Methods and kits for improving the fluorescent signal of dmb-labeled sialic acids

Abstract

This invention provides a method and kit for improving the signal from sialic acid labeled with the dye 1,2-diamino-4,5-methylenedioxybenzene (“DMB”). The method comprises labeling the sialic acid with DMB in an aqueous solution containing a glycine amino acid whose pH is adjusted to 1.5–3.2 by an acid such as phosphoric acid or hydrochloric acid, and a reducing agent.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 861,924, filed June 14, 2019, the contents of which are incorporated herein by reference for all purposes.

[0003] Federal Funding Statement

[0004] not applicable. Background Technology

[0005] This invention relates to the field of improving the signal from sialic acid (such as that released from glycoconjugates or reference standards) labeled with the dye 1,2-diamino-4,5-methyleneoxybenzene (“DMB”).

[0006] Multiple commercial and regulatory requirements necessitate the determination of the nature and quantity of glycans present on glycoproteins or glycopeptides, particularly for glycoproteins used as therapeutic agents. Since glycans attached to glycoproteins can influence characteristics crucial to their function, including pharmacokinetics, stability, biological activity, and immunogenicity, it is important to identify which glycans are present. The Food and Drug Administration (FDA) requires characterization of glycans attached to biological products (such as therapeutic glycoproteins and vaccines) to demonstrate consistency in composition and manufacturing, leading to the need for extensive product characterization. Carbohydrate profiling is also important for quality control in the production of both therapeutic and non-therapeutic recombinant proteins, where changes in carbohydrate profiles can indicate stress in the system, suggesting conditions that may require the disposal of contents from commercial-scale fermenters. Therefore, biochemists, clinical chemists, pharmaceutical manufacturers, and protein producers are of great interest in determining the distribution profile of glycans in biological samples, such as therapeutic glycoproteins.

[0007] Sialic acid is a family of 9-carbon carboxylated 2-keto sugars found on many glycans associated with glycoproteins. As Reiding et al., Analyt. Chem., 2014, 86:5784-93, "N-glycans on mammalian proteins often exhibit terminal sialic acids such as N-acetylneuraminic acid, which...show importance in cellular communication and determine protein half-life. Sialic acids are most frequently attached to a terminal galactose via α2,6 or α2,3 glycosidic linkages and show different functionality as a consequence." (Reiding et al., p. 5784). More than 25 sialic acid derivatives have been identified in nature and have shown to play roles in a variety of biological activities.

[0008] The presence and type of sialic acid attached to glycoproteins or other glycoconjugates can be determined by enzymatic digestion of sialic acid with sialatesase or by partial acid hydrolysis of glycoproteins or other glycoconjugates at 80°C in an acidic solution such as 2M acetic acid or 0.05N HCl for 1 to 3 hours. Once sialic acid is released from any glycoconjugate to which it is attached, it can be labeled with DMB and then detected by various analytical techniques. Typically, sialic acid is labeled with DMB in a solution containing approximately 1.5M acetic acid and at least one reducing agent (typically β-mercaptoethanol), the resulting DMB-labeled sialic acid is separated by liquid chromatography, and the separated labeled sialic acid is provided to a fluorescence detector, followed by quantification of the signal from the labeled sialic acid.

[0009] Unfortunately, acetic acid, perhaps the most commonly used acid in the acid hydrolysis of sialic acid and subsequent DMB labeling of sialic acid, has a strong odor and is highly volatile. If not kept tightly sealed, the volume of acid decreases, making it impossible to calculate the concentration. Furthermore, β-mercaptoethanol, a reducing agent commonly used in DMB labeling protocols, not only has a strong, unpleasant odor but is also a toxin that can be fatal if inhaled, swallowed, or absorbed through the skin of the user. Moreover, while DMB labeling is considered a sensitive and selective technique for detecting the presence of sialic acid in samples, some sialic acids are present only in small amounts on certain glycoconjugates, and some glycoconjugates are so expensive or difficult to produce that reducing the amount of glycoconjugate required to measure their sialylation would be useful. Increasing the signal from DMB-labeled sialic acid would help improve the ability to detect sialic acid in samples, even in small amounts.

[0010] There remains a need in the art for methods and kits that provide additional means of increasing the signal from DMB-labeled sialic acid. Furthermore, there remains a need in the art for methods using acids that are less volatile and less odorous than acetic acid, and for methods using reducing agents that are less toxic and less odorous than β-mercaptoethanol but allow for the increase of the signal obtained from DMB-labeled sialic acid. Surprisingly, the present invention satisfies these and other needs. Summary of the Invention

[0011] This invention provides methods and kits for improving signals from free sialic acid (such as those released from glycoconjugates or reference standards) labeled with the dye 1,2-diamino-4,5-methyleneoxybenzene (“DMB”). In a first set of embodiments, the invention provides an in vitro method for labeling free sialic acid with DMB and optionally for analyzing said DMB-labeled sialic acid, the method comprising the step (a) incubating said free sialic acid with an effective amount of DMB in an aqueous solution at a time and temperature sufficient to allow labeling, thereby labeling said free sialic acid with DMB, said aqueous solution comprising (i) an aqueous solution of glycine and an acid, said glycine being present at a molar concentration of 0.25 M to 3 M and said solution having a pH of 1.5-3.2, and (ii) a reducing agent selected from thioglycerol and β-mercaptoethanol (“BME”). In some embodiments, said solution further comprises sodium dithionite. In some embodiments, said acid is phosphoric acid. In some embodiments, said acid is hydrochloric acid. In some embodiments, the molar concentration of glycine is from 0.25M to 2.5M ± 0.25M. In some embodiments, the molar concentration of glycine is from 0.25M to 2.0M ± 0.25M. In some embodiments, the molar concentration of glycine is from 0.40M to 1.75M ± 0.25M. In some embodiments, the molar concentration of glycine is from 0.4M to 1.5M ± 0.25M. In some embodiments, the molar concentration of glycine is from 0.4M to 1.25M ± 0.25M. In some embodiments, the molar concentration of glycine is from 0.5M to 1M ± 0.25M. In some embodiments, the molar concentration of glycine is from 0.5M to 0.9M. In some embodiments, the molar concentration of glycine is from 0.6M to 0.8M ± 0.1M. In some embodiments, the molar concentration of glycine is 0.75M ± 0.1M. In some embodiments, the molar concentration of glycine is 0.75M ± 0.05M. In some embodiments, the solution has a pH of 2 to 3.2. In some embodiments, the solution has a pH of 2.5 ± 0.1 to 3. In some embodiments, the solution has a pH of 2.7 ± 0.25 to 2.9 ± 0.25. In some embodiments, the solution has a pH of 2.8 ± 0.25. In some embodiments, the acid is phosphoric acid. In some embodiments, the acid is hydrochloric acid. In some embodiments, the time sufficient to label the free sialic acid is 1-6 hours. In some embodiments, the time sufficient to label the free sialic acid is 2-5 hours. In some embodiments, the time sufficient to label the free sialic acid is 3 hours ± 30 minutes. In some embodiments, the time sufficient to label the free sialic acid is 2.5 hours ± 30 minutes.In some embodiments, the temperature sufficient to label the free sialic acid is 35°C-65°C. In some embodiments, the temperature sufficient to label the free sialic acid is 50°C ± 5°C. In some embodiments, the method further includes step (b) separating the free DMB-labeled sialic acid. In some embodiments, the free DMB-labeled sialic acid is separated by subjecting it to liquid chromatography. In some embodiments, the method further includes step (c) analyzing the separated free DMB-labeled sialic acid by subjecting it to an analytical method. In some embodiments, the analytical method is fluorescence detection. In some embodiments, the analytical method is UV detection.

