US009222913B2
(12) United States Patent (10) Patent No.: US 9.222,913 B2 Deschamps et al. (45) Date of Patent: Dec. 29, 2015
(54) ANALYSIS AND ASSAY OF GLYCATED FOREIGN PATENT DOCUMENTS HAEMOGLOBINS BY CAPILLARY ELECTROPHORESIS, BUFFER EP 1659 398 A1 5, 2006 COMPOSITIONS AND KITS FOR JP 6-5O2244 3, 1994 CAPLLARY ELECTROPHORESS (Continued) (75) Inventors: Gérald Deschamps, Ury (FR); Frédéric OTHER PUBLICATIONS Robert, Mennecy (FR); Denis Simonin, Evry (FR) Margo P. Cohen, Rex S. Clements, Measuring Glycated Proteins: Clinical and Methodological Aspects, Diabetes Technology & Thera (73) Assignee: SEBIA, Lisses (FR) peutics, vol. 1, No. 1, pp. 57-70 (1996).* (*) Notice: Subject to any disclaimer, the term of this (Continued) patent is extended or adjusted under 35 U.S.C. 154(b) by 199 days. Primary Examiner — J. Christopher Ball (21) Appl. No.: 12/827,795 Assistant Examiner — Maris R. Kessel (74) Attorney, Agent, or Firm — Birch, Stewart, Kolasch & (22) Filed: Jun. 30, 2010 Birch, LLP (65) Prior Publication Data US 2011 FOOOO788A1 Jan. 6, 2011 (57) ABSTRACT The invention relates to a method for analysis by capillary (30) Foreign Application Priority Data electrophoresis of glycated haemoglobins comprising at least Jul. 1, 2009 (FR) ...... O9 O322O one globin chain comprising a glucose residue bound to the amino acid in the N-terminal position, contained in a biologi (51) Int. Cl. cal sample, said method comprising using a buffer composi G0IN 27/447 (2006.01) tion comprising at least one compound which is capable of (52) U.S. Cl. specifically complexing glucose residues of one or several CPC ...... G0IN 2744747 (2013.01) glycated haemoglobin(s) and of providing said glycated hae moglobin(s)with several negative electric charges at an alka (58) Field of Classification Search line pH. By way of example this compound may be 3,4- or CPC ...... G01N 27/26: G01N 27/447; G01N 3,5-dicarboxyphenylboronic acid, preferably 3,5-dicarbox 27/44747; C07F 5/02 yphenylboronic acid. Said method may in particular be used USPC ...... 204/450 468, 600-616 to separate and assay haemoglobin HbA present in a bio See application file for complete search history. logical sample optionally comprising other haemoglobins, in (56) References Cited particular other minor fractions. The invention also concerns buffer compositions for use in said analysis, as well as kits for U.S. PATENT DOCUMENTS the analysis and for the assay of glycated haemoglobins by capillary electrophoresis. 4,861,728 A * 8/1989 Wagner ...... 436,501 5,120,413 A * 6/1992 Chen et al...... 204,451 (Continued) 15 Claims, 16 Drawing Sheets
DAD1 D, Sig=415,8Ref=off (2101101A1C00011.D) DAD1 D, Sig=415,8Ref=off (2101101A1C00003.D) DAD1 D, Sig=415,8Ref=off (2101101A1C00007.D) mAU AO 80 3,4-Dicarboxyphenylboronic Acid
A1a, A1b
10 12 14 16 18 min US 9.222,913 B2 Page 2
(56) References Cited Song et al., Sensors and Actuators B: Chemical, "Boronic acid modified thin film interface for specific binding of glycated hemo U.S. PATENT DOCUMENTS globin (HbAlc) and electrochemical biosensing, vol. 140, pp. 233 239, (2009). 5,599.433 A * 2, 1997 Keo et al...... 204,451 5,631,364 A 5/1997 Sundrehagen et al. 2002/0173044 A1 11/2002 Pachl et al. Chen et al., “Practical Method for the Synthesis of Substituted 2010.018711.0 A1 7/2010 Tanaka et al. Phenylboronic Acids”, Journal of Sichuan Normal University (Natu ral Science), vol. 23, No. 5 (2000) pp. 511-512. FOREIGN PATENT DOCUMENTS Hall, D.G., Boronic Acids: Preparation and Applications in Organic WO WO92,08984 5, 1992 Synthesis and Medicine (e.d. D.G. Hall), Wiley-VCHVerlag GmbH WO WO96,22524 A1 T 1996 WO WO98,40750 A1 9, 1998 & Co. KGaA. Weinheim, FRG, doi:10.1002/3527606548.ch1, pp. WO WO 2008/O29685 A1 3, 2008 1-57 and 79-80 only. WO WO 2008/O29885 A1 3, 2008 Menard et al., “Quantitative Determination of Glycosolated Hemo OTHER PUBLICATIONS globin A1 by Agar Gel Electrophoresis”. Clin. Chem, vol. 26, No. 11 (1980) pp. 1598-1602. French Preliminary Search Report mailed on Feb. 9, 2010 for the corresponding Priority French application No. 0903220. * cited by examiner U.S. Patent Dec. 29, 2015 Sheet 1 of 16 US 9.222,913 B2
Current Electropherogram(s) DAD1E,Sig=415,8 Ref-off (111207A1C00001.D) mAU
400 -
300 -
200 -
100 -
20 go 4o so in U.S. Patent Dec. 29, 2015 Sheet 2 of 16 US 9.222,913 B2
Current Electropherogram(s) DAD, E,Sig=415,8 Ref-off (11 1207W1C00007.D) *DAD1 E. Sig=415,8Ref=off (111207W1C00008.D)
150 -
125 -
100 75
25 -
30 325' 35 375 40 42.5 45 min Figure 2 U.S. Patent Dec. 29, 2015 Sheet 3 of 16 US 9.222,913 B2
Current Electropherogram(s) DAD1E,Sig=415,8Ref=off (131207W1C00003.D) *DAD1 E,Sig=415,8 Ref-off (131207W1C00004.D) mAU
175
150
125
100
75
50
25 - U.S. Patent Dec. 29, 2015 Sheet 4 of 16 US 9,222,913 B2
Current Electropherogram(s) DAD, E,Sig=415.8 Ref-off (131207A1C00005.D) *DAL)1 Esig=415,8 Ref-off (131207W1C00006.D) mAU -
175
150 125
100
75 so 25
2 2s so 32 34 36 38 min Figure 4 U.S. Patent Dec. 29, 2015 Sheet 5 of 16 US 9,222,913 B2
Current Electropherogram(s) - - - DAD1 E,Sig=415,8Ref=off (131207W1C00007.D) *DAD1E,Sig=415,8 Ref-off (131207W1C00008.D) mAU 140
120
100 .
80 - 60 40 20 - A1a, A1b
26 28 30 32 34 36 .38 min U.S. Patent Dec. 29, 2015 Sheet 6 of 16 US 9.222,913 B2
Current Electropherogram(s) DAD1 E,Sig=415,8 Ref-off (1312O7\A1C00009.D) *DAD1E,Sig=415,8 Ref-off (131207A1C0001O.D) mAU 140
120
100
80 60 40.
