Title of Invention

A METHOD FOR QUANTIFYING THE TEICHOIC ACIDS IN A PREPARATION COMPRISING ONE OR MORE CAPSULAR POLYSACCHARIDES OF STREPTOCOCCUS PNEUMONIAE

Abstract

The invention relates to a method for dosing teichoic acids most often present in residual form, in a preparation originating from a culture of Gram+ bacteria. This method firstly requires a hydrolysis controlled by hydrofluoric acid at a temperature lower than or equal to 40 °C in order to liberate the specific oligosaccharides from the teichoic acids. The dosing of the specific oligosaccharides can then be carried out by employing diverse techniques, particularly by high-performance chromatography coupled with a pulsed field amperometric detection (HPAEC-PAD). The inventive method can, in particular, serve to dose residual quantities of teichoic acids present in preparations containing capsular polysaccharides of Streptococcus pneumoniae that can be used as vaccines.

Full Text

Assaying teichoic acids of 6ram+ bacteria The invention relates to a method for assaying teichoic acids, in particular in preparations derived from Gram+ bacterial culture. Teichoic acids are constituents of the complex constituting the membrane and the wall of Gram+ bacteria. They are in close contact with the peptidoglycan of the wall. Covalent bonds reinforce this link. Teichoic acids which comprise a lipid or glycolipid end in their chemical structure can anchor themselves in the membrane via hydrophobic bonding. Two types of teichoic acids are mainly distinguished: the first type includes the teichoic acids which are linear polymers of ribitol phosphate or of glycerol phosphate linked by phosphodiester bonds. The free hydroxyl functions of the ribitols or of the glycerols can be substituted with residues such as amino acid, N-acetylglucosamine, glucose or N-acetylgalactosamine residues. These teichoic acids are listed as "alcohol polymers"; the second type includes teichoic acids which are polysaccharides made up of a chain of repeating units. The repeating unit generally consists of a chain of at least two monosaccharides followed by a ribitol phosphate or by a glycerol phosphate. The phosphate group is involved in the chain of the repeating units by means of phosphodiester bonds. The free hydroxyl functions of the repeating units can also be substituted with various residues. In addition, among teichoic acids, those which have an additional lipid or glycolipid chain are distinguished. This chain is connected to the end ribitol phosphate or end glycerol phosphate by means of a phosphodiester bond. The lipid component is usually a monoacyl glycerol chain or a diacyl glycerol chain. Depending on the bacterial species, the teichoic acids are of the "alcohol polymer" type as in the case of Staphylococcus aureus or of the "polysaccharide" type as in the case of Streptococcus pneumoniae. In this species, two ubiquitous teichoic acids are known: the C-polysaccharide and lipoteichoic acid (also referred to as Forssman antigen). The C-polysaccharide consists of a chain of repeating units, each made up of four monosaccharide residues, (a glucopyranose (Glcp) residue, a 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (AAT) residue and two N-i acetyl galactopyranose (GalpNAc) residues), followed by a ribitol-5-phosphate (ribitol-5-P) residue. At least one of the two GalpNAc residues is substituted with phosphocholine (P-Cho). The structural formula of this repeating unit is as follows: C. Karlsson et al., Eur. J. Biochem. (1999) 265: 1091 have shown that the repeating unit can exist in two i forms: (i) a form disubstituted with two phosphocholine residues, as represented above; and (ii) a form monosubstituted with a single phosphocholine residue. Consequently, the C-polysaccharide chain contains monosubstituted and disubstituted repeating units in > varying proportion. These proportions vary in particular as a function of the culturing conditions and of the pneumococcal serotype. As regards the Streptococcus pneumoniae lipoteichoic acid, it has the primary structure of the C-polysaccharide associated with a glycolipid, of formula (}-D-Glcp(l-4)-p-D-AAT (1-3)-a-D-Glcp (1-3) acyl2Gro, in which Glcp denotes a glucopyranose residue, AAT denotes a 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose, acyl denotes a fatty acid residue and Gro denotes a glycerol residue. This glycolipid is attached, via a phosphodiester bond, to the terminal ribitol of the chain. Some Gram+ bacteria, such as Streptococcus pneumoniae, have a capsule and/or are responsible for serious infections. Vaccines consisting of capsule polysaccharides in purified form exist against Streptococcus pneumoniae. The methods used to purify the capsular polysaccharides eliminate as much as possible the polysaccharides from the wall (teichoic acid) . In fact, although they are very immunogenic, teichoic acids are weakly protective and their presence in a vaccine needlessly increases