[0012] In a second set of embodiments, the present invention provides a method for releasing, labeling, and optionally analyzing sialic acid present on a glycoconjugate, the method comprising: (a) contacting a desired volume of the glycoconjugate with a first aqueous solution containing glycine, the glycine being acid-adjusted to a pH of 1.5-3.2; (b) incubating the glycoconjugate and the first aqueous solution together for a time and temperature sufficient to release the sialic acid from the glycoconjugate, thereby releasing the sialic acid from the glycoconjugate; and (c) cooling the released sialic acid in the first aqueous solution to 50°C ± (d) At a temperature of 10°C, the released sialic acid is contacted with a solution to form a sample / labeled mixture, the solution comprising: (i) an effective amount of 1,2-diamino-4,5-methylenedioxybenzene (“DMB”), (ii) a second aqueous solution containing glycine, the solution being adjusted to a pH of 1.5-3 with acid, and (iii) an effective amount of one or more reducing agents, and (e) the sample / labeled mixture is incubated at a time and temperature sufficient to label the released sialic acid in the mixture, thereby causing the released sialic acid to be labeled with DMB. In some embodiments, the glycoconjugate is a glycoprotein. In some embodiments, the glycoconjugate is a glycolipid or oligosaccharide. In some embodiments, the first aqueous solution is glycine from 0.25 to 2.0 M and the pH is adjusted to a pH of 1.5-3.2 with acid. In some embodiments, the first aqueous solution is glycine from 0.25 to 2.0 M and the pH is adjusted to a pH of 2-3 with acid. In some embodiments, the first aqueous solution is composed of 0.25 to 2.0 M glycine, and the pH is adjusted to 2.5-3 with acid. In some embodiments, the first aqueous solution is composed of 0.25 to 2.0 M glycine, and the pH is adjusted to 2.8 ± 0.1 with acid. In some embodiments, the second aqueous solution is composed of 0.25 to 2.0 M glycine, and the pH is adjusted to 1.5-3.2 with acid. In some embodiments, the second aqueous solution is composed of 0.25 to 2.0 M glycine, and the pH is adjusted to 2-3 with acid. In some embodiments, the second aqueous solution is composed of 0.25 to 2.0 M glycine, and the pH is adjusted to 2.5-3 with acid. In some embodiments, the second aqueous solution is composed of 0.25 to 2.0 M glycine, and the pH is adjusted to 2.8 ± 0.1 with acid. In some embodiments, the acid in the first aqueous solution is phosphoric acid. In some embodiments, the acid in the second aqueous solution is phosphoric acid. In some embodiments, the acid used to adjust the pH of the solutions in both the first and second aqueous solutions is phosphoric acid. In some embodiments, the acid in the first aqueous solution is hydrochloric acid. In some embodiments, the acid in the second aqueous solution used to adjust the pH of the solution is hydrochloric acid. In some embodiments, the acid in both the first and second aqueous solutions is hydrochloric acid.In some embodiments, the one or more reducing agents include β-mercaptoethanol (“BME”). In some embodiments, the one or more reducing agents include thioglycerol. In some embodiments, the one or more reducing agents include BME and sodium dithionite. In some embodiments, the one or more reducing agents include thioglycerol and sodium dithionite. In some embodiments, the time for releasing sialic acid in step (b) is 0.5-5 hours. In some embodiments, the time for releasing sialic acid in step (b) is 1.0-4 hours. In some embodiments, the time for releasing sialic acid in step (b) is 2 hours ± 30 minutes. In some embodiments, the temperature for releasing sialic acid in step (b) is 70°C-100°C. In some embodiments, the temperature for releasing sialic acid in step (b) is 70°C-90°C. In some embodiments, the temperature for releasing sialic acid in step (b) is 80°C ± 5°C. In some embodiments, the time for labeling the released sialic acid in step (e) is 1-6 hours. In some embodiments, the time sufficient to label the released sialic acid in step (e) is 2-5 hours. In some embodiments, the time sufficient to label the released sialic acid in step (e) is 3 hours ± 30 minutes. In some embodiments, the time sufficient to label the released sialic acid in step (e) is 2 hours ± 30 minutes. In some embodiments, the temperature sufficient to label the released sialic acid in step (e) is 35°C-65°C. In some embodiments, the temperature sufficient to label the released sialic acid in step (e) is 50°C ± 5°C. In some embodiments, the method further includes step (f), separating the released DMB-labeled sialic acid. In some of these embodiments, the separation of the released DMB-labeled sialic acid is performed by subjecting the released DMB-labeled sialic acid to liquid chromatography. In some embodiments where the released DMB-labeled sialic acid has been separated, the method further includes step (g), analyzing the separated DMB-labeled sialic acid by subjecting the separated DMB-labeled sialic acid to an analytical method. In some of these implementations, the analytical method is performed by detecting fluorescence.

[0013] In yet another set of embodiments, the present invention provides a kit for labeling free sialic acid with 1,2-diamino-4,5-methylenedioxybenzene (“DMB”). The kit comprises DMB, glycine, an acid, and a reducing agent. Conveniently, DMB, glycine, the acid, and the reducing agent can be arranged in one or more containers. In some embodiments, the acid is phosphoric acid. In some embodiments, the acid is hydrochloric acid. In some embodiments, the glycine is premixed with the acid in solution. In some embodiments, the solution containing the glycine mixed with the acid has a pH between 2 and 3. In some embodiments, the kit further comprises sodium dithionite. In some embodiments, the reducing agent is β-mercaptoethanol (“BME”). In some embodiments, the reducing agent is thioglycerol. In some embodiments, the acid is hydrochloric acid and the reducing agent is thioglycerol. In some embodiments, the acid is phosphoric acid and the reducing agent is thioglycerol. In some embodiments, the kit further comprises one or more sialic acid standards. In some embodiments, the kit further comprises one or more sialidases. In some embodiments, the kit comprises (a) glycine in solution, the solution adjusted to pH 2.8 with phosphoric acid, (b) thioglycerol, (c) DMB, and (d) sodium dithionite. In some of these embodiments, the thioglycerol is provided at 0.72 mol ± 0.1 mol, the DMB at 5 mmol ± 1 mmol, and the sodium dithionite at 56 mmol ± 10 mmol. Attached Figure Description

[0014] Figure 1 . Figure 1 The bar graph shows the results of a study comparing the fluorescence signals detected from an exemplary sialic acid standard after equal volumes of sialic acid were labeled with the dye 1,2-diamino-4,5-methylenedioxybenzene (“DMB”) in a labeling solution containing (a) an acidic buffer solution provided by one of the following: 1.5 M acetic acid, 1.5 M phosphoric acid titrated to pH 2.8 with sodium hydroxide (“sodium phosphate”), 1.5 M glycine solution adjusted to pH 2.8 with phosphoric acid (“glycine phosphate”), or 1.5 M glycine solution adjusted to pH 2.8 with hydrochloric acid (“glycine HCl”), (b) a reducing agent selected from β-mercaptoethanol (“mercaptoethanol”) or thioglycerol, and (c) sodium dithionite. To allow for comparison, the signal from sialic acid labeled in the presence of the standard reagents acetic acid, β-mercaptoethanol, and sodium dithionite is stated as 100%, as shown in the first bar from the left, while the signal from the same sialic acid labeled in the presence of other reagents listed below the bar is stated as a percentage of the signal from sialic acid labeled in the presence of the standard reagents.

