Analysis of eluates obtained from anion-exchange chromatography

FIELD: chemistry.

SUBSTANCE: sample analysis device has a column for anion-exchange chromatography, as well as a buffer for elution, which contains an ion formed from a group consisting of a nitrate and a chloride. The device also includes an amperometric sensor and a spectroscopic sensor. The two sensors are placed such that, an eluate is obtained from the column.

EFFECT: provision for additional and improved methods of and systems for determining characteristics of saccharides using anion-exchange chromatography.

14 cl, 21 dwg

 

All cited in the present description the document is included in its entirety by reference.

The technical field of the invention

The present invention relates to the field of analysis of sugars.

Prior art inventions

Immunogen containing capsule sacharine antigens conjugated to protein carriers well known in the art. Conjugation transforms T-independent antigens in T-dependent antigens, thereby enhancing the reaction with the creation of memory and allowing the formed protective immunity, and prototype conjugated vaccine was the vaccine against Haemophilus influenzae type b (Hib) [see, for example, Chapter 14 references i]. After creating a vaccine against Hib were developed conjugated sacharine vaccine to protect against Neisseria meningitidis (meningococcus) and Streptococcus pneumoniae (pneumococcus). Other microorganisms, the vaccine which is of interest, are Streptococcus agalactiae (group b Streptococcus), Pseudomonas aeruginosa and Staphylococcus aureus.

When included in the composition of vaccines and other biological products saccharides regulatory authorities generally require that their characteristics were determined. Characterization of saccharides is generally used anion-exchange chromatography and, in particular, high-performance anion-exchange x is omatography (NRAES), usually with pulsed amperometric detection (PAD) [ii, iii]. Suitable systems HPAEC-PAD supplied Dionex™ Corporation (Sunnyvale, CA), for example, BioLC system™. Such systems allow for the quantitative analysis of individual sugars in mixtures without the need for derivatives or separation prior to analysis, and characterization of saccharides can be used in the analysis of mixed sugars.

In the analysis of sugars the eluate from the column to NRAES, as a rule, is analyzed using a pulsed amperometric detector (PAD), i.e. the detected based on the electric current. When appropriate (high) pH carbohydrates can be electrocatalytically oxidized on the electrode surface by the application of positive potential. The current produced thereby is proportional to the concentration of carbohydrate that provides detection and quantity determination of carbohydrate by the method of amperometry. Unlike simple amperometric detection in accordance with the method of PAD along with the standard capabilities for the detection of short pulses are emitted purifying and regenerating capacity that avoids the difficulties associated with the poisoning of the electrode by oxidation products of the analyzed substances. In addition to use in NRAES-analysis PAD so the e is used for analysis by the method of high-performance cation-exchange chromatography [4], and other methods HPLC-separation.

For more analytical information, especially in the analysis amperometric inactive compounds and chemically modified compounds, the authors of the present invention decided to analyze eluate spectroscopic methods. Unfortunately, the high pH values used for NRAES analysis of capsular saccharides, means the presence in the eluate hydroxide ions and high values of absorption of these ions (especially in the ultraviolet region) indicate that additional spectroscopic analysis is a challenging task. Hydroxide ions can be removed using micromembrane chemical suppressor device, but this creates new problems, because the acetate is commonly used as a propellant is used for elution of the capsular saccharides are converted by the suppressor in strongly absorbing acetic acid.

The objective of the invention is the provision of additional and improved methods and systems for the characterization of sugars by the method of anion-exchange chromatography. In particular, the purpose is to overcome difficulties spectroscopic analysis of the eluates resulting from the presence of either hydroxide ions, or, after conversion to the hydroxide ion, acetic KIS is the notes.

Disclosure of inventions

To overcome the above mentioned difficulties caused by the use of eluents hydroxide-based/acetate, the inventors used two approaches. First, they made a choice in favour of using weak anion exchange media, which do not require the use of acetate displacer. Such a column is not currently used for analysis of carbohydrates and more widely used for the separation of a large number of inorganic anions and organic acids [5]. Secondly, they made a choice in favor of the use of displacers with weak spectroscopic absorption. Both of these approaches made it possible to combine spectroscopic detection with PAD-discovery. Moreover, they are also compatible with conventional protocols HPAEC-PAD and can be preferably used even in cases where the use of spectroscopic detection is considered undesirable, especially because they allow a better share of long chain oligosaccharides.