the antigenic load. Moreover, teichoic acids may give rise to undesirable inflammatory reactions (Infection & Immunity (2003) 71:5541). Nowadays, the health authorities demand that the content of a high quality vaccine be characterized with precision. It must be possible to assay not only the vaccine antigen, but also all the possible contaminants, among which are the teichoic acids, in particular with regard to capsular polysaccharide-based vaccines. The assaying of the contaminants is all the more difficult and tricky since it relates to very small quantities. Assaying methods must therefore be sensitive, efficient and specific. The latter requirement of specificity is particularly difficult to respect. In fact, strong structural similarities exist between the teichoic acids of the polysaccharide type and the capsular polysaccharides. For example, the repeating unit of the C-polysaccharide of Streptococcus pneumoniae contains a phosphate group just like that of the capsular polysaccharides of the serotypes 6B, 10A, 11A, 15B, 17F, 18C, 19A, 19F, 20, 23F. The phosphate group of the repeating unit of the C-polysaccharide is directly involved in the linking together of the repeating units via a phosphodiester bond, as are those of the capsular polysaccharides of serotype 6B, 10A, 17F, 19A, 19F and 20. The capsular polysaccharides of serotypes 6B and 10A are those which exhibit the strongest similarity with the pneumococcal teichoic acids since they have a ribitol phosphate involved in the linking together of the repeating units. Jones C. et al., have described, in Biologicals (1991) 19: 41, a method for assaying the C-polysaccharide by nuclear magnetic resonance (NMR). This method evaluates the resonance of the N-methyl radical of the phosphocholine groups of the C-polysaccharide. This technique is not precise since, as indicated above, the proportion of phosphocholine groups is not constant. Talaga P. et al. propose, in Vaccine (2001) 19: 2987, an assaying method based on quantification of the ribitol released during hydrolysis of the C-polysaccharide subsequent to two successive treatments: the first with hydrofluoric acid (HF) at 48% for 2 hours at 65°C, the second with 2N trifluoroacetic acid for 2 hours at 120°C. This double treatment brings about cleavage of the oside bonds. The hydrolysate contains essentially monosaccharides. The ribitol is then separated from the other constituents by high performance anion exchange chromatography (HPAEC) on a CarboPac™ MAI analytical chromatography column, using, for the elution, a 480 mM isocratic sodium hydroxide solution. The ribitol is then quantified by a pulsed amperometric detection (PAD) system. Even though this technique for assaying ribitol allows a correct and sensitive evaluation of the amount of C-polysaccharide, it is incompatible with the presence, in the medium, of a capsular polysaccharide which contains ribitol. Now, some capsular polysaccharides of interest in vaccines contain ribitol. As has just been seen, these are, for example, the capsular polysaccharides of serotype 6B and 10A of Streptococcus pneumoniae. The technique of Talaga et al. is not therefore suitable for assaying the teichoic acids in a preparation which contains these serotypes, for example in the commercial pneumococcus vaccines. Briefly, the methods for assaying polysaccharide-type teichoic acids which are known to date have drawbacks. No method yet exists which is precise, sensitive, reliable and applicable to any preparation derived from Gram+ bacterial culture. The present invention overcomes this shortage by proposing a novel method for assaying polysaccharide-type teichoic acids. This method searches for the oligosaccharides specific for teichoic acids released through cleavage of the phosphodiester bonds, during ' treatment with hydrofluoric acid (HF), according to the method of Jennings & Lugowski, Can. J. Chem. (1980) 58: 2610. When the repeating unit contains a p-D GalpNac molecule or a P~D GlcpNac molecule linked to a ribitol phosphate or glycerol phosphate molecule, a cleavage of i this bond may also occur with ribitol or glycerol being released. In other words, depending on the structure of the teichoic acid, the specific oligosaccharide will be substantially identical to the repeating unit or else will differ therefrom through the loss of ribitol or glycerol. Thus, in the case of the C-polysaccharide, the specific oligosaccharide has the formula: P-OGICpGa!pNAG-(1 *3) - P-D-GalpNAc After hydrolysis, specific oligosaccharides then remain which are subsequently separated from one another and assayed. Furthermore, it is indicated, in the interests of thoroughness, that this definition applies mutatis mutandis to the oligosaccharides specific for capsular polysaccharides having repeating units incorporating phosphate groups involved in the chain, such as the polysaccharides of pneumococcal serotypes 6B, 10A, 17F, 19A, 19F and 20. Thus, a subject