CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 61/422,056 filed December 10, 2010, which application is incorporated herein by reference in its entirety.

BACKGROUND

Technical Field

The present invention relates generally to a preparation of antibiotic G418 with low cell toxicity and high purity, and methods of producing such a preparation. More specifically, the present invention relates to an isolated antibiotic G418 with a purity of greater than 95% and no individual group of gentamicin impurities exceeding 3%, as measured by HPLC.

Description of the Related Art

The antibiotic known as "G418" is an aminoglycoside antibiotic and thus belongs to the class of antibiotics that includes gentamicin, neomycin, paromycin, sisomicin, kanamycin and the like. G418 is similar in structure to gentamicin Bl . G418 possesses three primary amino groups, of which one or more of the primary amino groups when charged may form salts, such as acid addition salts. Typically, G418 is in the form of a mono- or di-salt. Common forms of G418 are a monosulfate salt or a disulfate salt. These sulfate salt forms may be generated by exposing G418 base to sulfuric acid to convert the base form to a corresponding acid addition salt at one or more of the primary amino group positions on G418.

The biological activity of antibiotic G418 is to inhibit the elongation step in both prokaryotic and eukaryotic cells, such that peptide and polypeptide synthesis is blocked. In particular, G418 binds irreversibly the 80S ribosomal subunit and interferes with its function. G418 is not normally used as a standard antibiotic. Rather, it is normally used in laboratory research, for example, to select genetically engineered cells. G418 is commonly used for the selection of prokaryotic and eukaryotic cells stably transfected with neomycin resistance genes (neo); and for the maintenance of the (neor) phenotype of neomycin resistant cells. Resistance to neomycin is conferred by one of two dominant genes of bacterial origin, which can be expressed in eukaryotic cells. The resistance genes are APH (3') II or aminoglycoside phosphotransferase 3' (II) and aminoglycoside phosphotransferase 3' (I) or APH (3') I. Examples of sources of prokaryotic cells are bacteria. Examples of sources of eukaryotic cells are mammals, insects, algae, yeast, fungi, amoebae and plants. An example of an amoeba is dictyostelium. Examples of cells from mammals are cells from human and non-human mammals, such as rodents and non-human primates. Examples of sources of rodent cells are mice, rats and hamsters.

G418 is commonly used by researchers desiring a selection agent in gene transfer experiments. Antibiotic-resistant genes are used as markers to identify the successful transfer of other genes. The KanMX selectable marker is a marker commonly used in laboratory research to select genetically engineered cells. Transfection of the neomycin resistance genes (neo) into cells produces resistance to G418 and allows the cells to grow in media containing G418. In addition to its use in gene transfer experiments, G418 is used for the elimination of cells (such as fibroblasts) contaminating mixed cell cultures.

G418 is produced in nature by a species of the microorganism

Micromonospora. The species is M. rhodorangea. The preliminary characterization of G418 was described by Wagman et al. (Antimicrob. Ag. Chemother. 6: 144-149, 1974). Production of partially purified G418 is disclosed in U.S. Patent No. 3,997,403 (the '403 Patent), naming Weinstein, Wagman, Testa and Marquez as inventors.

The processes disclosed in the '403 Patent for producing a G418 preparation are the commonly used processes in the art. Example 2 of the '403 Patent describes initial isolation of G418 from fermentation broth obtained from tank fermentation (Example 1 of the '403 Patent) of Micromonospora rhodorangea. The initial isolation of G418 involves the use of Amberlite IRC-50 cation exchange resin. In Example 3 of the '403 Patent, the eluate (of the ion exchange resin of Example 2) that contains crude G418 is subjected, in part, to exposure to activated charcoal and later to Amberlite IRA-401S anion exchange resin. The partially purified G418 (of Example 3) is subjected to further purification by either of two processes, as described in Example 4 and Example 5 of the '403 Patent. In Example 4, the partially purified G418 (of Example 3) is subjected, in part, to activated charcoal for further purification. In Example 5, the partially purified G418 (of Example 3) is subjected, in part, to Dowex 1X2 anion exchange resin in the hydroxyl form.

The potency of killing gentamicin-sensitive cells is not the same for all gentamicins. Therefore, if a preparation of G418 contains other gentamicins or gentamicin-like aminoglycoside antibiotics (collectively part of "aminoglycoside antibiotic impurities"), the cell-killing potency will be different than that for high purity antibiotic G418. The problem is made worse due to either the level of impurities in general varying or the ratio of impurities relative to each other (or both level and ratio) varying, from batch to batch of G418.

