Methods for detecting contaminants in solutions containing glucose polymers

Abstract

The invention relates to a method for detecting contaminants of glucose polymers, said contaminants being capable of acting in synergy with one another so as to trigger an inflammatory reaction, characterized in that it comprises an in vitro inflammatory response test using modified cell lines.

Claims

1. A method for detecting a contaminant in a composition comprising glucose polymers, the method comprising: contacting a cell line expressing Toll-Like Receptor-2 (TLR2) and containing a reporter gene encoding a secreted form of alkaline phosphatase, wherein the reporter gene is a Nuclear Factor-kappaB (NF-κB)-inducible secreted embryonic alkaline phosphatase (SEAP), with said composition comprising glucose polymers; contacting a negative control cell line with said composition comprising glucose polymer, measuring the activity of the secreted form of alkaline phosphatase of the reporter gene; and identifying, based on a higher activity of the reporter gene in the cell line expressing TLR2 compared to the negative control cell line, the presence in the composition of a contaminant capable of activating TLR2 and of triggering an inflammatory reaction.

2. The method of claim 1, wherein the composition comprising glucose polymers is for peritoneal dialysis, for enteral feeding, for parenteral feeding or for feeding newborn babies.

3. The method of claim 1, wherein said method further comprises: a step of treating the composition comprising glucose polymers with a mutanolysin prior to said contacting with the cell line expressing TLR2; or a step of filtering the composition comprising glucose polymers with a cut-off threshold of between 30 kD and 150 kD prior to contacting the filtrate with the cell line expressing TLR2.

4. The method of claim 1, wherein the method comprises quantifying the contaminant by comparing to a previously established dose-response curve.

5. The method of claim 4, wherein said method further comprises: a step of treating the composition comprising glucose polymers with a mutanolysin, a step of incubating mutanolysin treated and untreated glucose polymer compositions with said cell line expressing TLR2, and a step of quantifying the contaminant by comparing to a previously established dose-response curve.

6. The method of claim 1, wherein the composition comprising glucose polymers has a polymer concentration from 5 to 50 mg/ml.

7. The method of claim 1, wherein the method further comprises: contacting the composition with at least one or more cell lines selected from the group consisting of: a cell line expressing the Nucleotide-binding Oligomerization Domain-containing protein-like receptor 2 (NOD2) and containing a reporter gene encoding a secreted form of alkaline phosphatase, wherein the reporter gene is a Nuclear Factor-kappaB (NF-κB)-inducible secreted embryonic alkaline phosphatase (SEAP); a cell line expressing TLR4 and containing a reporter gene encoding a secreted form of alkaline phosphatase, wherein the reporter gene is a Nuclear Factor-kappaB (NF-κB)-inducible secreted embryonic alkaline phosphatase (SEAP); and a cell line expressing each of TLR2, TLR4 and NOD2 and containing a single reporter gene encoding a secreted form of alkaline phosphatase, wherein the reporter gene is a Nuclear Factor-kappaB (NF-κB)-inducible secreted embryonic alkaline phosphatase (SEAP); and measuring the activity of the secreted form of alkaline phosphatase of the reporter gene of the at least one or more cell-lines; and detecting, based on the activity of the secreted form of alkaline phosphatase of the reporter gene(s), the presence in the composition of pro-inflammatory contaminants capable of activating the receptors expressed by said at least one or more cell lines and of triggering an inflammatory reaction.

8. The method of claim 1, wherein the cell line expressing TLR2 is an HEK293 cell line that stably co-expresses the human TLR2 and Nuclear Factor-kappaB (NF-κB)-inducible secreted embryonic alkaline phosphatase (SEAP).

9. The method of claim 1, wherein the negative control cell line is an HEK293 cell line expressing a Nuclear Factor-kappaB (NF-κB)-inducible secreted embryonic alkaline phosphatase (SEAP).

10. The method of claim 7, wherein said at least one or more cell lines is selected from the group consisting of: an HEK293 cell line that stably co-expresses the human NOD2 and NF-κB-inducible SEAP; an HEK293 cell line that stably co-expresses the human TLR4 and NF-κB-inducible SEAP; and a RAW 264.7 cell line that stably expresses NF-κB-inducible SEAP gene and that expresses TLR2, TLR4 and NOD2.

11. The method of claim 4, wherein the dose-response curve is produced with the same cells, under the same conditions, with increasing doses of a standard amount of contaminants.

12. The method of claim 4, wherein the dose-response curve is produced for the cells expressing TLR2 by measuring the reporter gene activation after exposure to increasing doses of a standard amount of peptidoglycan.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Production of RANTES in response to the PGN and to the LPS in THP-1 cells sensitized with MDP at 10 μg/ml.

(2) FIG. 2: Production of TNF-α in response to the PGN and to the LPS in THP-1 cells sensitized with MDP at 10 μg/ml.

(3) FIG. 3: Production of RANTES in response to the PGN in THP-1 cells sensitized with f-MLP (10 nM).

(4) FIG. 4: Production of RANTES in response to the PGN in THP-1 cells sensitized with LPS (25 μg/ml).

(5) FIG. 5: Effect of the glucose polymer concentration on the production of RANTES induced by the PGN in the THP-1 cells.

(6) FIG. 6: Effect of the THP-1 cell concentration on the production of RANTES induced by the PGN.

(7) FIG. 7: Production of RANTES by the sensitized THP-1 cells in response to the various glucose polymer samples.

(8) FIG. 8: HEK-BLUE cell response test. The cells were stimulated with PGN, MDP and TNF-α, which is a positive control for stimulation of the HEK-BLUE cells.

(9) FIG. 9: SEAP response as a function of the volume of culture supernatant added to the reaction medium. The activation of the cells (HEK-TLR2) was carried out with TNF-α.

(10) FIG. 10: Optimization of the reaction time between the SEAP and its substrate. The HEK-TLR2 (FIG. 10A) and RAW-BLUE (FIG. 10B) cells were stimulated in the presence of increasing concentrations of PGN. After 20 h of stimulation, the supernatants containing the SEAP were incubated in the presence of the QUANTI-BLUE solution at the times indicated, and then the absorbance was measured at 620 nm.