Thus, in accordance with the first aspect of the invention amperometric detection combined with spectroscopic detection, so use both methods of detection. Thus, the invention relates to a method of analysis of the eluate from the column for liquid chromatography, in which luat analyze how the method of amperometry, and according to the method of spectroscopy. The invention also relates to an apparatus for analysis of a sample, and the unit comprises (i) a column for liquid chromatography, (ii) amperometric detector and (iii) spectroscopic detector, and two detector positioned to receive the eluate from the column.

Reported separately using a PAD or UV detection for the analysis of eluates columns for NRAES [6], but the use and amperometric, and spectroscopic methods for the analysis of one of the eluate is new. The amount of information obtained from the use of both types of detection, it is advantageous than that received each of the two detection methods separately, for example, as shown in figure 1, some peaks are characterized by the best dynamic range according to UV detection, than according to the PAD, and some peaks do better according to the PAD, than according to UV-detection.

In the second aspect, the invention also relates to a method for elution of the analyzed capsular saccharide with anion-exchange chromatographic column, in which elute an analyte eluent containing anion other than acetate or hydroxide. Eluent preferably contains an anion selected from the group consisting of nitrate, chloride, carbonate and borate. It is convenient to use Natrii the e salts of these anions. This method is particularly applicable if the eluent is the primary (e.g., pH>9), and even more applicable when using chemical suppressor hydroxide ions and provides the best resolution of long-chain oligosaccharides. Moreover, this method is especially applicable for the analysis of acetylated saccharides.

In the third aspect, the invention also relates to a method of analysis of a saccharide according to the method of anion exchange chromatography, in which the chromatography is carried out with the use of the column, characterized by a capacity less than 50 mkacf. (for example, ≤40 mkacf., ≤30 mkacf., ≤20 mkacf. and so on). Use column low capacity means that the elution occurs rapidly and requiring the use of displacers no. Thus, the suppression of hydroxide can be carried out without the formation of acetic acid. As a rule, use besaratinia gidroksilsodyerzhascimi eluent, and the rejection of acetate ions are particularly useful in the analysis of acetylated saccharides. The use of columns low capacity allows us to better separate the long-chain oligosaccharides.

The second and third aspects of the invention can be advantageously used together, i.e. the invention relates to method of analysis analyzed capsular saccharide according to the method of anion exchange chromatography, and chromatography is carried out is the use of columns, characterized by a capacity less than 50 mkacf., and for elution from the column using eluent containing an ion selected from the group consisting of nitrate and chloride.

The invention also relates to the eluate obtained in accordance with the method according to the invention. The invention additionally relates to a pharmaceutical preparation containing as active ingredient a substance analyzed in accordance with the method according to the invention. In particular, the invention relates to immunogenic compositions, such as vaccine containing bacterial capsular saccharide analyzed in accordance with the method according to the invention.

Column for liquid chromatography

The first aspect of the invention is applicable for the analysis of the eluate from the column for liquid chromatography. This aspect can be applied to different columns for liquid chromatography, but preferably it is applicable to high-performance liquid chromatography (HPLC). The invention is particularly applicable to the analysis of the results of separation by the method of high-performance anion-exchange chromatography (NRAES) or by the method of high-performance cation-exchange chromatography (NRSAS).

Preferred speakers are speakers that spontaneously delay sugars so that the sugars have eluted from the column. Als the I with the chromatographic column can represent the isocratic elution or gradient elution. Eluent containing sodium hydroxide and/or sodium acetate, are typical suenami used in the analysis of saccharides on methods HPAEC-PAD. However, in accordance with the second aspect of the invention is used eluent containing nitrate and/or chloride solution (usually sodium salt), usually on the merits in the absence of any acetate eluent. In this case, the elution of the analyzed substances with anion-exchange columns, typically use a basic eluent, for example, the pH value is >8, >9, >10, >11, >12, >13, etc. To achieve the desired pH can be used hydroxides (e.g., NaOH), and anion-exchange eluent, as a rule, use the hydroxide ions.

Eluate can undergo chemical suppression of Oh-ions, especially if the ions hinder the implementation of the methods of analytical detection. Can be conveniently used micromembrane suppressor, such as MMS products from Dionex™. Product MMS III provides constant chemical suppression to increase the conductivity of the analyte by reducing the conductivity of the eluent and makes possible a direct determination of conductivity by applications of ion exchange using isocratic or gradient elution at a wide range of concentrations. The use of suppressors, abrazos the x acetic acid from acetate ions, preferably avoided when the acetate ions are part of the eluent and the resulting acetic acid prevents the implementation of the methods of analytical detection.