20 A1a, A1b A0 A1C c r s states
26 28 30 32 34 36 38 min Figure 6 U.S. Patent Dec. 29, 2015 Sheet 7 of 16 US 9.222,913 B2
Current Electropherogram(s) DAD1E,Sig=415.8 Ref-off (1911 07W1C00007.D) *DAD1E,Sig=415.8 Ref-off (1911 07W1C00008.D) mAU 175. 150 125 100
75 -
50 -
25 -
is 20 22 24, 26 28 min U.S. Patent Dec. 29, 2015 Sheet 8 of 16 US 9.222,913 B2
DAD1 D, Sig=415.8 Ref-off (21 0110\a1C00010.D) DAD1 D, Sig=415,8 Ref-off (21011OW1C00002.D) DAD1 D, Sig-A15,8Ref-off (21011 OW1C00006.D) mAU 80-3,5-Dicarboxyphenylboronic Acid
0 2 4 6 8 10 12 14 16 18 min Figure 8
DAD1 D, Sig=415,8. Ref-off (21011OW1C00011.D) DAD1 D, Sig=415,8Ref=off (2101101A1C00003.D) DAD1 D, Sig=415,8Ref=off (2101101A1C00007.D) mAU AO 80 . 3,4-Dicarboxyphenylboronic Acid
U.S. Patent Dec. 29, 2015 Sheet 9 of 16 US 9.222,913 B2
A1c Max O.D.: 0.0291
-T | ? A 2. it.a s= a-&SAs, assesseeast&--- 54O 26O (Seconds)
Figure 10 U.S. Patent Dec. 29, 2015 Sheet 10 of 16 US 9.222,913 B2
Max O. D. : 0.1961
300 210 (Seconds)
Max O. D. : 0.1961
Hib AC \ Hb A2 sea-arnawk 4s-led-N-a- 54 O' 260" (Seconds) Figure 11 U.S. Patent Dec. 29, 2015 Sheet 11 of 16 US 9.222,913 B2
(3,5-dCPBA) At , t Art Ao At/t at Al-tAo?t Ao (min)
0.16. 0.12. > i 0.08. 0.04
O 20 40 60 80 100 120 140 Concentration of 3,5-dCPBA in buffer (mM)
Figure 12 U.S. Patent Dec. 29, 2015 Sheet 12 of 16 US 9.222,913 B2
n Hb E i
Hb A1c \\ | Hit A2 3&s. -A- Cl-N-a --V sa-awaasaa-rr 54 O' 260" (Seconds Figure 13
Max O.D. : 0.0561 Hb A A
H S Hb.." F N C+A2 f V \ -as -a asHb . . -(N-ace. A1c - 2 i. A-Salis A 4|| - 54.0" 260" (Seconds) Figure 14 U.S. Patent Dec. 29, 2015 Sheet 13 of 16 US 9,222,913 B2
- A
- A2 540" m 260 it (Seconds) Figure 15 Max O. D. : 0.2301 Hb A
3 - A2 Hbac Sw is A 540". 260 (Seconds) Figure 16 U.S. Patent Dec. 29, 2015 Sheet 14 of 16 US 9.222,913 B2
sample % A.-HPlc T Gross values 4A - Ec s
55 m
corrected values: 1 2.W y = 0.9554x + 0.272s 1 O 10 R42 =- 0.9904 af
8 raw values SS 6 2. raw values: a corrected Values 4. y = 1.2532X - 2.8946 R2 = 0.9898 2 O 0 2 4 6 8 10 12 14 % A1C HPLC Figure 17 U.S. Patent Dec. 29, 2015 Sheet 15 of 16 US 9.222,913 B2
HYDRAGEL 15 Hb A1C
1 2 3 4 5 6 7 8 9 Figure 18A
Glucose Concentration (g/L) 96 A1c gel % A1c EC O (reference) 4.1 4.2 1 4.5 5 4.6 4.3 10 5.3 4.3 2O 7.8 4.1 30 8.6 4.1 50 14.8 4.1 Figure 18C U.S. Patent Dec. 29, 2015 Sheet 16 of 16 US 9.222,913 B2
Added glucose Ogil Added glucose: 20 gift. SbA.
D. O. Max: 0.249 D. Q. Max: 0.274
HbAlc 3, \ ibR2 SibAie 3 \ BibA2 e-A-si-. ...-- o-A-est. ---4----~~ S& Seconds 26 S$8 Secords 2 & 3 riaemoglobin eleciophoresis Haemoglobin eiectrophoresis
Added glucose 5 gfi. Added glucose: 30 gill EA
E. O. Max: 0.169i D. O. Max: 0.2777
T-sel-N-TRAct 3 iii.2 Ace 3 to 3 54 ) Secoids 2 & O S4C 5&is (Seconds}a \ A2 26 (3. aemoglobin elects opioiesis aemoglobin &lectopheresis
Added glucose: 0 g/l. Added glucose: 50 g.
iskA D. O. Max: 0.2151 P. O. Max: 0.271
Sibal.c. 3 k E.A. Ace 3 b2 ra-N-4 a.--e-N -a- 54: i. Seconds 260 5A Seconds 268 taeragiaixir; eisctrophoresis aerrogicain eiects ophoresis
Figure 8B US 9,222,913 B2 1. 2 ANALYSIS AND ASSAY OF GLYCATED the beta chains of haemoglobin A (HbA) is glucose: HAEMOGLOBINS BY CAPLLARY HbA is obtained by condensation of one molecule of glu ELECTROPHORESIS, BUFFER cose with the NH group of the N-terminal valine of the B COMPOSITIONS AND KITS FOR chain, to form a stable keto-amine, derived from a rearrange CAPLLARY ELECTROPHORESS ment (Amadori rearrangement) of a previously formed unstable aldimine (designated as labile HbA, or preA). The present application concerns the field of the analysis Finally, the term haemoglobin Ao (HbA) generally and assay of glycated haemoglobins, in particular haemoglo encompasses non-glycated A haemoglobins and glycated A bin A, by capillary electrophoresis. haemoglobins not comprising an ose bound to the amino acid The invention pertains to a method for analysis by capillary 10 electrophoresis of a biological sample comprising one or in the N-terminal position of the B chains. several haemoglobin(s) and in particular one or several gly Protein glycation (including glycated haemoglobin and in cated haemoglobin(s), as well as appropriate buffer compo particular haemoglobin A, among many others) is caused by sitions for said analysis, and kits for analysis and for assay of a too high concentration of Sugars in the blood (as is the case glycated haemoglobins by capillary electrophoresis. 15 with diabetes). The glycation of proteins alters their func Haemoglobin (Hb) is a globular molecule generally com tions, causing cell lesions, tissue lesions and vascular ageing, prising four Sub-units each constituted by a polypeptide chain and participates in the development of several diseases Such conjugated to a heme portion. The polypeptide chains are as arteriosclerosis, renal insufficiency, diabetic retinopathy collectively designated by the name “globin portion of the and cataracts. Once glycated, haemoglobin transports oxygen haemoglobin molecule. less well. All human haemoglobins are constituted by four polypep Included among all of the groups and Sub-groups of hae tide chains in two groups of two identical chains. In an adult moglobin, HbA is of particular interest as it serves as a human, three types of haemoglobin are normally present: long-lasting marker of the diabetic state of patients. The for haemoglobin A (HbA), which is the vast majority (it gener mation of HbA is dependent on glycaemia (concentration of ally represents approximately 97% of the haemoglobin in an 25 glucose in the blood). It intervenes throughout the lifespan of adult), constituted by 2 alpha chains and two beta chains the red blood cells, which is normally 120 days. The blood (haemoglobin CB), haemoglobin A2 (Hb A2, which gener concentration of HbA, thus indicates the average glycaemia ally represents approximately 1-3% of the haemoglobin in an over the 2-3 months preceding assay. Elevation of the blood adult) constituted by two alpha chains and two delta chains concentration of HbA is indicative of prolonged hypergly (haemoglobin Cö), and foetal haemoglobin (Hbf), consti 30 caemia during that period. The blood concentration of HbA. tuted by two alpha chains and two gamma chains (haemoglo is not influenced by short-term fluctuations in the levels of bin CY), which only subsists in trace amounts (generally blood glucose. Thus, by measuring the HbA level between less than 1% of the haemoglobin in an adult) and remains two dates, the evolution of diabetes can be monitored. limited to a restricted cell population (“F cells”). In a diabetic patient, the aim is to obtain a HbA level that Haemoglobin contains species known as minor species; 35 is as close as possible to the figures characterizing a good they are glycated haemoglobins. balance of glycaemia. The reference value in a non-diabetic Glycated haemoglobin (also known as glycosylated hae human with a normal glycaemia is 4% to 6% HbA in the moglobin or glycohaemoglobin) corresponds to the set of blood compared with the total concentration of A haemoglo haemoglobin molecules modified by binding of oses, princi bins (glycated haemoglobins and non-glycated haemoglo pally glucose, on the amine functions (NH) of the globin; the 40 bins) (i.e. 20 to 42 mmol/moles; Panteghini et al., 2007). NH group of a haemoglobin condenses with an aldehyde The detection of glycated haemoglobins, in particular the group derived from a reducing Sugar. This reaction, which is detection of an increase in the HbA level, is thus of manifest non-enzymatic, is then stabilized by an Amadori rearrange interest for the diagnosis of diabetes (type 1 or type 2) or ment. Potential glycation sites are the N-terminal amino acids monitoring diabetic patients, in particular to evaluate the of the four globin chains of haemoglobin (C. chains and B, Ö, 45 efficacy of a treatment against diabetes (type 1 or type 2). or Y chains depending on the type of haemoglobin) and the However, interpreting the results may occasionally be dif free amino-epsilon groups (in particular those of the lysine ficult, since many factors may falsify the results, in particular residues) in the globin chains of the haemoglobin. the presence of abnormal haemoglobins. Many forms of glycated haemoglobin exist; they depend Indeed, haemoglobins termed 'abnormal' or variant, in on the number of bound oses, the nature of the globin chains 50 particular denoted by the letters S, C, D, E, H and J. are and the oses bound on said chains and on the position of said encountered in certain patients. Haemoglobin A is partially or oses on the globin chains. completely replaced by one or several abnormal haemoglo The term “total glycated haemoglobin' is used when con bins. They originate in particular from reduced synthesis of sidering the molecules of glycated haemoglobin on any NH certain globin chains and/or from a modification of the struc residue. 