WO2016087524A1

WO2016087524A1 - Saccharolipids labelling

Info

Publication number: WO2016087524A1
Application number: PCT/EP2015/078378
Authority: WO
Inventors: Simona ROTELLA
Original assignee: Ares Trading S.A.
Priority date: 2014-12-05
Filing date: 2015-12-02
Publication date: 2016-06-09

Abstract

The present invention relates to the use of 2-aminobenzamide (2-AB) for labelling saccharolipids, as well as to methods of labelling saccharolipids with 2-AB. It also describes methods of identifying, detecting, characterizing or analysing saccharolipids in a sample, once labelled with 2-AB. Such uses and methods have their place as analytical methods when it is needed to analyse saccharolipids containing medicinal, biological or cosmetic samples, for instance. The use and methods herein described aim at facilitating identification and analysis of saccharolipids. The saccharolipid compounds contain four to seven fatty acyl chains.

Description

Saccharolipids labelling

Field of Invention

The present invention relates to the use of 2-aminobenzamide (2-AB) for labelling saccharolipids, as well as to methods of labelling saccharolipids with 2-AB. It also describes methods of identifying, detecting, characterazing or analysing saccharolipids in a sample, once labelled with 2-AB. Such uses and methods have their place as analytical methods when it is needed to analyse saccharolipids containing medicinal, biological or cosmetic samples, for instance. The use and methods herein described aim at facilitating identification and analysis of saccharolipids.

Background of the invention

The saccharolipids are compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. They are also known as "acylaminosugars". Natural lipid A belongs to the saccharolipid family. It is the lipid-carbohydrate component of the lipopolysaccharide (LPS) found in the cell wall of gram-negative bacteria. Monophosphoryl lipid A (MPLA or MPL®) is a detoxified lipid A derivative, lacking an acyl chain as well as a phosphorylation group. It is prepared from the cell wall of a bacterium and consists of structurally similar β-1 ',6 linked disaccharides of 2-deoxy-2 aminoglucose residues, phosphorylated at the 4' position. It contains variable numbers and types of fatty acid (acyl) groups at various position. The heterogeneous substitution results in MPL being composed of a mixture of at least 8 forms (also called congeners) consisting of various tetra, penta, hexa and hepta acyl forms. MPLA is known as an endotoxic TLR4 agonist. MPL is a low-toxicity derivative of LPS having interesting immunostimulatory properties. It is approved as a human vaccine adjuvant, and has been incorporated in liposomal formulation for several vaccines (Alving et al., 2012). For instance, the Fendrix® Hepatitis B vaccine, from GSK, comprises this adjuvant. Similarly, MPL enters in the composition of stimuvax, from Merck KgaA, a therapeutic cancer vaccine in development.

Current analytical panel of a drug product after formulation foresees the testing and identification of all components content in such product, for instance for assessing stability. For instance, in stimuvax, components includes the peptide BLP-25 (also known as tecemotide) and the liposomal compounds DMPG (1 ,2-Dimyristoyl-sn-glycero-3-[phospho-rac-(1 -glycerol)], DPPC (1 ,2-Dipalmitoyl-sn-glycero-3- phosphocholine), cholesterol and MPL. In particular, MPL degradation pattern is tested analyzing its congeners distribution.

Saccharolipids typically have no or low absorbivity in both UV and visible light wavelenghts. Therefore, the detection systems associated with most of the common analytical techniques require special detectors, that do not distinguish common lipids from MPL or the lipid allowing sensitive and quantitative detection. Using reductive amination chemistry, the free reducing end of lipids may be labelled with fluorescent tags, and then the resulting labelled lipids may be injected directly in RP- HPLC. One of the common analytical procedure for testing MPL and its congeners involves a RP-HPLC analysis after MPL derivatization with DNBA in pyridine, followed by RP-HPLC analysis and UV detection (Hagen et al., 1997).

While analytical methods for saccharolipids have been described in the art, there remains a need for other methods, possibly less toxic than the current methods.