[0015] Figure 2 . Figure 2 This is a chromatogram (read from a fluorescence detector) of sialic acid (Neu5Ac) released from the chimeric monoclonal antibody rituximab and labeled with DMB in a solution containing acetic acid, β-mercaptoethanol (“mercaptoethanol”), and sodium dithionite. The shaded area is the peak of sialic acid Neu5Ac. The small peak at retention time 1.5 min is the peak of sialic acid Neu5Gc. The peaks at retention times of 0.5–0.6 min are artifacts.

[0016] Figure 3 . Figure 3 This is a chromatogram of sialic acid released from the chimeric monoclonal antibody rituximab and labeled with DMB in solution containing glycine (“glycine phosphate”) adjusted to pH 2.8 with phosphate, thioglycerol, and sodium dithionite. The shaded area is the peak of sialic acid Neu5Ac. The small peak at retention time 1.5 min is the peak of sialic acid Neu5Gc. The peaks at retention times of 0.5–0.6 min are artifacts. Detailed Implementation

[0017] introduction

[0018] As described in the background section, the analysis of the types and quantities of glycans attached to glycoconjugates such as glycoproteins and glycolipids is important for various regulatory and quality control purposes. In particular, the analysis of the types of glycans attached to therapeutic glycoproteins (such as monoclonal antibodies) and the amount of each type of glycan has become an important quality control measurement in the production of such glycoproteins and in confirming that they will have the desired pharmacological activity.

[0019] Sialic acid is a family of N- and O-derived forms of neuraminic acid (a nonacarbonic acid) found at the ends of glycan chains attached to glycoproteins and glycosides. Glycoconjugates such as glycoproteins can have different half-lives and biological activities depending on the presence and, if present, of sialic acid. Therefore, determining the amount and type of sialic acid present on the target glycoconjugate is important for determining the biological properties of the glycoconjugate. For glycoproteins intended for use as therapeutic agents, such as fusion proteins, confirming that the sialylation of the glycoprotein remains unchanged throughout the manufacturing process is important for maintaining consistency of the expected biological effects and for regulatory approval.

[0020] Sialic acid is typically released from glycoconjugates via partial acid hydrolysis or enzymatic digestion of the glycoconjugates by any of a variety of sialic acidases. Sialic acid not attached to the glycoconjugate, such as sialic acid released from the glycoconjugate by any of these methods or sialic acid provided as a reference standard, is sometimes referred to herein as “free sialic acid.” Free sialic acid is typically labeled with the dye 1,2-diamino-4,5-methylenedioxybenzene (“DMB”) in acidic aqueous solution in the presence of one or more reducing agents (the labeling of free sialic acid with DMB will sometimes be referred to herein as the “labeling reaction”). A typical DMB labeling protocol requires incubating sialic acid with DMB in acidic aqueous solution at 50°C for two to three hours. Following the labeling reaction, the DMB-labeled sialic acid is typically separated by liquid chromatography such as high-performance liquid chromatography (“HPLC”), and then analyzed by providing the separated DMB-labeled sialic acid to a fluorescence detector or a UV detector. The release of sialic acid from glycoproteins via partial acid hydrolysis, the labeling of sialic acid with DMB, and the detection of the labeled sialic acid by fluorescence detection have been used since at least the early 1990s (see, for example, Lagana et al., Anal. Biochem, 1993, 215(2):266-272; Hayakawa et al., J Chromatography B: Biomed Sciand Applns, 1993, 620(1):25-31; Kawano et al., J Biol Chem, 1995, 270(27):16458-16463). It is assumed that practitioners are familiar with the current techniques and conditions used for the release of sialic acid from glycoconjugates, the DMB labeling of free sialic acid (whether released from glycoconjugates or reference standards), and the subsequent isolation and detection of DMB-labeled sialic acid. It should also be noted that, conventionally in the art, sialic acid labeled with DMB is referred to as "DMB-labeled sialic acid," regardless of whether it falls within the chemical definition of sialic acid after labeling. This usage will be followed in this document.

[0021] Although DMB labeling has been used for the detection of sialic acid for decades, there is a desire to further improve the sensitivity of DMB-labeled sialic acid detection by increasing the signal from labeled sialic acid. Furthermore, current methods have several drawbacks. In particular, the acid typically used to maintain the acidic conditions required for the labeling reaction is acetic acid, which has a strong odor and is volatile. Volatility can lead to acid loss and consequent changes in molar concentration and reagent volume if the container containing the acid is not kept sealed before and during the labeling reaction. Moreover, the most commonly used reducing agent in the labeling procedure is β-mercaptoethanol (also referred to herein as “BME”). Unfortunately, BME is not only a toxin but also has a strong and unpleasant odor. Alternative reducing agents with less unpleasant odor and potentially lower toxicity are desired.

[0022] Surprisingly, it has now been found that not only can acetic acid, the acid used for decades in the process of labeling free sialic acid with DMB, be replaced, but replacing acetic acid with glycine, an amphoteric amino acid adjusted to the desired pH by a strong acid, results in a significant increase in the signal of exemplary free sialic acid labeled with DMB in the presence of acid-buffered glycine compared to the same free sialic acid when labeled with DMB in the presence of standard acetic acid in the art. A glycine solution adjusted to the desired pH by an acid is sometimes referred to herein as “acid-buffered glycine,” and the resulting glycine solution in an acidic solution is sometimes referred to herein as “glycine buffer” or “acidic glycine buffer.”

[0023] The increased signal is expected to significantly increase the sensitivity of the assay and improve the ability to detect free sialic acid in a sample, whether the free sialic acid is a reference standard, obtained by enzymatic release from a glycoconjugate via sialidase, or obtained from a glycoconjugate through partial acid hydrolysis. The increased signal is also expected to significantly improve the ability to detect sialic acid present only in small amounts on a target glycoconjugate and to determine the presence of sialic acid on a target glycoconjugate (wherein only a small amount of the glycoconjugate is available). In a preferred embodiment, the glycoconjugate is a glycoprotein.

[0024] Even more surprisingly, it has been found that using acid-buffered glycine significantly reduces artifacts observed when subsequently analyzing DMB-labeled free sialic acid by fluorescence detection. This reduction in artifacts makes it much easier to detect the presence of sialic acid in samples containing only small amounts. The combination of increased signal intensity and reduced artifacts results in a new combination of reagents, procedures, and kits using them that are surprisingly more sensitive than labeling reactions currently used in the art in their ability to acquire and analyze signals from DMB-labeled sialic acid.

[0025] Furthermore, it has been found that the reducing agent BME, which has been used for many years in the DMB labeling scheme of sialic acid, can be replaced by thioglycerol, which has a lower odor and less toxicity than BME. Studies based on this invention show that thioglycerol results in DMB labeling that is at least comparable to that of sialic acid in the presence of BME, and can even result in a stronger signal compared to DMB labeling of the same sialic acid in the presence of BME.

[0026] Using acid-buffered glycine increases the DMB-labeled signal from sialic acid.