55 ture of the C. B. Y or ö chains, by substitution of one amino The term “haemoglobin A1 (Hb A1) is reserved for mol acid by another. Thus, a modification in the B chain may, for ecules of haemoglobin Ahaving one molecule of ose bound to example, give rise to S or Chaemoglobins (C.B. haemoglo the N-terminal amino acid of one or two beta chains of the bins in which the glutamic acid in position 6 in the 3 chain is protein. The A1 fraction, which is heterogeneous, comprises respectively substituted by a valine or a lysine). in particular the minor fractions A, A, and especially A. 60 These abnormal haemoglobins are also susceptible to gly Haemoglobin A (HbA) includes haemoglobin A cation. Thus, haemoglobins S (HbS) and C (HbO) (HbA), in which the ose bound to the N-terminal amino exist which, like HbA, comprise one molecule of glucose acid is fructose 1-6 diphosphate, and haemoglobin A bound to the N-terminal valine on the ? chains (Abraham et (HbA), in which the ose bound to the N-terminal amino al, 1984). acid is glucose-6-phosphate. In the case of haemoglobin A, 65 Methods for analysis and assay of glycated haemoglobin (HbA), the ose bound to the N-terminal amino acid is pyru and in particular haemoglobin HbA may be classified into 2 vate. Finally, the ose bound to the N-terminal amino acid of broad categories. US 9,222,913 B2 3 4 The first category, the major category, includes methods Analysis by isoelectric focussing (Molteni, 1994; Hempe, Such as immunoassay or affinity chromatography. It is based 1994) is carried out in coated capillaries. Methylcellulose is on the structural characteristics of the Sugar molecule bound used for the coating. The catholyte used is sodium hydroxide to the haemoglobin. As an example, the publication by Wilson (NaOH) and the anolyte is phosphoric acid (HPO). By etal, 1993 and U.S. Pat. No. 6,162,645 describe a method for 5 using ampholytes, this analysis enables separation of various the assay of glycated haemoglobin found in a sample of variants of haemoglobin, including HbA, the peak of which human blood by affinity chromatography. Said method is is very close to that of HbA (Ap-0.10). Although this method based on the use of a Solid positively charge phase, coupled functions well, it is not simple to carry out routinely and via a polyanionic compound (for example polyacrylic acid), necessitates great precision, in particular as regards the pH to boronic acid, phenylboronic acid, boric acid or a similar 10 range of the ampholytes (the isoelectric point (pl) of HbA is boronate compound. When a blood sample to be analyzed is very close to that of HbA). incubated with that solid phase, the glucose residues of the Free solution analysis using an anti-HbA antibody (U.S. glycated haemoglobin molecules present in that sample are Pat. No. 5,431,793) is carried out in aborate buffer at a basic complexed with the boronate compound. The glycated hae pH (pH of 8 to 10), using samples subjected to a reaction with moglobin molecules are thus grafted onto the Solid phase. The 15 a anti-HbA monoclonal antibody. More precisely, a first proportion of glycated haemoglobin immobilized on the Solid electropherogram is produced using the working sample phase compared with the proportion of non-immobilizedhae without prior reaction with an anti-HbA antibody. A single moglobin can then be quantified. That assay is carried out peak is obtained, with area X, containing all of the haemoglo either by measuring the extinction of fluorescence (Wilson et bins (including HbA). A second electropherogram is al, 1993), or by using labelled antibodies directed against obtained by analyzing the working sample which has been human haemoglobin (U.S. Pat. No. 6,162,645). brought into contact with the anti-HbA antibody. The non The second category of methods for the analysis and assay bound anti-HbA antibody, the anti-HbA antibody/HbA. of glycated haemoglobin includes ion exchange chromatog complex and the haemoglobins not complexed by the anti raphy or electrophoresis. It exploits the physico-chemical body are thus separated. The quantity of HbA is then deter characteristics of the molecules and is based on the charge 25 mined by the difference of the area under the peaks between difference existing between glycated proteins and non-gly the peak for the first electropherogram and that of the second cated proteins. relative to the haemoglobins not complexed by the anti Of the various analytical methods allowing glycated hae HbA antibody, with area y, i.e. a quantity X-y. This method moglobin to be assayed that are described in the literature, thus proves to be long and complicated in implementation. electrophoretic methods are divided into several categories: 30 The free solution analysis method using a naked capillary gel analyses include the polyacrylamide gel isoelectric focus (U.S. Pat. No. 5,599.433) involves a sugar complexing agent ing analysis (Beccaria, 1978; Simon, 1982: Stickland, 1982: bound to haemoglobin (borate) and a Zwitterionic buffer Bosisio, 1994) and agarose gel analysis (U.S. Pat. Nos. 5.246, (CAPS) at a basic pH (pH of 9 to 12). The profile obtained 558; 4.222,836; Menard, 1980). Capillary electrophoresis (shown in FIG. 1 of the present application) under the condi analyses include isoelectric focusing analysis (Molteni, 35 tions described show a rather small separation of the peaks for 1994; Hempe, 1994), free solution analysis using an anti-A HbA (described as being the peak for HbA in patent U.S. antibody (U.S. Pat. No. 5.431,793), free solution analysis Pat. No. 5,599.433) and Hb Ao (see Example 1). Further, using a naked capillary (U.S. Pat. No. 5,599.433) and free analysis of the purified minor fractions shows that the solution analysis using a dynamic coating (EP 0733 900 A2; HbA, fractions co-migrate with HbA, interfering with U.S. Pat. No. 5,611,903). 40 the final assay result (see Example 2 and FIG. 2 of the present Analysis by electrophoresis of a biological sample can application). In fact, that technique separates the Hb Ao and separate the various proteins present in the sample, in particu HbA fractions, but does not separate the HbA fractions lar the various haemoglobins, and enables determination of themselves. the quantity and/or proportion of one or several protein(s) of The technique for capillary analysis with a dynamic double interest in the biological sample. In capillary electrophoresis, 45 coating (EP 0733900 A2; U.S. Pat. No. 5,611,903) is used in in a capillary filled with an electrolyte, the proteins of a the only test based on capillary electrophoresis which is com biological sample migrate under the effect of an electric field mercially available (Analis CEofixTM HbA kit). It consists from the anode towards the cathode as a function of their mass of a first wash of the capillary with an “initiator containing a and their charge. They thus produce an electrophoretic migra polycation (in Solution, pH 4.6) resulting in coating the wall tion profile comprising a series of peaks (also termed frac 50 of the capillary with said polycation. A second wash is then tions), each of them corresponding to one or several proteins. carried out with the analysis buffer containing a polyanion Abnormal haemoglobins such as HbS, HbC and HbE have a (pH 4.6), which has the effect of providing a second coating different electrophoretic mobility from that of HbA. Further, layer by interaction with the polycation. The quantity of nega because of the ose residuebound to the N-terminal amino acid tive electrical charges then present on the internal wall of the of the beta chains, the HbA has a reduced isoelectric point 55 capillary is even higher than with a naked capillary, resulting and, as a result, an electric charge that is slightly different in a much higher electroosmotic flux. The resolution obtained from that of the other HbA type haemoglobins. Thus, in an between the various forms of haemoglobin analyzed (includ electrophoretic field or in an ion exchange resin, the migra ing glycated Al haemoglobins) is satisfactory, with short tion rate of HbA is different from that of Hb Ao, which analysis times. From a practical viewpoint, this double coat enables separating HbA from HbA, which has a different 60 must be re-applied between each analysis using a precise charge. procedure provided by the kits manufacturer but which is The advantage of capillary electrophoresis also resides in only described for a mono-capillary apparatus ((P/ACE, the fact that only very small quantities of the biological Beckman) not adapted to routine analyses. sample to be analyzed are necessary. Further, separation by Thus, there is a need for reagents and a simple method of this technique may be very rapid, since high Voltages can be 65 implementation that