Summary of the invention

Herein described is a method for labelling saccharolipid compounds in a sample, comprising the steps of: a. providing a sample to be analysed; b. diluting said sample in a solvent; and c. adding a fluorescent label to the diluted sample of step b, wherein the fluorescent label is 2-aminobenzamide (2-AB).

It is another object of the present invention to describe a method for detecting, monitoring or localizing saccharolipid compounds in a sample, comprising the steps of: a. labelling each saccharolipid compound in the sample, wherein the fluorescent label is 2-aminobenzamide (2-AB) and; b. detecting the emitted fluorescence.

In another aspect of the invention, herein is provided a method for identifying or characterising saccharolipid compounds in a sample, comprising the steps of: a. labelling each saccharolipid compounds in the sample with a fluorescent label, wherein the fluorescent label is 2-aminobenzamide (2-AB); b. separating the different saccharolipid compounds using at least one analytical method and; c. detecting the emitted fluorescence for any labelled separated saccharolipid compounds of step b. In a further aspect, the present invention provide a method for analysing a sample containing a plurality of saccharolipid compounds which comprises the steps of: a. labelling the saccharolipid compounds in the sample with a detectable fluorescent label, wherein the label is 2-aminobenzamide (2-AB); b. separating the labelled saccharolipid compounds using at least one analytical method, before or after labelling; and determining the presence of the separated labelled saccharolipid compounds by detecting the emitted fluorescence.

It is also an object of the present invention to describe a method of characterizing or identifying individual species within a sample comprising saccharolipid compounds, said method comprising steps of: (i) providing a sample comprising saccharolipid compounds, wherein the saccharolipid compounds includes at least one free reducing sugar; (ii) labelling the saccharolipid compounds with a detectable fluorescent label, the label being 2-aminobenzamide (2-AB); (iii) separating the glysaccharolipid compounds using at least one analytical method and at least, thereby characterizing individual saccharolipid compounds within the sample by detecting the emitted fluorescence.

In another aspect, the present invention provides the use of a fluorescent label for detecting a saccharolipid compound, wherein the fluorescent label is 2-aminobenzamide (2-AB).

The saccharolipids to be detected in the sample should have at least one free reducing sugar.

Saccharolipids, or saccharolipid compounds, according to the invention includes precursors, derivatives or congeners of the saccharolipid of interest. Preferably, the saccharolipid contains four to seven fatty acyl chains and is for instance Lipid A, one or more precursor(s), one or more derivative(s) or one or more congener(s) thereof. More preferably, the saccharolipid is a monophosphoryl lipid A (MPL), one or more precursor(s), one or more derivative(s) or one or more congener(s) thereof. The sample according to any one of the methods herein described is any sample susceptible to comprise saccharolipids. Preferably it is a medicinal, a biological or a cosmetic sample. More preferably, it is a sample comprising liposomes, virosomes, saponins or emulsions. Although not limitating, more preferably the sample is a vaccine.

Definitions

- The terms "saccharolipid(s)", "saccharolipid compounds()", "glycolipid(s)" or "acylaminosugars" describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a sugar substitutes for the glycerol backbone that is present in glycerolipids and glycerophospholipids. Saccharolipids can occur as glycans or as phosphorylated derivatives. Some saccharolipid structures are shown in Fig. 3. The most familiar saccharolipids are the acylated glucosamine precursors of the lipid A component of the lipopolysaccharades in Gram-negative bacteria. (Fahy et al 2005). Other common saccharolipids include O-acylated glucose derivatives of plants. LPS is classified as a saccharolipid glycan. For any saccharolipid, the attached acylic chains are heterogeneous mixture of various congeners of tetra, penta, hexa and hepta acylic forms for instance. The various forms of a given saccharolipid are called "congeners".

