Patent Application
20070128702
Not Applicable
Not Applicable.
The present invention relates to methods of making and using consumable bacterial strains, as well as compositions containing them.
Historical Background
After a weekend vacation, Alexander Fleming returned to his laboratory to discover that one of his cultures of bacteria had been contaminated with mold. Not only was the plate contaminated, but the bacterial cells, Staphylococcus aureus, had lysed. Instead of throwing the contaminated plates away, Fleming observed that bacterial cell lysis occurred in an area next to the mold and hypothesized that the mold had made a product responsible for the death of the bacteria. He later was able to extract the diffusible substance from the mold, and penicillin was born.
Because antibiotics as a class of drugs are able to kill a broad spectrum of harmful bacterial pathogens, their use has revolutionized medicine, trivializing many diseases that had before taken millions of lives. For example, the plague, caused by infection with the Yersinias pestis bacterium, has laid claim to nearly 200 million lives and has brought about monumental changes, such as the end of the Dark Ages and the advancement of clinical research in medicine. Gentamycin and streptomycin are used to treat patients infected with plague, thus increasing the likelihood of survival. Erythromycins are used to treat respiratory tract and Chlamydia infections, diptheria, Legionnaires' disease, syphilis, anthrax and acne vulgaris. Erythromycins are also used to prevent Streptococcal infections in patients with a history of rheumatic heart disease.
Biological weapons are a real and current threat. Antibiotics are an important defense against the possible devastation such weapons can bring.
Antibiotics, among a vast array of other therapeutic and nutritional products, can be produced in enormous quantities thanks to genetic engineering of cells, especially of bacterial cells. Other important products that can be made by such fermentations include polypeptides, such as antigens for vaccines, antibodies and other therapeutic polypeptides; small molecules and small molecule inhibitors; and nutritional and industrial products, including isoflavones, sugars and alcohols.
For example, strain improvement technology for antibiotic producing strains began during the golden-age of antibiotic discovery in the 1940's. An empirical process of random mutation followed by large scale screening was used to find higher producing strains. Many of the antibiotic-producing strains in use today are descendants of the strains isolated during this early period.
The technology developed through the cooperation of industry and government during this period continued to be practiced relatively unchanged for many decades by private pharmaceutical companies in their strain improvement programs. Highly improved strains developed in-house were highly guarded from distribution to other companies.
Since the 1990's, strain improvement programs and other natural product related research at major pharmaceutical companies has been severely reduced or eliminated. However, the competition among big companies for the best strain for existing products, such as the antibiotic erythromycin, still continues. Today a larger percentage of the strain improvement work is out-sourced to specialty strain improvement.
The business of creating and selling biotech strain improvement technology for natural-product producing fermentations is in its early stages. Developers of this technology are historically pre-disposed to create strains with permanently improved trains, but if a strain can be created that has the same or nearly the same quality of strain improvement in a consumable format, it would be more beneficial to the biotech company to pursue this business approach.
The Problems with Out-Sourcing from the Biotech Company Perspective
The principal problems with out-sourcing, from the point of view of the small biotech (developer) company, are in control and distribution of the strain improvement technology. Distributing strains to countries that do not recognize US intellectual property rights also presents a problem. Pirating of strains is also a potential problem for both big and small companies.
The Problems with Out-Sourcing from the Pharmaceutical Company Perspective
Pharmaceutical companies do not want to pay a large up-front costs for technology that does not benefit their pharmaceutical process. Therefore they would like to test the new technology with low, up-front costs in case the technology does not work in their process. If the technology works, then payments to the developer company are justified.
In a first aspect, the invention provides for methods of producing strains that have a target characteristic that the strain loses over time.
In a second aspect, the invention provides compositions containing strains that have target characteristic that the strain loses over time.
In a third aspect, the invention provides methods of doing business, wherein candidate strains that have target characteristics that the strain loses over time are provided to an end-user for evaluation.
In a fourth aspect, the invention provides methods of doing business wherein candidate strains that have target characteristics that the strain loses over time that have been evaluated by an end-user are repeatedly supplied to the end-user.
