 mL of Phytone™ peptone medium containing 0.9 mL of a 1:10 diluted phosphate solution (32% (w/v)) and 0.45 mL of 1:2 diluted calcium chloride solution (53 % (w/v)). The culture was incubated at 36°C, 200 rpm for 24 hours. Five mL of the culture was transfesrred to a secondary shake flask culture containing 250 mL of Phytone™ peptone medium containing 2.5 mL of a 1:10 diluted phosphate solution (32 % (w/v)) and L25 mL of a 1:2 calcium chloride solution (53 % (w/v)). The culture was incubated at 36°C for a further 24-28 hours. Ten mL of the above secondary shake flask culture was dispensed into five 50 mL sterile screw capped centrifuge tubes and centrifuged at 6 000 xg for 10 minutes at 4°C.
The supernatant was decanted and the pellet of each tube, re-suspended in 5 mL of one of the following lyophilization media:
a) 10% (w/v) skim milk (Animal Control)
b) 10% (w/v) yeast extract
c) 10% (w/v) Phytone™ peptone
d) " 5% (w/v) monosodium glutamate + 10% (w/v) yeast extract
e) 10% (w/v) Phytone™ peptone>10% (w/v) yeast extract + 0.25% (w/v) agar
The cultures in the above lyophilization medium were dispensed in 0.25 mL amounts in 1 mL glass vials and freeze dried as follows.
Freeze-Drying cycle
The product temperature was allowed to reach -30 °C and held at that temperature for about 10-12 hours under a vacuum of 120 mT. After 10-12 hours, the product temperature was increased and maintained at 20 °C under a vacuum of 120mT. The vials are sealed under vacuum and stored at 4°C. The freeze dried cultures were analyzed for viability-by measuring colony forming units (CFU/mL) on Columbia blood agar plates.
The results of CFUs obtained before and after fteeze-drying for C. diptitheriae strain are shown tabulated in Tables 4, 5,6 and 7.

Table 4: Comparison of CFU counts of the freeze dried cultures of G diphtheriae in skim milfr and animal component-free lyophilization medium. The CFU count before freeze-drying of C. diphtheriae was 6.0 x 109 CFU/mL

Table 5: Comparison of CFU counts of the freeze dried cultures of C diphtheriae in skim milk and animal component-free lyophilization medium as a function of time. (C. diphtheriae strain)

Table 6: Screening of the animal component-free lyophilization medium and their respective CFU counts in comparison to animal component lyophilization medium after freeze-drying.

Table 7: Comparison of CFU counts of the freeze dried cultures of C. diphtheriae in animal component and animal component-free lyophilization medium.

The most stable mixture for freeze-drying is the mixture of Yeast extract (10% w/v) with mono sodium glutamate (5%w/v), as shown in Tables 4-7..

SUMMARY OF THE DISCLOSURE
In summary of this disclosure, there is provide, a lyophilization medium for a microorganism wherein the medium is substantially free of animal-derived products and comprises yeast extract and monosodium ghitamate and uses thereof. Modifications are possible within the scope of the invention.

We claim:
1. . A lyophilization medium for a microorganism wherein the medium is substantiality
free of animal-derived products and comprises yeast extract and monosodium
2. The lyophilization medium or claim 1, comprsing about 1-10% (w/v) monosodium
glutamate and about 1-10% (w/v) yeast extract
3. The lyophilization medium of claim 2, comprsing about 5 % (w/v) monosodium
glutamate and about 10% (w/v) yeast extract.
4. The lyophilization medium of claim 1 or 2 or 3 wherein the microorganism is a strain
of bacteria.
5. The lyophilization medium of claim 4 wherein the strain of bacteria is
Corynebacterium diphtheriae
6. A method for preparing a freeze-dried culture of a microorganism comprising the
steps of:
providing a quantity of the microorganism;
mixing said quanity with a lyophilization medium wherein the medium is
substantiality free of animal-derived products and comprises* yeast extract and
monosodium glutamate to provide a mixture; and
freeze-drying said mixture.
7. The method of claim 4, wherein the lyophilization medium of comprses about 5 %
(w/v) monosodium glutamate and about 10% (w/v) yeast extract.

8.. The method of claim 5, wherein the lyophilization medium of comprses about 1-10% (w/v) monosodium glutamate and about 1-10% (w/v) yeast extract
9. The method of claim 6 or 7 or 8 wherein freeze-drying of said" mixture comprises
steps of:
(a) achieving a first temperature of about -30 °C for said mixture to provide a
cooled mixture;
(b) maintaining said cooled mixture in a vacuum for a time until said cooled
mixture is substantially dry to provide a dried mixture.

10. The method of claim 7 wherein the vacuum is about 120 mT.
11. The method of claim 8 wherein the time is between about 10 and about 12 hours.
12. The method of claim 7 wherein the step of maintaining said cooled mixture in a
vacuum for a time until said cooled mixture is substantially dry to provide a dried
mixture comprises:

(a) maintaining said cooled mixture in a vacuum for a time of between about 10
and about 12 hours; and
(b) increasing said temperature of about -30 °C to a second temperature of about
+20 °C.

13. The method of claim 10 wherein the vacuum is about 120 mT.
14. The method of claim 6 or 7 or 8 wherein the microorganism is a strain of bacteria.
15. The method of claim 14 wherein the strain of bacteria is a strain of Corynebacterium
diphtheriae

16. A free-dried lyophile comprising cells of a microorganism and a lyophilization
medium wherein the medium is substantiality free of animal-derived products and
comprises yeast extract and monosodium glutamate.
17. The freeze-dried lyophile of claim 12, wherein the medium comprises about 1-10%
(w/v) monosodium glutamate and about 1-10% (w/v) yeast extract
18. The freeze-dried lyophile of claim 13, wherein the medium comprises about 5 %
(w/v) monosodium glutamate and about 10% (w/v) yeast extract.
19. The freeze-dried lyophile of claim 16 or 17 or 18 wherein the microorganism is a
strain of bacteria.
20. The freeze-dried lyophile of claim 19 wherein the strain of bacteria is a strain of
Corynebacterium diphtheriae.