- The term "natural lipid A", or "lipid A" corresponds to the lipid-carbohydrate component of the lipopolysaccharide found in the cell wall of gram-negative bacteria. This component is composed of a series of structurally similar β1 -6 linked disaccharides of 2-deoxy-2-aminoglucose residues phosphorylated at the 1 and 4' positions of the disaccharide. It contains variable numbers and types of acyl chains at the 2, 3, 2' and 3' positions of the sugars. Acyl chains are of defined lengths between 12 and 16 carbons depending on their position (leading to various congeners of lipid A). The 2, 2' and 3' positions of the disaccharide contain 1 or 2 acyl chains, while the 3 position contains only a single acyl chain (Figure 1 a). In some bacteria the glucosamine backbone of lipid A is replaced by 2,3- diamino-2,3-dideoxyglucose.

- The term "MPL", "MPL®" or "MPLA" stends for monophosphoryl lipid A ( MPL® GSK). It is a detoxified lipid A derivative, consists of a glucosamine (carbohydrate/sugar) unit linked directely with acyl chains

(fatty acids) and it is represented by different congeners in term of number of acyl chains. Normally, it contains one phosphoryl group on each carbohydrate. It is detoxified by selective hydrolysis of the 1 position phosphoryl group as well as by deacylating the 3 position acyl chain (Fig.1 b). The optimal immune activating lipid A structure is believed to contain 6 acyl chains. Four acylic chains are attached directly to the glucosamine sugars; these chains are β hydroxy acylic usually between 10 and 16 carbons length. Two additional acylic chains are often attached to the β hydroxy group. The attached acylic chains are heterogeneous mixture of various congeners of tetra, penta, hexa and hepta acylic forms. This heterogeneous substitution results in MPL® being composed of a mixture of at least 8 forms (called congeners) consisting of various tetra, penta, hexa and hepta acyl forms as in Fig. 2. - The term "BPL25" refers to a synthetic 25-amino acid sequence of the cancer-associated marker MUC-1 ("STAPPAHGVTSAPDTRPAPGSTAPP", see SEQ ID NO.1 ). It is also known as tecemotide or emepepimut-S. A palmitoyi lysine residue is included at the carboxy terminal to enhance incorporation of the lipopeptide into liposome particles. BPL25 is thus a lipopeptide.

- The term "L-BLP25" refers to a cancer vaccine comprising BPL25 as active ingredient encapsulated in a specially designed liposomal delivery system. Liposomes, which are fat droplets smaller than red blood cells, are believed to enhance immune recognition of cancer cells when used in medicinal product, demonstrating stronger protection from tumor challenge and increased cellular immune responses. L-BLP25 drug product components are BLP25 lipopeptide (i.e. antigen) and Monophosphoryl lipid A® (i.e adjuvant). Antigen and adjuvant are incorporated into the lipid bilayer of the liposomes formed by three lipids: Cholesterol, 1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine (i.e. DPPC; other names include dipalmitoylphosphatidylcholine or 1 ,2 dipalmitoyl glycerophosphocholine) and 1 ,2-Dimyristoyl-sn-glycero-3-[phospho-rac-(1 -glycerol)] (i.e. DMPG; other names include dimyristoylphosphatidylglycerol, dimyristoyl-L-a-phosphatidylglycerol or 1 ,2-dimyristoyl-sn-glycero-3- phosphoglycerol). This cancer vaccine is also known as Stimuvax.

- The term "2-AB" refers to 2-aminobenzamide, a fluorescent label. It has been described for the first time as a label for carbohydrates in US5747347.

- The term "pattern" or "saccharolipid pattern" refers to the pattern of distribution of each components we are interested in. Should we refer to a MPL pattern, this would refer for instance to the distribution of the different congeners, eg tetrad, pentacyl, hexacyl and heptacyl form of the MPL.

Detailed description of the invention

Because of the complexity of saccharolipids, it was found that using the currently known techniques may not provide the best resolution of individual saccharolipid components.