can effectively separate glycated haemo used without causing the sample to heat up too much during globins, in particular HbA, from other haemoglobins, vari separation. ants, interfering forms (labile forms, acetylated forms, car US 9,222,913 B2 5 6 bamylated forms) and from other minor fractions (especially bin (including the minor fractions), whether they be normal or HbA, and Hb A.) present in a biological sample containing abnormal, as well as the many variants of said haemoglobins other haemoglobins. Ideally, such a method can produce a (at least 1000 variants of haemoglobin have been described). semi-quantitative or quantitative assay of glycated haemoglo The term “glycated haemoglobin' as used in the context of bins, in particular of HbA, directly from the electrophoretic the present application means any haemoglobin modified by profile obtained. binding one or several Sugar(s), in particular one or several The present invention proposes an alternative capillary glucose, glucose-6-phosphate, fructose 1-6 diphosphate or electrophoresis method, which enables performing a semi pyruvate, to one or several globin chain(s) (whatever the quantitative or quantitative analysis of one or several glycated globin chain). The Sugar(s) may be bound to the amino acid in haemoglobin(s) comprising at least one globin chain com 10 prising a glucose residue bound to the amino acid in the the N-terminal position on a globin chain and/or to an amino N-terminal position, contained in a biological sample in par acid with a free amino acid group (for example lysine). ticular comprising other haemoglobins (glycated and/or non The term “globin chain” as used in the present application glycated). Said method of the invention exploits both the means any globin chain known to the skilled person, in par structural characteristics of the glucose molecules bound to 15 ticular a chain selected from alpha (C), beta (B), delta (8) and said glycated haemoglobins and the charge differences exist gamma (Y) chains, whether normal or abnormal, or a variant ing between the various haemoglobins of the sample. of one of said chains. According to a particular embodiment, The inventors have shown that by using a particular buffer said “globin chain” is a beta chain, for example a beta chain of composition, it is possible to obtain greatly improved sepa a A haemoglobin, in particular A, S or C. ration of glycated haemoglobin(s) comprising a glucose resi When reference is made in the embodiments of the inven due bound to the amino acid in the N-terminal position on at tion to “glycated haemoglobin', it should be understood that least one globin chain, and in particular on the beta chains, said embodiments are of particular application to each par and more particularly a greatly improved separation of ticular type of glycated haemoglobin cited in the present HbA. application. One of the advantages of the method of the invention is that 25 Unless otherwise indicated, each embodiment indicated in because said buffer composition is used, the electrophoresis this application is applicable independently of and/or in com peak corresponding to one or several types of glycated bination with any or several of the other described embodi haemoglobin(s) of interest (one or several types of haemo mentS. globin(s) comprising a glucose residue bound to the amino The capillary electrophoresis analysis method of the inven acid in the N-terminal position on at least one globin chain, 30 tion is applicable to a biological sample comprising at least for example HbA) is displaced, when it is present in a one glycated haemoglobin comprising one or several glucose biological sample, with respect to the position of the peak residue(s) and necessarily comprising, on one or several which would be obtained without the buffer composition. globin chain(s) and in particular on the beta chains, a glucose This displacement does not interfere with the separation of residue bound to the amino acid in the N-terminal position. the other proteins, in particular the other haemoglobins (gly 35 These haemoglobins are designated as "haemoglobins gly cated and/or non-glycated) of the sample. This in particular cated in the N-terminal position' in the present application. enables separation of HbA. from other minor HbA (in par Said method enables separation of one or several haemo ticular HbA, and HbA). As a consequence, means that the globins glycated in the N-terminal position from other gly results of the analysis can be read reliably and allows the cated haemoglobins (i.e. glycated haemoglobins wherein the accurate assay of one or several glycated haemoglobin(s) of 40 globin chains are devoid of a glucose residue bound to the interest comprising one or several glucose residue(s) with one amino acid in the N-terminal position) or from non-glycated glucose bound to the amino acid in the N-terminal position of haemoglobins, which may be present in the analyzed biologi at least one globin chain (for example haemoglobin A). cal sample. The method of the invention thus constitutes a method of According to a particular embodiment, the amino acid in choice with a view to establishing a diagnosis or of monitor 45 the N-terminal position on the beta chain(s) is valine. ing diabetic patients, in particular to evaluate the effective In a preferred embodiment, the analysis method of the ness of an anti-diabetic treatment. invention is intended for the analysis of a biological sample Thus, the present application provides a method for analy comprising haemoglobin A. In particular, it is appropriate sis, by capillary electrophoresis, of glycated haemoglobins for separating haemoglobin A. from other haemoglobins or (one or several glycated haemoglobin(s)) comprising one or 50 from the other haemoglobins (differently glycated or non several glucose residue(s) contained in a biological sample glycated) which may be present in the analyzed biological comprising one or several haemoglobin(s), said method com sample, and in particular from other minor fractions, for prising using a buffer composition comprising at least one example HbA, and from HbA. compound which is capable of specifically complexing glu According to a particular embodiment, the analysis cose residues (one or several residue(s)) of glycated haemo 55 method of the invention can separate haemoglobin Sand/or globins (one or several glycated haemoglobin(s)) of the bio haemoglobin C from the other haemoglobins (differently logical sample and also of providing said glycated glycated or non-glycated) which may be present in the ana haemoglobins with several negative electrical charge(s) at an lyzed biological sample. alkaline pH (i.e. a pH of more than 7). The term “buffer composition” means a composition, in The term “several as used in the context of the present 60 particular a solution, which retains approximately the same application means at least two, i.e. two or more than two, for pH despite the addition of small quantities of an acid or a base example, two, three, four, five, six, seven, eight, nine, ten or or despite dilution. more than ten. The term “to complex” or “complexing is used in the The term “at least one' as used in the context of the present present application to signify that an association (chemical or application means one or several. 65 simply physical) occurs between two entities, in particular The term “haemoglobin' as used in the context of the between two molecules (for example a small molecule and a present application means any form or fraction of haemoglo macromolecule) via functional groups. US 9,222,913 B2 7 8 In a preferred embodiment, one of these two entities (the in the sense of the present invention may be used in the compound which is capable of complexing glucose residues) context of the present invention. An appropriate antibody may combines with (for example reacts with) a cis-diol group, in be generated using any method which is known to the skilled particular with two vicinal hydroxyl groups of a glucose person and may, for example, be a polyclonal or monoclonal residue from the other entity (a glycated haemoglobin). antibody, a chimeric antibody or a fragment of antibody, for As can be seen in the “examples' section, “specifically example a Fab fragment. complexing the glucose residue(s) of one or several glycated As can be seen in the “examples' section, boric acid is not haemoglobin(s) and providing said glycated haemoglobin(s) a chemical compound which is capable of specifically com with negative charges at an alkaline pH in the present appli plexing glucose residues and of providing negative charges at cation means that the compound used in the context of the 10 an alkaline pH in the sense of the present invention as it does present invention allows to displace the electrophoretic not enable