of the present invention is a method for assaying the polysaccharide-type teichoic acids in a preparation derived from Gram+ bacterial culture, according to which: (i) the preparation is treated with hydrofluoric acid (HF) at a temperature of less than or equal to 40 °C, in order to release the oligosaccharides specific for the teichoic acids present in the preparation; and (ii) the specific oligosaccharides obtained in (i) are assayed. The expression "preparation derived from Gram+ bacterial culture" is intended to mean: (i) a preparation of whole bacteria; (ii) a bacterial lysate that is obtained in particular by treating the Gram+ bacteria with chemical detergents (deoxycholate, tween, ether, etc.) or by using mechanical methods such as osmotic shock or sonication; (iii) a bacterial fractionation product that is obtained, for example, by using the fractionated alcoholic precipitation method; (iv) a Gram+ bacterial culture supernatant; and (v) a preparation comprising one or more Gram+ bacterial components (antigens), in purified form. Preferably, the preparation of Gram+ bacteria is a Gram+ bacterial culture supernatant or a preparation comprising one or more purified antigens; the latter being most particularly preferred. The treatment with hydrofluoric acid (HF) is advantageously carried out at a temperature ranging from -70°C to 40°C, preferably ranging from 0°C to 20°C, even more preferably between 4°C and 10°C. In general, the higher the temperature, the lower the selectivity of action of the hydrofluoric acid on the phosphodiester bonds. For this reason, it is important not to exceed 40°C. Typically, the hydrofluoric acid is used at a final concentration of between 10 and 73% (weight/weight), limits included; preferably between 40 and 60%; entirely preferably between 45 and 50%; e.g. at a concentration of 48%. The duration of treatment with hydrofluoric acid is not critical. It is inversely proportional to the temperature. Those skilled in the art are capable of adjusting this duration as a function of the temperature chosen and of the concentration at which the hydrofluoric acid is used. However, it is indicated that, the higher the temperature, the shorter the duration of treatment must be in order to ensure as much as possible the integrity of the oside bonds. In general, it is preferable for the treatment not to exceed 96 hours. When the temperature is between 20 and 40°C, a duration of treatment not exceeding 1 hour is recommended; between 0 and 10 °C, a duration of treatment not exceeding 2 hours is recommended. Conversely, when the temperature is less than 0°C, the duration of treatment may exceed 24 hours. Good results are obtained when the HF is used at a concentration of 48%, at 4°C for 48 hours. Under these conditions, the rate of recovery of the specific oligosaccharide can reach 90% of the theoretical value. To assay the teichoic acid-specific oligosaccharide(s) obtained after treatment with HF, it is advantageously advisable to separate it or them from the other constituents and to characterize it or them. Separation, characterization and quantification can be carried out according to various techniques, e.g. biochemical techniques, available to those skilled in the art. It is indicated, however, that methods known to allow the separation and/or the assaying of monosaccharides are most particularly suitable. According to a particular method, use is made of a high performance anion exchange chromatography (HPAEC) technique, optionally coupled with pulsed amperometric detection (PAD). To this end, a suitable chromatography support should allow good resolution and should be compatible with a medium having a very high pH (> 12). In practice, under these pH conditions, the support should remain intact, free of degradation. It may consist of a resin, e.g. made of sulfonated polystyrene-divinylbenzene, having for example a degree of crosslinking ranging between 1 and 5%. The resin is advantageously in the form of microbeads, the diameter of which preferably ranges between 450 and 550 nm. If the medium derived from the treatment with HF contains numerous products, it may be useful to optimize the resolution. To this end, the resin, e.g. made of sulfonated polystyrene-divinylbenzene may be packaged in the form of a column and may be completed, in its upper portion, with a layer of a material bearing positive charges, e.g. a primary amine salt, a secondary amine salt, a tertiary amine salt, a quaternary ammonium salt, or an ammonium group. This material may be in the form of beads advantageously having a diameter of 5 to 15 \xsxi, e.g. approximately 10 pm. This material may be porous or nonporous. By way of example, it is indicated that a suitable material may consist of latex advantageously having a degree of crosslinking of 3 to 7%, e.g. approximately 5%. The chromatography material (e.g. analytic column) marketed by the companies Dionex and Metrohm under the respective