An innovative application based on saccharolipid labelling with 2-AB has been established. In addition to this, chromatographic analysis for separating labelled saccharolipid congeners was set up as well. The innovative method consists in labelling saccharolipid compounds not as lipids, as currently done, but as glycans, with 2-AB (2-Aminobenzamide), one of the most widely used detectable fluorescent labels for glycosylation analysis. It has been found that a saccharolipid can be chemically functionalized with the fluorescent probe 2-aminobenzamide (2-AB), exploiting its carbohydrate part. As shown in the example section, is was possible to actually label a saccharolipid compound, and congenersthereof, using MPL and L-BPL25 as examples (see examples section).

Herein described is a method for labelling saccharolipid compounds in a sample, comprising the steps of: a. providing a sample to be analysed; b. diluting said sample in a solvent; and c. adding a detectable fluorescent label to the diluted sample of step b, wherein said detectable fluorescent label is 2- aminobenzamide (2-AB). Any suitable solvent can be used for the dilution step. Once labelled, the saccharolipid compounds can be detected, via the emitted fluorescence, by any known methods. It is another object of the present invention to describe a method for detecting, monitoring or localizing saccharolipid compounds in a sample, comprising the steps of: a. labelling each saccharolipid compound in the sample with a detectable fluorescent label, wherein said detectable fluorescent label is 2-aminobenzamide (2-AB) and; b. detecting the emitted fluorescence. The saccharolipid compounds can be labelled according to the method above described. The labelled saccharolipids compounds can be detected, via the emitted fluorescence, by any known methods. They can be detected in situ, for instance, using microscopy such as a confocal microscopy.

In another aspect of the invention, herein is provided a method for identifying or characterising saccharolipid compounds in a sample, comprising the steps of: a. labelling each saccharolipid compounds in the sample with a detectable fluorescent label, wherein said detectable fluorescent label is 2-aminobenzamide (2-AB); b. separating the different labelled saccharolipid compounds using at least one analytical method and; c. detecting the emitted fluorescence for any labelled separated saccharolipid compounds. The saccharolipid compounds can be labelled according to the method above described. The labelled saccharolipids compounds can be separated by any known analytical methods. The labelled saccharolipids compounds can be detected, via the emitted fluorescence, by any known methods.

In a further aspect, the present invention provide a method for analysing a sample containing a plurality of saccharolipid compounds which comprises the steps of: a. labelling each saccharolipid compounds in the sample with a detectable fluorescent label, wherein said detectable fluorescent label is 2- aminobenzamide (2-AB); b. separating the labelled saccharolipid compounds using at least one analytical method, before or after labelling; and c. determining the presence of the separated labelled saccharolipid compounds by detecting the emitted fluorescence. The saccharolipid compounds can be labelled according to the method above described. The labelled saccharolipids compounds can be separated by any known analytical methods. The labelled saccharolipids compounds can be detected, via the emitted fluorescence, by any known methods.

It is also an object of the present invention to describe a method of characterizing individual species within a sample comprising saccharolipid compounds, said method comprising steps of; (i) providing a sample comprising saccharolipid compounds, wheren the sample has been previously diluted in a suitable solvent; (ii) labelling the saccharolipid compounds with a detectable fluorescent label, said detectable fluorescent label being 2-aminobenzamide (2-AB); (iii) separating the glysaccharolipid compounds using at least one analytical method and at least, thereby characterizing individual saccharolipid compounds within the sample by detecting the emitted fluorescence. The saccharolipid compounds can be labelled according to the method above described. The labelled saccharolipids compounds can be separated by any known analytical methods. The labelled saccharolipids compounds can be detected, via the emitted fluorescence, by any known methods.

In a further aspect, herein is disclosed a method for monitoring the presence of saccharolipids compounds in a sample, the method comprising the steps of: providing a marker for saccharolipid compounds in the sample, said marker being detectable through fluorescence and said marker being 2-aminobenzamide (2-AB); labelling the saccharolipid compounds with said detectable fluorescent label, and detecting fluorescence emitted from the sample; and analyzing or detecting said fluorescence to monitor presence of saccharolipids compounds in the sample. In another aspect, the present invention provides the use of a detectable fluorescent label for detecting a saccharolipid compound in a sample, wherein the detectable fluorescent label is 2-aminobenzamide (2-AB).