(11) FIG. 11: Effect of the glucose polymer concentration on the production of SEAP by the HEK-TLR2 (FIG. 11A) and RAW-BLUE (FIG. 11B) cells in response to increasing concentrations of PGN.

(12) FIG. 12: Comparison of the SEAP responses of the HEK-NOD2 cells cultured according to the method provided by the supplier versus the improved procedure without a subculturing step before stimulation.

(13) FIG. 13: Production of SEAP in response to the PGN in HEK-TLR2 and HEK-Null cells.

(14) FIG. 14: Production of SEAP in response to the LTA in HEK-TLR2 and HEK-Null cells.

(15) FIG. 15: Production of SEAP in response to the PGN and to the LPS in RAW-BLUE cells.

(16) FIG. 16: Production of SEAP in response to the MDP in HEK-NOD2 cells.

(17) FIG. 17: SEAP activity in HEK-TLR2 cells in response to the various glucose polymer samples.

(18) FIG. 18: SEAP activity in RAW-BLUE cells in response to the various glucose polymer samples.

(19) FIG. 19: SEAP activity in HEK-NOD2 cells in response to the various glucose polymer samples.

(20) FIG. 20: SEAP production in RAW-BLUE and HEK-TLR2 cells in response to the various glucose polymer samples before (Total) and after ultrafiltration at 30 kDa (Filtrate).

(21) FIG. 21: Calibration curve of the cell response as a function of the level of S. aureus PGN. FIG. 21A, theoretical curve. FIG. 21B, curve obtained with HEK-BLUE-hTLR2 cells.

(22) FIG. 22: SEAP activity in HEK-TLR2 cells in response to various glucose polymer samples before and after treatment with the mutanolysin.

(23) FIG. 23: SEAP production by HEK-TLR2 cells in response to the PGN before and after treatment with the mutanolysin.

EXAMPLE 1

Preparation of the Glucose Polymers for Peritoneal Dialysis

(24) The raw material for obtaining the glucose polymers according to the invention is produced from waxy corn starch in the following way: cleaning of the corn so as to keep exclusively the whole corn grains, steeping of the corn thus cleaned, in the presence of lactic acid so as to soften the grains, wet milling, then separation of the various constituents, i.e. germ, cellulose husk, proteins and starch, cleaning of the starch in countercurrent mode with purified water so as to purify the starch both physicochemically and bacteriologically, centrifugation and drying of the starch, suspension of the starch in purified water at a final dry matter content of 40% and at a temperature of 45° C. to 50° C., acidification of the starch suspension by addition of HCl at a pH <2, and raising of the temperature to 115 to 120° C. for 6 to 8 minutes, flocculation of the proteins and of the fats at this pH, neutralization of the suspension at pH 5, filtration of the suspension through diatomaceous earth (so as to retain the residual proteins, fats and cellulose), demineralization on strong cationic resin and weak anionic resin, treatment with activated carbon at a temperature of 70-80° C. and at a pH of from 4 to 4.5; which removes the colored impurities and reduces the level of microbiological impurities.

(25) The activated carbon powder which is added at a concentration between 0.2% and 0.5% on a dry basis is retained on a 10 μm ceramic filter loaded beforehand with a filtering agent, concentration by passing through a falling film evaporator, spray-drying of the concentrated solution in an MSD spray dryer sold by the company Niro.

(26) This starch hydrolysate complies with the monograph of the European Pharmacopeia (ref Maltodextrins: 1542). pH: 4.0-7.0 for a solution at 10%, Id.: complies, Loss on drying: 6% max, DE: <20 Sulfated ash: 0.5% max SO.sub.2. 20 ppm max Heavy metals: <10 ppm E. coli: absent/g Salmonellae: absent/10 g Total viable microorganisms: 100 CFU/g max (EP 1000 CFU/g) Molds: 100 CFU/g max

(27) In addition to this, the batches produced are analyzed on the basis of the values of: yeast+mold contamination: 150 CFU/10 g max, i.e. 15/g max aerobic microorganisms: 500 CFU/10 g max, i.e. 50/g max endotoxins (endpoint gel clot LAL test): 20 EU/g max peptidoglycans: 2700 ng/g max

(28) The conditions for obtaining the glucose polymers in accordance with the invention from the starch hydrolysate thus obtained are the following:

(29) 1) Water preparation/water quality purification of the water by filtration through 3 μm; treatment on activated carbon, demineralization on cations and anion exchange resins, and filtration again (UA), two tanks used: 10 m.sup.3 for dissolving the starch hydrolysate, and the spray-dry rinsing and cleaning steps, 60 m.sup.3 for the main process (cleaning of the tanks, its suspensions of activated carbon and chromatography).

(30) 3) Chromatography solubilization of the starch hydrolysate with purified water so as to obtain a dry matter content of 35-45% at a temperature between 60-85° C., sterilizing filtration of the starch hydrolysate by passing it through 0.45 μm and then 0.22 μm, carried out at a □□P<3 bar, size exclusion chromatography (SEC) separation carried out using a continuous system composed of six series of double plates, each of 1 m.sup.3 of resin. The resin used is a PCR145K sold by the company PUROLITE.

(31) The solution which passes through this resin has a temperature between 75 and 85° C. at 35-45% dry matter content.

(32) The duration of each sequence defines the process.

(33) In the present case, the duration of each sequence is 15 minutes.

(34) The control is carried out by analysis of molecular weight distribution and analysis of the chromatography yield, in the following way: (Amount of dry matter of the desired fraction)/(Amount of dry matter of the feed).

(35) The lowest molecular weights interact with the resin and the high molecular weights are eluted with purified water.

(36) The concentration is carried out by falling film evaporation at a dry matter content of 35-45%.

(37) A heat treatment is carried out at a temperature of 120° C. for 2 minutes.

(38) Activated carbon is added between 0.5% and 1.5% of the total weight of the starch hydrolysate at 75° C. with cationic resins (1 to 3 l) for controlling the pH (4-4.5) and anionic resins (5 to 10 l) for controlling the pH (5.5-6).

(39) Filtration is carried out through polypropylene bag filters with □P<5 bar, in 5 to 6 hours per batch.

(40) A second and third filtration through 1.5 and 0.45 μm, and then through 0.22 and 0.1 μm, and ultrafiltration through a membrane with a cut-off threshold of about 40 000 Da are carried out.