The problem the developer of improved strains is leveraging fair value from strain improvement technology because of poor control of product distribution. The invention addresses the basic business problem with a technology-driven solution through the development of a strain improvement product in a “consumable” format.
Definitions
Biotech strain improvement technology refers to strain improvement introduced by recombinant DNA technology as opposed to being empirically introduced by random mutagenesis. The improvement can be transient or permanent.
Co-expressed, in the context of gene expression, means that two target traits are transcribed or translated or active simultaneously at some point during the life of the cell. The term is usually applied to two or more introduced genes. However, in some instances, co-expression will not occur unless the gene(s) is activated (e.g., a gene operably-linked to an inducible promoter); in this case, co-expression is applied in the context of when the inducible molecule(s) is present in the culture medium. Co-expressed genes can be genetically linked, but need not be.
Consumable refers to an item for sale intended to be used up and then replaced.
Consumable strain improvement product is defined by the characteristic of at least one transient genomic modification A reversible genetic modification introduced temporarily into the chromosome, for example, brought about by the introduction of a single-crossover event of an insertion plasmid. Also referred to as a plasmid-insertion construct; used in the consumable strain improvement format
End-user refers to a person or entity who uses a product. For the purposes of this application, an end-user may be the same as a customer, who might buy the product but does not necessarily use it, and thus has a more contractual meaning, wherein the term refers simply to a non-reseller.
Marker refers to one or more genes that are introduced exogenously into a cell. They are often carried by an insertion plasmid to indicate the presence of the plasmid in the genome. Markers are commonly genes that confer drug-resistance. A selectable marker is one that is to distinguish cells that have been successfully transformed, wherein exogenous DNA has been introduced, and confers upon the cells the ability to survive culture conditions in which their non-transformed counterparts die. Examples of selectable markers include antibiotic resistance marker genes, herbicide tolerant marker genes and metabolic marker genes. Screenable markers allow for the identification of successfully transformed cells, but do not confer a trait to the cells that allows for the survival of the cells from their non-transformed counterparts. Selection medium refers to a culture medium that contains a substance that allows for the identification of a cell carrying a selectable marker, while its non-transformed counterparts do not grow or die; screening medium allows for the identification of transformants, but does not prevent or kill non-transformed cells.
Prototype refers to a first or preliminary model of something.
Reverse engineering refers to the reproduction of another manufacturer's product following detail examination of its construction of composition.
Target trait means a characteristic that is brought about by a genetic modification, transient or permanent. Examples of target traits include those modifications that express or improve the expression of pharmaceutical or otherwise therapeutic substances (e.g., antibiotics, small molecules, small molecule inhibitors, antigens useful in vaccine formulations, therapeutic polypeptides (e.g., insulin, antibodies, portions of antibodies, engineered antibodies), etc.), or commercially important nutritional products (e.g., isoflavones, alcohols, sugars, etc.).
Traditional strain improvement product is defined by the characteristic of at least one permanent genomic modification A genetic modification introduced permanently into the chromosome, for example created by a double-crossover event following the introduction of an insertion plasmid. Also referred to as a gene-replacement construct. Two other types of permanent genomic modifications often used are transposon insertions or phage insertions (Kieser et al., 2000; Davison, 2002).
In the methods of the invention, strain improvement technology is usually introduced by recombinant DNA technology into a candidate organism. The strain has a temporal shelf-life. Once the improved strain is constructed, it is presented to the end-user for testing. Usually the end-user will test the strain on a small scale, and if successful, test the strain for compatibility in large-scale production formats. When microbial strains are being tested, testing often proceeds from shake flasks, followed by pilot plants, and finally the strain must show the improved production trait in the large scale commercial fermentors.
If after testing, the improved strain is not desired by the end-user, there will be no loss-control problems to consider since the improved strain loses its improvement effect and returns to its original pre-improvement condition during normal handling of the strain. An added benefit of the consumable format is that if the strain is inadvertently released into the environment and allowed to propagate, it reverts back to its natural, non-recombinant form.
If the end-user wishes to continue its commercial fermentations with the strain improvement effect, it simply purchases more modified strains from the developer company in the desired lot size. The developer company has an actual product to produce and sell on a periodic basis, rather than in the traditional method having a single product to sell in a one time deal. This keeps the two companies working together in a vendor-end-user relationship which fosters greater business interactions between the developer and the manufacturing companies.