trademarks CarboPac™ and Metrosep Carb satisfy the required criteria. This material is commonly referred to as being "of the CarboPack™ type". The CarboPack™ PA1 column is most particularly preferred. Once the preparation derived from the treatment with HF has been loaded onto the chromatography column, elution is carried out with an eluting solution having a pH > 12, in particular so that the hydroxyl functions of the sugars ionize and are in the form of oxyanions which can be separated by anion exchange chromatography. If the PAD technique is subsequently used for detection, the eluting solution must also be compatible with this detection. In this regard, the eluting solution is advantageously free of carbonate. Advantageously, the eluting solution is a sodium hydroxide solution, the molarity of which is between 10 and 300 mM, preferably between 20 and 150 mM, even more preferably between 40 and 100 mM. The flow rate of the eluting solution depends on the type of column used, but is generally between 0.1 and 4 ml/min. The specific oligosaccharides are detected on the chromatogram in the form of chromatography peaks having characteristic retention times under given operating conditions (same chromatography column, same solution and rate of elution, etc). Finally, the oligosaccharide specific for the teichoic acid intended to be assayed is sought and quantified using a detection system, for example pulsed amperometric detection (PAD). This method of detection is based on the oxidation of the sugars on a working electrode, leading to the formation of an electric current which is measured. Regenerating and cleaning potentials are often applied to the working electrode. To determine the absolute amount of the teichoic acid intended to be assayed, results are advantageously related to a calibration curve established using a purified preparation of the teichoic acid in question, under the same conditions of treatment and analysis. According to a particular embodiment, the method according to the invention applies to the assaying of teichoic acids in a preparation derived from a culture of capsulated Gram+ bacteria i.e. a preparation of Gram+ bacteria which comprises one or more capsular polysaccharides. The method is particularly suitable for assaying teichoic acids in a culture supernatant of capsulated Gram+ bacteria or in a preparation of purified capsular polysaccharide(s). In fact, due to the method of purification of these polysaccharides generally carried out using a bacterial lysate or a culture supernatant, the preparations which are obtained contain residual amounts of teichoic acids which it is advisable to assay. The preparations of purified capsular polysaccharides may contain one or more polysaccharides which are in their free native form or in a form modified by partial depolymerization, activation or conjugation to a carrier peptide or protein such as tetanus toxoid or diphtheria toxoid. According to an embodiment of particular interest, the method according to the invention applies to the assaying of teichoic acids of Streptoccocus pneumoniae; and, in particular, in a preparation containing one or more capsular polysaccharides of Streptococcus pneumoniae. Pneumococci {Streptococcus pneumoniae) are capsulated Gram+ bacteria responsible for infectious pathological conditions, in particular for meningitis, bronchitis, rhinitis and otitis with complications in adults as in children. The pneumococci are divided up into serotypes depending on the structure of the polysaccharides which form the capsule. Pneumococcal serotyping is carried out using a battery of immune sera, each immune serum being specific for a single type of capsular polysaccharide (monospecific immune sera). More than 90 different serotypes have been listed to date. The vaccines against pneumococcus which are currently marketed all contain capsular polysaccharides. As the polysaccharides are purified using a bacterial lysate these vaccines contain residual amounts of teichoic acids which it is advisable to assay. The capsular polysaccharides of Streptococcus pneumoniae are in particular chosen from those which characterize the 23 serotypes (valences) usually encountered in humans; namely serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. By virtue of the assaying method according to the invention, it is in particular possible to quantify the pneumococcocal teichoic acids in any preparation containing one or more capsular polysaccharide(s), whatever the number and the serotype. In particular, the preparation may contain one or more purified capsular polysaccharides in native or modified form, the structure of which exhibits similarities with those of the teichoic acids. These are the polysaccharides of the serotypes 6B, 10A, 17F, 19A, 19F and 20. In fact, the presence of these polysaccharides, the hydrolysis of which under the conditions of the invention leads to the production of specific