In the context of the invention, the saccharolipids compounds in the sample should have at least one free reducing sugar.

Saccharolipids, or saccharolipid compounds, according to the invention, includes precursors, derivatives or congeners. Preferably, the saccharolipid or saccharolipid compound contains four to seven fatty acyl chains and is for instance Lipid A, one or more precursor(s), one or more derivative(s) or one or more congener(s) thereof. More preferably, the saccharolipid is a monophosphoryl lipid A (MPL), one or more precursor(s), one or more derivative(s) or one or more congener(s) thereof.

Any one of the methods or uses described herein can be used with any sample that includes or that is susceptible to include at least one saccharolipid compound, irrespective of the presence and/or the nature of any further components. Preferably, the sample is a medicinal, a biological or a cosmetic sample. More preferably, it is a sample comprising liposomes, virosomes, saponins or emulsions. Although not limitating, more preferably the sample is a vaccine, such as a vaccine comprising liposomes. The sample can be in any state. For instant, the sample can be in a liquid state, in a solid state or in a lyophilised (i.e. freeze-dried) state.

As an example, should the labelled saccharolipid compounds be part of liposomes, they can be detected or localized directly within the liposomes, according to one of the methods or uses of the present invetion. Such a method would allow, for instance, to test that the saccharolipids are correctly integrated within the liposomes. Such a method will also allow to detect or monitor the presence of saccharolipid compounds in a sample.

During the labelling step, any suitable solvent can be used for the dilution. Due to the lipidic nature of saccharolipids, said solvent is preferably an alcohol or acetone or comprises at least an alcohol or acetone. More preferably, the solvent is or comprises at least ethanol. The solvent can also be a mixture of solvents, such as a mixture comprising both an alcohol (for instance ethanol) and an aquous solvent (such as water).

When a separation step using at least one analytical method is required according to the present invention, such separation step can be performed either before or after the labelling step. Preferably, the separation step is performed after the labelling step.

In the context of the invention as a whole, the labelled saccharolipid compounds can be separated by any known analytical methods. Preferably, the separation is on the basis of charge or weight of said compounds and with a fluorescent detector. The at least one analytical method is preferably a chromatographic method, for instance selected from the group consisting of, but not limited to, reversed phase liquid chromatography (RP), High-performance liquid chromatography , Ultra Performance Liquid Chromatography, normal phase liquid chromatography (NP), hydrophilic interaction chromatography (HILIC), ion-pairing reverse phase chromatography (IP-RP), size exclusion chromatography, affinity chromatography (AQ, capillary electrophoresis (CE); fluorophore- assisted carbohydrate electrophoresis (FACE); and electrochromatography. The labelled saccharolipid compounds according to any one of the methods or uses herein described can be analysed or detected via the emitted fluorescence, by any known methods. The detection can be performed either in situ, in the sample as such, or after separation of the labelled saccharolipid compounds by analytical methods, by any known methods. For instance, the analysis/detection can be performed in situ using a microscopy such as a confocal microscopy, fluorescence resonance energy transfer (FRET), Co-Localization Microscopy, Multiphoton microscopy, fluorescence recovery after photobleaching (FRAP), Two/Three-Dimensional Scanning X-ray fluorescence (2D/3D-XRF), confocal X-ray fluorescence (XRF), in general all fluorescence microscopy. In an alternative, the analysis or detection can be performed after separation via analytic chromatography techniques such as above reported. In a further alternative, the analysis or detection can be performed by mass spectrometry. The analysis or detection of the emitted fluorescence can be performed in real time, at the time of labelling or at the time of separation. Alternatively, the analysis or detection of the emitted fluorescence can be performed in a further step, after the labelling step or after the separation step. 2-AB labelled lipids may be studied by a number of different analytical methods such as HPLC/UPLC, mass spectrometry and fluorescence microscopy.