(41) For the spray-drying: feed at 500 kgs/h with a solution at 40% dry matter content and at 250° C. in an MSD spray dryer sold by the company Niro.

(42) The spray-dried product has, on exiting, a moisture content of less than 6%.

(43) The product is then cooled in a fluidized air bed comprising three cooling zones fed with air at 40, 30 and 20° C. The product obtained is then sieved through 800 μm in order to remove the aggregates.

(44) About 500 kgs of final product are obtained from 800 kgs of starting maltodextrins, i.e. a yield of about 60%.

(45) The determination of the possible contamination of the circuit is carried out by analysis of the peptidoglycan and endotoxin content on the final product.

(46) For example, the contents usually observed and measured on the batches of the final product (expressed per g of glucose polymer) are, for the criteria specified above, the following: Yeasts and molds: 0/g Aerobic microorganisms: 0/g Endotoxins (endpoint gel clot LAL test): ≤0.3 EU/g Peptidoglycans: <3 ng/g B. acidocaldarius: 1/g

EXAMPLE 2

Use of the “Sensitized” THP-1 Cell Line for Detecting Pro-Inflammatory Contaminants

(47) Materials & Methods

(48) The THP-1 cells (88081201, ECACC) are cultured routinely in the laboratory.

(49) For the pro-inflammatory activation experiments, the THP-1 cells are differentiated for 3 days in the presence of phorbol ester (PMA). In particular, the cells are placed in culture in 200 μl of complete medium in the presence of 20 nM of PMA for 72 h (final cell density: 0.75×10.sup.6 cells/ml).

(50) The glucose polymer samples are prepared according to example 1.

(51) TABLE-US-00001 TABLE 1 Glucose polymer samples I- I- I- I- MM- MM- MP- MP- 10.01 10.02 10.03 11.12 10.04 10.05 10.06 10.07 LAL Test <0.3 0.3 <0.3 0.6 1.2 9.6 <0.3 38.4 (EU/g) Standard <20 755 27 530 <20 2755 <20 4613 SLP Test (ng/g) SLP-HS <3 253 12 393 <3 501 <3 645 Test (ng/g)

(52) The standard molecules for establishing the calibration ranges are, for the: LAL test: E. coli O55B5 LPS Standard SLP test: M. luteus PGN—Wako SLP-HS test: S. aureus PGN—Wako.

(53) The assayed PGN values differ from one Wako test to another owing to the different reactivity and sensitivity of these tests and potentially to their limited and relative specificity (in particular, possible response to β-glucans).

(54) The optimization studies are carried out with I-10.01 glucose polymer solutions (table 1) artificially loaded with standard inflammatory molecules: PGN and MDP (source: S. aureus), LPS (source: E. coli), f-MLP (synthetic peptide).

(55) The solution for dilution of the standards is I-10-01 with <0.3 EU/g of LPS (LAL test), <20 ng/g of PGN (standard SLP test) and <3 ng/g (SLP-HS test) and used at the final concentration of 5 mg/ml.

(56) The analyses are then carried out on a first series of samples corresponding to various batches selected on the basis of the levels of contamination with impurities measured using the LAL and SLP tests (PGN, LPS and β-glucans).

(57) The ELISA kits for assaying TNF-α and CCL5/RANTES are purchased from AbCys, the standard agonists (PGN, LPS, f-MLP and MDP) from Sigma Aldrich and InvivoGen.

(58) The differentiated THP-1 cells (0.75×10.sup.6 cells/ml) are placed in culture in 200 μl of complete medium, and then incubated in the presence of the various test samples.

(59) Each analysis is carried out in triplicate.

(60) The cell supernatants are collected in order to assay the secreted cytokines after 8 h of stimulation for TNF-α, and 20 h for RANTES.

(61) The ELISA assays are carried out according to the indications given by the supplier.

(62) Results

(63) The first tests consisted in testing the synergistic potential of MDP and of f-MLP, and also the PGN/LPS combination, on the production of pro-inflammatory cytokines by the differentiated THP-1 cells.

(64) The MDP was tested at the doses of 1, 10 and 100 μg/ml. The minimum dose does not induce a significant synergistic effect. On the other hand, the doses of 10 and 100 μg/ml have a similar synergistic activity on the production of RANTES and of TNF-α in response to the PGN and LPS.

(65) The results presented in FIGS. 1 and 2 clearly show a synergistic effect of MDP on the production of RANTES. On the other hand, this synergistic effect is not very marked for TNF-α. Furthermore, the detection threshold (amount of PGN or of LPS giving a response greater than three times the SD of the background noise) is lower for RANTES (see Table 2).

(66) The f-MLP was used at the doses of 1 nM, 10 nM and 100 nM. However, no synergistic effect was observed in the THP-1 cells (FIG. 3).

(67) The synergistic effect of the LPS was analyzed by adding a sub-optimal dose (25 pg/ml) to increasing doses of PGN (FIG. 4). The synergistic effect of the two agonists is visible after assaying the two cytokines. However, the LPS-induced synergistic effect on the production of RANTES remains less than that induced by MDP.

(68) The response was quantified by measuring the production of RANTES and of TNF-α. In all cases, synergy is clearly visible for the RANTES assay, with a high sensitivity. This assay will therefore be preferred and retained for the rest of the experiments.

(69) These results show that the addition of MDP at 10 μg/ml to THP-1 cells, with in return assaying of RANTES, is the most effective mode of sensitization for detecting low levels of PGN and of LPS. In theory, these detection thresholds should make it possible to detect the contaminants present in manufactured products.

(70) These first analyses were carried out by diluting the standard inflammatory molecules in the presence of the I-10.01 polymer. The solutions were added to the THP-1 cells so as to obtain a final glucose polymer concentration of 5 mg/ml and a final cell density of 0.75×10.sup.6 cells/ml. In order to increase the sensitivity of the assay, the tests were carried out while varying these two parameters.

(71) The presence of the glucose polymer does not hamper the production of RANTES for concentrations less than or equal to 25 mg/ml. This increase in sensitivity is not linked to a pro-inflammatory effect of the polymer on the cells, since the response is identical to the background noise in the absence of PGN (FIG. 5).

(72) On the other hand, increasing the cell density beyond 0.75×10.sup.6/ml reduces the production of RANTES in response to PGN (exhaustion of the culture medium or cell modification) (FIG. 6).