[0027] In one aspect of the invention, it has been found that (1) replacing acetic acid, traditionally used to provide mild acidic conditions for DMB labeling reactions, with acid-buffered glycine results in a surprisingly better signal from labeled sialic acid, and (2) the reducing agent traditionally used for DMB labeling can be replaced with a reducing agent that is less odorous and less toxic while still yielding an equivalent signal. (For clarity, note that all the following studies include the second reducing agent sodium dithionite, also known as sodium thiosulfate. Previous studies have shown that DMB labeling is better when both BME and sodium dithionite are used than when either is used alone.)

[0028] In studies based on this invention, the fluorescence signal of an exemplary sialic acid Neu5Ac labeled with DMB in an aqueous solution where acidic conditions are provided by acetic acid and the reducing agent is BME is compared with the signal of the same sialic acid when labeled in an aqueous solution where acidic conditions are provided by phosphoric acid adjusted to pH 2.8 by adding sodium hydroxide or by glycine adjusted to pH 2.8 by phosphoric acid or hydrochloric acid. After DMB labeling, the solution is subjected to HPLC separation and then fluorescence detection to quantify the signal from the labeled Neu5Ac.

[0029] Figure 1 Results obtained using the reagent label Neu5Ac discussed above are presented. For ease of comparison, peak areas obtained using the conventional reagents acetic acid and BME are normalized to 100%, and signals from other reagent combinations are shown as percentages relative to the stated signal. Those skilled in the art should recognize that peak area is the integral of the area under the curve and is a quantification of the signal.

[0030] refer to Figure 1The signal intensity produced by labeling sialic acid under acidic conditions provided by phosphoric acid (shown as "sodium phosphate" in the figure) adjusted to pH 2.8 with sodium hydroxide was only 74% of the signal intensity obtained when labeling sialic acid under acidic conditions provided by acetic acid. However, when sialic acid was labeled under acidic conditions provided by glycine ("glycine phosphate") adjusted to pH 2.8 with phosphoric acid, the signal was 143% of the signal obtained using standard acid acetic acid, as shown in the third bar from the left. (As discussed in the examples below,) Figure 1 The solution used in the study shown was prepared by starting with an aqueous solution of 3M glycine and adjusting the pH of the solution with acid. However, the same solution can be prepared by taking the amount of the selected acid added to the glycine solution in the previous sentence and adding it to the same amount of 3M glycine solution. The beneficial effects of using glycine adjusted to the desired pH by acid were confirmed by performing the same measurements, except for a 3M glycine solution adjusted to pH 2.8 with hydrochloric acid (this solution is sometimes referred to herein as "glycine hydrochloride"). Figure 1 As shown in the fifth bar from the left, the signal generated by DMB labeling of sialic acid in the presence of glycine hydrochloride and BME is 123% of the signal of the same sample labeled in the presence of standard acid acetic acid and the same reducing agent.

[0031] Furthermore, the study tested whether thioglycerol (3-mercaptopropane-1,2-diol, sometimes referred to herein as "TG") (a reducing agent with a weaker odor intensity and lower toxicity than BME) could replace BME as the preferred reducing agent for DMB-labeled free sialic acid. DMB labeling of exemplary sialic acid under acidic conditions provided by glycine adjusted to pH 2.8 with phosphoric acid, but using thioglycerol as the reducing agent, increased the signal to 146% of the signal of the same sialic acid when labeled under acidic conditions provided by acetic acid and in BME, which is even better than the results obtained when labeling the same sialic acid in the presence of glycine adjusted to pH 2.8 with phosphoric acid and in the presence of BME. Furthermore, DMB labeling of exemplary free sialic acid in the presence of glycine adjusted to pH 2.8 with hydrochloric acid and in the presence of thioglycerol increased the signal to 123% of the signal of the same sialic acid when labeled in the presence of acetic acid and BME. The results showed that BME, which is used as a reducing agent in DMB labeling, can be replaced by thioglycerol, which is a reducing agent with less unpleasant odor and less toxicity than BME, while still achieving equivalence and, in some cases, better labeling signals.

[0032] Changes in acidity significantly reduce artifacts.

[0033] As noted above, research based on this invention shows that labeling sialic acid with acid-buffered glycine and a reducing agent dramatically reduces artifacts compared to labeling sialic acid with the standard acid and reducing agent acetic acid and BME used in DMB labeling. Figure 2 The chromatogram of sialic acid released from rituximab, a commercially important therapeutic chimeric monoclonal antibody used to treat, among other conditions, particularly leukemia and lymphoma, is shown. The released sialic acid was labeled in a standard buffer containing acetic acid and BME (sodium dithionite was also present, as in other labeled solutions reported herein), separated on an HPLC column, and passed through and detected by a fluorescence detector. The Y-axis of the figure plots the relative response (in %), and the X-axis plots the retention time in minutes as the sample components passed through the fluorescence detector. In this chromatogram, the highest peak shows a value of almost 57.5, reflecting the presence of sialic acid Neu5Ac in the sample, with a retention time between approximately 1.85 min and 2.15 min. A second largest peak, with a measured value of almost 25, or approximately 43% of the sialic acid Neu5Ac peak, appears at a retention time between 0.5 min and 0.6 min. However, this peak is an artifact, as no sialic acid escaped from the HPLC column at that time. At a retention time of 1.5 minutes, a smaller peak of approximately 4 was observed. This peak represents sialic acid Neu5Gc and is approximately 16% of the artifact peak height.

[0034] Figure 3 A chromatogram of the same sample containing sialic acid is shown, but in which sialic acid is labeled in a solution provided by glycine phosphate under acidic conditions. BME is also used as a reducing agent (as in other studies reported here, sodium dithionite is also present in the solution). [Axis as...] Figure 2 The peak of sialic acid Neu5Ac in this chromatogram shows a value of almost 95. The peak reflecting artifacts at retention times from 0.5 to 0.6 has approximately 13% or 14% of the height of the Neu5Ac peak, while... Figure 2 In the image, the artifact peak is 43% of the Neu5Ac peak. The smaller peak representing the sialic acid Neu5Gc peak, seen at retention time 1.5 minutes, is again approximately 4, but now represents about 31% of the artifact peak height. Figure 2 In this study, the peak of sialic acid was only 16% of the artifact peak height. Therefore, the change from acetic acid to a glycine solution with a pH adjusted by adding a strong acid surprisingly reduced the artifacts seen in the fluorescence detection of DMB-labeled sialic acid and made it much easier to identify the presence of sialic acid compared to the artifacts.

[0035] Acids that can be used for partial acid hydrolysis, acids that can be used together with glycine for DMB labeling of sialic acid, or both.

[0036] Some sialic acids, such as the abundant sialic acid N-acetylneuraminic acid (Neu5Ac or NANA), are acetylated. In the past, the use of strong acids to analyze sialic acids was often avoided because strong acids can deacetylate sialic acids, causing them to "break down" and resulting in the loss of information about the presence of sialic acid on the starting glycoconjugate. Perhaps partly for this reason, the protocols for the partial acid hydrolysis of sialic acids often use acetic acid in DMB-labeled reactions, which is considered a weak acid.

[0037] like Figure 1 As shown, studies based on this specification using glycine, an amphoteric amino acid obtained by adding acid to a selected pH suitable for DMB labeling, resulted in a significantly higher signal from labeling with the exemplary acetylated sialic acid (Neu5Ac) than under acidic conditions provided by standard reagent acetic acid or phosphoric acid adjusted to the same selected pH with sodium hydroxide.

