STABILIZATION OF GLUTAMATE DEHYDROGENASE IN AN AQUEOUS SOLUTION

Abstract

An aqueous composition includes (i) glutamate dehydrogenase from a bacterium of the Clostridium genus, (ii) a stabilizing compound that is a carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two COOH groups, or a salt thereof, and (iii) any of a monosaccharide polyol, disaccharide polyol, or polymeric macromolecule in addition to the glutamate dehydrogenase. A process for stabilizing the glutamate dehydrogenase in order to maintain antigenic properties of the glutamate dehydrogenase includes stabilizing the glutamate dehydrogenase in the aqueous composition and maintaining the antigenic properties of the glutamate dehydrogenase during storage of the aqueous composition.

Claims

1. A process for stabilizing glutamate dehydrogenase from a bacterium of the Clostridium genus in order to maintain antigenic properties of the glutamate dehydrogenase, comprising: stabilizing the glutamate dehydrogenase in an aqueous solution comprising (i) a stabilizing compound that is a carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two COOH groups, or a salt thereof, and (ii) any of a monosaccharide polyol, disaccharide polyol, or polymeric macromolecule in addition to the glutamate dehydrogenase; and maintaining the antigenic properties of the glutamate dehydrogenase during storage of the aqueous solution, wherein: the glutamate dehydrogenase is in the aqueous solution at a concentration ranging from 0.75 to 10 ng/ml; and the aqueous composition has a pH of between 4.5 and 7.

2. The process as claimed in claim 1, wherein the carboxylic acid contains a carbon-based chain of 4, 5 or 6 carbon atoms.

3. The process as claimed in claim 1, wherein the stabilizing compound is chosen from the following carboxylic acids and salts thereof: (i) carboxylic acids containing a carbon-based chain of four carbon atoms having at least two COOH groups and CX groups chosen independently from =CH, CH.sub.2 and C(H)OH, and (ii) carboxylic acids containing a carbon-based chain of five carbon atoms having at least two COOH groups and CX groups chosen from CH.sub.2 and C(H)NH.sub.2, and (iii) carboxylic acids containing a carbon-based chain of six carbon atoms having at least two COOH groups and CX groups chosen from CH.sub.2, C(H)OH and C(O)NH.sub.2.

4. The process as claimed in claim 1, wherein the carboxylic acid contains a double bond in its carbon-based chain and is an E isomer.

5. The process as claimed in claim 1, wherein the carboxylic acid contains two COOH groups.

6. The process as claimed in claim 1, wherein the stabilizing compound is chosen from fumaric acid, succinic acid, malic acid, glutaric acid, citric acid, tartaric acid, N-(2-acetamido)iminodiacetic acid, glutamic acid, adipic acid, aspartic acid, pimelic acid and malonic acid, and salts thereof.

7. The process as claimed in claim 1, wherein the stabilizing compound is chosen from succinic acid and fumaric acid, and salts thereof.

8. The process as claimed in claim 1, wherein the stabilizing compound is chosen from malic acid, glutaric acid, citric acid, N-(2-acetamido)iminodiacetic acid, glutamic acid, adipic acid, pimelic acid and malonic acid, and salts thereof.

9. The process as claimed in claim 1, wherein the aqueous solution comprises at least 50% by volume of water.

10. The process as claimed in claim 1, wherein the aqueous solution comprises any of glycerol, erythritol, xylitol, arabitol, ribitol, sorbitol, dulcitol, mannitol, or volemitol as the monosaccharide polyol.

11. The process as claimed in claim 1, wherein the aqueous solution comprises any of maltitol, isomaltitol, or lactitol as the disaccharide polyol.

12. The process as claimed in claim 1, wherein the aqueous solution comprises a filler protein as the polymeric macromolecule.

13. The process as claimed in claim 1, wherein the aqueous solution comprises a dextran or polyethylene glycol polymer as the polymeric macromolecule.

14. The process as claimed in claim 1, further comprising binding a binding partner to the glutamate dehydrogenase to detect or quantify the glutamate dehydrogenase as part of a glutamate dehydrogenase immunoassay.

15. The process as claimed in claim 14, wherein the glutamate dehydrogenase is detected or quantified as a control, a calibrator, or for establishing a standard concentration range.

16. An aqueous composition comprising: (i) glutamate dehydrogenase from a bacterium of the Clostridium genus; (ii) a stabilizing compound that is a carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two COOH groups, or a salt thereof; and (iii) any of a monosaccharide polyol, disaccharide polyol, or polymeric macromolecule in addition to the glutamate dehydrogenase, wherein: the glutamate dehydrogenase is in the aqueous composition at a concentration ranging from 0.75 to 10 ng/ml; and the aqueous composition has a pH of between 4.5 and 7.

17. A kit for detecting the presence of a bacterium of the Clostridium genus in a biological sample that may contain the bacterium, comprising the aqueous composition as claimed in claim 16 and compounds required for carrying out a process for detecting the presence of the bacterium of the Clostridium genus.

