# US 10,119,933 B2 · Analysis and assay of glycated haemoglobins by capillary electrophoresis, buffer compositions and kits for capillary electrophoresis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,119,933 B2 - Document ID: US10119933B2 - Status: Granted - Kind: B2 - Application number: 14/945,108 - Filed: 2015-11-18 - Published: 2016-03-17 - Granted: 2018-11-06 - Earliest claimed: 2009-07-01 - Assignees: Sebia - Applicants: Sebia - Inventors: Gérald Deschamps; Frédéric Robert; Denis Simonin - Web page: https://patentgrep.com/patent/US10119933B2 ## CPC - G01N 27/44747: {Composition of gel or of carrier mixture} - G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)} - G01N 33/723: {Glycosylated haemoglobin} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The invention relates to a method for analysis by capillary electrophoresis of glycated haemoglobins comprising at least one globin chain comprising a glucose residue bound to the amino acid in the N-terminal position, contained in a biological sample, said method comprising using a buffer composition comprising at least one compound which is capable of specifically complexing glucose residues of one or several glycated haemoglobin(s) and of providing said glycated haemoglobin(s) with several negative electric charges at an alkaline pH. By way of example, this compound may be 3,4- or 3,5-dicarboxyphenylboronic acid, preferably 3,5-dicarboxyphenylboronic acid. Said method may in particular be used to separate and assay haemoglobin HbA1c present in a biological sample optionally comprising other haemoglobins, in particular other minor fractions. The invention also concerns buffer compositions for use in said analysis, as well as kits for the analysis and for the assay of glycated haemoglobins by capillary electrophoresis. ## Claims ### Claim 1 A method for analysis by capillary electrophoresis of glycated hemoglobin A1c comprising at least one beta globin chain and a glucose residue bound to the amino acid in the N-terminal position of the beta globin chain, contained in a biological sample, said method comprising the following steps: a. introducing a buffer composition and biological sample into an electrophoresis capillary, said buffer composition comprising at least one compound which specifically complexes the glucose residue(s) bound to an amino acid in the N-terminal position in hemoglobin A1c of the biological sample, and provides said glycated hemoglobin A1c with several negative electric charges at an alkaline pH, and wherein said compound which specifically complexes the glucose residue(s) of hemoglobin A1c of the biological sample and provides negative charges at an alkaline pH comprises two or more than two functional groups, at least one of said functional group(s) specifically complexing one or several glucose residue(s), thereby providing one negative electric charge per complexed glucose residue, the other, one of the others or all of the other functional group(s), which do not complex said glucose residue(s), providing each of said glucose residue with one or several additional negative electric charge(s) at an alkaline pH and; b. performing capillary electrophoresis on the buffered biological sample for separating the constituents of said biological sample, thereby producing an electrophoretic profile comprising a series of fractions, wherein the glycated hemoglobin A1c fraction is separated from other hemogloblins including hemoglobin A1a and hemoglobin A1b found in the biological sample, and; c. performing a step for detection of hemoglobin A1c present in the biological sample. ### Claim 2 (depends on 1) The method according to claim 1, wherein the functional group(s) specifically complexing one or several glucose residue(s) interacts with two vicinal hydroxyl groups of a glucose residue. ### Claim 3 (depends on 1) The method according to claim 1, in which the compound which specifically complexes glucose residues of hemoglobin A1c of the biological sample and provides negative charges at an alkaline pH comprises one or several groups which are anionisables at an alkaline pH, that are one or several carboxylate(s), carboxyl(s), sulphonate(s) and/or sulphonyl(s). ### Claim 4 (depends on 1) The method according to claim 1, in which the compound which specifically complexes glucose residues of hemoglobin A1c of the biological sample and provides negative charges at an alkaline pH comprises one (or more) boronyl and/or boronate group(s), and is: (i) a boronate compound with general formula RB(OH)2 or RB(OH)3−, in which the group R comprises at least one aryl and/or alkyl (linear, branched or cyclic) and/or an aralkyl and/or other functional groups or heteroatoms, and/or a combination thereof, and said group R provides glycated hemoglobins with one or several negative electric charge(s) at an alkaline pH for each glucose residue complexed with the boronate group; or (ii) a salt of said boronate compound. ### Claim 5 (depends on 4) The method according to claim 4, in which the boronate compound is a polysubstituted phenylboronate that is a phenylboronate which is disubstituted with carboxyl and/or sulphonyl groups. ### Claim 6 (depends on 4) The method according to claim 4, in which the boronate compound is a dicarboxyphenylboronic acid. ### Claim 7 (depends on 6) The method according to claim 6, in which the boronate compound is a dicarboxyphenylboronic acid selected from 3,4-dicarboxyphenylboronic acid and 3,5-dicarboxyphenylboronic acid. ### Claim 8 (depends on 1) The method according to claim 1, in which the concentration in the buffer composition of the compound which specifically complexes glucose residues of glycated hemoglobin A1c of the biological sample and provides negative charges at an alkaline pH is in stoechiometric excess with respect to the total quantity of proteins, compared