Preferred speakers for NRAES for use in accordance with the first and second aspects of the invention are column CarboPac sold Dionex, such as PA1 column [diameter of 10 μm, polystyrene substrate, crosslinked with 2% divinylbenzene, agglomerating with 500 nm functionalized Quaternary ammonium latex MicroBead (cross-linked 5%)], RA, RA, PARADISE [diameter of 10 μm, ethylvinylbenzene substrate made of 55% divinylbenzene, agglomerating with 460 nm defunctionalization ion of the Quaternary ammonium MicroBead (cross-linked 5%)], RA or MA. Preferred speakers for NURSES speakers are lonPac, also sold Dionex, including CS10 column.

As an alternative to using liquid chromatography first aspect of the invention can be used to analyze the output of the fractions separated by the method of capillary electrophoresis systems, for example, Beckman-Coulter P/ACE.

In accordance with a third aspect of the invention, the anion exchange column is characterized by ion-exchange capacity less than 50 mkacf. (microequivalents charge), for example, <40 mkacf., <30 mkacf., <20 mkacf. etc. Preferred speakers for NRAES awsomeherovision column lonPac AS sold Dionex, such as AS11 column with alkanolamine cybertechnologies functional groups. When used in analytical format 2×250 mm column AS11 characterized by capacity 11 mkacf., rising to 45 mkacf. in format 4×250 mm are Preferred hydroxyisoleucine column low capacity.

Amperometric and spectroscopic detection

In accordance with the first aspect of the invention, the eluate from the column for liquid chromatography analyze both amperometric and by a spectroscope. Substance aliremove from the column may be divided so that one part was analyzed amperometric, and the other part was analyzed by a spectroscope. Alternatively, the substance aliremove from the column can be analyzed sequentially without separation or amperometric, and then by a spectroscope or by a spectroscope, and then amperometric. These two different General methods illustrated by figure 2 and 3. If using serial analysis, it may be preferable to carry out spectroscopic detection before amperometric detection, especially if the spectroscopic detection is non-destructive in comparison with amperometric detection.

Amperometric detection is s preferably represents pulsed amperometric detection (PAD). When conducting PAD can be used for different pulse shape [7], including any of those profiles, shown in figure 4. It is preferable to use a negative potential to clean the electrode. The pulse shape on figa includes a negative potential to clean the electrode and improves as the long-term reproducibility and reduces the wear of the electrode.

Preferably, the electrode used for amperometric detection is a gold electrode.

Spectroscopic detection is preferably based on the absorption and/or emission of electromagnetic radiation, preferably with a wavelength from 100 nm to 900 nm (for example, with a wavelength in the range of 100-200 nm, 150-250 nm, 200-300 nm, 250-350 nm 300-400 nm, 350-450 nm 400-500 nm, 450-550 nm, 500-600 nm, 550-650 nm, 600-700 nm, 650-750 nm, 700-800 nm, 750-850 nm, 800-900 nm). The used wavelength can be selected depending on the subject detection period(s) of substance(s). Two particularly preferred methods for the analysis of saccharides are absorption spectroscopy in the ultraviolet (UV) range (for example, at 220 nm) and absorption spectroscopy in the visible light range.

Alternatively, the use of amperometric detection in accordance with the invention can be used in the detection p is ultimate (including supressirovano detection by conductivity).

Analyte

The invention is used for the analysis of the eluate from the column for liquid chromatography. The eluate is the result of chromatographic separation of one or more of the analyzed substances in the sample.

The invention is particularly applicable for analysis sharidny substances. They can be a polysaccharide (e.g., characterized by the degree of polymerization comprising at least 10, for example, 20, 30, 40, 50, 60 or more), oligosaccharides (e.g., characterized by the degree of polymerization comprising from 2 to 10) or monosaccharides. Oligosaccharides and monosaccharides can be the result of depolymerization and/or hydrolysis of the original polysaccharide, for example, an analyte can be sharedstorage fragment of a larger saccharide.