Any one of the methods or uses according to the present invention requesting a step of separation of the labelled compounds by at least an analytical method has the advantage to further allow relative quantitation of each saccharolipid compounds, precursor, derivatives or congeners thereof, by comparison of fluorescence peak areas of each species that are resolved by chromatography.

The methods and uses according to the present invention can be applied to saccharolipids contained in a wide variety of sources including, but not limited to, medicinal, biological or cosmetic samples. Any sample of interest may undergo one or more analysis methods and/or purification steps prior to or after being used according to any of the methods and uses of the present invention.

Any one of the methods or uses described herein can be used in any of a variety of applications. preferably, they can be used as analytical tools or analytical methods. For instance, they can be integrated as an analytical tool in the panel of assays needed for testing medicinal sample(s) or for identifying components content in such medicinal sample(s). Similarly, any one of the methods and use herein provided can be used as an analytical tool in the panel of assays needed for testing biological or cosmetic sample(s) or for identifying components content in such biological or cosmetic sample(s).

When it is refered to a medicinal or a cosmetic product on the market, it may be desirable to assess the quality or the stability of said product, in view of the known initial pattern or the known degradation pattern of the reference product. Samples of medicinal or cosmetic products can be taken at any time, for instance at any time during production or at any time said product is on the market, in order to perform such an assessment (for instance to compare the pattern of said smple to the one of the reference product. For example, when saccharolipid compounds are contained in a medicinal product, it may be desirable to monitor samples of said medicinal products in order to check that said samples meet as much as possible the established saccharolipid pattern of said medicinal product. Indeed, saccharolipids are known to degradate in various congeners. In the context of the invention, a sample will meet the established saccharolipid pattern, if said the saccharolipid pattern, either initial pattern or degradation pattern in the sample is at least 90% identical to the one of the reference product at the same "stage". Preferably, the saccharolipid pattern in the sample is at least 95%, 98% or 99% identical to the one of the reference product

Description of the figures:

Figure 1 : Structure of Natural Lipid A. It is composed of a series of structurally similar β1 -6 linked disaccharides of 2-deoxy-2-aminoglucose residues phosphorylated at the 1 and 4' positions of the disaccharide. Natural lipid A contains variable numbers and types of acyl chains at the 2, 3, 2' and 3' positions of the sugars. Acyl chains are of defined lengths between 12 and 16 carbons depending on their position. The 2, 2' and 3' positions of the disaccharide contain 1 or 2 acyl chains, while the 3 position contains only a single acyl chain

Figure 2: Different congeners of MPL

Figure 3: Representative structures for saccharolipids (Fahy et al 2005)

Figure 4: Labelling of a glycan with 2-aminobenzamide acid (2-AB) by reductive amination

Figure 5: RP-UPLC profile of MPL labelled with 2-AB (Aex=330 nm; Aem = 420 nm)

Figure 6: The chromatographic pattern obtained for MPL labelled with pyridine in DNBA (Fig. 6a) or with 2-AB (fig.6b)

Figure 7: detection of 2-AB labelled MPL compounds in liposome membrane by means of confocal microscopy. Figure 7a shows the detection of 2-AB fluoresecence in a sample, figure 7b shows the same sample but using the technic of merging bright field wih 2-AB fluorescence.

Description of the sequences:

SEQ ID N0.1 : BPL25 amino acid sequence

Examples

Material

A lyophilised sample of MPL was used in the below examples.

Prozyme 2-AB kit ; ref. GKK-404 was used to label MPL.

Example 1

The saccharolipid samples are prepared by diluting the lyophilized 2 mg/mL MPL sample in 100 μί of 90% ethanol (absolute for analysis): water (milliQ). Fresh dye labelling solution is then prepared by mixing reagents in the kit (Prozyme 2-AB kit ; ref. GKK-404) according the following procedure taking into account the reactive lifetime of 1 hour:

Add 150 μΙ glacial Acetic Acid to the vial of DMSO (reactive from 2-AB kit) and mix by pipette action.