[0038] refer to Figure 1 The first bar from the left shows the signal from a sample of Neu5Ac labeled with DMB using BME as a reducing agent under acidic conditions provided by acetic acid, and the signal was set to 100% to provide a comparison with the signal from the same amount of sialic acid labeled using other reagents shown below the bar. As shown in the second bar from the left, the signal produced by sialic acid labeled with phosphate adjusted to pH 2.8 with BME as a reducing agent is only 74% of the signal produced by labeling with acetic acid and the same reducing agent (first bar). However, the third bar from the left shows that the signal from the same amount of DMB-labeled sialic acid in the presence of glycine adjusted to pH 2.8 by adding phosphate gave a signal that was 43% higher than the signal produced by labeling the sialic acid in the presence of acetic acid and almost twice the signal of the same acid adjusted to the same pH (but achieved by adding a strong base). Similarly, the signal generated by DMB labeling of the same sialic acid in the presence of glycine adjusted to pH 2.8 by adding hydrochloric acid was 123% of the signal of the same sialic acid sample labeled in the presence of acetic acid, indicating that glycine adjusted to the appropriate pH by a strong acid can be used in the methods and kits of the present invention.

[0039] Based on these results, it is anticipated that glycine adjusted to pH 1.5–3.2 with other strong acids (such as nitric acid, sulfuric acid, trifluoroacetic acid (“TFA”), hydrobromic acid, hydroiodic acid, perchloric acid, and chloric acid) will provide a similar improvement in the signal intensity of DMB labeling of free sialic acid compared to DMB labeling using acetic acid as a standard in the art, and will be usable in the methods and kits of the present invention. In some embodiments, the pH of the glycine solution is adjusted to a certain pH, preferably to a pH of about 2 to 3.2 (where “about” means ±0.25), in some embodiments, preferably to a pH of about 2.5 to 3 (where “about” means ±0.25), in some embodiments, more preferably to a pH of about 2.7 to 3.2 (where “about” means ±0.2), in some embodiments, more preferably to a pH of 2.7-2.9, even more preferably to a pH of 2.8 ±0.5, in some embodiments, even more preferably to a pH of 2.8 ±0.2, and most preferably to a pH of 2.8.

[0040] Corrosive acids (including hydrochloric acid) can be used in the methods and kits of the present invention, but care must be taken during their use to prevent accidental or unintentional contact with the acid by personnel performing labeling.

[0041] The concentration of the acid is preferably from 0.25M to about 2M, and "about" with respect to the acid concentration means ±0.25M.

[0042] As those skilled in the art will know, the pH of the labeling reaction affects the time required for the labeling reaction to proceed. At pH values ​​above 3.2, the labeling reaction proceeds more slowly and therefore requires a longer time. Since obtaining results more quickly is generally preferred than obtaining results after a longer period of time, it is preferred that the pH of the labeling reagent be 3.2 or lower. Suitable pH ranges are as described above. In the study reported in the examples, the solutions tested were prepared by mixing sufficient glycine into water to form a 3M solution, and then adjusting the pH of the glycine solution until the desired pH was achieved by adding a certain amount of acid as mentioned in the examples.

[0043] Glycine is amphoteric; in water, glycine has a pH of approximately 7. Practitioners do not need to begin with a 3M solution; some studies conducted during the work discussed herein used a 3M solution so that when other reagents are added, the resulting mixture will have a pre-selected glycine concentration in an acidic solution to allow for efficient DMB labeling. Practitioners can readily choose other starting concentrations of glycine to mix with acid to obtain solutions with the desired pH and concentration for use in the methods and kits of this invention.

[0044] Any specific acid and any specific target acid concentration (which, for convenience, may be referred to as "test acid" or "test concentration," respectively) can be readily tested for its suitability for DMB labeling of sialic acid by two tests. First, the test acid or test concentration can be added to an aqueous solution of 3M glycine to determine whether the test acid or test concentration can lower the pH of glycine to 1.5-3.2. If not, the test acid or test concentration is not suitable for the methods and kits of the present invention. Second, the test acid or test concentration and 1.5M acetic acid can be run in parallel assays with BME to label the same amount of known sialic acid with DMB, and the fluorescence signal from sialic acid labeled in the presence of acetic acid is compared with the fluorescence signal from sialic acid labeled in the presence of the test acid or test concentration. If the signal produced by sialic acid labeled in the presence of the test acid or test concentration, or both, is at least 10%, preferably 20%, higher than the signal from sialic acid labeled in the presence of 1.5M acetic acid, then the test acid or test concentration, or both, are suitable for the methods and kits of the present invention.

[0045] In the partial acid hydrolysis release step, glycine adjusted to a pH between 1.5 and 3.2 with a first acid can be used. The same combination of glycine and the first acid can also be used in the DMB labeling step, or the DMB labeling step can use glycine adjusted to the desired pH with a second acid. For example, the partial acid hydrolysis step can be performed with glycine adjusted to the desired pH with phosphoric acid (the first acid), while the DMB labeling step can be performed with glycine adjusted to the desired pH with hydrochloric acid (the second acid). However, for convenience, practitioners will generally avoid preparing two different acid solutions, instead selecting one acid to adjust the pH of the glycine solution, preparing a glycine solution / acid mixture at the desired pH, and using aliquots of this mixture in both steps. As reported in the examples, acid hydrolysis is performed using a glycine solution adjusted to pH 2.8 with an acid, and then mixed with a labeling solution containing glycine buffered to the same pH with the same acid for the labeling step.

[0046] In some embodiments, one acid may be used in the acid hydrolysis step, and another acid may be used to adjust the pH of the glycine solution to a desired pH. For example, (a) acetic acid may be used to release sialic acid via acid hydrolysis, and in said step, a glycine phosphate solution may be used to label the sialic acid released via acid hydrolysis with DMB; (b) hydrochloric acid may be used to release sialic acid via acid hydrolysis, while a glycine phosphate solution may be used to provide the acidic conditions required for the DMB-labeled sialic acid release; or (c) phosphoric acid may be used to release sialic acid via acid hydrolysis, while a glycine hydrochloride solution may be used to provide the acidic conditions required for the DMB-labeled sialic acid release.

[0047] Sialic acid is released from the glycoconjugate using sialidase.

[0048] As an alternative to releasing sialic acid from the target glycoconjugate, sialic acid can be released by enzymatic digestion. As stated by Juge et al., Biochem Soc Trans., 2016, 44(1):166-175: "Sialidases (also commonly referred to as neuraminidases) are a large group of enzymes, the majority of which are exo-sialidases catalyzing the cleavage of terminal sialic acids from complex carbohydrates on glycoproteins or glycolipids." Many sialidases are known, and many are commercially available. For example, MilliporeSigma (St. Louis, Missouri) sells a variety of sialidases with different specificities, which were originally isolated from many microorganisms, including Clostridium perfringens, Vibrio cholera, Arthrobacter ureafaciens, and Streptococcus pneumoniae.

[0049] Once the practitioner has selected a sialidase to release the target sialic acid and desialylated the glycoconjugate, the sialic acid released from the glycoconjugate can be labeled by aliquoting the sample into containers, adding DMB, buffer or acid and reducing agent, and labeling it as described elsewhere in this disclosure. The DMB-labeled sialic acid can then be isolated and analyzed.