Preferred analyzed sharedname substances are bacterial sugars and especially bacterial capsular saccharides, for example, Neisseria meningitidis (serogroups a, b, C, W135 or Y), Streptococcus pneumoniae (serotypes 4, 6B, 9V, 14, 18C, 19F, or 23F), Streptococcus agalactiae (types Ia, Ib, II, III, IV, V, VI, VII or VIII), Haemophilus influenzae (tierreich strains a, b, C, d, e or f), Pseudomonas aeruginosa, Staphylococcus aureus, etc. Other analyzed sacharine substances include glucans (e.g., glucan of fungi, such as Candida albicans glucan) and capsular saccharide is mushrooms, for example, capsules of Cryptococcus neoformans. Capsular saccharide from N. meningitidis serogroup a is a homopolymer of (α1→6-linked N-acetyl-D-mannosamine-1-phosphate. Capsular saccharide from N. meningitidis serogroup b is a homopolymer (α2 8)-linked sialic acids. Capsular saccharide from N. meningitidis serogroup is a homopolymer of (α2 9)-linked sialic acids. Saccharide from N. meningitidis serogroup W135 is a polymer composed of disaccharide glycosides links sialic acid-galactose [4)-D-Neup5Ac(7/9OAc)-α-(2 6)-D-Gal-α-(1]. Saccharide from N. meningitidis serogroup Y similar to the saccharide of serogroup W135, except that the repeating disaccharide glycosides link contains glucose instead of galactose [4)-D-Neup5Ac(7/9OAc)-α-(2 6)-D-Glc-α-(1]. Capsular saccharide N. influenzae type b is a polymer of ribose, ribitol and phosphate ['PRP', (pOli-3-β-D-pibot-(1,1)-D-pEBITA-5-phosphate)].

Addition of applicability for the analysis of a full-sized capsular saccharides of the invention can be applied to their oligosaccharide fragments.

Other preferred sharedname antigens are antigens derived from glycoconjugates, for example, from sahariana-protein antigens conjugated vaccines. Of the three conjugated vaccines against N. meningitidis serogroup C, approved for use in humans, Menjugate™ [8] and Meningitec™ - based oligosaccharides, tor is and how NeisVac-C™ used full sized polysaccharide.

Other preferred sharedname antigens are sugars eukaryotes, for example, sugars fungi, plant sugars, sugars person (for example, cancer antigens), etc.

Preferred analyzed substances are sugars, charged (e.g., anions) at a neutral pH value. Sacharine analyte with multiple phosphate and/or multiple carboxylate groups can be analysed in accordance with the methods of the invention. Thus, the invention is particularly applicable for the analysis of polyanionic sharidny connections.

Other preferred analyzed substances are lipopolysaccharides and lipooligosaccharides.

The invention is particularly applicable to the analyzed substances containing various sugars of different lengths, for example, different portions of a single source of saccharide.

The analyzed substance, usually in aqueous solution, and this solution is characterized by a high pH value and high concentration of salts as a result of NRES.

Typical of the analyzed substances are substances that can be detected as amperometric and spectroscopic methods.

In addition to the interest(s) analyzed(s) substance(s)subject to the analysis of substances in order to win other substances. They can resist or not to resist the chromatographic column and, accordingly, may or may not be present in the eluate. Typically, such components are not associated with the column.

Thus, eluate analyzed in accordance with methods according to the invention contain this analyte or are supposedly.

An analyte can be a product that you want to test before release (for example, in the production process or quality control), or can be a product to be checked after the release (for example, to assess the stability, shelf life etc).

Additional stages

Before analysis of the eluate from the column for liquid chromatography method according to the invention may include the application of containing an analyte (or suspected of containing an analyte) of sample on the column. Thus, the invention relates to a method for detecting the presence of analyte in the sample, which includes the following stages: (a) applying the sample to such a column for liquid chromatography that an analyte from the sample is retained by the column; (b) elution of the analyte from the column; and (C) analysis of the eluate as described above.

For the analysis of saccharides mo is et to be desirable to filter, at least some unparsed substances from the sample before application to the column and Dionex™ produces for these purposes predalone and traps, for example, aminoacetyl to remove amino acids, borate trap etc.

After elution and analysis of the invention may include the additional step of determining the characteristics of the detected analyte, for example, DP (typically, the average value of DP), its molecular weight, purity, etc.

After passing through amperometric and spectroscopic detectors the eluate can be analyzed by the mass spectrometer, for example, FAB/MS or ESI/MS.

The use of the invention for selecting the desired sugars

The invention is particularly applicable to conjugation to the stage where you need to be sure to obtain a conjugate selected sacharine chain of appropriate size.