(73) The sensitivity tests with MDP at 10 μg/ml were reproduced three times for LPS and six times for PGN with THP-1 cells originating from distinct preparations. The data obtained made it possible to determine the detection thresholds and the EC50s (Table 2). For the estimation of the sensitivity, the values were brought to per g of polymer, with the concentration of 25 mg/ml being considered.

(74) The synergistic effect induced by MDP is identical for LPS and PGN, since it makes it possible to increase the sensitivity of the THP-1 cells by a factor of 5 in both cases.

(75) TABLE-US-00002 TABLE 2 Detection thresholds and EC50 for PGN and LPS in the sensitized THP-1 model. PGN PGN + MDP LPS LPS + MDP Detection 14.5 ± 8.5 2.8 ± 1.2 10 ± 7 2 ± 1 threshold ng/ml ng/ml pg/ml pg/ml EC50 1.2 ± 0.7 0.4 ± 0.2 0.9 ± 0.1 0.3 ± 0.1 μg/ml μg/ml ng/ml ng/ml Sensitivity 580 ± 340 112 ± 48 400 ± 280 80 ± 40 ng/g ng/g pg/g pg/g

(76) These studies show that the sensitized THP-1 cells can be used to develop a sensitive inflammatory response test for detecting traces of contaminants in glucose polymer preparations.

(77) The following experimental conditions are selected: final concentration of differentiated THP-1 cells: 0.75×10.sup.6 cells/ml, sensitizing agent: MDP (S. aureus) at 10 μg/ml, final glucose polymer concentration: 25 mg/ml, response: ELISA assay of the production of RANTES after 20 h of stimulation.

(78) In order to validate the method, the tests with the MDP-sensitized THP-1 cells were carried out with the samples presented in table 1.

(79) The test based on the production of RANTES by sensitized THP-1 cells makes it possible to detect the samples of polymers contaminated with LPS: polymers referenced I-11.12, MP-10.07 and, to a lesser extent, MM-10.04 and MM-10.05 (FIG. 7).

(80) On the other hand, the samples contaminated only with PGN (e.g. I 10-02) do not give a response, thereby indicating that this cell test is more suitable for the detection of endotoxins. However, the amplitude of the responses is not directly proportional to the level of LPS measured using the LAL test (see, for example, the responses obtained with I-11.12 compared with MM-10.05), thereby suggesting that contaminants other than PGN probably act with LPS on the response of the THP-1 cells.

(81) The THP-1 cells respond to the solutions of control PGN, but barely or not at all to the samples originating from batches contaminated only with natural PGN.

(82) The size of the PGNs is very heterogeneous, and some of these molecules can reach impressive weights (>10.sup.6 Da). The latter have low solubility, which affects their pro-inflammatory power but promotes their removal by filtration. On the other hand, the PGNs which are soluble, and consequently active, have an average weight of 120 kDa and are not removed by filtration. It is therefore possible to envision that the two Wako SLP tests enable an overall assaying of all the PGNs, whereas the cell test detects only the active PGNs.

EXAMPLE 3

Use of the HEK-BLUE (hTLR2, hNOD2, Null2) and RAW-BLUE (InvivoGen) Cell Lines for Detecting Contaminants

(83) Materials & Methods

(84) The HEK-BLUE cell lines (InvivoGen) are lines modified by stable transfection with vectors encoding innate immunity receptors. These cells are also cotransfected with a reporter gene which produces a secreted form of alkaline phosphatase (SEAP: secreted embryonic alkaline phosphatase), the synthesis of which is under the direct control of the signaling pathway associated with the receptor(s) expressed in the same cell line.

(85) For the experiments relating to the detection of inflammatory contaminants, four lines are used:

(86) HEK-BLUE hTLR2 line (HEK-TLR2): this line responds specifically to TLR2 agonists (in particular PGN and the majority of glycolipids and lipopeptides),

(87) HEK-BLUE hNOD2 line (HEK-NOD2): this line responds to MDP and related molecules, such as monomeric PGNs,

(88) HEK-BLUENull2 line (HEK-Null): this is a control line, the use of which is necessary in order to verify that the glucose polymer solutions do not induce the production of the enzyme via an intrinsic mechanism,

(89) RAW-BLUE line: this is a mouse macrophage line transfected with SEAP. This line, which naturally expresses virtually all the innate immunity receptors, is used as a positive control in the tests.

(90) The RAW-BLUE and HEK-BLUE cells are cultured according to the supplier's recommendations. In particular, the selection pressure for the plasmids encoding the inflammatory molecule receptors (TLR2 or NOD2) and encoding the SEAP is provided by adding, to the culture medium, the HEK-BLUE Selection/blasticidin antibiotics. At 75% confluence, the cells are resuspended at a cell density of 0.28×10.sup.6 cells/ml. Before stimulation, 180 μl of the cell suspension are distributed into the culture wells (96-well plate), i.e. 50 000 cells/well. The cells are then stimulated for 24 h by adding 20 μl of the test samples (in ten-times concentrated form). The stimulation lasts from 16 to 24 h.

(91) The production of SEAP in response to the contaminated molecules is estimated by measuring the phosphatase activity according to the protocol supplied by the manufacturer: 20 μl of culture supernatant are diluted in 180 μl of QUANTI-BLUE. The color develops at 37° C. and the reading is carried out, at various times, at 620 nm. The data are expressed as absorbance after subtraction of the background noise, obtained by adding the same volume of nonconditioned culture medium to the reaction medium of the enzyme.

(92) The optimization studies are carried out with I-10.01 glucose polymer solutions (table 1) artificially loaded with standard inflammatory molecules: PGN, LTA and MDP (source: S. aureus) and LPS (source: E. coli). The analyses are then carried out on the series of samples corresponding to various batches selected on the basis of the levels of contamination with PGN and LPS (Table 1).

(93) Results

(94) The HEK-BLUE cells are received in the form of frozen vials. Before beginning the inflammatory response tests, it is necessary to be sure of the resumption of growth of the cells and of their capacity to respond to inflammatory molecules. Furthermore, these transfected cells rapidly degenerate after several passages, which can result in a loss of the expression vectors for the innate immunity receptors, or even of the vector encoding the SEAP.