In order for the selection of sensitive versus resistant cells to work, the cytotoxicity of the G418 plus its impurities (which possess different cell-killing potencies) has to be very different for sensitive cells and resistant cells. For each batch of G418 with varying impurity profiles (e.g., type, level or ratio, or combinations thereof), a researchist must re -optimize the concentration of G418 containing impurities so that at least 90% of the sensitive cells are killed and at least 90% of the resistant cells survive without much damage. The re-optimization process requires expenditures of time and resources. Further, the presence of aminoglycoside antibiobiotic impurities in commercially available G418 causes the loss of resistant cells, unless low concentrations are used which then significantly increases the time to kill the sensitive cells.

Accordingly, there is a need in the art of in vitro cell selection for an improved preparation of antibiotic G418 that is of higher purity than the preparations currently commercially available. The present invention fulfills this need and further provides other related advantages. BRIEF SUMMARY

Briefly stated, a preparation of antibiotic G418 with low cell toxicity and high purity, and methods of producing such a preparation, are provided. This highly pure antibiotic G418 with low cell toxicity may be used for a variety of purposes including, for example, for in vitro cell selection.

In an embodiment, the present invention provides an isolated antibiotic G418 with low cell toxicity having a purity of greater than 95% and no individual group of gentamicin impurities exceeding 3%, wherein the purity and the impurities are measured by high performance liquid chromatography (HPLC), and G418 having the formula:

In an embodiment, the isolated antibiotic G418 is in combination with a carrier or diluent acceptable to one or more cell type.

In an embodiment, the present invention provides a method of producing antibiotic G418 with low cell toxicity having a purity of greater than 95% and no individual group of gentamicin impurities exceeding 3%, wherein the purity and the impurities are measured by HPLC, and G418 having the formula set forth herein, comprising the steps of:

(a) suspending a preparation of G418 base containing aminoglycoside antibiotic impurities in a solvent mixture composed of methanol:chloroform:concentrated ammonium hydroxide (1 :2: 1 v/v), thereby forming a suspension; (b) filtering the suspension of step (a), to produce a filtrate and insolubles;

(c) concentrating the filtrate of step (b), to produce a concentrated filtrate;

filtrate of step (c) to silica gel, thereby of step (d) with a solvent mixture composed of methanol: chloroform: concentrated ammonium hydroxide (1 : 1 : 1 v/v) to desorb G418 thereby separating G418 from some impurities contained in the preparation of step (a) to yield G418 with low cell toxicity having a purity of greater than 95% and no individual group of gentamicin impurities exceeding 3%, as measured by HPLC.

These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a chromatogram from HPLC of commercial grade G418 (Invitrogen, Carlsbad, CA). The chromatogram shows the amount of G418 relative to aminoglycoside antibiotic impurities, such as gentamicin A and gentamicin X2, in the preparation of commercial grade G418.

Figure 2 is a chromatogram from HPLC of high purity grade G418 (prepared according to the methods disclosed herein). HPLC was performed in a manner identical to that of the HPLC used to produce the chromatogram of Figure 1. DETAILED DESCRIPTION

As noted above, the present invention provides a preparation of antibiotic G418 with low cell toxicity and high purity, and methods of producing such a preparation. This highly pure antibiotic G418 with low cell toxicity has a variety of in vitro uses. As used herein, the term "antibiotic G418" (also referred to simply as "G418" herein) includes salts thereof, such as acid addition salts. For example, monosulfate and disulfate salts of G418 are included. The non-salt form of G418 is "G418 base" and is where all three primary amino groups of G418 are unprotonated. Sulfate salts of G418 may be prepared by reaction of G418 base with sulfuric acid. Alternatively, sulfuric acid may be substituted with an equivalent of other inorganic or organic acids to form salts other than sulfate salts. As used herein, the phrase "low cell toxicity" refers to low toxicity to cells resistant to G418.

The percent purity of antibiotic G418 and the percent impurity of individual groups of gentamicin (for example, all gentamicin C fractions as one such individual group) within a G418 preparation may be measured by high performance liquid chromatography (herein referred to as "HPLC"). The specific protocol used herein to assay G418 purity and gentamicin impurities is that described by Anhalt and Brown in Clin. Chem. 24: 1940-1947 (1978), which disclosure is specifically incorporated by reference rather than reproduced herein since it is part of the art and well known to those in the art. As used herein, the phrases "wherein the purity and the impurities are measured by high performance liquid chromatography" or "wherein the purity and the impurities are measured by HPLC" or "as measured by high performance liquid chromatography" or "as measured by HPLC" or "using HPLC" or the like, each refer to the HPLC methodology described by the Anhalt and Brown journal article (as cited above).