performing a reliable analysis and in particular a migration peak corresponding to a haemoglobin glycated in reliable assay of glycated haemoglobins and in particular of the N-terminal position by glucose, beyond the electro HbA. Examples of appropriate chemical compounds are phoretic migration Zone corresponding to haemoglobins gly defined in more detail below. cated in the N-terminal position by another sugar (for 15 Each glucose residue complexed with the compound example glucose-6-phosphate, fructose 1-6 di-phosphate, or which is capable of specifically complexing glucose residues pyruvate), and preferably outside the Zone of the electro interacts with a different molecule of compound. phoretic migration peaks corresponding to other haemoglo According to a particular embodiment, when it is present in bins of the sample. Thus, the minor fractions which do not sufficient quantity in the buffer composition of the invention, comprise any glucose residue bond to the N-terminal amino said compound is in particular capable of specifically com acid of the beta chains, in particular the A and A, fractions, plexing the N-terminal glucose residue(s) of a glycated hae do not interfere with the analysis of the HbA according to moglobin, i.e. for each globin chain (in particular beta) com the method of the invention. prising a glucose residue bond to the amino acid in the As an example, the capacity of a compound to specifically N-terminal position, of specifically complexing said glucose complex glucose residues of one or several glycated haemo 25 residue. Said compound is thus capable of specifically com globin(s) and to provide negative charges at an alkaline pH in plexing each glucose residue which is bound in the N-termi the context of the invention may be demonstrated using the nal position on the beta chains of a A haemoglobin or of a following test: a reference sample (for example “HbA. S. or Chaemoglobin. HbA, mixture', 'A' and “A” from Exocell, USA), con According to a particular embodiment, when it is present in taining various purified fractions of glycated haemoglobin 30 sufficient quantity in the buffer composition of the invention, and in particular the Ao and A fractions or the Ao, A, and said compound is capable of specifically complexing all the A fractions, is introduced into a capillary electrophoresis glucose residues of the haemoglobins glycated in the N-ter capillary containing the following buffer composition: 200 minal position by a glucose. mM of CHES, 20 mM of putrescine, between 10 and 120 mM According to a particular embodiment, when it is present in (for example 30 mM or 50 mM) of test compound, if neces 35 sufficient quantity in the buffer composition of the invention, sary 2.5 g/l of sodium chloride, water, and if necessary, a base said compound is capable of specifically complexing all of to adjust the pH to a value greater than 9, for example 9.4. the glucose residues of a haemoglobin, regardless of their Naked capillary free solution capillary electrophoresis is then position on the globin chain(s). Thus, when said compound is carried out, for example using a Capillaries(R (Sebia) appa present in sufficient quantity in the buffer composition of the ratus, at a wavelength of 415 nm. Next, the electrophoretic 40 invention, a haemoglobin molecule comprising a number X of profile obtained is studied; if the separation between the peak glucose residues can bind a number X of molecules of said corresponding to HbA and the peak corresponding to HbA compound. (or, depending on the type of sample used, between the peak By specifically complexing with one or several glucose corresponding to HbA and the peaks corresponding to residue(s) of glycated haemoglobin(s) of a biological sample, Hb Ao, HbA, and Hb A) is complete, then the test com 45 said compound provides said glycated haemoglobin or hae pound is considered to be capable of specifically complexing moglobins with several negative electrical charges at an alka glucose residues and of providing negative charges at an line pH, i.e. enables Surcharging these glycated haemo alkaline pH in the sense of the invention. In contrast, if the globin(s) with negative electric charges at an alkaline pH. peak corresponding to HbA and that which corresponds to Said compound provides each glucose residue of the same HbA, (or, depending on the type of sample used, the peak 50 haemoglobin complexed thereby with several negative elec corresponding to HbA and the peaks corresponding to tric charges at an alkaline pH. Because of this provision of Hb Ao, HbA, or HbA), are Superimposed or overlap, then negative electric charges at an alkaline pH, the electro the test compound is not considered to be appropriate for phoretic mobility of the haemoglobins comprising at least carrying out the invention. one glucose residue is modified as a result of their complexing The concentration of the test compound in the test indi 55 with said compound. This is manifested on an electrophoretic cated above is given solely by way of indication; if a concen profile by the displacement of the electrophoresis peak(s) tration of 10 to 120 mM of test compound cannot produce corresponding to the glycated haemoglobins comprising one Sufficient separation between the peak corresponding to or several glucose residue(s) which are present in the ana HbA and the peak corresponding to the other haemoglobins lyzed biological sample. of the sample in the context of the test indicated above, it is 60 All of the haemoglobins comprising at least one glucose possible to go beyond this concentration range in order to residue which are present in the biological sample form a obtain optimal separation of the peak corresponding to complex with the compound complexing glucose and provid HbA. ing negative electric charges at an alkaline pH (of course Any chemical compound (i.e. generally an organic mol providing that said compound is present in Sufficient quantity ecule), any antibody, polypeptide, peptide, or any other com 65 to complex with each of these haemoglobins). However, the pound which is capable of specifically complexing glucose different haemoglobins and in particular the different haemo residues and of providing negative charges at an alkaline pH globins comprising at least one glucose residue all have a US 9,222,913 B2 9 10 different isoelectric point (due to the nature of their globin Thus, in the case of HbA, in general the percentage of chains, the number and the position of the glucose residue(s) HbA is measured with respect to the quantity of one or on those globins chains and the number, nature and position several other A haemoglobin(s); in general, the area of the of other Sugars that may be bound to said haemoglobins). electrophoretic peak corresponding to HbA is divided by Because of the negative electric charges at an alkaline pH the area of the peak corresponding to HbA or by the sum provided by the complexing of each glucose residue with said (area of peak corresponding to HbA+area of peak corre compound, the difference in charge between these various sponding to HbA), by the sum (area of all of the HbA peaks) haemoglobins is even more marked when they are in the form or by the sum (area of peak corresponding to HbA+area of which is complexed with said compound. peak corresponding to HbA+area of peak corresponding to The glycated haemoglobins which include a glucose resi 10 HbA), or even by the total surface area of the electrophoretic due bound to the amino acid in the N-terminal position on at profile. least one of their globin chains (in particular on the beta chains) thus have at least one glucose more than the other The assay may also be quantitative; the results are then haemoglobins (differently glycated or non-glycated). In par expressed in millimoles (mmoles) of a given haemoglobin per ticular, haemoglobin A has at least one more glucose on the 15 mole of another haemoglobin or other haemoglobin(s) beta chains than the other minor fractions of haemoglobin A. present in said sample, for example in mmoles of HbA per As a consequence, when they are complexed according to the mole of HbA. invention, these haemoglobins glycated in the N-terminal According to a particular embodiment of the invention, the position are more heavily loaded with negative electric compound specifically complexing glucose residue(s) of gly charges at an alkaline pH by said compound than the other cated haemoglobin(s) and providing said glycated haemoglo haemoglobins (the glycated haemoglobins not comprising a bins with negative charges at an alkaline pH comprises sev glucose residue bound to the N-terminal amino acid of the eral functional groups, in particular two or more than two globin chains or the non-glycated haemoglobins). functional groups. Thus, by increasing the difference in electrophoretic