Add 100 μΙ of the DMSO-acetic acid mixture to a vial of 2-AB. Dye and mix until the dye is dissolved. Add the dissolved dye to a vial of Sodium Cyanoborohydride (reductant) and mix by pipette action until the reductant is completely dissolved to make the final labelling reagent.

Add 50 μΙ of labelling reagent to each dried glycan sample, cap the microtube, mix thoroughly, and then gently tap to ensure the labelling solution is at the bottom of the vial.

- Place the reaction vials in a heating block and incubate the samples for 2 hours at 65°C to allow the labelling reaction to progress. Post-labelling sample clean-up is not necessary. After the incubation period remove the samples, centrifuge the microtubes briefly, and then allow them to cool completely to room temperature. Dilute 15 μΙ_ of sample at Vfm 100 μΙ_ with a solution of 45% methanol: 45% chloroform: 10% H2O.

2-AB labelled lipids may be studied by a number of different analytical methods such as HPLC/UPLC, mass spectrometry and fluorescence microscopy. In this example, 2-AB labelled lipids were analyzed by means of RP-HPLC.The RP-HPLC parameters are reported in Table 1. The chromatographic method is based on RP-HPLC for separating the labelled MPL congeners, revealed by a fluorimentric detector.

Results: The chromatographic pattern obtained for MPL labelled with 2-AB (figure 5) showed the same pattern and similar percentage to those observed with pyridine labelling (figure 6). As shown in this example, MPL congeners can be detected after labelling with the fluorescent label 2-AB, which is usually employed for glycans derivatization. This happens because MPL belongs to the Saccharolipids compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers.

Conclusions: The labelling with a marker usually employed for glycans (i.e. 2-AB) is an innovative approach for revealing other components preventing the use of reagents which are not safe. Among the current approaches, one foresees the use of pyridine to label MPL, but pyridine is harmful and carcinogenic if inhaled, swallowed or absorbed through the skin. Pyridine might also have minor neurotoxic, genotoxic, and clastogenic effects. Once labelled, MPL can be selectively detected in presence of other components or traced by other fluorescence based technologies and this allows studying other aspects related to the use of MPL, such as being part of Liposomes structure.

Its labelling with 2-AB facilitates the evaluation of the exposure on Liposomes surface by using confocal microscopy.

Example 2

MPL samples were labelled following a similar method as the one described in example 1 and then encapsulated in L-BLP25 in oder to localize the MPL into the liposome membrane.

An example of detection of MPL within L-BLP25 sample is reported in figure 7. In particular, Figure 7b shows that in optimized conditions, it is possible to visualize the presence of labelled MPL in the liposome membranes of the medicinal product. MPL appears homogeneously distributed in the liposome membranes. As shown in this figure, the presence of free labelled MPL does not interfere with the analysis.

Tables

Table 1 RP-HPLC parameters

Cited references

1 ) Alving et al., 2012, Current Opinion in Immunology, 24:310-315

2) Fahy et al., 2005, J Lipid Res., 46(5):839-61

3) US5747347

4) Hagen et al., 1997, Journal of Chrom. A, 767: 53-61

Claims

1. A method for labelling saccharolipid compounds in a sample, comprising the steps of: a. providing a sample to be analysed; b. diluting said sample in a solvent; and c. adding a detectable fluorescent label to the diluted sample of step b, wherein said detectable fluorescent label is 2-aminobenzamide (2-AB), wherein the saccharolipid compounds contain four to seven fatty acyl chains.

2. A method for detecting saccharolipid compounds in a sample, comprising the steps of: a. labelling each saccharolipid compound in the sample with a detectable fluorescent label, wherein said detectable fluorescent label is 2-aminobenzamide (2-AB) and; b. detecting the emitted fluorescence for any labelled saccharolipid compounds, wherein the saccharolipid compounds contain four to seven fatty acyl chains.

3. A method for identifying saccharolipid compounds in a sample, comprising the steps of: a. labelling each saccharolipid compounds in the sample with a detectable fluorescent label, wherein said detectable fluorescent label is 2-aminobenzamide (2-AB); b. separating the different labelled saccharolipid compounds using at least one analytical method and; c. detecting the emitted fluorescence for any labelled separated saccharolipid compounds, wherein the saccharolipid compounds contain four to seven fatty acyl chains.