The invention allows to monitor or track the progress of fragmentation of a full-sized polysaccharide prior to conjugation. If it is desirable to obtain oligosaccharides of a specific length (or interval lengths), it is important that the fragmentation of the polysaccharide was not so deep that the depolymerization went over the desired point (for example, in the extreme case, with the receipt of monosaccharides). The invention provides for the monitoring of leakage it is partial depolymerization by measuring the chain length of the saccharide in time. Thus, the invention relates to a method of analysis of saccharide(s) in the composition, which includes the following stages: (a) beginning of depolymerization of the saccharide(s) in the composition; and one or more time points thereafter (b) analysis of saccharide(s)as described above. In the initial experiments are usually analyzed at several time points to determine progress over time, and after the standard conditions defined, usually analyzes certain point in time to confirm. Upon reaching the desired endpoint, the method may include the following stage: (C) stop depolymerization, for example, by washing, separation, cooling, etc. the Method may also include the additional step of conjugating depolimerizovannogo saccharide to a protein carrier after chemical activation if necessary.

The invention also allows selection of the desired oligosaccharide chains after fragmentation. Thus, the invention relates to a method for the selection of saccharides for use in obtaining glycoconjugate, which includes the following stages: (a) preparation of a composition containing a mixture of various polysaccharide fragments; (b) separation of the mixture on podsieci; (C) the analysis of one or more podsosa in accordance with SP the way described above; and (d) use of results stage (s) for selecting one or more podsosa for use in the conjugation. The method can include the fragmentation of the polysaccharide before stage (a) or may begin with a pre-prepared mixture. Fragments may represent fragments of the same polysaccharide, for example, saccharide of the same serogroup. After stage (d) may provide the phase conjugation with protein carrier after chemical activation if necessary.

For saccharide before conjugation is quite common chemical activation with the aim of introducing a functional group capable of reacting with the carrier. Enable conditions saccharide can cause hydrolysis, and, respectively, after activation, it is useful to conduct an analysis of the saccharide. The term "saccharide" should, where appropriate, be understood as covering these activated sugars. Moreover, the invention relates to a method for producing an activated saccharide for use in obtaining glycoconjugate, which includes the following stages: (a) receive saccharide; (b) chemical activation of the saccharide with the aim of introducing a functional group capable of reacting with protein carrier; and (C) analysis of the product of stage (b), described above. The method may include the additional step (a) interaction of the activated saccharide to a protein carrier (which itself can be activated with getting glycoconjugate. The method can include the fragmentation of the polysaccharide before stage (a) or may begin with a pre-prepared mixture.

The invention can also be used after conjugation. After conjugation of the composition can be analyzed using the invention in three ways: first, it can be measured the total content of saccharides in the composition, for example, before mixing different conjugates or before the release of the vaccine in order to comply with regulations or quality control); second, can be measured content of free unconjugated saccharide in the composition, for example, to control an incomplete conjugation, or to monitor the hydrolysis of conjugates by monitoring the increase in the content of free saccharide over time; third, for the same purpose can be measured content of conjugated saccharide in the composition. For the first and third methods require that the saccharide was released from the conjugate prior analysis. For the separate assessment of conjugated and unconjugated saccharides they must be separated. Free (i.e. unconjugated) saccharide in water composition may be separated from the conjugated saccharide in a variety of ways. The conjugation reaction modifies various chemical and physical parameters the s saccharide, and differences can be used for separation. For example, for the separation of free or conjugated saccharides can be used for separation by size, because the conjugated substance is characterized by a higher weight due to the presence of carrier protein. The preferred method of separation by size is ultrafiltration. As a further alternative, if the conjugates adsorbed on Freund, centrifugation allows you to separate the adsorbed conjugate (in the sediment) from the free saccharide (in the supernatant), which is desorbed after hydrolysis.

The invention relates to method of analysis glycoconjugate, which includes the following stages: (a) processing glycoconjugate for the release of sugar from the media; and (b) analysis of released saccharide as described above. The invention relates to a method of analyzing the composition of glycoconjugate, which includes the following stages: (a) the Department of unconjugated saccharide in the composition from the conjugated saccharide; and (b) analysis of unconjugated and/or conjugated saccharide as described above.

The invention also relates to a method of production of vaccines for clinical use, which includes the following stages: (a) production of vaccines, providing the analysis stage, as described above; and e is whether the results of stage (a) indicate the acceptability of vaccines for clinical use, (b) the production of vaccines for clinical use. Stage (a) can be carried out on Packed vaccine, unpackaged vaccine before packing, sugars before conjugation, etc.

The invention also relates to a batch of vaccine, and one vaccine batch analyzed in accordance with the method according to the invention.