At least one of said functional groups specifically com mobility existing between the various haemoglobins 25 plexes a glucose residue (in particular a glucose residue bond comprising glucose and/or between one or several haemoglo to the amino acid in the N-terminal position on a globin chain) bin (s) comprising glucose and the other haemoglobins by interacting, for example, with a cis-diol group, in particu present in the analyzed biological sample, better separation is lar with two vicinal hydroxyl groups, of a glucose residue. As achieved between one or several haemoglobin(s) glycated in an example, this(these) group(s) may be a boronate group, in the N-terminal position and the other haemoglobin(s) which 30 may be present in said sample, in particular between haemo particular a boronate group as defined in the present applica globin A and the other minor fractions of haemoglobin A tion. haemoglobin which may be present in said sample. Said glucose complexing group(s) may provide glycated Thus, the method of the invention can effectively separate haemoglobin with one negative electric charge at an alkaline haemoglobins glycated in the N-terminal position, in particu 35 pH per complexed glucose residue, i.e. one or several nega lar HbA, from other haemoglobins, from certain variants tive charge(s) at an alkaline pH per complexed haemoglobin and from other minor fractions present in a biological sample molecule (one negative charge if the haemoglobin molecule containing other haemoglobins. Furthermore, conventional comprises only one glucose residue and n negative charges if interfering molecules such as labile, acetylated or carbamy the haemoglobin molecule comprises n glucose residues). lated forms do not interfere with the assay of HbA, using the 40 The other, one of the others, or the other functional method of the invention. In particular, the method of the group(s) of said compound (i.e. the functional group(s) which invention enables to avoid interferences which may result, in do not complex with glucose) provide(s) glycated haemoglo the absence of negative electric charges at an alkaline pH bin with one or several negative electric charge(s) at an alka provided by complexing, from the co-migration of haemo line pH for each complexed residue of glucose of a molecule globin A and by other minor fractions of haemoglobin A 45 of said haemoglobin. (including HbA, and HbA) present in the biological Thus, in this particular embodiment of the invention, each sample, during the electrophoretic separation step. molecule of compound specifically complexing glucose resi The separated haemoglobin(s) glycated in the N-terminal dues according to the invention comprises at least one func position may then be assayed in the presence of other or of the tional group which is capable of complexing a glucose resi other haemoglobins which may be present in the analyzed 50 due of a glycated haemoglobin (and in particulara N-terminal biological sample. Thus, as an example, HbA may be glucose residue of a haemoglobin glycated in the N-terminal assayed in the presence of other minor fractions of haemo position) and at least one functional group which does not globin A and in particular in the presence of HbA and/or complex glucose but enables Surcharging said glycated hae HbA, without these other minor fractions present in the moglobin with negative charges at an alkaline pH since it analyzed biological sample interfering with that assay. 55 provides said glycated haemoglobin with one or several The term “to assay” or “assay” in the present application Supplemental negative charge(s) at an alkaline pH. The func means that the quantity of haemoglobin or haemoglobins of tional group which is capable of complexing glucose may interest, possibly in the presence of one or several other also provide a negative electric charge at an alkaline pH. haemoglobin(s) of the analyzed biological sample, is deter According to a particular embodiment of the invention, the mined and/or the proportion of said haemoglobin(s) of inter 60 compound specifically complexing glucose residues and pro est is determined with respect to the total quantity of haemo viding negative charges at an alkaline pH (more precisely globin or with respect to the quantity of certain haemoglobins each molecule of said compound) provides a glycated hae present in said sample. moglobin molecule, and in particular to a haemoglobin gly The assay carried out in the context of the invention may be cated in the N-terminal position, with several negative elec semi-quantitative; thus, the percentage of a given haemoglo 65 tric charges at an alkaline pH, for each complexed residue of bin is measured with respect to the quantity of another hae glucose of said glycated haemoglobin molecule. Preferably, moglobin or the other haemoglobin(s) present in said sample. for each complexed glucose residue, it provides one glycated US 9,222,913 B2 11 12 haemoglobin molecule with at least two (in particular two, In the context of the present application, the term "com three, four, five or six) negative electric charges at an alkaline pound with general formula RB(OH), also means any ionic pH. forminequilibrium therewith, in particular RB(OH), or any According to a particular embodiment of the invention, the form that is capable of being inequilibrium therewith depend functional group or at least one of the functional groups ing on the conditions of the medium. specifically complexing glucose residues is negatively In the context of the present application, the term "salt” charged at an alkaline pH. denotes any salt and in particular a sodium, lithium or potas According to a particular embodiment of the invention, the sium salt. compound which is capable of specifically complexing glu The group R of the boronate compound of the invention cose residues of glycated haemoglobins and of providing said 10 may also include other functions and/or heteroatoms, in addi glycated haemoglobins with negative charges at an alkaline tion to the aryl, alkyl, aralkyl function(s) or one of their pH comprises one or several groups which are ionisable (in combinations. particular anionisables) at an alkaline pH. Said compound According to a particular embodiment, for each glucose may be of various types. For example, the compound may residue complexed with the boronyl or boronate group of the comprise one or several carboxylate(s), carboxyl(s), Sulpho 15 boronate compound of the invention or its salt, the group R nate(s) and/or Sulphonyl(s). Said compound may in particular provides two or more (preferably two) negative electric be a polycarboxylic acid, in particular a dicarboxylic or tri charges at an alkaline pH. carboxylic acid. Said compound may also be a polysulphonic According to a particular embodiment, the group R con acid, in particular a disulphonic or trisulphonic acid. sists of one or several aryl(s), alkyl(s) (linear, branched or According to a particular embodiment of the invention, the cyclic) and/or aralkyl(s) and/or a combination thereof. group, one of the groups or the groups providing a glycated According to a particular embodiment, the aryl(s), alkyl(s), haemoglobin molecule with one or several negative electric aralkyl(s) or other functional groups and/or heteroatoms or charge(s) at an alkaline pH for each complexed glucose resi their combinations present in the group R of the boronate due of said glycated haemoglobin molecule comprises or compound of the invention may be substituted. consists of one or several group(s) which are ionisable at an 25 The term “substituted as used in the present application alkaline pH as defined in the present application, and in may signify mono-Substituted or, in contrast, poly Substituted, particular one or several (in particular two or three) carboxy in particular di-, tri-, tetra-, penta- or hexa-Substituted. late, carboxyl, Sulphonate and/or Sulphonyl group(s). The Substituents may in particular be groups which are According to a particular embodiment of the invention, the ionizable (in particular anionisables) at an alkaline pH as group, one of the groups or the groups specifically complex 30 defined in the present application. The group R may include ing glucose residues comprise or consist of one or several one or several heteroatoms, or other functional groups. boronate group(s). According to a particular embodiment of the invention, the In the present application, the term “boronate group'. group R of the boronate compound provides a glycated hae means a group with formula: moglobin molecule with one or several negative electric 35 chargesatanalkaline pH for each complexed glucose residue. Preferably, for each complexed glucose residue, it provides a -B-D glycated haemoglobin molecule with one, two, three or four negative electric charges at