(95) It is therefore recommended to verify the capacity of the cells to respond to inflammatory factors at the beginning of the placing in culture, and then after several subculturing steps. These tests are carried out with PGN for HEK-TLR2, MDP for HEK-NOD2 and TNF-α, the latter being a powerful activator of the NF-κB pathway. Indeed, HEK cells naturally possess the receptor of this cytokine, and will therefore produce SEAP (the expression vector of which is under the control of the NF-κB pathway) in response to TNF-α.

(96) The results presented in FIG. 8 show the tests carried out in order to verify the resumption of growth of the three lines. As expected, the HEK-TLR2 and HEK-Null cells respond to TNF-α, with an equivalent production of SEAP. Furthermore, the HEK-Null line is insensitive to PGN and to MDP, whereas the HEK-TLR2 cell responds effectively only to PGN. These two lines have therefore acquired their phenotypic characteristics and it will be possible to use them for the rest of the experiments. In this example, the HEK-NOD2 line responds only very weakly to TNF-α and to MDP, thereby indicating that it has not acquired the expected characteristics.

(97) The response of the HEK-BLUE and RAW-BLUE cells to the pro-inflammatory molecules is directly linked to the production of SEAP. The supplier recommends adding 20 μl of culture supernatant to 180 μl of SEAP substrate. The experiments were therefore carried out under these conditions, and then optimized by increasing the volume of culture supernatant, and therefore the amount of enzyme in solution (FIG. 9).

(98) The results show that the 50/150 ratio gives a higher response than the ratio recommended by the supplier. On the other hand, the ratio 100/100 is not more effective, probably due to a lack of SEAP substrate in the reaction medium.

(99) The intensity of the coloration (620 nm) is proportional to the amount of SEAP secreted by the cells, but also to the time for hydrolysis of the substrate by the enzyme. Various visualization times were therefore tested using the same supernatants of the PGN-activated HEK-TLR2 and RAW-BLUE cells (FIG. 10).

(100) The optimum coloration is achieved starting from 60 min of reaction between the SEAP and its substrate for the HEK-TLR2 cells. A slight increase is also observed at 90 min for the RAW-BLUE cells, but this increase is probably linked to the fact that these cells produce less SEAP.

(101) The effect of the glucose polymer concentration on the cell responses was analyzed by stimulating the HEK-TLR2 and RAW-BLUE cells with standard PGN diluted in medium supplemented with the I-10.01 polymer. The solutions were then added to the cells so as to obtain final polymer concentrations ranging from 5 to 50 mg/ml (FIG. 11).

(102) The concentrations up to 37.5 mg/ml do not significantly modify the amplitude of the response to PGN in the HEK-TLR2 line. An improvement in the response to low concentrations of PGN is even noted for polymer concentrations above 25 mg/ml, probably linked to a better dispersion of the contaminant. As regards the RAW-BLUE cells, the production of SEAP is not modified, regardless of the glucose polymer concentration.

(103) The HEK-NOD2 line shows a low amplitude of response to MDP and even to TNF-α, which appears to be linked to a high background noise. In these cells, the SEAP gene is under the control of a weak promoter, which is more sensitive to cell stress than that used for the other HEK-BLUE cells. In order to reduce the background noise and to increase the amplitude of the response to the contaminants, the culture conditions were modified so as to reduce the stress of the cells.

(104) According to the supplier's recommendations, the cells at 75% confluence are resuspended at a density of 0.28×10.sup.6 cells/ml, and then 180 μL of the cell suspension are distributed into the culture well (96-well plate), i.e. 50 000 cells/well, before stimulation in the course of the day.

(105) In the new procedure referred to as without subculturing, the HEK-NOD2 cells are conditioned in wells (10 000/well) and cultured for three days. Before stimulation, the culture medium is removed and replaced with a medium containing 1% of FCS and the solutions to be assayed. In this case, the SEAP response is 5-6 times greater than the negative control, which is compatible with a test for detecting MDP in contaminated solutions (FIG. 12).

(106) These first studies show that the HEK-BLUE and RAW-BLUE cells can be used to develop sensitive inflammatory response tests for detecting traces of contaminants in glucose polymer preparations.

(107) The following experimental conditions were selected: final glucose polymer concentration: 37.5 mg/ml for the HEK-BLUE cells, and 50 mg/ml for the RAW-BLUE cells, enzymatic reaction: 50 μl of conditioned medium+150 μl of SEAP reagent, minimum reaction time: 60 min.

(108) FIGS. 13-16 show examples of assays of the SEAP activity produced by the HEK-TLR2 and RAW-BLUE cells in response to various inflammatory molecules.

(109) The HEK-TLR2 cells respond very effectively to PGN, whereas the response to LTA is weaker. The responses are, however, more effective than those observed with cells of monocyte/macrophage type. In addition, the glucose polymer has no direct effect on the production of SEAP, since no response is observed in the supernatants of HEK-Null cells.

(110) The RAW-BLUE cells respond well to PGN, but are less sensitive than the HEK-TLR2 cells. The response to LPS is weak and remains much lower than that observed when measuring the production of RANTES by the THP-1 cells.

(111) Contrary to the other cell lines, the HEK-NOD2 cells respond effectively to MDP, which can be taken advantage of for detecting the PGN degradation products.

(112) The experiments with the standard PGNs, LPS and MDP were reproduced with different cell preparations, which made it possible to determine the characteristics presented in table 3. For the estimation of the sensitivity, the values were brought to per g of polymer, with the concentration of 37.5 mg/ml being considered for the HEK-BLUE cells and 50 mg/ml for the RAW-BLUE cells.

(113) TABLE-US-00003 TABLE 3 Detection thresholds and EC50 for PGN, LPS and MDP in the HEK-BLUE and RAW-BLUE models HEK-TLR2 HEK-NOD2 line line RAW-BLUE line PGN MDP PGN LPS Detection 0.07 ± 0.04 1.5 ± 0.5 2.1 ± 1.5 0.24 ± 0.06 threshold ng/ml ng/ml ng/ml ng/ml EC50 3.1 ± 2.5 12 ± 6 210 ± 75 >10 ng/ml ng/ml ng/ml ng/ml Sensitivity 1.9 ± 1.1 40 ± 13 42 ± 30 4.8 ± 1.2 ng/g ng/g ng/g ng/g

(114) The HEK-TLR2 and RAW-BLUE lines are very effective for detecting the PGNs at low concentrations. In particular, the HEK-TLR2 line detects PGN levels below 2 ng/g of glucose polymer, i.e. a sensitivity threshold which is 50 times greater than that obtained with the sensitized THP-1 cells. The RAW-BLUE line effectively detects small traces of PGN, but it is not very reactive with respect to LPS, with a detection threshold approximately 50 times greater than that of the sensitized THP-1 cells.