Many manufacturers of antibiotic G418 use the process of making G418 that is disclosed in U.S. Patent No. 3,997,403 (the '403 Patent), which is described in the BACKGROUND section herein. When a commercial grade G418 was tested in the context of the present invention using HPLC, the commercial grade of G418 had only a maximum of less than 92% purity (Figure 1). Many of the commercial grade G418 have over 10%, and some over 30%>, impurities. In the commercial grade of G418, there are aminoglycoside antibiotic impurities, including gentamicin family members such as gentamicin A, gentamicin C fractions and gentamicin X2. Relative to G418, most gentamicin family members have reduced or no antibiotic activity relevant to, for example, cell selection. Thus, the potency of killing sensitive versus resistant cell lines is not the same among different gentamicins. Since the levels of gentamicins present vary relative to G418, and the ratio of particular gentamicins vary among themselves, from batch to batch of commercial grade G418, the concentration of a particular G418 preparation needed to effect about 90% killing of sensitive cells with 90%> survival of resistant cells will vary relative to other G418 preparations.

The present invention provides isolated antibiotic G418 with low cell toxicity and high purity. The purity is greater than 95% and no individual group of gentamicin impurities exceed 3%, as measured by HPLC. This is a significantly greater purity than most commercially available preparations and better purity than even the best commercial grade G418 currently available. This antibiotic G418 with low cell toxicity having high purity is an unexpected improvement in the art.

In the present invention, the production of antibiotic G418 with low cell toxicity having high purity is effected (as described in detail in the EXAMPLES section herein) by the use of silica gel in combination with the use of certain solvent mixtures with particular ratios which are specific to dissolution and loading versus elution of G418 from the silica gel preparation.

The isolated antibiotic G418 of the present invention with low cell toxicity having a purity of greater than 95% and no individual group of gentamicin impurities exceeding 3%, as measured by HPLC, has a variety of in vitro uses. One of the uses is for cell selection. For example, this high purity G418 may be used for selection and maintenance of prokaryotic and eukaryotic cells stably transfected with neomycin resistance genes. This methodology is commonly used in vitro in gene transfer experiments. Another use of this high purity G418 may be for the elimination of cells contaminating mixed cell cultures. An example of cells that may be desired to be eliminated are fibroblasts. The above uses and others are well known to those in the art.

The following Examples are offered by way of illustration and not by way of limitations. EXAMPLES

EXAMPLE 1

TESTING OF DIFFERENT BATCHES OF COMMERCIALLY AVAILABLE G418

Different batches of G418 sulfate (from Invitrogen, Carlsbad, CA) are tested for ED90 (leads to 90% death) for both sensitive cells and the survive rate recorded for the resistant cells at the same concentration of G418 sulfate. This is shown in the following table:

Table 1

Selectivity properties of different batches of G418 sulfate ED90

All batches of G418 sulfate have been vacuum dried at 60°C for two hours in order to reach the same water content.

The purity as measured by HPLC and the results shown as impurity profiles of these batches are listed in the following table:

Table 2

A B C D

Gentamicin x 5.5% 7.6% 6.2% 6.3%

Gentamicin A 3.1% 4.2% 6.1% 4.7%

Gentax C 0.7% 6.4% 7.7% 2.1%

G418 90.7% 81.8% 80% 86.9%

Water content 11.3% 12% 9.8% 8.3%

From the above tables (Table 1 and Table 2), it can be seen that the G418 sulfate varies from 80%> to 90%> purity, but the survive rate changes even more dramatically. In order to reach 99% death rate of the sensitive cells, the concentration of G418 sulfate varies for sensitive cells, but the survive rate of resistant cells varies from 58% to 93%. The death rate of the resistant cells may be caused by different impurities. Prior to the present invention, high purity G418 sulfate has not been previously available to test its toxicity to different cell lines and at the same time to prepare its impurities to test the selectivity properties. The present invention makes a G418 sulfate product having purity greater than 95% and gentamicin C contamination to be less than 0.5%>.

"High purity" (i.e., of the present invention) G418 Sulfate (99.5%) was prepared as well as all its impurities, like Gentamicin A (99.1%), Gentamicin X2 (99.8%) and Gentamicin Cla (99.6%), Clb(99.1%), and C2 (99.75%), by preparative HPLC, in order to test the toxicity of G418 sulfate. The concentration of LD10 ("LD" refers to "Lethal Dosage" and "LD10" to the lethal dosage causing 10% death) of the resistant cells for different cell lines and impurities is in the following table: Table 3

LD10 concentration for different cell lines and

different component related to G418

From the above table, it is seen that the gentamicin impurities are much more toxic to the common cell lines. The toxicity level varies from 4 times (gentamicin x2 vs G418 to CHO cells ) to about 120 times (gentamicin clb vs G418 to NIH 373).

The high concentration of high purity G418 sulfate will kill the sensitive cells quicker than low concentration and the resistant surviving rate is much higher, thus the selection process is done quicker than with presently available commercial grade G418 sulfate. EXAMPLE 2

PROCESS OF PREPARING THE OVER 95% PURE G418 SULFATE The G418 made by using the method desclosed in U.S. Patent No. 3,997,403 (the '403 Patent) has been sold by many manufacturers. The purity of those G418 preparations has never exceeded 92%.