oligosaccharides, has no effect on the assaying of the teichoic acids. The method according to the invention therefore applies to the assaying of teichoic acids in a preparation comprising one or more capsular polysaccharides of serotypes 6B, 10A, 17F, 19A, 19F or 20. The preparation may also contain one or more polysaccharides of serotypes 11A, 15B, 18C and 23F. Briefly, the preparation may contain the polysaccharides of the 23 most widespread serotypes or any possible combination based on these 23 serotypes. By way of example, such a preparation may contain the polysaccharides of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F. It may also contain one or more additional polysaccharides resulting from serotypes 1, 3, 5 and 7F. By way of indication, the following remarks are submitted: The oligosaccharides specific for the C-polysaccharide and for lipoteichoic acid, which are released during the treatment with HF under cold conditions according to the invention, are identical. In fact, the two teichoic acids have the same repeating unit. It is not therefore possible to assay the C-polysaccharide and lipoteichoic acid separately. The capsular polysaccharides of serotypes 6B, 10A, 17F, 19A, 19F and 20, in which the linking together of the repeating units involves phosphodiester bonds, are degraded essentially in the form of specific oligosaccharides. The capsular polysaccharides of serotypes 11A, 15B, 18C and 23F, the phosphodiester bonds of which are not directly involved in the linking together of the repeating units, cannot be hydrolyzed in the form of specific oligosaccharides. When subjected to the treatment with HF, they can however partially and randomly depolymerize to heterogeneous products, including in particular molecules such as glycerol or choline, and monosaccharides. Finally, the other capsular polysaccharides which do not contain any phosphate groups are not depolymerized or are partially and randomly depolymerized to give, after treatment with HF, heterogeneous products, mainly of high molecular weight, which are nonidentifiable and nonquantifiable, and also a minor component of monomers and monosaccharides. In fact, the medium derived from the treatment with HF may be more or less complex depending on the number and the type of capsular polysaccharide(s) present at the start. It is therefore advisable to separate the teichoic acid-specific oligosaccharide from the other hydrolysis products (monomers, monosaccharides, unhydrolyzed or partially hydrolyzed polysaccharides, etc.). Surprisingly, the oligosaccharide specific for the teichoic acids of Streptococcus pneumoniae behaves like a monosaccharide when it is analyzed and separated by the HPAEC-PAD technology and differs from the oligosaccharides of the capsular polysaccharides. In order to separate it and quantify it by HPAEC-PAD with particular resolution, the use of a CarboPac PA1™ chromatography column and a 7 5 mM isocratic sodium hydroxide solution are recommended. Under these conditions, the chromatography peak corresponds to a retention time of 4.30 min ± 10% when the flow rate of the eluting solution is 1 ml/min. The surface area of the chromatography peak reflects the relative amount of C-polysaccharide and of lipoteichoic acid present in the preparation. Using the method for assaying the teichoic acids of the invention, it was discovered that S. pneumoniae serotype 5 does not possess any C-polysaccharide. On the other hand, a teichoic acid, referred to as C5-polysaccharide, is found, which substitutes for this. The formula of its repeating units differs slightly from that of the C-polysaccharide. It is as follows: For this reason, according to a very particular method, the invention also applies to the assaying of the C5-polysaccharide, in particular in a preparation containing the capsular polysaccharide of S. pneumoniae serotype 5. The particularly resolvent procedure described above for assaying the C-polysaccharide is also suitable for assaying the C5-polysaccharide. Its chromatography peak has, under these conditions, a retention time of 3.30 min ± 10%. To determine the residual absolute amounts of teichoic acid, reference is made to standard curves produced from "stock" preparations expressing known amounts as a function of the surface areas of the chromatography peaks. For example, the residual absolute amounts of C-polysaccharide and of lipoteichoic acid can be determined using a preparation of purified C-polysaccharide, such as that provided by Staten Serum Institute, Denmark (cf. example 1). In order to assay the residual absolute amounts of C5-polysaccharide, a purified C5-polysaccharide preparation prepared from a pneumococcus serotype 5 lysate is preferably used as stock preparation. Initially, the polysaccharides are extracted by means of one or more fractionated alcoholic precipitations. The purification is completed by removing the main contaminants, i.e. the proteins and the nucleic acids, and