(115) In order to validate these tests, the assays were therefore carried out with glucose polymer samples.

(116) The HEK-TLR2 line makes it possible to detect contaminations in the I-10.02, I-batches (the response observed with MM-10.04 is not significant in comparison with I-10.01 and is not therefore retained).

(117) On the other hand, the I-10.03 sample is not detected, which can be correlated with the very low level of PGN, close to the limit of detection of the SLP-Wako test (FIG. 17).

(118) While the positive responses obtained with I-10.02, I-11.12, MM-10.05 and MP-10.07 were expected, given the high level of PGN present in these four samples, it may be noted that there is no proportionality between the concentration given by the SLP test and the response of the cells.

(119) Indeed, the I-10.02 and I-11.12 glucose polymers give the highest responses, whereas the PGN levels are less than 1 μg/g. Conversely, the MP-10.07 sample, which is the one with the highest PGN load, gives a response close to the background noise. PGNs are macromolecules of very variable weight, and it has been demonstrated that their reactivity is inversely proportional to their molecular weight. It can therefore be envisioned that the PGNs of I-10.02 and I-11.12 are smaller in size than the PGNs present in the MM-10.05 and MP-10.07 samples, which would explain their higher pro-inflammatory potential.

(120) It is also surprising to see a response with the MP 10-06 batch, even though it does not contain PGN. However, the HEK-TLR2 cells react with other inflammatory molecules, such as lipopeptides, LTAs or LAMs (lipoarabinomannans), which are all TLR2 agonists. Thus, this result suggests that this sample which is uncontaminated in terms of absence of LPS and of PGN contains other pro-inflammatory molecules which are TLR2 agonists.

(121) With the exception of the I-10.01 and I-10.03 samples, all the samples give a positive response with the RAW-BLUE cells (FIG. 18).

(122) The level of LPS present in the I-10.02 sample is close to the detection threshold of the LAL test, which indicates that the response observed is due to the presence of PGN. This result confirms that, contrary to the THP-1 cells which do not respond to this sample, the RAW-BLUE cells are effective for detecting small contaminations with PGN. The strong response of the I-11.12, MM-10.05 and MP-10.07 batches therefore appears to be due to the presence of the two contaminants (PGN and LPS).

(123) Like the HEK-TLR2 cells, the RAW-BLUE cells respond positively to MP-10.06, which confirms the presence of a contaminant other than LPS and PGNs in this sample.

(124) Finally, the HEK-NOD2 cells react strongly in the presence of MP-10.07, and give a weak but significant response with the I-10.03, MM-10.04 and MP-10.06 samples, which indicates that these samples were contaminated with PGNs during a step of their production, and that the latter were partially degraded during the course of production of the product (FIG. 19).

(125) The HEK-TLR2 and RAW-BLUE cells are effective for detecting traces of inflammatory contaminants in products of I-10.01 and I-10.03 type.

(126) They even have characteristics that are complementary to the sensitized THP-1 cells. Indeed, the tests using the three of them make it possible to detect the majority of inflammatory contaminants that may be present in glucose polymer samples. THP-1: any inflammatory contaminant with a high reactivity for LPS. RAW-BLUE: any inflammatory contaminant with a high reactivity for PGNs. HEK-TLR2: specific for TLR2 agonists with a high reactivity for PGNs. HEK-NOD2: specific for MDP and consequently for PGN degradation products. As for the THP-1 cells, an absence of proportionality between the levels of PGN and/or of LPS and the amplitudes of the cell responses is also noted.

(127) This problem is doubtless linked to the size of these macromolecules and/or to their presence in the form of aggregates, which influences their reactivity with respect to the cells. Thus, it was observed that passing the standard PGN through a 0.22 μm filter reduces by approximately 50% the reactivity thereof for the HEK-TLR2 cells. Consequently, in all the tests, the glucose polymer solutions are prepared under aseptic conditions but without a step of filtration of the standard molecules.

EXAMPLE 4

Characterization of the Contaminants by Using the Sensitized THP-1, the HEK-BLUE (hTLR2, hNOD2) and the RAW-BLUE Cell Lines

(128) Examples 2 and 3 show that the sensitized THP-1, the HEK-TLR2, the HEK-NOD2 and the RAW-BLUE lines are effective for detecting traces of inflammatory contaminants in the glucose polymer samples. In addition to LPS and MDP, the presence of which is detected via the TLR4 and NOD2 receptors, the other contaminants that may be present in the samples are predominantly TLR2 ligands (PGN, LTA and lipopeptides). Consequently, the lines do not make it possible to establish the contribution of the PGNs in the TLR2-specific response. However, PGNs are macromolecules of which the weight ranges from ˜100 kDa to several million Da. Conversely, LTAs and lipopeptides have low weights, less than 15 kDa. Thus, the introduction of an ultrafiltration step (30 kDa) should make it possible to retain the PGNs and to measure only the response to TLR2 ligands of small sizes.

(129) Experimental Procedures

(130) For the experiments relating to this example, the five cell lines presented in examples 2 and 3 are used: THP-1 monocyte line: response to any inflammatory contaminant with a high reactivity for LPS, RAW-BLUE line: response to any inflammatory contaminant with a high reactivity for PGNs, HEK-BLUE hTLR2 line: specific for TLR2 agonists with a high reactivity for PGNs, HEK-BLUE hNOD2 line: specific for PGN total depolymerization products (MDP), HEK-BLUE Null line: negative response control.

(131) The glucose polymer samples are presented in example 2 (Table 1). These samples are prepared in solution at the concentrations described in examples 2 and 3. The cell responses (production of RANTES for the THP-1 cells and secretion of SEAP for the BLUE cells) are analyzed either with the nonfiltered samples: Total response, or with the filtrates obtained by ultrafiltration on a microconcentrator with a cut-off threshold at 30 kDa (Sartorius): Filtrate response.