Common definitions

The term "comprising" encompasses the term "comprising"and "consisting of", for example, a composition "comprising" X may consist exclusively of X or may include something different, for example, X+y

The phrase "on the merits" does not exclude values "fully" or "completely"e.g. a composition which essentially does not contain" Y can absolutely not contain a y If necessary, the phrase "on the merits" can be omitted from the definition according to the invention.

The term "about" in relation to a numerical value x means, for example, x±10%.

The methods according to the invention can be used in analytical and/or preparative purposes. The reference to "analysis", "analysis", etc. should not be understood as excluding preparative methods.

The degree of polymerization (DP) of the saccharide is defined as the number of repeating units in the saccharide. Thus, for homopolymer DP represents the same as the number of monosaccharide SV is Nievo. However, for heteropolymer DP equal to the number of monosaccharide units in the chain divided by the number of monosaccharide units in the minimum repeating part of, for example, DP (Glc-Gal)10equal to 10, not 20, a DP (Glc-Gal-Neu)10equal to 10, not 30.

Brief description of drawings

Figure 1 shows the output signal of NRAES analyzed amperometric method (top) and UV200nmspectroscopy (lower profile). Units in the upper profile are NCP; in the lower profile used relative units.

Figure 2 shows a serial arrangement of amperometric and spectroscopic detections, while figure 3 shows a parallel arrangement.

Figure 4 shows three pulse shape PAD. On x axis is time in seconds. On the y-axis pending potential in volts relative to the Ag/AgCl reference electrode. All three forms of impulse contain the delay period (the first period), the detection period (second period), and then the cleaning period.

Figure 5 illustrates the analysis of the eluate from the column PA1, and the detection was carried out according to the method of PAD (bottom curve) or by the method of UV-spectroscopy (upper curve).

Figure 6 illustrates the analysis of the eluate sample saccharide of serogroup A, stored in various conditions, PA1 column. The elution program is identical to that of Fig.

N is 7 and 8 illustrate the analysis of the eluate saccharides serogroup W135 in column R using different eluents. Figure 9 illustrates the elution of the same analyte on column PA1, and figure 10 illustrates the elution of the same analyte on column RA.

Figure 11 illustrates the analysis of the eluate Hib oligosaccharide on column RA.

On Fig illustrated by the analysis of the eluate pool hydrolyzed Hib saccharide on the AS11 column.

On Fig illustrated by the analysis of the eluate polysaccharide serogroup on the AS11 column. The same analysis after hydrolysis of the saccharide is illustrated in Fig.

On Fig shows the results of mass spectrometry MALDI-TOF standard saccharide of serogroup C, and Fig illustrated by the analysis of the eluate of the same standard on PA1 column. Also analyzed the elution of standard AS11 column, as shown in Fig.

On Fig illustrated analysis of the elution hydrolyzed polysaccharide serogroup C in AS11 column.

On Fig shows the elution profile of the water measured at 214 nm using acetate/hydroxide and nitrate/hydroxide eluents for column PA1 with micromembrane suppression. The elution program is identical to that of Fig.6.

Ways of carrying out the invention

Detection in the ultraviolet range in the eluate NRAES

High pH value used in the analysis of NRAES capsular saccharides, means that the eluate contains hydroxide ions, and these ions feature is resultsa strong absorption in the ultraviolet range. As shown in Fig, using a standard acetate/hydroxide eluent with plain water on a column CarboPac PA1 allows to obtain the elution profile in the UV range, which is not detected(are) no(s) of interest(s) analyzed(s) substance(s). Thus, for introduction into the analysis program eluents NRAES detection in the UV range requires a different strategy.

United PAD and UV-spectroscopic analysis of the eluate NRAES

The capsular saccharide of serogroup a meningococcus were subjected to depolymerization with obtaining the pool of charged due to the phosphate of mannosamine-1-phosphate monomers) saccharides variable length (with high value of DP). The mixture was separated using column PA1 for NRAES-separation, and through the speaker lonPac AS11 from Dionex with manufacturer's recommended by precolonial using as eluent a gradient of sodium hydroxide (rate of elution of 1.0 ml/min). The eluate from the column to NRAES analyzed sequentially using electrochemical (PAD, the gold electrode) and UV detectors. The output signal resulting from the integrated amperometric detection, shown in the upper profile in figure 1, and the output signal resulting from detection in the UV-visible range (200 nm), shown at the bottom of the profile.

