Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
1999-07-16
2003-05-06
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C435S252700, C435S253600, C435S842000
Reexamination Certificate
active
06558926
ABSTRACT:
BACKGROUND
Tetanus is a life-threatening disease caused by infection with
Clostridium tetani
(
C. tetani
), a ubiquitous anaerobic spore-forming soil microbe.
C. tetani
causes disease by releasing a peptide toxin, Tetanus Toxin, that enters the nerve cells of the infected host and blocks release of neurotransmitters at inhibitory synapses. This blockage produces unregulated excitation of certain host neurons, resulting in uncontrollable muscle contraction and paralysis, typically of facial and back muscles. As recently as 1989, 10-40% of
C. tetani
infections of non-immunized hosts resulted in death.
For the past 50 years, widespread immunization programs have been in effect to protect against the effects of
C. tetani
infection. Vaccines are prepared from Tetanus Toxin that has been inactivated, usually by exposure to formalin (Descomby,
Can. Roy. Soc. Biol
. 91:239, 1924; Plotkin et al.,
Vaccines
, 2nd. Edition, W. B. Saunders, 1994). The inactivated Toxin is known as Tetanus Toxoid.
The
C. tetani
vaccination effort, although it has achieved significant success, has been hampered by certain complications associated with preparation of the Tetanus Toxoid. Because Tetanus Toxin is an extremely potent and dangerous compound (the estimated lethal dose for a human is only 2.5 ng/Kg), it is necessary to inactivate the Toxin very early in its purification process so that the risks of exposure to even partially purified active Toxin is minimized. As mentioned above, Tetanus Toxin is usually inactivated by exposure to formalin, which destroys the activity of the peptide toxin by generating inter-peptide cross-links and adducts. Unfortunately, formalin reactivity is not specific to Tetanus Toxin. A large number of other microbial and media proteins are also cross-linked by formalin. Because the Tetanus Toxin must be treated with formalin at an early stage in its purification, the treatment produces a complex, heterogenous composition containing not only formalin-inactivated Tetanus Toxin, but also formalin adducts of other peptides and proteins that were present in the formalin-treated mixture.
Tetanus Toxin is usually prepared from
C. tetani
cells that have been grown in media containing animal and dairy by-products (e.g., casein digests, meat extracts) as sources of the proteins, peptides, and amino acids that are required for growth or that stimulate growth. Tetanus Toxoid preparations generated from such cells necessarily contain some amount of formalin adducts of animal proteins. A possible consequence of this fact is that some Toxoid preparations could contain carry-over amounts of undesirable contaminants, such as the protein agent (prion) that causes Bovine Spongioform Encephalopathy (BSE), or antigenic peptides that stimulate undesired immune reactions (e.g., anaphylactic reactions) in immunized subjects.
The medium used to grow
C. tetani
for Tetanus Toxoid preparation has a variety of additional problems as well. The standard medium, known as “MM”, was developed in 1954 by Mueller and Miller (
J. Bacteriol
. 67:271, 1954) and contains glucose, beef heart infusion (BHI), NZ-Case or NZ-Case TT (an enzymatic digest of casein), some amino acids and vitamins, uracil, and inorganic salts. The BHI component has proven to be particularly problematic, both because of the risk that it will contain undesirable products that may be carried over into the final Toxoid preparation (see, for example, Robb,
Proc
. 4
th Internat. Conf. Tetanus
., Dankar, Senegal, pp. 735-43, 1975) and because of its variability from lot to lot.
The NZ Case component of MM medium has also caused problems due to its variability (see, for example, Stainer,
Proc
. 4
th Internat. Conf Tetanus
., Dakar, Senegal, pp. 745-54, 1975). It is now common practice for producers of Tetanus Toxoid preparations to screen numerous lots of NZ Case to identify a particular lot that results in high level Tetanus Toxin production. Once such a lot is identified, the Toxoid producer will typically purchase the entire lot.
Yet another problem with the MM medium typically used to culture
C. tetani
for Tetanus Toxoid preparation is that the medium has proven to be very sensitive to “cooking,” in a manner that is independent of the requirement for sterilization. Apparently, extended cooking protocols can produce Maillard adducts and/or can degrade medium components such as proteins and peptides, so that a less effective growth medium is produced when a particular batch of medium is imperfectly cooked.
Finally, the process of
C. tetani
fermentation in MM medium generates H
2
S, which is toxic to
C. tetani
, and may also generate some alcohols that can similarly be toxic.
C. tetani
growth in this medium, not to mention Toxin production, can be dramatically affected by the rate at which the head space in a fermenter is purged or gas exchanged, creating yet another point at which the extent and quality of Toxoid preparation can vary significantly from one batch to another.
There is a need for the development of an improved system for preparing Tetanus Toxoid that minimizes these risks and problems. Preferably, the system should allow for reproducible, high levels of Toxin production, and should minimize the dangers associated with formation of animal- or dairy-product adducts. There is a particular need for the development of a
C. tetani
culture medium and growth protocol that does not utilize meat or dairy by-products.
SUMMARY OF THE INVENTION
The present invention provides a system for the growth of
C. tetani
and production of Tetanus Toxin for use in formulating Tetanus Toxoid preparations. The inventive system allows production of high levels of Tetanus Toxin and preferably utilizes at least one growth medium that contains significantly reduced levels of meat or dairy by-products as compared with MM medium; preferred media embodiments are substantially free of such components.
In one aspect, the present invention provides media that contain reduced levels of animal or dairy byproducts and are preferably substantially free of animal or dairy byproducts. For the purpose of the present invention, animal or dairy byproducts means any compound or collection of compounds that was produced in or by an animal cell, whether in a living organism or in vitro. Preferred non-animal sources of media ingredients such as proteins, amino acids, and nitrogen, include but are not limited to vegetables, microbes (such as yeast) and synthetic compounds.
In another aspect, the present invention provides methods of preparing Tetanus Toxin using at least one medium that is substantially free of animal or dairy byproducts. In one embodiment, Tetanus Toxin is produced by culturing an organism of the genus Clostridium in a fermentation medium substantially free of animal products.
In another embodiment of the present invention, Tetanus Toxin is produced by culturing an organism of the genus Clostridium in a fermentation medium substantially free of animal products and containing vegetable-derived products.
In yet another embodiment, Tetanus Toxin is produced by culturing an organism of the genus Clostridium in a fermentation medium substantially free of animal products and containing soy-based products.
In yet another preferred embodiment, Tetanus Toxin is produced by culturing
Clostridium tetani
in a fermentation medium substantially free of animal products and containing hydrolyzed soy as a substitute for animal-derived products. Preferably, growth in a fermentation medium proceeds until at least cell lysis occurs. The source of
C. tetani
used for inoculation of the fermentation medium may be obtained from a seed medium containing
C. tetani
. Preferably,
C. tetani
grown in a seed medium and used as an inoculant for a fermentation medium has not undergone cell lysis. The source of
C. tetani
used for inoculation of the seed medium may be obtained from a lyophilized culture.
C. tetani
may be lyophilized as a culture in animal milk or soy milk. Preferably
C. tetani
is lyophilized as a culture in soy milk.
The present inventi
Demain Arnold L.
Fang Aiqi
Afremova Vera
Choate Hall & Stewart
Saucier Sandra E.
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