[0050] After incubation with DMB and the labeling reagent, the labeling reaction is typically terminated by diluting the dye with excess water. The DMB-labeled sialic acid is then typically separated by liquid chromatography, such as high-performance liquid chromatography (“HPLC”) or ultra-high-performance liquid chromatography (“UHPLC”). The separated DMB-labeled sialic acid is then typically detected by a fluorescence detector. In some embodiments, the labeled sialic acid can be separated and then analyzed by detecting the presence of the DMB label. In some embodiments, detection is performed using a fluorescence detector such as an Agilent 1260 Infinity II fluorescence detector (Agilent Technologies, Santa Clara, CA). DMB can be detected using an excitation frequency of 373 nm and an emission frequency of 448 nm. In some embodiments, detection is performed using an ultraviolet (“UV”) detector.

[0051] Reagent test kit

[0052] In some embodiments, the present invention further provides kits for the release of sialic acid from glycoconjugates and for the labeling of sialic acid (whether the sialic acid is released from the glycoconjugate or is free sialic acid, such as known sialic acids that can be separately provided as analytical and quantitative standards in assays) with DMB labeling.

[0053] The kit preferably contains a selected acid, glycine, an acid to adjust the pH of glycine in solution, DMB, and one or more selected reducing agents. In some embodiments, glycine and the acid may be provided in a pre-mixed solution to provide glycine at a desired pH and concentration. In some embodiments, the acid is phosphoric acid. In some embodiments, the acid is hydrochloric acid. The reagents in the kit are typically provided in one or more containers.

[0054] In some embodiments, the kit contains glycine phosphate or glycine hydrochloride with a pH between 1.5 and 3.2, more preferably 2-3, and still more preferably 2.8 ± 0.1, for partial acid hydrolysis of the glycoconjugate. In some embodiments, the glycine phosphate or glycine hydrochloride is provided in 3M in 10 μl equal parts. In some embodiments, the kit contains one or more sialic acid enzymes for releasing sialic acid from the glycoconjugate by enzymatic digestion.

[0055] In some embodiments, the kit further comprises a DMB labeling mixture for labeling free sialic acid. The DMB labeling mixture preferably comprises DMB; glycine adjusted by acid to a pH between 1 and 3.2, more preferably 2-3, still more preferably 2.8 ± 0.1; and one or more reducing agents. In some embodiments, the reducing agent is BME. In some embodiments, the reducing agent is thioglycerol. In some embodiments, the kit further comprises sodium dithionite.

[0056] In some embodiments, the DMB-labeled mixture comprises 0.3 M glycine phosphate pH 2.8, 0.72 mol thioglycerol, 20 mmol DMB, and 225 mmol sodium dithionite.

[0057] In some embodiments, the kit may contain 10 μl aliquots of a DMB-labeled mixture containing 0.3 M glycine phosphate pH 2.8, 0.72 mol thioglycerol, 20 mmol DMB, and 225 mmol sodium dithionite.

[0058] Example

[0059] Example 1

[0060] This embodiment illustrates a scheme for testing the effects of different acid and reducing agent formulations on signals from DMB-labeled sialic acid in their presence.

[0061] One thousand picomoles of Neu5Ac (USP) were analyzed in each labeling test. Four replicate analyses were performed for each labeling condition. Each reaction (40 μL per reaction) was incubated in a sealed container at 50 °C for 3 h and then diluted with deionized water to 200 μL. Five μL of each diluted sample were analyzed by HPLC using an Agilent Poroshell 120EC-C18 column (2.1 mm diameter x 75 mm length, 2.7 μm particle size). The mobile phase used for HPLC was 4% methanol and 8% acetonitrile in water at a flow rate of 0.4 mL / min.

[0062] Example 2

[0063] This embodiment illustrates some combinations of different acids and reducing agents, wherein sialic acid is labeled with DMB in the working process described in this specification.

[0064] "Acetic acid mercaptoethanol": 1.5 mol acetic acid (CAS 64-19-7), 0.75 mol β-mercaptoethanol ("BME" CAS60-24-2), 14 mmol sodium dithionite (CAS7575-14-6), 7 mmol DMB (CAS81864-15-5).

[0065] "Sodium phosphate pH 2.8 mercaptoethanol": 1.5 mol sodium phosphate pH 2.8, 0.75 mol β-mercaptoethanol, 14 mmol sodium dithionite, and 7 mmol DMB. 3 mol (2x strength) of sodium phosphate was prepared by adjusting the pH of phosphoric acid to 2.8 with sodium hydroxide.

[0066] "Glycine phosphate pH 2.8 mercaptoethanol": 1.5 mol glycine phosphate pH 2.8, 0.75 mol β-mercaptoethanol, 14 mmol sodium dithionite, and 7 mmol DMB. 3 mol (2x strength) glycine phosphate was prepared by adjusting the pH of glycine to 2.8 with phosphoric acid.

[0067] "Glycine phosphate pH 2.8 thioglycerol": 1.5 mol glycine phosphate pH 2.8, 0.75 mol thioglycerol (CAS 96-27-5), 14 mmol sodium dithionite, 7 mmol DMB. 3 mol (2x strength) glycine phosphate was prepared by adjusting the pH to 2.8 with phosphoric acid.

[0068] "Glycine HCl pH 2.8 β-mercaptoethanol": 1.5 mol glycine hydrochloride pH 2.8, 0.75 mol β-mercaptoethanol, 14 mmol sodium dithionite, 7 mmol DMB. 3 mol (2x strength) glycine hydrochloride was prepared by adjusting the pH of glycine to 2.8 with hydrochloric acid.

[0069] "Glycine HCl pH 2.8 Thioglycerol": 1.5 mol glycine HCl pH 2.8, 0.75 mol thioglycerol (CAS 96-27-5), 14 mmol sodium dithionite, 7 mmol DMB. 3 mol (2x strength) glycine hydrochloride was prepared by adjusting the pH of glycine to 2.8 with hydrochloric acid.

[0070] Example 3

[0071] This embodiment reports the results of a study that tested the reagent combination described in Example 2 following the protocol described in Example 1. The results are as follows: Figure 1 The text is presented graphically. The terms in quotation marks in Example 2 correspond to... Figure 1 The bars in the diagram represent different preparation methods.

[0072] The signals from the sample of Neu5Ac labeled with DMB in the presence of acetic acid, BME, and sodium dithionite are shown in the first bar from the left (“Acetic Acid / BME Signal”). Since the standard labeling scheme uses acetic acid to provide acidic conditions for labeling and BME as a reducing agent, the signal provided by this formulation is used as the basis for comparison. For ease of comparison, the signal from this labeled sample is set to 100%, and the signals from samples labeled under other test conditions are stated as a percentage of the signal shown in the first bar.

[0073] As shown in the second bar from the left, the Neu5Ac-like sample labeled in the presence of sodium phosphate pH 2.8 mercaptoethanol formulation produced a signal that was only 74% of the acetic acid / BME signal. In contrast, as shown in the third bar from the left, the Neu5Ac-like sample labeled in the presence of glycine phosphate pH 2.8 mercaptoethanol formulation produced a signal that was 143% of the signal from the standard acetic acid / BME formulation. Even better signals were seen in the samples labeled in the presence of glycine phosphate pH 2.8 thioglycerol formulation. Switching the acid to hydrochloric acid in the glycine phosphate pH 2.8 mercaptoethanol formulation produced a signal that was 123% of the acetic acid / BME signal, as shown in the fifth bar from the left. Furthermore, as shown in the last bar from the left, switching the reducing agent in the glycine phosphate pH 2.8 thioglycerol formulation to thioglycerol again resulted in a signal that was 123% of the acetic acid / BME signal.