Column AS11 is characterized by low capacity (11 mcak is.). The same analyzed substance was applied on the column, high capacity CarboPac PA1 from Dionex (100 mkacf.), as displacer in eluent used sodium chloride. As shown in figure 5, the eluate from the column PA1 is not amenable to analysis by the method of PAD (bottom curve), but allows the detection method UV-spectroscopy (upper curve). Detection method is UV-spectroscopy, thus, allows the use of low column capacity for AES in the analysis of the capsular saccharides.

Comparison of nitrate and acetate eluents

A pool of oligosaccharide fragments of the capsular saccharide of Neisseria meningitides serogroup W135 were analyzed according to the standard Protocol HPAEC-PAD using a column CarboPac RA with gradient elution with sodium acetate 100 mm sodium hydroxide. The results are shown in Fig.7.

For comparison conducted the same analysis, but using eluent sodium nitrate instead of sodium acetate. As shown in Fig, the dynamic range of the output signal was much larger and the resolution of fragments with a high value of DP was better. Succumbed to the detection of fragments with DP 50.

Replacement column on the CarboPac PA1 increased dynamic range even more (Fig.9), and the detection yielded fragments with DP 40. When using 5 μm column RA could be observed fragments with DP 80 (figure 10).

Thus, neither the rata applicable for elution of the capsular saccharides of different lengths with anion-exchange columns.

Nitrate eluent to determine the DP in time

Charigny component of conjugate vaccines may be subject to gradual hydrolysis, and this leads to decrease over time, the values of DP and fewer saccharide associated with protein carrier. Monitoring depolymerization saccharides serogroup a meningococcus under different conditions of pH was performed according to the method of HPAEC-PAD using column CarboPac PA1. Eluent was a mixture of sodium acetate and sodium nitrate in 100 mm sodium hydroxide at a speed of elution of 1.0 ml/min

Used three different sets of storage conditions: (1) pH ~9 at 37°C for 4 days; (2) pH ~4 at 37°C for 4 days; and (3) pH ~7 at -20°C, i.e. under recommended storage conditions. As shown in Fig.6, adding in eluent nitrate allows for the eluate PAD and to detect fragments with low value DP after depolymerization. Short fragments were observed in the matter, which was kept at 37°C, in the areas in which the substance was kept at low temperatures, the peaks were observed (upper line).

Analysis of saccharides N. influenzae using low column capacity

Pool Hib oligosaccharide (DP 2-6) were analyzed according to the standard Protocol HPAEC-PAD on a column CarboPac PA100. Eluent was a gradient of sodium acetate from 30 mm to 100 mm with 100 mm NaOH in those who tell 24 minutes at a speed of elution of 0.8 ml/min The results are shown figure 11.

Only short-chain oligomers were loirevalley with a CarboPac PA-100, and elution was required for the high percentage of sodium acetate as a displacer. And even then, oligomers with DP-5 and DP 6 was badly divided and with low sensitivity. Thus, CarboPac PA-100 is not suitable for determining the profile of elution pool Hib oligosaccharide with a DP>4. Therefore, investigated the possibility of using other chromatographic column.

Column lonPac AS11 used to analyze pool hydrolyzed Hib saccharide, which is characterized by the average value of DP, amounting to 12.44. This column is characterized by a high selectivity with respect to the hydroxide, thereby allowing eluted highly charged anions at lower concentrations of hydroxide than used previously for strongly held polianionov (for example, fragments Hib). The elution was performed from 3 mm to 150 mm NaOH for 15 minutes. The results are shown in Fig, again suggesting that for the analysis of bacterial capsular saccharides can be used speakers low capacity. Elution took place very quickly and require only the gradient hydroxide. All components of the pool Hib can be separated in the chromatographic process session lasting less than 10 minutes.

Analysis of saccharide of serogroup a meningococcus with POM is using low column capacity

Column lonPac AS11 used for analysis of the distribution of molecular masses of the capsular polysaccharide of Neisseria meningitides serogroup A. In Fig shows the chromatogram obtained for sample polysaccharide with DP ~200 by using a hydroxide gradient from 10 to 150 mm in 19 minutes. It was unexpectedly found that by using this column, you can separate this high molecular weight polyanion, and that the column efficiency is so high that an analyte was loirevalley just 17 minutes in the form of a relatively narrow peak. This result is far superior to the results obtained by methods such as gel chromatography.

The same column was used for determining the profile of elution of oligosaccharides obtained by acid hydrolysis of the polysaccharide. As shown in Fig, oligosaccharides can be separated by ascending values of DP with the presence of correlation between the value of DP and retention time with HPAEC-PAD.