then the polysaccharide extract from which these main contaminants have been removed is subsequently subjected to partition chromatography or to ion exchange chromatography so as to separate the C5-poly-saccharide from the other polysaccharides. The chromatographic method as described in example 1 can in particular be used. The methods for extracting the polysaccharides by fractionated alcoholic precipitation, along with the methods for removing the protein and nucleic acid contaminants, are methods well known to those skilled in the art. Reference may in particular be made to the methods described in US 4,686,102. The preparation of C5-polysaccharide thus purified can be used as a standard for determining the residual absolute amounts of C5-polysaccharide by the method according to the invention; however, more generally, it can be used in any method of assaying the C5-polysaccharide that refers to a standard curve. Finally, a subject of the invention is the use of a purified C5-polysaccharide having a repeating unit of formula: for establishing a standard curve intended for assaying the C5-polysaccharide, in a preparation containing a capsular polysaccharide of Streptococcus pneumoniae serotype 5. The present invention will be understood more clearly in light of the following examples which serve to illustrate the invention without, however, limiting the content thereof. Figure 1 represents the HPAEC-PAD chromatogram, after treatment with HF, of the Pneumo 23™ vaccine containing the capsular polysaccharides of serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. The peaks with an above them correspond to the monosaccharides originating from the hydrolysis of the capsular polysaccharides. The Rib peak corresponds to ribitol. The Fru peak corresponds to fructose (internal standard). The oligo C and oligo C5 peaks correspond, respectively, to the specific oligosaccharides C and C5. Figure 2 represents the HPAEC-PAD chromatogram, after treatment with HF, of a vaccine formulation F3 as described in WO 98/51339, containing the Tt and Dt conjugates of the capsular polysaccharides of serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F of pneumococcus. i 1.1. Stock solutions for standard range 1.1.1. C5-polysaccharide: i This polysaccharide is purified as described in the following paragraph, from a preparation of polysaccharides of pneumococcus serotype 5, obtained by fractionated alcoholic precipitation of a pneumococcus serotype 5 lysate, followed by a phenolic extraction to I remove the proteins, by a fractionated precipitation in the presence of calcium chloride to remove the nucleic acids and, finally, by a second fractionated alcoholic precipitation. The precipitate obtained is washed with absolute alcohol and then dried under vacuum. The > desiccate is resolubilized at a concentration of 10 mg/ml in a 200 mM NaCl buffer. 4 ml of this solution are loaded onto a chromatography column (90 cm x 1.6 cm in diameter) containing sepharose-CL 4B gel. The column is subjected a flow of 200 mM NaCl solution at a flow rate of 0.6 ml/min in order to separate the C5-polysaccharide from the capsular polysaccharide. The eluate is collected in fractions. The UV absorbance at 206 nm of each fraction is measured. This makes it possible to identify two series of clearly distinct fractions: the first containing the type 5 capsular polysaccharide; the second, eluted, containing the C5-polysaccharide. This second series of fractions is dialyzed against distilled water, concentrated using a rotary evaporator, and then conserved in the form of a lyophilisate. The degree of purity of the lyophilisate is approximately 7 0%. Subsequently, the exact amount of C5-polysaccharide in the preparation thus obtained is determined by assaying the recital released after hydrolysis. The lyophilized is resolubilized in ultrafiltered purified water, in a proportion of 10 u.g/ml (dry weight) . In parallel, a standard range of ribitol of 0 to 4 ug/ml is prepared in ultrafiltered purified water. To control the reproducibility of the chromatography, a fixed amount of mannose, which acts as an internal standard, can be added to all the samples. 400 (il of the solution of C5-polysaccharide prepared above are dried under nitrogen. The desiccate is treated with 200 p.1 of 48% hydrofluoric acid for 2 hours at 65°C. The mixture is dried and 400 ul of 2N trifluoroacetic acid are added for 2 hours at 135°C. The mixture is dried under a stream of nitrogen. For analysis by HPAEC-PAD chromatography, the desiccates obtained in the preceding step are dissolved in 400 ul of ultrafiltered purified water. An aliquot of 100 p.1 of each of the solutions is injected onto a CARBOPAC MAI analytical column (4 x 250 mm) (DIONEX # 44066) pre-equilibrated with a 480 mM sodium hydroxide solution. The column is subjected to a flow of 48 0 mM sodium hydroxide solution for 60 minutes at a flow rate of 0.4 ml/min in order to elute the neutral monosaccharides such as ribitol and mannose. The column temperature is maintained at 30°C. Under these conditions, the chromatography peak corresponding to the ribitol appears at 19.9 ± 5% min, while the peak corresponding to the mannose (internal standard) appears at 25.2 ± 5% min. The standard curve (amount of ribitol as a function of the surface area of the peaks) is then established. The amount of ribitol contained in the starting preparation is determined by interpolation. Next, the exact amount of C5-polysaccharide is then deduced, in the knowledge that the repeating unit of the C5-polysaccharide consists of 11.1% (w/w) of ribitol. 