[0074] Example 4

[0075] This embodiment describes a scheme for releasing sialic acid from an exemplary target glycoprotein, labeling it with DMB using two different acids, and then analyzing the results to see the effect of the acid change on the fluorescence signal of the DMB-labeled sialic acid.

[0076] Rituximab, a therapeutically important chimeric anti-CD20 monoclonal antibody, was used as the target glycoconjugate from which sialic acid was obtained for analysis. Two hundred μL of rituximab were analyzed in each reaction. In the “Acid Release” step, rituximab was incubated at 80°C for 2 hours under one of the following acidic conditions. This was followed by a “DMB Labeling” step, in which the sialic acid released from rituximab in the acid release step was incubated at 50°C for 3 hours in a sealed container using the appropriate DMB labeling mixture. Following the DMB labeling step was an “Analysis” step, in which each sample was diluted to 200 μL with deionized water. Five μL of each diluted sample was analyzed by HPLC using an Agilent Poroshell 120EC-C18 column (2.1 mm diameter x 75 mm length, 2.7 μm particle size). The mobile phase used for HPLC was 4% methanol and 8% acetonitrile in water at a flow rate of 0.4 mL / min.

[0077] "Acetic acid mercaptoethanol":

[0078] Acid release procedure: Incubate each rituximab sample with 2 moles of acetic acid at 80°C for 2 hours.

[0079] DMB labeling procedure: Add 1.5 mol of acetic acid, 0.75 mol of β-mercaptoethanol, 14 mmol of sodium dithionite and 7 mmol of DMB to each sample.

[0080] "Glycine phosphate pH 2.8 mercaptoethanol":

[0081] Acid release: Each sample was incubated with 1 mole of glycine phosphate at pH 2.8 at 80°C for 2 hours.

[0082] DMB labeling: 0.75 mol glycine phosphate pH 2.8, 0.75 mol β-mercaptoethanol, 14 mmol sodium dithionite, 7 mmol DMB. 3 mol (2x strength) glycine phosphate was prepared by adjusting the pH to 2.8 with phosphoric acid.

[0083] Example 5

[0084] This embodiment illustrates the research results described in Embodiment 4.

[0085] Figure 2This is a chromatogram that graphically shows the trace of sialic acid released and labeled (as with other labeled solutions reported herein, also containing sodium dithionite), separated on an HPLC column, and flowed through and detected by a fluorescence detector. The Y-axis of the figure plots the relative response (in %), and the X-axis plots the retention time in minutes as the sample components flowed through the fluorescence detector. In this chromatogram, the highest peak shows a value of almost 57.5, reflecting the presence of sialic acid Neu5Ac in the sample, with a retention time between approximately 1.85 min and 2.15 min. A second largest peak, with a measurement of almost 25, or approximately 43% of the sialic acid Neu5Ac peak, appears at a retention time from 0.5 min to 0.6 min. However, this peak is an artifact, as no sialic acid elutes from the HPLC column at that time. At a retention time of 1.5 min, a smaller peak with a measurement of approximately 4 is observed. This peak represents sialic acid Neu5Gc and is approximately 16% of the height of the artifact peak.

[0086] Figure 3 A chromatogram of the same sample containing sialic acid is shown, but the sialic acid was labeled in a labeled solution provided by glycine phosphate under acidic conditions. BME was also used as a reducing agent (as in other studies reported herein, sodium dithionite was also present in the labeled solution). [Axis as...] Figure 2 The peak of sialic acid Neu5Ac in this chromatogram shows a value of almost 95. The peak reflecting artifacts at retention times from 0.5 to 0.6 has approximately 13% or 14% of the height of the Neu5Ac peak, while... Figure 2 In the image, the artifact peak is 43% of the Neu5Ac peak. The smaller peak representing the sialic acid Neu5Gc peak, seen at retention time 1.5 minutes, is again approximately 4, but now represents about 31% of the artifact peak height. Figure 2 In this study, the peak of sialic acid was only 16% of the artifact peak height. Therefore, the change in acidity surprisingly reduced the artifacts seen in the fluorescence detection of DMB-labeled sialic acid and made it much easier to identify the presence of sialic acid compared to the artifacts.

[0087] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or changes made based on them will be known to those skilled in the art and should be included within the spirit and scope of this application and the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. An in vitro method for labeling free sialic acid with 1,2-diamino-4,5- methylenedioxybenzene ("DMB") and, optionally, for analyzing the DMB labeled sialic acid, the method comprising the step of (a) incubating the free sialic acid with an effective amount of DMB in an aqueous solution comprising (i) an aqueous solution of glycine and an acid, wherein the glycine is present at a molar concentration of 0.25 M to 3 M and the solution has a pH of 1.5-3.2, and (ii) a reducing agent selected from the group consisting of thio-glycerol and beta-mercaptoethanol ("BME"), at a time and temperature sufficient to allow the labeling, thereby causing the free sialic acid to be labeled with DMB.

2. The method of claim 1, further wherein the solution comprises sodium dithionite.

3. The method of claim 1, wherein the acid is phosphoric acid.

4. The method of claim 1, wherein the acid is hydrochloric acid.

5. The method of claim 1, wherein the molar concentration of the glycine is from 0.25 M to 2.5 M ± 0.25 M.

6. The method of claim 1, wherein the molar concentration of the glycine is from 0.25 M to 2.0 M ± 0.25 M.

7. The method of claim 1, wherein the molar concentration of the glycine is from 0.40 M to 1.75 M ± 0.25 M.

8. The method of claim 1, wherein the molar concentration of the glycine is from 0.4 M to 1.5 M ± 0.25 M.

9. The method of claim 1, wherein the molar concentration of the glycine is from 0.4 M to 1.25 M ± 0.25 M.

10. The method of claim 1, wherein the molar concentration of the glycine is from 0.5 M to 1 M ± 0.25 M.

11. The method of claim 1, wherein the molar concentration of the glycine is from 0.5 M to 0.9 M.

12. The method of claim 1, wherein the molar concentration of the glycine is from 0.6 M to 0.8 M ± 0.1 M.

13. The method of claim 1, wherein the molar concentration of the glycine is 0.75 M ± 0.1 M.

14. The method of claim 1, wherein the molar concentration of the glycine is 0.75 M ± 0.05 M.

15. The method of claim 1, wherein the solution has a pH of 2 to 3.

2.

16. The method of claim 1, wherein the solution has a pH of 2.5 ± 0.1 to 3.

17. The method of claim 1, wherein the solution has a pH of 2.7 ± 0.25 to 2.9 ± 0.

25.

18. The method of claim 1, wherein the solution has a pH of 2.8 ± 0.

25.

19. The method of claim 1, wherein the time sufficient to label the free sialic acid is 1-6 hours.

20. The method of claim 1, wherein the time sufficient to label the free sialic acid is 2-5 hours.

21. The method of claim 1, wherein the time sufficient to label the free sialic acid is 3 hours ± 30 minutes.

22. The method of claim 1, wherein the time sufficient to label the free sialic acid is 2.5 hours ± 30 minutes.

23. The method of claim 1, wherein the temperature sufficient to label the free sialic acid is 35°C - 65°C.

24. The method of claim 1, wherein the temperature sufficient to label the free sialic acid is 50°C ± 5°C.

25. The method of claim 1, further comprising step (b), isolating the free DMB-labeled sialic acid.

26. The method of claim 25, wherein the isolation of the free DMB-labeled sialic acid is performed by subjecting the free DMB-labeled sialic acid to liquid chromatography.