4. A method for analysing a sample containing a plurality of saccharolipid compounds which comprises the steps of: a. labelling the saccharolipid compounds in the sample with a detectable fluorescent label, wherein said detectable fluorescent label is 2-aminobenzamide (2-AB); b. separating the labelled saccharolipid compounds using at least one analytical method, before or after labelling; and c. determining the presence of the separated labelled saccharolipid compounds by detecting the emitted fluorescence, wherein the saccharolipid compounds contain four to seven fatty acyl chains.

5. A method of characterizing individual species within a sample comprising saccharolipid compounds, said method comprising steps of; (i) providing a sample comprising saccharolipid compounds; (ii) labelling the saccharolipid compounds with a detectable fluorescent label, said detectable fluorescent label being 2-aminobenzamide (2-AB); (iii) separating the glysaccharolipid compounds using at least one analytical method and at least, thereby characterizing individual saccharolipid compounds within the sample by detecting the emitted fluorescence, wherein the saccharolipid compounds contain four to seven fatty acyl chains.

6. The method according to any one of claims 1 to 5, wherein the saccharolipid compound is a lipid A, a precursor, derivative or a congener thereof.

7. The method according to any one of claims 1 to 5, wherein the saccharolipid compound is a monophosphoryl lipid A (MPL) , a precursor, derivative or a congener thereof.

8. The method according to claim any one of the preceding claims, wherein the saccharolipid compound has at least one free reducing sugar.

9. The method according to any one of the preceding claims, wherein the sample is a medicinal, a biological or a cosmetic sample.

10. The method according to claim 9, wherein the medicinal, the biological or the cosmetic sample comprising liposomes, virosomes, saponins or emulsions.

1 1. The method according to claim 9 or 10, wherein the sample is a vaccine

12 . The method according to claim 1 , wherein the solvent is an alcohol or acetone or comprises at least an alcohol or acetone.

13. The method according to claim 12, wherein the alcohol is ethanol

14. Use of a detectable fluorescent label for detecting a saccharolipid compound in a sample, wherein said detectable fluorescent label is 2-aminobenzamide (2-AB).

15. The use according to claim 14, wherein the saccharolipid compound is detected in real time.

16. The use according to claim 14 or claim 15, wherein the saccharolipid compound is a lipid A, a precursor, derivative or a congener thereof.

17. The use according to claim 14 or claim 15, wherein the saccharolipid compound is a monophosphoryl lipid A (MPL) , a precursor, derivative or a congener thereof.

18. The use according to any one of claims 14 to 17, wherein the saccharolipid compound has at least one free reducing sugar.

19. A method according to any one of claims 3 to 5, wherein the separation is on the basis of charge or weight.

20. The method of any one of claims 3-5, wherein the at least one analytical method is a chromatographic method which is selected from the group consisting of reversed phase liquid chromatography (RP), High-performance liquid chromatography, Ultra Performance Liquid Chromatography, normal phase liquid chromatography (NP), hydrophilic interaction chromatography (HILIC), ion-pairing reverse phase chromatography (IP-RP), size exclusion chromatography, affinity chromatography (AQ, capillary electrophoresis (CE); fluorophore-assisted carbohydrate electrophoresis (FACE); and electrochromatography.

21. The method according to any one of claims 2-5, wherein said emitted fluorescence is detected with a microscope.

22. The method according to claim 21 , wherein the microsope is a confocal microscopy, fluorescence resonance energy transfer (FRET), Co-Localization Microscopy, Multiphoton microscopy, fluorescence recovery after photobleaching (FRAP), Two/Three-Dimensional Scanning X-ray fluorescence (2D/3D-XRF), confocal X-ray fluorescence (XRF)

23. The method of any of claims 2-5 , wherein said emitted fluoresence is performed in real time.