1.1.2. (^-polysaccharide: The purified C-polysaccharide powder (Staten Serum Institute, Denmark) is dissolved in ultrapurified water, and then its concentration is adjusted to 10 u.g/ml on the basis of the ribitol assay carried out according to the same method as that for which details are given in paragraph 1.1.1. 1.2. Standard ranges: From 0 to 5 jag/ml of precisely assayed teichoic acids, prepared from the stock solutions by dilution in ultrafiltered purified water. The samples for the range are then dried under nitrogen. 1.3. Preparation of the sample to be assayed 3 vaccine doses (1.5 ml) are dialyzed against distilled water, and the dialysate is then lyophilized. The lyophilizate is then taken up with 1.5 ml of ultrafiltered purified water, from which an aliquot of 40 n-1 is taken, which is then dried under nitrogen. To control the reproducibility of the chromatography, a fixed amount of fructose, which serves as an internal standard, can be added to the sample to be analyzed. 1.4. Hydrolysis with HF All the desiccates (sample to be assayed and samples for the standard range) are treated with 4.00 JJ,1 of 48% hydrofluoric acid for 48 hours at 5°C. Drying is carried out under a stream of nitrogen and the dried material is taken up with 400 p.1 of ultrafiltered purified water at the time of analysis. 1.5. Analysis by HPAEC-PAD chromatography 100 }il of each of the hydrolysates are injected onto a CARBOPAC PA1 analytical column (4 x 250 mm) (DIONEX #35391) pre-equilibrated with a 75 mM sodium hydroxide solution. The column is subjected to a flow of 7 5 mM sodium hydroxide solution for 10 min at a flow rate of 1 ml/min in order to elute the C-polysaccharide-specific and C5-polysaccharide-specific oligosaccharides and also the monosaccharides which are neutral at the pH of the analysis. The column temperature is maintained at 30°C. To finish off the chromatography, a 7 5 mM sodium hydroxide solution containing sodium acetate is gradually added in order to elute all the remaining monosaccharides, oligosaccharides and polysaccharides. Under these conditions, the chromatography peak corresponding to the ribitol released during hydrolysis of the C-polysaccharide, of the C5-polysaccharide and of the 6B and 10A capsular polysaccharides appears at 2.65 ± 5% min. The peaks corresponding to the C5-polysaccharide-specific and C-polysaccharide specific oligosaccharides, and also the fructose peak (internal standard), appear, respectively, at 3.30 + 5% min, 4.30 ± 5% and 8.5 + 5% min (see Figure 1). The standard curves (amount of C-polysaccharide or C5-polysaccharide as a function of the surface area of the peaks) is established. The amounts of C-polysaccharide and C5-polysaccharide present in the starting preparation are determined by interpolation. Example 2: Assaying of the residual amounts of C-polysaccharide and C5-polysaccharide in the vaccine formulation F3 as described in WO 98/51339, containing the Dt and/or Tt conjugates of the capsular polysaccharides of serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F of pneumococcus The same protocol as that described in paragraph 1.3 is used to prepare the sample to be assayed. The volume of the aliquot taken is 1 ml here. The chromatographic profile obtained is that shown in Figure 2. Claims 1. A method for assaying the polysaccharide-type teichoic acids in a preparation derived from Gram+ bacterial culture, according to which: (1) the preparation is treated with hydrofluoric acid (HF) at a temperature of less than or equal to 40°C, in order to release the oligosaccharides specific for the teichoic acids present in the preparation; and (2) the specific oligosaccharides obtained in (1) are assayed. 2. The assaying method as claimed in claim 1, in which the preparation is a culture supernatant. 3. The assaying method as claimed in claim 1, in which the preparation comprises one or more purified antigens. 4. The assaying method as claimed in one of claims 1 to 3, in which the preparation is treated with hydrofluoric acid (HF) at a temperature of between 0 and 20°C, limits inclusive. 5. The assaying method as claimed in claim 4, in which the preparation is treated with hydrofluoric acid (HF) at a temperature of between 4 and 10°C, limits inclusive. 