(132) Before use, the microconcentrators were treated with a saline solution (150 mM NaCl) prepared with non-pyrogenic water. The retentates and filtrates were tested with the cell lines, and only the filters giving a negative response in each test were retained for the analyses with the glucose polymer samples.

(133) Results

(134) The results presented in FIG. 20 show the responses of the HEK-TLR2 and RAW-BLUE cells obtained in the presence of the various samples before and after ultrafiltration.

(135) The Total responses are similar to those observed in example 3 (FIGS. 17 and 18).

(136) The Filtrate responses are greatly reduced for the I-10.02 and I-11.12 samples in the two cell types, with values close or equal to the detection thresholds. These data confirm that these two samples are contaminated with PGN, which was retained by the filter.

(137) The filtrate response of MM-10.05 is reduced in the HEK-TLR2 cells, but not in the RAW-BLUE cells, indicating that this sample is contaminated with a combination of several molecules, with a considerable part contributed by PGN.

(138) The MM-10.04, MP-10.06 and MP-10.07 samples are not significantly contaminated with PGN, since the Total and Filtrate responses are identical in the HEK-TLR2 cells. On the other hand, a 50% decrease in the Filtrate response can be noted for MP-10.07 in the RAW-BLUE cells. The LAL test showed that this sample is loaded with LPS. Although the weight of endotoxins is less than 30 kDa, these molecules are capable of forming aggregates, which may account for the loss of response after filtration.

(139) The Total and Filtrate responses were analyzed in the THP-1, RAW-BLUE, HEK-TLR2 and HEK-NOD2 cell types. The results are given in Table 4.

(140) For the HEK-NOD2 cells, the Total and Filtrate responses are identical, which was expected since MDP and the related molecules have a weight much less than 30 kDa (MW-500 Da). Only the Total responses are reproduced in Table 4.

(141) TABLE-US-00004 TABLE 4 Characterization of the contaminants by analysis of the cell responses. The contamination levels are expressed as a function of the threshold limits of detection (LOD) and of the EC50s defined in examples 2 and 3 for each cell type (Tables 2 and 3), with the dose-response curves with respect to LPS for the sensitized THP-1 cells, with respect to PGN for the RAW-BLUE and HEK-TLR2 lines, and with respect to MDP for the HEK-NOD2 line: (−): level < LOD; (±): LOD ≤ level < 3 × LOD; (+): 3 × LOD ≤ level < 0.3 × EC50; (++): 0.3 × EC50 ≤ level < 3 × EC50; (+++): level ≥ 3 × EC50 I- I- MM- MM- MP- MP- Response I-10.01 10.02 I-10.03 11.12 10.04 10.05 10.06 10.07 THP-1 − − ± + + ± ± + <30 kDa − − ± ± ± ± ± ± RAW- − + ± + ± + ± + BLUE <30 kDa − − − − ± ± ± + HEK- − ++ ± +++ ± ++ ± ± TLR2 <30 kDa − − ± − ± ± ± ± HEK- − − ± − + − + ++ NOD2 Major negative PGN residues PGN residues PGN residues LPS effect including and including and MDP residues MDP residues

(142) The data make it possible to characterize the types of contaminants present in each glucose polymer solution and to establish their contribution in the inflammatory response:

(143) I-10.01: sample not contaminated.

(144) I-10.02: sample strongly contaminated with PGN. The absence of response of the THP-1 and HEK-NOD2 cells and of the Filtrates indicates that the contribution of the other contaminants is negligible.

(145) I-10.03: sample weakly contaminated with residues probably originating from degradation products of small size. Indeed, the Total and Filtrate responses are at the limit of the background noise in all the tests.

(146) I-11.12: sample strongly contaminated with PGN. The weak response of the THP-1 cells also points to traces of LPS.

(147) MM-10.04: sample weakly contaminated with LPS and TLR2-activating molecules of small size (LTA, lipopeptides). The presence of MDP also points to PGN degradation products.

(148) MM-10.05: sample contaminated with PGN and other molecules of small sizes. The weak responses of the other tests suggest the presence of traces of LPS and of other TLR2 ligands.

(149) MP-10.06: sample contaminated with numerous small molecules. On the other hand, the weak THP-1 and HEK-TLR2 responses indicate the absence of PGN and of LPS.

(150) MP-10.07: sample contaminated with numerous small molecules, with a high proportion of LPS and of MDP.

(151) It may be noted that the results match the Wako SLP-HS assays for the I-10.01, I-10.02, I-10.03 and I-11.12 samples, which are final products, and the MM-10.04 and MP-10.06 samples, which were identified as being devoid of PGN.

(152) On the other hand, the two MM-10.05 and MP-10.07 samples give PGN responses that are weaker than those expected with the data of the SLP tests. However, it is possible that these samples gave false positives with the SLP tests, via crossreaction with β-glucans, for example. Another possibility is that these samples contain very large PGNs, the low solubility of which prevents the triggering of a response in the cell tests.

EXAMPLE 5

Peptidoglycan Assay Method

(153) The assay is based on the specific recognition of PGNs by a line expressing the TLR2 receptor and on the production of an enzymatic activity that is measurable via the activation of the signaling pathway associated with TLR2.

(154) Experimental Procedures

(155) For the experiments relating to this assay, two lines are used: HEK-BLUE hTLR2 line; HEK-BLUENull2 line.

(156) The two lines are presented in example 3.

(157) Establishment of the Dose-Response Curve

(158) The dose-response curve was produced with standard S. aureus PGN (FIG. 21).

(159) The HEK-BLUE cells are incubated with increasing concentrations of standard, and the cell response is measured by quantifying the enzymatic activity produced.

(160) The result is a conventional sigmoid cell response curve: part (A) corresponds to the responses obtained with low concentrations of PGN, below those which give effective activation of TLR2. This nonlinear zone therefore corresponds to the threshold limit of detection of the method. In such a way as to include the variability of the method, this detection threshold is estimated at three times the value of the background noise (response obtained in the absence of stimulus). part (B) is the most interesting since a linear response is observed. This effective-response zone makes it possible to determine a direct relationship between the cell response and the level of PGN. It is therefore the assaying zone; part (C) corresponds to a saturation of the cell response in the presence of PGN concentrations which are too high. There is in fact a saturation of the TLR2 receptors.