Analysis of saccharide of Neisseria meningitides serogroup using low column capacity

The polysaccharide of Neisseria meningitides serogroup is a partially O-acetylated a homopolymer of α-2,9-linked N-acetylneuraminic acids. Thus, it is polycarboxylate anion. Determination of the chromatographic profile of the saccharide of serogroup With complicated by the presence of O-acetyl groups, which convey each oligomer determines the presence of a number of peaks depending on the distribution in the structure of acetyl groups.

The purified standard with DP 5 was analyzed by the method of mass spectrometry MALDI-TOF and found that it contains many different oligomers (Fig). Analysis of the same standard method HPAEC-PAD on a column CarboPac PA1 illustrated in Fig. For elution from the column was required stringent conditions (500 mm sodium acetate, 100 mm sodium hydroxide), and the differences between Fig and 16 indicate that such processing acetate alters the natural distribution of acetyl groups in the saccharide. Thus, the use of powerful speakers for AES high capacity for analysis of acetylated saccharides cannot be considered as optimal.

So, instead, used a column lonPac AS11, coupled with detection supressirovano conductivity. As shown in Fig, using a gradient of sodium hydroxide from 10 mm to 60 mm (without acetate) allowed eluted standard with DP 5 on the profile, showing the different oligosaccharides and distribution of O-acetyl groups. Interpretation of the chromatographic profile is much easier than the profile obtained by the method of MALDI-TOF, which is complicated by the presence of various amounts of sodium counterions, characterized by a molecular weight equal to half the weight of acetyl groups.

The elution profile obtained for the standard DP-5, was also repeated for different lengths of product hydroly is and capsular polysaccharide (Fig).

It should be understood that the invention is described for illustrative purposes only, and may be made of modifications falling within the scope and essence of the invention.

Links

(the contents of which are incorporated into this description by reference)

1. Vaccines (eds. Plotkin et al.) 4th edition, ISBN: 0721696880.

2. Hardy et al. (1988) Anal Biochem 170:54-62.

3. Wang et al. (1990) Anal Biochem 190:182-187.

4. Mellor et al. (2000) Anal Biochem 284:136-142.

5. Product Manual for lonPac™ ASH column. Dionex™ document 034791, revision 08.

6. Gert-Jan et al. (2002) JBiol Chem 277:25929-25936.

7. LaCourse & Johnson (1993) Anal Chem 65:50-55.

8. Jones (2001) Curr Opin Investig Drugs 2:47-49.

1. The method of detecting the presence of the analyzed saccharide in the sample, which includes the following stages: (a) the sample application in this column for anion-exchange chromatography that an analyte from the sample is retained by the column; (b) elution of the analyte from the column using eluent containing an ion selected from the group consisting of nitrate and chloride; and (C) analysis of the eluate by the method of amperometry and spectroscopy method.

2. The method according to claim 1, in which anion-exchange column chromatography is a column for high-performance anion-exchange chromatography (NRAES).

3. The method according to claim 1, in which the applied pulsed amperometric detection (PAD).

4. The method according to claim 1, in which the used absorption spectroscopy in ultra is Fioletovo (UV) range and/or absorption spectroscopy in the visible light range.

5. The method according to any of the preceding paragraphs, in which the eluate contains a bacterial capsular saccharide or sharedstorage fragment of a bacterial capsular saccharide.

6. The method according to claim 5, in which the bacterial capsular saccharide is a saccharide of Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, Pseudomonas aeruginosa or Staphylococcus aureus.

7. The method according to any one of claims 1 to 4, in which the chromatography is carried out with application of the column, characterized by a capacity less than 50 mkacf.

8. The method according to claim 5, in which the chromatography is carried out with application of the column, characterized by a capacity less than 50 mkacf.

9. The method according to any one of claims 1 to 4, and in accordance with the method used selective in respect of the hydroxide anion-exchange chromatographic column.

10. The method according to claim 5, and in accordance with the method used selective in respect of the hydroxide anion-exchange chromatographic column.

11. Installation for analysis of the sample, and the unit comprises (i) anion-exchange column chromatography, (ii) a buffer for elution containing an ion selected from the group consisting of nitrate and chloride; (iii) amperometric detector and (iv) spectroscopic detector, and two detector positioned to receive the eluate from the column.

12. Installation according to claim 11, in which anion-exchange column is cromatografia is a column for NRAES.

13. Installation according to claim 11, in which the amperometric detector is a PAD.

14. Installation according to any one of § § 11 to 13, in which the spectroscopic detector is a UV detector.



 

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