The basis for “consumable” strain improvement technology relies on what has traditionally been seen as an unwanted feature of a widely used genetic technology called “insertion vectors” or “integration vectors”. Insertion vectors are plasmids or other circular DNA molecules such as phage, that have conditional replication combined with a selectable marker and at least one target DNA fragment for insertion of the plasmid into the chromosome. Such technology is applicable to almost all organisms, including prokaryotes (phages, viruses, bacteria) and eukaryotes (e.g, those numbering among the Opishtokonta, Amoebozoa, Plantae, Chromalveolate, Rhizaria and Excavata; Simpson and Roger, 2004). Examples include, but is certainly not limited to, Pseudomonas, Escherichia, Streptomyces, Saccharopolyspora, Bacillus, Ralstonia eutropha, Alcaligenes, Chromatium, Thiocystis, Saccharomyces, Yeasts, Clostridium, Thermobacillus, etc. Other examples of prokaryotes can be found in Balows, A., et al. (eds.). The Prokaryotes, 2nd ed. Springer-Verlag, New York. 1992, which is herein incorporated by reference.
The plasmid-based system, which relies on a Campbell-type homologous recombination, was originally developed and used in Bacillus subtilis in 1980 (Haldenwang et al.) and later adapted to S. erythraea in 1988 (Weber and Losick) and is still used today as a rapid method of generating genetic modifications in many different organisms, particularly bacteria.
Since their first use in 1980 for mapping the spoVG gene in B. subtilis, five additional new uses for integration vectors have been developed. Integration vectors are now also used for (1) gene knockouts, (2) gene amplifications, (3) chromosome walking, (4) gene fusions, and (5) ectopic (second-site) integration (Zeigler, 2002; Keiser et al., 2002).
Insertion vectors have proven useful for research purposes despite the unwanted feature of being inherently unstable in the bacterial chromosome. That is, insertion plasmids excise themselves spontaneously from the chromosome in a reversible reaction, leaving behind the original strain in its unaltered condition.
Molecular biologists have developed simple methods for handling strains carrying insertion plasmids to minimize the problems associated with their instability. One way to reduce the problems associated with instability to maintain selective drug pressure on the strain during an experiment, so that only individual cells that carry the insertion plasmid can survive. The cells that have lost the insertion plasmid are killed by the drug selective agent.
In a commercial environment, however, it is impractical (for cost and quality control issues) to maintain drug-selective pressure in large scale formats. While cells are generated during the course of the fermentation that have lost the plasmid conferring a desired trait, the half-life of plasmid loss is relatively long, the effect on yield is usually low. The end-user will not have knowledge of the drug-selection agent and so he is unable to propagate the improved strain for long until the newly propagated cells have all lost the plasmid and thus lost the desired trait. In order to regain the potency of the fully improved strain the end-user obtains them from the supplier.
Anti-Reverse Engineering
To protect the strain having a target characteristic, it must not being easily reverse engineered—an important aspect for the methods of the invention. The features of insertion plasmids that are most susceptible to reverse engineering are (1) the identification of the drug-resistance markers and (2) the identification of the target DNA sequences.
The use of novel and/or rarely used drug-resistance markers is the first line of defense. Examples of drugs that can be used as markers, or developed to be used as markers, is shown in Table 1. A preferred rarely used drug-resistance marker is viomycin resistance. Table 2 presents appropriate drugs and metabolites, as well as some providers and relative costs.
Suppliers of antibiotics: the blocks (▪) indicate the relative prices of the antibiotics; some of the prices are very high only because the quantities sold are small and the demand for the substances is low. Samples for research may be obtainable from the antibiotic producers directly.
The second line of defense is to remove those elements that facilitate recovering the polynucleotide fragment that contains the target characteristic(s). For example, in bacteria, if the E. coli origin of replication if left in the strain facilitates rapid recovery of the plasmid from the partially digested chromosome and subsequent transformation of the ligated DNA with ampicillin selection into E. coli where the plasmid could be easily characterized. Alternatively, the E. coli origin of replication could be left in the construct as long as the traditional resistance gene was removed and replaced with an alternate drug resistance marker such as viomycin resistance or an entirely new marker.