with the total quantity of all of the hemoglobins present in the biological sample or compared with the total quantity of all of the hemoglobins comprising glucose present in the biological sample. ### Claim 9 (depends on 1) The method according to claim 1, in which the concentration in the buffer composition of the compound which specifically complexes glucose residues of glycated hemoglobin A1c of the biological sample and provides negative charges at an alkaline pH is in the range 0.10 to 100 mM. ### Claim 10 (depends on 1) The method according to claim 1, in which the buffer composition further comprises a flow retardant. ### Claim 11 (depends on 10) The method according to claim 10, in which the concentration of flow retardant in the buffer composition is in the range 0.10 to 40 mM. ### Claim 12 (depends on 1) The method according to claim 1, in which the buffer composition further comprises: a buffer compound having a pKa in the range 8.0 to 11.0; and/or a base; and/or a salt, and/or a diluting solution, for example water. ### Claim 13 (depends on 12) The method according to claim 12, in which the buffer compound is a zwitterionic compound. ### Claim 14 (depends on 12) The method according to claim 12, in which the concentration of buffer compound in the buffer composition is in the range 20 to 500 mM. ### Claim 15 (depends on 1) The method according to claim 1, in which the buffer composition has a pH of 9 or more. ### Claim 16 (depends on 1) The method according to claim 1, further comprising a step for generating an electropherogram from a detection signal which is proportional to the quantity of hemoglobin A1c detected. ### Claim 17 (depends on 1) The method according to claim 1, further comprising a step for determining the quantity of one or several hemoglobin (s) present in the biological sample and/or of the proportion of one or several hemoglobin (s) present in the biological sample with respect to the total quantity of proteins, the total quantity of hemoglobin or the quantity of hemoglobin A1c present in the biological sample. ### Claim 18 (depends on 1) The method according to claim 1, further comprising a step for quantification of one or several hemoglobin (s) present in the biological sample compared with one or several standardized calibrator(s). ### Claim 19 (depends on 1) The method according to claim 1, in which the biological sample is a blood sample. ### Claim 20 (depends on 1) The method according to claim 1, in which the biological sample is diluted in a haemolyzing solution. ### Claim 21 (depends on 1) A method of diagnosing diabetes in a biological sample of human or non-human mammal and/or monitoring the glycaemic balance in a biological sample of human or non-human mammal, which comprise the following steps: a. performing the method of claim 1 on a biological sample of human or non-human mammal, wherein the at least one compound which specifically complexes glucose residue(s) comprises one boronyl and/or boronate group(s), and b. performing a step of diagnosing diabetes and/or monitoring the glycaemic balance based on results from step a. ### Claim 22 (depends on 21) The method for diagnosing diabetes in a human or non-human mammal and/or monitoring the glycaemic balance in a human or non-human mammal according to claim 21, wherein the compound comprising one (or more) boronyl and/or boronate group(s), is (i) a boronate compound with general formula RB(OH)2 or RB(OH)3−, in which the group R comprises at least one aryl and/or alkyl (linear, branched or cyclic) and/or an aralkyl and/or other functional groups or heteroatoms, and/or a combination thereof, and said group R provides glycated hemoglobins with one or several negative electric charge(s) at an alkaline pH for each glucose residue complexed with the boronate group; or (ii) a salt of said boronate compound. ### Claim 23 The process for separating, by capillary electrophoresis glycated hemoglobin A1c comprising at least one beta globin chain comprising a glucose residue bound to the amino acid in the N-terminal position of the beta globin chain, from other hemoglobins including hemoglobin A1a and hemoglobin A1b present in a biological sample, which comprises: a. contacting a biological sample with a compound which specifically complexes the glucose residue(s) of glycated hemoglobin A1c in said biological sample, and provides said glycated hemoglobin A1c with several negative charges at an alkaline pH, said compound comprising two or more than two functional groups, at least one of said functional group(s) specifically complexing one or several glucose residue(s), the other, one of the others or all of the other functional group(s), which do not complex said glucose residue(s), providing each of said glucose residue with one or several additional negative electric charge(s) at an alkaline pH, and said compound comprising one (or more) boronyl and/or boronate group(s), and; b. a step of performing capillary electrophoresis on the biological sample obtained from step a., thereby displacing the electrophoretic migration peak corresponding to said glycated hemoglobin A1c from the other hemoglobins including hemoglobin A1a and hemoglobin A1b, and detecting said glycated hemoglobin A1c. ### Claim 24 (depends on 23) The process according to claim 23, wherein the compound comprising one (or more) boronyl and/or boronate group(s), is (i) a boronate compound with general formula RB(OH)2 or RB(OH)3−, in which the group R comprises at least one aryl and/or alkyl (linear, branched or cyclic) and/or an aralkyl and/or other functional groups or heteroatoms, and/or a combination thereof, and said group R provides glycated hemoglobins with one or several negative electric charge(s) at an alkaline pH for each glucose residue complexed with the boronate group; or (ii) a salt of said boronate compound.