27. The method of claim 26, further comprising step (c), analyzing the free isolated DMB-labeled sialic acid by providing the free isolated DMB-labeled sialic acid to an analytical device.

28. The method of claim 27, further wherein the analytical device is a fluorescence detector.

29. The method of claim 27, further wherein the analytical device is a UV detector.

30. An in vitro method of releasing, labeling, and optionally analyzing sialic acid present on a glycoconjugate, the method comprising (a) contacting a desired volume of the glycoconjugate with a first aqueous solution comprising glycine, the glycine adjusted to a pH of 1.5 - 3.2 with acid, (b) incubating the glycoconjugate with the first aqueous solution for a time and temperature sufficient to release the sialic acid from the glycoconjugate, thereby releasing the sialic acid from the glycoconjugate, (c) cooling the released sialic acid in the first aqueous solution to a temperature of 50°C ± 10°, (d) contacting the released sialic acids with a solution, thereby forming a sample / label mixture, the solution consisting of: (i) an effective amount of 1,2-diamino-4,5-methylenedioxybenzene ("DMB”), (ii) a second aqueous solution comprising glycine, wherein the pH of the solution is adjusted to a pH of 1.5 - 3 with acid, and (iii) an effective amount of one or more reducing agents, and (e) incubating the sample / labeled mixture for a time and temperature sufficient to label the released sialic acid in the mixture, thereby causing the released sialic acid to be labeled with DMB.

31. The method of claim 30, wherein the glycoconjugate is a glycoprotein.

32. The method of claim 30, wherein the glycoconjugate is a glycolipid or an oligosaccharide.

33. The method of claim 30, wherein the first aqueous solution is glycine from 0.25 to 2.0 M and adjusted to a pH of 1.5 - 3.2 with acid.

34. The method of claim 30, wherein the first aqueous solution is glycine from 0.25 to 2.0 M and adjusted to a pH of 2-3 with acid.

35. The method of claim 30, wherein the first aqueous solution is glycine from 0.25 to 2.0 M and adjusted to a pH of 2.5-3 with acid.

36. The method of claim 30, wherein the first aqueous solution is glycine from 0.25 to 2.0 M and adjusted to a pH of 2.8 ± 0.1 with acid.

37. The method of claim 30, wherein the second aqueous solution is glycine from 0.25 to 2.0 M and adjusted to a pH of 1.5-3.2 with acid.

38. The method of claim 30, wherein the second aqueous solution is glycine from 0.25 to 2.0 M and adjusted to a pH of 2-3 with acid.

39. The method of claim 30, wherein the second aqueous solution is glycine from 0.25 to 2.0 M and adjusted to a pH of 2.5-3 with acid.

40. The method of claim 30, wherein the second aqueous solution is glycine from 0.25 to 2.0 M and adjusted to a pH of 2.8 ± 0.1 with acid.

41. The method of claim 30, wherein the acid in the first aqueous solution is phosphoric acid.

42. The method of claim 30, wherein the acid in the second aqueous solution is phosphoric acid.

43. The method of claim 30, wherein the acid that adjusts the pH of the solution in the first aqueous solution and the second aqueous solution is phosphoric acid.

44. The method of claim 30, wherein the acid in the first aqueous solution is hydrochloric acid.

45. The method of claim 30, wherein the acid that adjusts the pH of the solution in the second aqueous solution is hydrochloric acid.

46. The method of claim 30, wherein the acid in the first aqueous solution and the second aqueous solution is hydrochloric acid.

47. The method of claim 30, wherein the one or more reducing agents comprise BME.

48. The method of claim 30, wherein the one or more reducing agents comprise thioglycerol.

49. The method of claim 30, wherein the one or more reducing agents comprise BME and sodium hydrosulfite.

50. The method of claim 30, wherein the one or more reducing agents comprise thioglycerol and sodium hydrosulfite.

51. The method of claim 30, wherein the time sufficient to release the sialic acid in step (b) is 0.5-5 hours.

52. The method of claim 30, wherein the time sufficient to release the sialic acid in step (b) is 1.0-4 hours.

53. The method of claim 30, wherein the time sufficient to release the sialic acid in step (b) is 2 hours ± 30 minutes.

54. The method of claim 30, wherein the temperature sufficient to release the sialic acid in step (b) is 70°C - 100°C.

55. The method of claim 30, wherein the temperature sufficient to release the sialic acid in step (b) is 70°C - 90°C.

56. The method of claim 30, wherein the temperature sufficient to release the sialic acid in step (b) is 80°C ± 5°C.

57. The method of claim 30, wherein the time sufficient to label the released sialic acid in step (e) is 1 - 6 hours.

58. The method of claim 30, wherein the time sufficient to label the released sialic acid in step (e) is 2 - 5 hours.

59. The method of claim 30, wherein the time sufficient to label the released sialic acid in step (e) is 3 hours ± 30 minutes.

60. The method of claim 30, wherein the time sufficient to label the released sialic acid in step (e) is 2 hours ± 30 minutes.

61. The method of claim 30, wherein the temperature sufficient to label the released sialic acid in step (e) is 35°C - 65°C.

62. The method of claim 30, wherein the temperature sufficient to label the released sialic acid in step (e) is 50°C ± 5°C.

63. The method of claim 30, further comprising step (f), isolating the released DMB-labeled sialic acid.

64. The method of claim 63, wherein the isolation of the released DMB-labeled sialic acid is performed by subjecting the released DMB-labeled sialic acid to liquid chromatography.

65. The method of claim 64, further comprising step (g), analyzing the released isolated DMB-labeled sialic acid by providing the released isolated DMB-labeled sialic acid to an analytical device.

66. The method of claim 65, further wherein the analytical device is a fluorescence detector.

67. A kit for labeling free sialic acid with 1,2-diamino-4,5-methylenedioxybenzene ("DMB"), the kit comprising (a) DMB, (b) an aqueous solution of glycine and an acid, wherein the solution has a pH of 1.5 to 3.2, and (c) a reducing agent.

68. The kit of claim 67, wherein the acid is phosphoric acid.

69. The kit of claim 67, wherein the acid is hydrochloric acid.

70. The kit of claim 67, wherein the glycine is in solution with the acid.

71. The kit of claim 70, wherein the solution comprising the glycine mixed with the acid has a pH of between 2 and 3.

72. The kit of claim 67, further comprising sodium hydrosulfite.

73. The kit of claim 67, wherein the reducing agent is beta-mercaptoethanol ("BME").

74. The kit of claim 67, wherein the reducing agent is thioglycerol.

75. The kit of claim 67, wherein the acid is hydrochloric acid and the reducing agent is thioglycerol.

76. The kit of claim 67, wherein the acid is phosphoric acid and the reducing agent is thioglycerol.

77. The kit of claim 67, further comprising one or more sialic acid standards.

78. The kit of claim 67, further comprising one or more sialidases.

79. The kit of claim 67, wherein the kit comprises (a) glycine in solution, the pH of the solution adjusted to a pH of 2.8 with phosphoric acid, (b) thioglycerol, (c) DMB, and (d) sodium hydrosulfite.

80. The kit of claim 79, further wherein the thioglycerol is provided at 0.72 moles ± 0.1 moles, the DMB is provided at 5 millimoles ± 1 millimoles, and the sodium hydrosulfite is provided at 56 millimoles ± 10 millimoles.

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