Other mechanisms to foster protection from reverse engineering can be used. For example, if the strain improvement effect is exerted through the concerted transcription of two to five genes, their arrangement in the genome can be altered. For example, if the genes are usually clustered, they could be separated and re-inserted into the chromosome at ectopic (neutral second) sites, and each expressed separately and held in place through selective pressure, each with a different antibiotic. If any one of the insertions is lost, the desired characteristic is also lost.
Other types of modifications can be used; falling under the six uses of insertion plasmids described above. For example, insertion plasmids can create gene knockouts, and gene amplifications. Just about any modification can be created through the use of an insertion plasmid.
Determining the Stability of Strains Carrying Insertion Plasmids
The stability of the insertion plasmid in the chromosome may affect the quality of the strain improvement effect. Experiments are performed to determine the stability of varying size insertion plasmids and then determine how much of an effect stability plays on the target characteristic. The rate of spontaneous excision (i.e., the instability) of insertion plasmids from the chromosome depends upon the size of the target DNA fragment in the insertion plasmid, the organism, and the insertion site (Metzenberg et al., 1991).
Method
The methods for analyzing for drug resistance and the target characteristic are known to those of skill in the art and vary according to organism and the effect of the target characteristic (Ausubel et al., 1987).
Comparing Improved Strains in the Consumable vs. the Permanent Format
Construction of a permanently modified strain is done using known techniques (Ausubel et al., 1987).
Method
In the methods of the invention, consumable product strains are about 25%-100% efficient as their permanent counterparts (that is, one improved using traditional methods). More preferably, the consumable product strains are at least 50% efficient, and more preferably, at least 75% efficient as the permanent counterpart.
Protecting Against Reverse Engineering of the Consumable Strain Improvement Technology
The consumable strain improvement product is designed to give the owner of the strain improvement technology control over its sale and distribution. The key to maintaining the strain without its losing its strain improvement effect is by propagating the strain in the presence of the proper drug-selection agent. Other key information that could be gained by reverse engineering regards the identity of the cloned target DNA sequences and the origins of replication on the plasmid.
As an example, the modification of a bacterial strain is provided. In this strategy, the commonly used resistance genes are substituted for the less commonly used viomycin resistance gene. The ColE1 origin of replication (ori) is replaced by conditional R6Kγ E. coli ori. Plasmids containing this origin of replication do not replicate in the commonly used E. coli host strains, but require a special E. coli host strain, R6Kγ-5.
Other more stringent protections include eliminating the E. coli origins of replication completely and create plasmid constructions exclusively in the host organism.
Kits
The consumable products can be included in a kit, container, pack, or dispenser together with instructions for growth and, if appropriate, induction of the target characteristic. When supplied as a kit, the different components can be packaged in separate containers. Such packaging can permit long-term storage without losing the activity of the components. For example, such kits can contain containers of spores and powdered media. In other embodiments, the kits include the consumable product with instructions for use; in other embodiments the kits include an order form to re-order the product, or directions to obtaining additional product, either from a website, via electronic mail, telephone, facsimile, or any other appropriate channel of communication.
Kits may also include reagents in separate containers that facilitate the execution of a specific test, such as tests for the target characteristic.
Containers or vessels The reagents included in the kits can be supplied in containers of any sort such that the life of the different components are preserved and are not adsorbed or altered by the materials of the container. For example, sealed glass ampoules may contain lyophilized buffer that has been packaged under a neutral non-reacting gas, such as nitrogen. Ampoules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampoules, and envelopes, that may consist of foil-lined interiors, such as aluminum or an alloy. Examples of containers include test tubes, vials, flasks, bottles and syringes. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, etc.