18. A process for detecting the presence of a bacterium of the Clostridium genus in a biological sample that may contain the bacterium, comprising the steps of: (i) carrying out a process for detecting the presence of the bacterium of the Clostridium genus by detecting or quantifying the presence of glutamate dehydrogenase in said sample using the aqueous composition as claimed in claim 16, and (ii) concluding that the bacterium is present if a positive result is obtained in step (i).

19. A process for detecting the presence of a toxigenic bacterium of the Clostridium genus which produces at least one toxin, in a biological sample that may contain the bacterium and the at least one toxin, comprising the steps of: (i) carrying out a process for detecting the presence of the bacterium of the Clostridium genus by detecting or quantifying the presence of glutamate dehydrogenase in said sample using the aqueous composition as claimed in claim 16, and (ii) concluding that the bacterium is absent if a negative result is obtained in step (i), or (ii) if the process of step (i) is positive, carrying out a process for detecting or quantifying the at least one toxin that may be released by said bacterium of the Clostridium genus, in the same biological sample, or in a new biological sample from the same individual, and concluding that the bacterium is toxigenic and produces said at least one toxin if said at least one toxin is present.

20. The process as claimed in claim 19, wherein the bacterium is Clostridium difficile and said at least one toxin comprises toxin A, toxin B or both.

Description

[0117] The invention will be understood more clearly by means of the following examples which are given by way of nonlimiting illustration, and also by means of FIGS. 1 and 2, in which:

[0118] FIG. 1 gives examples of stabilizing compounds used in the aqueous compositions according to the invention, and also the chemical formula thereof, and

[0119] FIG. 2 is a graph giving the change in the fluorescent signal obtained during an immunoassay with the VIDAS GDH kit (bioMrieux) emitted by aqueous compositions according to the invention or comparative compositions, as a function of time, when these compositions are maintained at 37 C.

EXAMPLES

Example 1: Preparation of Recombinant GDH from Clostridium difficile

[0120] The gluD gene encoding the GDH from Clostridium difficile (Genbank accession No. M65250), with a sequencing encoding a HIS tag added thereto, is cloned into the pMR78 vector (bioMrieux, France). The expression plasmid thus constructed is introduced into E. coli BL21 bacteria and derivatives (Stratagene, Agilent Technologies). The cultures are carried out in 2YT medium (Difco), in the presence of ampicillin, at 37 C. with shaking. The expression of the protein is induced by adding 1 mM of IPTG (isopropyl beta-D-1-thiogalactopyranoside). The bacteria are collected by centrifugation at the end of culturing.

[0121] The bacterial pellets are taken up in 2PBS buffer (phosphate buffered saline) and lysed. The lysates are centrifuged at 3000 g for 30 min at 4 C. The supernatant contains the soluble proteins, including the recombinant GDH to be purified.

[0122] The purification of the protein is carried out by one-step metal chelate affinity chromatography. The supernatant obtained after centrifugation is loaded onto an Ni-NTA-Agarose resin (Qiagen). After a washing cycle, the protein is eluted in the presence of an imidazol gradient. The protein is dialyzed in a 20 mM phosphate buffer.

Example 2: Demonstration of the Stabilization of the Antigenic Properties of GDH Using Stabilizing Compounds According to the Invention

[0123] The recombinant GDH, prepared in example 1, is diluted to 3 ng/ml in the following formulations, according to the indications relating to the calibrator and control (S1/C1) given in the information sheet for the VIDAS GDH reagent (Ref 30125, bioMrieux, France): [0124] Comparative composition: 100 mM phosphate+50 g/l BSA, pH 5.8 (formulation of the calibration solution of the Vidas kit, GDH in lyophilized form, taken up in demineralized water), [0125] Composition according to the invention 1:100 mM N-(2-acetamido)iminodiacetic acid (ADA)+50 g/l BSA, pH 5.8 (ADA composition), [0126] Composition according to the invention 2:100 mM succinic acid+50 g/l BSA, pH 5.8 (succinate composition), [0127] Composition according to the invention 3:100 mM disodium fumarate+50 g/l BSA, pH 5.8 (fumarate composition).

[0128] Each composition (comparative and ADA, succinate and fumarate) was prepared beforehand as follows: each stabilizing compound (1.18 g of succinic acid Merck, 1.6 g of dibasic sodium fumarateSigma, 1.90 g of ADASigma or 0.78 g of phosphate NaH.sub.2PO.sub.4.2H.sub.2O+1.79 g of Na.sub.2HPO.sub.4.12H.sub.2O) was mixed with demineralized water so as to obtain 50 ml. 10 N NaOH was then added so as to adjust the pH to 5.8. Five g of BSA (Millipore) were added. Finally, demineralized water was added to as to obtain a solution of 100 ml.

[0129] The four compositions containing the GDH are then aliquoted into fractions of 1 ml and then stored at 37+/1 C. The impact of the formulation on the stability of the antigenic properties of the recombinant GDH protein is evaluated by carrying out several GDH assays over a period of 91 days with the VIDAS GDH kit (Ref 30125) and the VIDAS instrument according to the instructions of the manufacturer (bioMrieux, France).