an alkaline pH. D2 Aboronate compound as defined above in particular com 40 prises boronic acids, and in particular phenylboronic acids. in which D and D are selected, independently of each other, According to a particular embodiment of the invention, the from a hydroxyl group and a group Susceptible of being compound which is capable of specifically complexing glu hydrolyzed to produce a hydroxyl group in aqueous solution, cose residues of glycated haemoglobins and of providing said in particular at an alkaline pH. glycated haemoglobins with negative charges at an alkaline In a particular embodiment of the invention, said boronate 45 pH is a poly Substituted phenylboronate, in particular a disub group is the group —B(OH), (also known as a boronyl stituted phenylboronate, for example disubstituted with car group) or an ionized form, in particular —B(OH). boxyl and/or Sulphonyl groups. In a particular embodiment of the invention, the compound According to a particular embodiment of the invention, the which is capable of specifically complexing glucose residues group R of the boronate compound or its salt is a diacid, in of glycated haemoglobins and providing said glycated hae 50 particular a dicarboxylic acid. According to a particular moglobins with negative charges at an alkaline pH is: embodiment of the invention, the boronate compound is a (i) a boronate compound, with general formula RB(OH) dicarboxyphenylboronic acid, preferably selected from 3.5- (compound also known as boronic acid), or with general dicarboxyphenylboronic acid and 3,4-dicarboxyphenylbo formula RB(OH), wherein the group R comprises at ronic acid. least one aryland/oran alkyl (linear, branched or cyclic) 55 Thus, by way of example, the boronate compound and/or an aralkyl and/or a combination thereof, and said employed may be 3,5-dicarboxyphenylboronic acid (3.5- group R provides glycated haemoglobins with one or dCPBA). This compound is commercially available, in par several negative electric charge(s) at an alkaline pH for ticular from the supplier Combi-blocks Inc. (San Diego, each glucose residue complexed with the boronate USA), under the trade name 3,5-dicarboxyphenylboronic group; or 60 acid and from Apollo Scientific Ltd (Cheshire, United King (ii) a salt of said boronate compound. dom), under the trade name 3,5-dicarboxybenzeneboronic Thus, at an alkaline pH, in addition to the negative electric acid. charge at an alkaline pH provided by the boronyl or boronate In the buffer composition of the invention, the concentra of the boronate compound or its salt, the group R provides tion of the compound which is capable of specifically com glycated haemoglobins with one or several Supplemental 65 plexing glucose residues of glycated haemoglobins and of negative electric charge(s) for each glucose residue com providing said glycated haemoglobins with negative charges plexed with the boronyl or boronate group. at an alkaline pH is generally in Stoichiometric excess with US 9,222,913 B2 13 14 respect to the total quantity of proteins, in particular with 1.5-diaminopentane (cadaverine), 1-6-diaminohexane, sper respect to the total quantity of all of the haemoglobins present mine and DETA, or a derivative of said aliphatic diamine or a in the biological sample or with respect to the total quantity of mixture thereof. all of the haemoglobins comprising glucose present in the The term "derivative' as used herein means an aliphatic biological sample. Thus, the quantity of this compound in the polyamine, a cyclic polyamine, a salt (for example a sodium buffer composition is greater than the quantity necessary for salt) or one of their mixtures. all of the glucose residues of the haemoglobins present in the According to a particular embodiment of the invention, the sample to be complexed by said compound when the sample flow retardant is selected from putrescine, its derivatives and or an aliquot of that sample is diluted in the buffer composi mixtures thereof. 10 According to a particular embodiment of the invention, the tion. Thus, for each molecule of glycated haemoglobin com flow retardant is putrescine. In particular, putrescine is in the prising glucose present in the analyzed biological sample, pure form, a salt being optionally added. there are at least as many molecules of that compound in the According to another particular embodiment of the inven buffer composition as there are glucose residues present in tion, the flow retardant is putrescine hydrochloride (or this glycated haemoglobin molecule. This enables achieving 15 putrescine-2HCl). a complete separation of the glycated haemoglobin or gly In the buffer composition of the invention, the concentra cated haemoglobins of interest from the other haemoglobins tion offlow retardant is advantageously in the range 0.10 to 40 also present in the analyzed biological sample. mM, preferably in the range 10 to 30 mM and more preferably According to a particular embodiment of the invention, in in the range 15 to 25 mM, for example 20 mM. the buffer composition of the invention, the concentration of The base optionally added to the buffer composition allows compound which is capable of specifically complexing glu the pH of said composition to be adjusted. In particular, a base cose residues of glycated haemoglobins and of providing said belonging to the hydroxide family may be used, in particular glycated haemoglobins with negative charges at an alkaline a base selected from lithium hydroxide, sodium hydroxide, pH is in the range 0.10 to 100 mM, preferably in the range 10 potassium hydroxide, rubidium hydroxide, caesium hydrox to 60 mM, and more preferably in the range 20 to 50 mM, for 25 ide, francium hydroxide, a mono-, di-, tri- or tetra-alkyl example 30 mM or 50 mM. ammonium hydroxide, and mixtures thereof. The expression “in the range X to y' as used in the present According to a particular embodiment of the invention, the application means that the limits X and y are included in the base optionally added to the buffer composition is sodium indicated range X-y. hydroxide. According to a particular embodiment, the buffer compo 30 Advantageously, sufficient base is added to the buffer com sition of the invention further comprises: position so that its pH is 9.0 or more, preferably in the range a buffer compound with a pKa in the range 8.0 to 11.0: 9.0 to 11.0 and more preferably in the range 9.0 to 10.0, for and/or example in the range 9.3 to 9.5, and still more preferably a pH a flow retardant; and/or of 9.4 or 9.5. An alkaline pH of more than 9 can produce negatively charged haemoglobin fractions (the isoelectric a base; and/or 35 point of haemoglobins being in the range 6.05 to 7.63). a salt; and/or According to a particular embodiment of the invention, the an appropriate dilution Solution, for example water. salt which is optionally present in the buffer composition of Thus, the buffer composition of the invention comprises or the invention is a sodium salt, preferably sodium chloride. consists of (i) a compound which is capable of specifically The buffer composition may comprise for example 2.5 g/l of complexing glucose residues of glycated haemoglobins and 40 sodium chloride. of providing said glycated haemoglobins with negative According to a particular embodiment of the invention, charges at an alkaline pH, and (ii) one or several and in when the buffer composition comprises putrescine, in par particular all of the compounds selected from: a buffer com ticular pure putrescine, it further comprises a salt (in particu pound having a pKa in the range 8.0 to 11.0, a flow retardant, lar a sodium salt), preferably sodium chloride, for example a base, a salt, an appropriate diluting Solution (for example 45 2.5 g/l of sodium chloride. water) and mixtures thereof. According to another particular embodiment of the inven The buffer compound is preferably a Zwitterionic com tion, the buffer composition comprises putrescine hydrochlo pound. It may in particular be selected from AMP, AMPD, ride, does not comprise sodium chloride and preferably does AMPSO, bicine, CABS, CAPS, CAPSO, CHES, HEPBS, not comprise any salt. methylamine, TABS, TAPS, taurine, tricine and Tris; in par 50 According to a particular embodiment of the invention, the ticular, CAPS, CAPSO or CHES are selected, preferably buffer composition comprises or consists of CHES. These compounds have a high buffering power at the 200 mM of CAPSO; target pH (pH 8-11) and are particularly suitable for obtaining 10 mM of putrescine (for example either putrescine-2HCl good focussing of the haemoglobins. or pure putrescine and Sodium chloride); The concentration of buffer compound in the buffer com 50 mM of 3,5-dicarboxyphenylboronic acid; position of the invention is generally in the range 20 to 500 55 water, and mM, preferably in the range 