Instructional materials Kits can also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, mini-disc, cassette tape or provided by calling a prescribed telephone number. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
Business Methods
In one embodiment, a potential end-user requests the consumable product for testing for her purposes. The supplier supplies the consumable product, either as live cultures, frozen cultures, or in an inactive state (e.g., spores, seeds, etc.). The end-user then tries the product in her application until either the strain no longer expresses the desired characteristic, the application is determined to be inappropriate for the strain, or the strain is applicable to the application and more strain is requested. In the case of the latter, the end-user then orders the quantities she needs for her application, replacing the strain as its target characteristic wanes. If the strain is inappropriate, the end-user is free to use the strain (subject to any agreements, such as contracts or licenses, etc.), until the desired characteristic dissipates, or, more usual, the strain is appropriately disposed of.
The following examples exemplify a specific embodiment wherein S. erythraea is modified according to the methods of the invention to increase production of erythromycin.
Background Plasmid pFL2212 contains a 6.8 kb fragment of the S. erythraea chromosome (Fermalogic, Inc.; Chicago, Ill.). It is the insertion plasmid for the consumable strain improvement product, strain FL2385 (Fermalogic). Plasmid pFL2212 contains the mutAB region of the S. erythraea chromosome. This plasmid contains the 6.8 kb methylmalonyl-CoA mutase region from S. erythraea. The plasmid also contains the Streptomyces origin of replication, pIJ101, which has lost the ability to replicate autonomously in S. erythraea. The tsr gene encodes the thiostrepton-resistance gene is also on the plasmid and is used in the selection of transformants after protoplast transformation. neo encodes a promoter-less neomycin resistance gene, also included on the plasmid. ColE1 is the E. coli replication origin; Apr, represents the ampicillin resistance gene
Method A cosmid library of S. erythraea chromosomal DNA was prepared in a SuperCos cosmid vector (Stratagene, La Jolla, Calif.). Approximately 600 recombinant cosmids were screened by PCR for the presence of the mutAB region using primers designed based on the DNA sequence information deposited by Luz-Madrigal et al., 2002 (Genbank accession no. AY117133 (SEQ ID NO:1)). The primer sequences were: gntRF1-5′-GTCGAATTCGCCGTCACCGTCGACCCCAA-3′ (SEQ ID NO:2) and gntR1-5′-GTCGGATCC CAGCATCAGCGCTCCCGGA-3′ (SEQ ID NO:3). Two cosmids, 5G10 and 6E7, were found containing the mutAB operon. Cosmid 6E7 was used for DNA sequencing of the mutAB flanking regions using a primer-walking method. Cosmid 6E7 was also used to sub-clone a fragment of DNA containing the five genes shown in FIG. 4between the EcoRI and BamHI sites. This fragment was cloned into pFL8 to create plasmid pFL2212. S. erythraea protoplasts were transformed with pFL2212 with selection for thiostrepton resistance. Single thiostrepton-resistant colonies were isolated tested in shake flask fermentation. These strains were designated FL2385.
Method Fermentations were performed in un-baffled 250-ml Erlenmeyer flasks with milk-filter closures. The flasks were incubated at 32.5° C. and 65% humidity on an Infors Multitron Shaker having 1-inch circular displacement. Seed cultures containing a carbohydrate-based medium (and no thiostrepton) were prepared on the same shaker under the same growth conditions. Seed cultures were inoculated from fresh spores prepared on fresh sporulation agar plates containing thiostrepton at 10 micrograms/ml. Fermentations were inoculated with 1.25-ml of a 42 h seed culture into 25-ml of oil-based media. Thiostrepton was not added to any seed or fermentation media. Fermentations were grown for 5 days; their volumes were then corrected for evaporation through the addition of water before being further analyzed.
Results Strain FL2385 produced erythromycin in excess of 35% above that which can be achieved with the wild-type (white) S. erythraea strain when grown under optimal fermentation conditions including using an oil-based medium.
Conclusions The strain improvement trait of FL2385 was significant and occurred in the absence of drug selection in the fermentation or seed media. The only point at which FL2385 was exposed to drug selection was in the preparation of the spores used to inoculate the fermentation seed medium. The insertion plasmid pFL2212 therefore was sufficiently stable in the chromosome to produce a significant strain improvement effect.
A new strain, FL2385P, will be created that is genetically equivalent to the current prototype consumable strain FL2385. This strain will have a duplication of the 1st generation gene cluster region, but will be a permanent genomic modification created by a gene-replacement technique rather than a single-crossover technique.