(161) The standard curve for response of the HEK-TLR2 cells to PGN exhibits a linearity zone for concentrations between 0.07 and 10 ng/ml (i.e. between 2 and 267 ng/g).

(162) In the case of samples that may be highly contaminated with PGN, it will be necessary to perform several serial dilutions so as always to be in the linearity zone. Conversely, low PGN concentrations require a step of concentrating the sample if it is desired to increase the sensitivity of the assay.

(163) Sample Preparation

(164) The PGN assays are carried out on glucose polymer solutions. The sample requiring the PGN quantification is incubated with HEK-BLUE hTLR2 cells, and the cell response is measured by quantifying the enzymatic activity produced. The amount of PGN contained in the sample can be determined by referring to the dose-response curves.

(165) The first tests were carried out with contaminated samples originating from manufactured glucose polymer batches (example 3, FIG. 15).

(166) The HEK-TLR2 cells make it possible to detect the presence of PGN in the majority of the samples. On the other hand, it was observed that there is no correlation between the PGN levels measured by the SLP-Wako method and the amplitudes of the cell responses to PGN. Indeed, the samples with the highest PGN load are not those which induce the strongest production of SEAP.

(167) By way of illustration, the MP-10.07 sample, which is the one most highly loaded with PGN (645 ng/mg), does not give a marked cell response with the HEK-TLR2 cells. Conversely, the I-10.02 and I-11.12 samples are less contaminated (253 and 393 ng/g, respectively), but are the most reactive in terms of cell response.

(168) PGNs have very heterogeneous sizes. The heaviest forms (>10.sup.6 Da) have low solubility and are therefore not very reactive in the cell tests. On the other hand, they are assayed by the SLP-Wako test. The PGNs of intermediate size (˜100 kDa) are very soluble and are powerful inducers of TLR2. Despite low levels, they are capable of triggering a strong inflammatory response. This size dispersion and therefore activity dispersion is a major drawback for relating the contamination level to the risk of developing an inflammatory response when conventional quantitative assays are used (LAL and SLP-Wako).

(169) The PGNs present in the I-10.02 and I-11.12 samples are soluble and therefore very reactive. Conversely, the MP-10.07 sample probably contains PGNs of large size, which will be removed in the course of the production of the final product.

(170) On the basis of these first results, the PGN assay method based on the use of the HEK-TLR2 cells would therefore make it possible to quantify the biologically active PGNs capable of causing inflammatory reactions in vivo.

(171) A particularly advantageous procedure for assaying the PGNs capable of triggering an inflammatory response is to reduce their size, and therefore increase their solubility for the in vitro cell response test.

(172) Mutanolysin is an enzyme which, through its muramidase activity, is capable of depolymerizing PGNs. In order to test its activity, solutions of standard (S. aureus) PGN were prepared by diluting the molecule in culture medium in the absence or in the presence of the I-10.01 polymer (noncontaminated polymer) at the concentrations of 7.5% and 37.5% (weight/volume).

(173) The samples were treated in the presence of 2500 U/ml of mutanolysin for 16 h at 37° C., and then added to the HEK-TLR2 cells. The cell response was measured by following the activity of the SEAP produced, according to the conditions described in example 3.

(174) In the absence of glucose polymer, the treatment with mutanolysin for 16 h reduces the response of the HEK-TLR2 cells by more than 50%, indicating that the depolymerization was too strong and reduced the reactivity of the PGN with respect to the cells. Conversely, the presence of the glucose polymer reduces the activity of the enzyme, since the response of the cells is even improved in the presence of 7.5% of polymer, whereas it is unchanged in the presence of 37.5% of polymer.

(175) Mutanolysin alone does not induce any cell response, indicating that it is not contaminated and that it has no activating effect on the cells.

(176) In order to verify the effect of this treatment, the I-10.01, I-10.02, I-10.03, I-11.12, MM-10.05 and MP-10 0.07 polymers were diluted to the concentration of 7.5% and then treated for 16 h at 37° C. in the absence or in the presence of 2500 U/ml of mutanolysin.

(177) The cell response was induced by adding 40 μl to 160 μl of cell suspension (final polymer concentration: 15 mg/ml).

(178) Results

(179) The responses of the HEK-TLR2 cells in the presence of the untreated polymers are weak, which was expected given the lower polymer concentration compared with example 3.

(180) After mutanolysin treatment, the responses to the glucose polymer solutions are clearly increased (FIG. 22). In addition, it may be noted that the I-10.03 polymer gives a response equivalent to the I-10.02 and MM-10.05 polymers, even though it was not reactive in the previous tests.

(181) These results indicate that the mutanolysin partially depolymerized and therefore solubilized PGNs which were totally or partially insoluble owing to their excessively large size.

(182) The absorbance values were reported on the calibration curves established under the same conditions with the standard PGN (FIG. 23), so as to determine the PGN concentrations present in the glucose polymers.

(183) The results expressed as ng of PGN/g of glucose polymer are reported in table 5. After mutanolysin treatment, the values are lower than those obtained with the SLP-Wako tests, but they reflect the load of the glucose polymers in terms of PGN with pro-inflammatory activity (Table 1).

(184) The I-10.03 polymer gives a high value, close to I-10.02, after mutanolysin treatment. This piece of data is interesting since the two polymers have been the subject of complaint owing to episodes of aseptic peritonitis. The mutanolysin treatment of I-10.03 therefore made it possible to reveal the active PGN load, probably by enabling the contaminant to be solubilized.

(185) The values of the MM-10.05 and MP-10.07 samples remain lower than those obtained with the SLP tests. However, these samples are contaminated with other inflammatory molecules other than PGNs. These molecules, and in particular the β-glucans, probably interfered in the SLP assays, increasing the response of the SLP tests.

(186) TABLE-US-00005 TABLE 5 Assaying of PGNs present in the glucose polymers before and after mutanolysin treatment. The values are expressed in ng S. aureus PGN equivalent/g of polymer. MM- MP- I-10.01 I-10.02 I-10.03 I-11.12 10.05 10.07 without <2 13.5 <2 17.5 11.5 3.5 treatment with <2 33.5 36.8 113.5 36 13.5 treatment