6. The assaying method as claimed in one of claims 1 to 5, in which the preparation is treated with hydrofluoric acid (HF) at a final concentration of between 10 and 73% (weight/weight), limits inclusive. 7. The assaying method as claimed in claim 6, in which the preparation is treated with hydrofluoric acid (HF) at a final concentration of between 40 and 60%. 8. The assaying method as claimed in claim 7, in which the preparation is treated with hydrofluoric acid (HF) at a final concentration of between 45 and 50%. 9. The assaying method as claimed in one of claims 1 to 8, in which the specific oligosaccharides are assayed using a technique which makes it possible to separate and/or to assay the monosaccharides. 10. The assaying method as claimed in claim 9, in which the specific oligosaccharides are assayed using a technique which is high performance anion exchange chromatography (HPAEC), optionally coupled with pulsed amperometric detection (PAD). 11. The method as claimed in claim 10, in which the HPAEC uses a support compatible with a pH greater than or equal to 12. 12. The method as claimed in claim 11, in which the support consists of sulfonated polystyrene-divinylbenzene. 13. The method as claimed in claim 12, in which the sulfonated polystyrene-divinylbenzene has a degree of crosslinking of 1 to 5%. 14. The method as claimed in claim 12 or 13, in which the sulfonated polystyrene-divinylbenzene is in the form of microbeads having a diameter of 450 to 550 nm. 15. The method as claimed in one of claims 11 to 14, in which the chromatography support is packaged in the form of a column and is completed, in its upper part, by a layer of a material bearing positive charges for optimizing the column resolution. 16. The method as claimed in claim 15, in which the material for optimizing the resolution consists of material bearing a primary amine salt, a secondary amine salt, a tertiary amine salt, a quaternary ammonium salt, or an ammonium group. 17. The method as claimed in claim 16, in which the material is latex having a degree of crosslinking of 3 to 7%. 18. The method as claimed in one of claims 10 to 17, in which the HPAEC uses a material of the CarboPac™ type. 19. The method as claimed in claim 18, in which the material is a CarboPac™ PAl chromatographic column. 20. The method as claimed in claim 10, in which the HPAEC uses an eluting solution having a pH > 12. 21. The method as claimed in claim 20, in which the eluting solution is free of carbonate. 22. The method as claimed in claim 20 or 21, in which the eluting solution is a sodium hydroxide solution, the molarity of which is between 10 and 300 mM. 23. The method as claimed in claim 22, in which the sodium hydroxide solution has a molarity of between 40 and 100 mM. 24. The method as claimed in one of claims 1 to 23, in which the preparation derived from a Gram+ bacterial culture comprises one or more capsular polysaccharides. 25. The method as claimed in claim 24, in which the preparation comprises one or more capsular polysaccharides of Streptococcus pneumoniae. 26. The method as claimed in claim 25, in which the preparation comprises one or more capsular polysaccharides of Streptococcus pneumoniae serotype 6B, 10A, 17F, 19A, 19F or 20. 27. The method as claimed in claim 26, in which the preparation comprises the capsular polysaccharides of Streptococcus pneumoniae serotypes 6B, 10A, 17F, 19A, 19F and 20. 28. The method as claimed in claim 25, in which: (i) the preparation is treated with hydrofluoric acid (HF) at a temperature of less than or equal to 40°C, in order to release the teichoic acid-specific oligosaccharides present in the preparation, said specific oligosaccharides comprising the oligosaccharide specific for the C5-polysaccharide; and (ii) the oligosaccharide specific for the C5-polysaccharide obtained in (i) is assayed. 29. The method as claimed in claim 28, in which the preparation comprises a capsular polysaccharide of Streptococcus pneumoniae serotype 5. 30. A purified C5-polysaccharide having a repeating unit of formula: 6 6 I I O)-P-Ch0 0)-P-Cho 31. The use of the C5-polysaccharide as claimed in claim 30, for establishing a standard curve intended for assaying the C5-polysaccharide, in a preparation comprising a capsular polysaccharide of Streptococcus pneumoniae serotype 5.

Documents:

0066-chenp-2006 abstract duplicate.pdf

0066-chenp-2006 claims duplicate.pdf

0066-chenp-2006 description(complete) duplicate.pdf

0066-chenp-2006 drawings duplicate.pdf

66-CHENP-2006 CORRESPONDENCE OTHERS.pdf

66-CHENP-2006 CORRESPONDENCE PO.pdf

66-CHENP-2006 FORM-1.pdf

66-CHENP-2006 FORM-3.pdf

66-CHENP-2006 PETITIONS.pdf

66-CHENP-2006 POWER OF ATTORNEY.pdf

66-chenp-2006-abstract.pdf

66-chenp-2006-claims.pdf

66-chenp-2006-correspondence others.pdf

66-chenp-2006-correspondence po.pdf

66-chenp-2006-discription complete.pdf

66-chenp-2006-drawings.pdf

66-chenp-2006-form 1.pdf

66-chenp-2006-form 18.pdf

66-chenp-2006-form 3.pdf

66-chenp-2006-form 5.pdf

66-chenp-2006-pct.pdf