[0130] The implementation of the VIDAS GDH test adheres to the protocol of the kit sold: [0131] introduction of 200 l of sample+1 ml of the R1 pretreatment reagent [0132] homogenization by vortexing [0133] introduction of 300 l of this dilution to 1/6 into the sample well of the cartridge of the VIDAS GDH kit.

[0134] Each aliquot is used for only one monitoring timepoint, but is systematically used in duplicate. The VIDAS instrument measures a fluorescence signal and the results are expressed as relative fluorescence value or RFV.

[0135] The RFV results obtained for the two measurements in duplicate (1 and 2) and also the D/D0 ratio (RFV on day D relative to RFV on day 0) are given in table 4 below and are also reproduced on the graph of FIG. 2.

TABLE-US-00007 TABLE 4 Comparative ADA Succinate Fumarate Days composition composition composition composition 37 C. 1 2 D/D0 1 2 D/D0 1 2 D/D0 1 2 D/D0 0 417 445 1 428 451 1 419 462 1 446 428 1 7 132 137 0.31 355 357 0.81 339 371 0.81 371 364 0.84 14 25 25 0.06 278 311 0.67 343 340 0.78 375 378 0.86 28 8 12 0.02 249 221 0.53 336 354 0.78 329 329 0.75 63 NA NA NA 124 108 0.26 258 260 0.59 322 302 0.71 91 NA NA NA 59 64 0.14 209 210 0.48 297 302 0.69 NA = not applicable

[0136] The results demonstrate that, at 37 C., the use of stabilizing compounds consisting of carboxylic acids having a carbon-based chain of at least three carbon atoms and comprising at least two COOH groups makes it possible to very substantially improve the storage time of an aqueous solution containing GDH since, for the comparative composition, there is no longer any signal at 28 days, whereas the D/D0 ratio at this date for the compositions according to the invention is at least equal to 0.5.

Example 3: Monitoring of the Antigenic Properties of GDH in an Aqueous Solution Using Citric Acid as Stabilizing Compound

[0137] The procedure of example 2 was repeated, except for the fact that citric acid (100 mM) was used as stabilizing compound and that the aqueous solutions were stored for a longer period of time, at 2-8 C. and at 37 C.

[0138] The RFV results obtained for the two measurements in duplicate (1 and 2) and also the D/D0 ratio are given in table 5 below.

TABLE-US-00008 TABLE 5 Citrate composition-37 C. Citrate composition-2-8 C. Days 1 2 D/D0 1 2 D/D0 0 713 677 1 713 677 1 1 697 757 1.05 653 722 0.99 7 717 683 1 636 694 0.96 14 666 678 0.97 646 700 0.97 29 521 565 0.78 692 680 0.99 51 421 418 0.6 613 641 0.9 78 233 222 0.33 701 680 0.99 124 67 67 0.1 648 690 0.96 184 6 10 0.01 739 774 1.09 275 NA NA NA 657 663 0.95 369 NA NA NA 664 612 0.92 552 NA NA NA 523 558 0.78 NA: not applicable

[0139] The above results demonstrate that the addition of citric acid allows very good stability associated with the preservation of the antigenic properties of the GDH, with a virtually optimal stabilization, even after 18 months, when the aqueous composition is stored between 2-8 C.

Example 4: Monitoring of the Antigenic Properties of GDH in an Aqueous Solution Using Succinic Acid and Sorbitol

[0140] The procedure of example 2 was repeated, except for the fact that 10% of sorbitol was also added to a succinate composition and that the aqueous solutions were stored for a longer period of time, at 2-8 C. and at 37 C.

[0141] The RFV results obtained for the two measurements in duplicate (1 and 2) and also the D/D0 ratio are given in table 6 below.

TABLE-US-00009 TABLE 6 Succinate composition with Succinate composition with sorbitol-37 C. sorbitol-2-8 C. Days 1 2 D/D0 1 2 D/D0 0 726 710 1 726 710 1 1 740 714 1 744 700 1.01 7 702 696 0.97 725 764 1.04 14 689 655 0.94 720 765 1.03 29 624 658 0.89 690 708 0.97 51 653 646 0.9 603 668 0.89 78 606 591 0.83 670 720 0.97 124 529 556 0.76 709 691 0.97 184 379 376 0.53 656 785 1 275 267 286 0.39 659 710 0.95 369 156 161 0.22 692 663 0.94 552 48 48 0.07 663 710 0.96 891 NC NC NC 638 681 0.92 NC: not calculated

[0142] The above results demonstrate that succinic acid also allows a lengthy stabilization of the antigenic properties of the GDH in an aqueous solution, the addition of sorbitol not modifying this stabilization, with a stabilization which is virtually optimal, even after approximately 30 months, when the aqueous solution is stored between 2-8 C.

LITERATURE REFERENCES

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