The process of the present invention is as follows. Suspend 6 kg of silica gel (silica gel 60, 200-400 mesh, 43-60 micron; EMD Chemicals, Inc., Gibbstown, NJ, a part of Merck KGaA, Darmstadt, Germany) in the lower phase of a solvent mixture composed of methonal:chloroform:concentrated ammonia hydroxide (1 :2: 1 v/v). Transfer the suspension into a chromatographic column having an inner diameter of about 10 cm. Suspend 100 g of antibiotic G418 base (obtained by the procedure of Example 3 of the '403 Patent), which has main impurities of gentamicin x2 and A, in about 1.0 liter of the lower phase of the solvent system used to prepare the chromatographic column. Filter the so obtained suspension and retain the insolubles for further processing. Concentrate the filtrate to approximately 1.0 liter prior to placing atop the column. The ratio of elution solvents is set to 1 : 1 : 1 (methonal:chloroform:concentrated ammonia hydroxide) and elution is continued until the remaining material is desorbed. Generally, in effecting the separation of the antibiotic complex on a column of this size, the number of fractions collected is from about 250 to about 350. Spot each fraction on filter paper and test with ninhydrin reagent to determine the presence or absence of antibiotic (i.e., ninhydrin positive material). Subject those fractions containing antibiotic substances to paper chromatography in a system consisting of chloroform: methanol: 17% ammonium hydroxide (2: 1 :1 v/v) followed by bioautography against Staphylococcus aureus (ATCC 6538P), to determine which fractions contain the same antibiotic. Combine the fractions in accordance with the determination made by chromatography and bioautography, concentrate the combined fractions in vacuo to about 300 ml and adjust the pH by adding a 3 mol solution of sulfuric acid to obtain a pH of 5.4, and then lyophilize. If different purity fractions are collected, different purity of over 95% to 99.5% is reached. The distribution of antibiotics from the column is substantially as follows:

Fractions antibiotics

1-118 Inactive organic materials or nihydran negative fractions

119- 259 Antibiotic G418

259- end Gentamicin A and X2

See Figure 2 for an HPLC chromatogram of high purity G418 sulfate. EXAMPLE 3

COMPARISON OF COMMERCIALLY AVAILABLE G418 VERSUS HIGH PURITY G418

A. "Normal" (i.e., commercially available) G418 sulfate ( around 80% purity) versus 99% pure G418 sulfate

A normal grade of G418 sulfate was chosen to compare to the 99% pure G418 sulfate of the present invention, at different dates at various concentrations. The results of this comparison are shown in the following table:

Table 4

Selectivity properties of different batches of commercially available G418 sulfate comparing with the high purity G418 sulfate

Surviving rate of Resistance NIH3T3 98% 95% 92% 90%

All batches of G418 sulfate have been vacuum dried at 60°C for two hours in order to reach the same water content.

From the above table, it is seen that the concentration of the high purity G418 sulfate of the present invention can be increased and does not cause the loss of resistant cells while the increase of concentration of commercial grade G418 sulfate (including Geneticin) causes the loss of resistant cells. At low concentration, the sensitive cells take a long time to be killed. Assuming the death rate of 10% for the resistant cells is a successful selection, the normal G418 sulfate still has around 10%> of the sensitive cells surviving. Assuming a 70%> resistant cell surviving rate as successful selection, the concentration of high purity G418 sulfate of the present invention can be very high and the selection process can be done within a week as compared to three weeks.

B. Geneticin ( around 90%> purity) vs 97% pure G418 sulfate Geneticin is compared with 97% pure G418 sulfate (gentamicin X2

1.1%, gentamicin Al .7% Gentamicin C 0.2%) at different dates at various concentrations. The results of this comparison are shown in the following table:

Table 5

Selectivity properties of geneticin comparing with

the high purity G418 sulfate (97%)

Surviving rate of Resistance NIH3T3 98% 95% 91% 87%

All batches of G418 sulfate have been vacuum dried at 60°C for two hours in order to reach the same water content.

From the above table it is seen that the concentration of the high purity G418 sulfate of the present invention can be increased and does not cause the loss of resistant cells while the increase of Geneticin causes the loss of resistant cells. At low concentration, the sensitive cells take a long time to be killed. Assuming the death rate of 10%) for the resistant cells is a successful selection, the Geneticin needs 28 days to complete the selection, while a 97% G418 sulfate preparation of the present invention takes 14 days to complete the selection. This reduction in time to complete the selection is of significant benefit to researchers in industry and academia. Thus, the surprisingly highly purified G418 preparation of the present invention will be of assistance to the progress of research. The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.