Next a bank of plasmids will be created with different sized target DNA sequences that will allow for the measurement of the half-life of the strain improvement effect in strains made with the transient (consumable) technology.
Rationale The stability of the insertion plasmid in the chromosome can affect the quality of the strain improvement effect. Experiments can be performed to determine the stability of varying size insertion plasmids and then determine how much of an effect stability plays on erythromycin production during the course of the fermentation. The rate of spontaneous excision (i.e. the instability) of insertion plasmids from the chromosome depends upon the size of the target DNA fragment in the insertion plasmid, the organism, and the insertion site (Metzenberg et al., 1991)
Method
1. Construct insertion plasmids of different sizes, pFL2212 A-G.
2. Transform plasmids pFL2212 A-G into wild type S. erythraea (strain FL11635), using thiostrepton selection, to create strains FL2385 A-G, respectively.
3. Produce thiostrepton-resistant spores of Strains FL2385 and FL2385 A-G
4. Analyze the spores of each strain to show that the spores are 100% thiostrepton resistant, and therefore all carrying the insertion plasmid.
5. Perform shake flask fermentations on each of the seven strains. See Preliminary Results for a description of the fermentation method. Seed cultures and fermentation cultures will not contain thiostrepton since in the commercial setting it would be impractical to add these drugs to large-scale fermentors.
6. Seed cultures and fermentations will be sampled daily and a time course analysis will be performed to determine the proportion of cells carrying the insertion plasmid. The stability analysis will be performed by plating the culture samples on sporulation agar and incubating the plates at 32° C. for 10 days until the plates is fully sporulated. Spores will be harvested and diluted in 20% aqueous glycerol to obtain single colonies on sporulation agar plates. A sample of one hundred colonies will be screened for the presence of the insertion plasmid by patching colonies on agar containing thiostrepton at 10 micrograms/ml, and also on plates containing no thiostrepton. Alternatively, equivalent volumes of diluted spore suspensions will be plated on thiostrepton and no-thiostrepton agar to calculate plasmid loss from the difference in colony numbers on the two plates. Additionally, it may prove useful to place a visual marker gene on the insertion plasmid to aid in the counting of colonies.
7. The proportion of thiostrepton-resistant colonies per unit volume will be plotted vs. time to determine the half-life of the insertion plasmid in the chromosome.
To know whether the consumable strain improvement product with the transient genomic modification can achieve the level of strain improvement that is attainable by a permanently modified strain, equivalent strains will be constructed in both formats and then compared them directly in fermentations.
Construction of the permanently modified strain will be done using the gene replacement technique using standard techniques. An earlier study (Reeves et al., 2002) described the eryCI-flanking region; this is a suitable site for insertion of the second copy of the mutAB gene pair by a gene replacement.
Method
The consumable strain improvement product is designed to give the owner of the strain improvement technology control over its sale and distribution. The key to maintaining the strain without its losing its strain improvement effect is by propagating the strain in the presence of the proper drug-selection agent. Other key information that could be gained by reverse engineering regards the identity of the cloned target DNA sequences and the origins of replication on the plasmid. Our initial protections for our first generation strain improvement technology described here will involve substituting the commonly used thiostrepton-resistance gene and kanamycin resistance gene for the less commonly used viomycin-1-resistance gene. The ColE1 origin of replication (ori) will also be replaced by conditional R6Kγ-4 E. coli ori. Plasmids containing this origin of replication do not replicate in the commonly used E. coli host strains, but require a special E. coli host strain, R6Kγ-5. Also the ampicillin-resistance gene will be eliminated because the viomycin resistance gene is a useful selectable marker for both Streptomyces and E. coli.
Method
The examples here presented are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, formulations and/or methods of use of the invention, may be made without departing from the spirit and scope thereof.
This application claims priority to U.S. Ser. No. 60/742,094, filed Dec. 1, 2005, entitled, “METHODS OF CREATING CONSUMABLE BACTERIAL STRAINS AND COMPOSITIONS THEREOF,” and is incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60742094 | Dec 2005 | US |