50 to 400 mM, and more pref if necessary, a base, for example sodium hydroxide, to erably in the range 100 to 350 mM or in the range 150 to 300 adjust the pH, for example to a value greater than 9 or 10, mM, for example approximately 200 mM or approximately and preferably to a value of 10.2: 300 mM. or, more preferably, of: The flow retardant is intended to strengthen the effect of the 60 200 mM of CAPSO; differences in electric charge in order to obtain a good reso 15 mM of putrescine (for example either putrescine-2HCl lution of separation between the various haemoglobins. This or pure putrescine and Sodium chloride); type of compound acts by reducing the electro-osmotic flux, 100 mM of 3,5-dicarboxyphenylboronic acid; which retards migration of the various fractions and can water, and increase their separation. The flow retardant used may be an 65 if necessary, a base, for example sodium hydroxide, to aliphatic diamine, in particular an aliphatic diamine selected adjust the pH, for example to a value greater than 9 or 10, from 1,3-diaminopropane, 1,4-diaminobutane (putrescine), and preferably to a value of 10.2: US 9,222,913 B2 15 16 or, still more preferably, of: tion Capillaries(R Hemoglobin(e) from Sebia, the haemolyz 200 mM of CHES; ing solution MINICAP(R) Hemoglobin(e) from Sebia, the 20 mM of putrescine (for example either putrescine-2HCl haemolyzing solution Hydragel(R) HbA from Sebia, pure or pure putrescine and Sodium chloride); water, water Supplemented with Surfactant, in particular 30 mM of 3,5-dicarboxyphenylboronic acid; water supplemented with Triton X 100 (for example 1 g/L of water, and Triton X100), and mixtures thereof. if necessary, a base, for example sodium hydroxide, to According to a particular embodiment, the method for adjust the pH, for example to a value greater than 9, and analysis by capillary electrophoresis of the invention com preferably to a value of 9.4. prises the following steps: According to another particular embodiment of the inven 10 a) introducing the buffer composition of the invention and tion, the buffer composition comprises or consists of the the biological sample into an electrophoresis capillary; components indicated above in the concentrations indicated above, and one or several salt(s), in particular a sodium salt, and especially sodium chloride, for example 2.5 g/l of sodium b) separating the constituents of said biological sample by chloride. 15 capillary electrophoresis. As illustrated in the example section, such buffer compo These two steps are generally preceded by a step for dilu sitions enable achieving a perfect separation of haemoglobin tion of the biological sample, for example in a haemolyzing HbA from the other haemoglobins present in the biological Solution. This dilution step may in particular be carried out in sample analyzed, as well as mutual separation of the various the sample support, for example in a Capillaries(R) (Sebia) minor fractions (including HbA, HbA, and HbA) in a dilution segment or in the MINICAP(R) (Sebia) sample cup. manner unequalled in the field of free solution capillary elec In step a), the buffer composition of the invention and the trophoresis. biological sample may be introduced separately (simulta The term “biological sample' as used in the present appli neously or not) into the same electrophoresis capillary, the cation means any biological fluid comprising red blood cells. mixture then being produced in the capillary. As an example, Said biological fluid may derive from a healthy or sick human 25 it is possible to introduce the buffer composition of the inven patient (for example a diabetic patient) or be of animal origin, tion first, then the sample into the electrophoresis capillary. in particular deriving from a non-human mammal (healthy or Alternatively, the biological sample may be introduced sick). Said biological liquid (human or non-human mammal) into an electrophoresis capillary in step a) in the form of a may, for example, be blood, in particular normal or non mixture with the buffer composition of the invention. normal blood, washed, decanted, centrifuged, haemolysed or 30 Depending on the type of capillary electrophoresis appa whole blood. The biological samples may also be of synthetic ratus used and as a function of the number of analyses to be or purified origin. carried out, a single capillary or several capillaries in parallel The invention is of particular application in the analysis of are used. When a single capillary is used, this capillary is blood samples, in particular for the analysis of a blood sample generally used several times in Succession, in order to carry deriving from a diabetic patient or from a diabetic non-human 35 out several analyses. Of course, when several capillaries are mammal. used, if necessary several electrophoretic migrations can be According to a particular embodiment, said biological carried out in Succession, during which several capillaries are sample comprises or consists of several haemoglobins, in used in parallel. particular several glycated haemoglobins comprising a glu The step for separating the constituents of the biological cose residue bound to the amino acid in the N-terminal posi 40 sample by capillary electrophoresis in particular consists of tion on at least one globin chain (for example on the beta applying to the capillary(ies) an electric field with a sufficient chains), and more particularly haemoglobin A. Voltage to allow the separation of one or several glycated The biological sample used may be diluted prior to analysis haemoglobin(s) of interest from other proteins and in particu by capillary electrophoresis with a suitable diluting solution, lar from other haemoglobins which may be present in the for example a haemolyzing solution and/or an analyzing 45 biological sample. buffer solution, in particular the buffer composition of the The conditions for carrying out liquid capillary electro invention. Preferably, the analysis is carried out using a phoresis are the conditions generally used by the skilled per sample of haemolyzed blood. son for the steps of introducing the sample and the buffer According to another particular embodiment, the biologi composition into the capillary(ies) and separating the con cal sample is initially diluted in a haemolyzing solution, then 50 stituents of the sample by electrophoretic migration. The in the buffer composition of the invention. electric field applied may, for example, be approximately 400 The term “haemolyzing solution' as used in the present V/cm. They usually comprise washing the capillaries with a application designates a solution capable of causing washing solution, washing with the buffer composition used haemolysis of red blood cells, i.e. the destruction of red blood for the analysis, optionally diluting the sample one or more cells, and thus of liberating haemoglobin. Depending on its 55 times, introducing the sample into the capillary(ies), migra composition, it can carry out total lysis of the red blood cells, tion and detection. These steps may be carried out by auto optionally by employing a small additional mechanical action mated machines. (Vortex, stirring, etc). The haemolyzing solution may include The conditions for carrying out capillary electrophoresis the usual additives for cell lysis, such as Triton X100, which are, for example, conditions Suitable for using the Capillar is usually used in a concentration of 1 g/L. The haemolyzing 60 ies(R) automated machine from Sebia or the MINICAPR) from Solution may also optionally include a compound which is Sebia. capable of specifically complexing glucose residues of gly The step for separating the constituents of the biological cated haemoglobins and of providing said glycated haemo sample is generally followed by a step for detection of one or globins with negative charges at an alkaline pH as defined in several protein(s), in particular one or several haemoglobins the present application. 65 present in the biological sample, and more particularly one or By way of example, the haemolyzing Solution may be several glycated haemoglobins of interest present in the bio selected from the group constituted by the haemolyzing solu logical sample, for example haemoglobin A. US 9,222,913 B2 17 18 This detection step may in particular be carried out by As an example, in a particular embodiment, the electro measuring the absorbance, for example at a wavelength of phoresis capillary(ies) is (are) of fused silica. approximately 415 nm, which is specific to haemoglobin; the The present invention also pertains to the use of the capil haemoglobins may be analyzed at a wavelength of approxi lary electrophoresis analysis method according to the inven mately 200 nm, but in order to avoid interference with plasma tion for separating one or several glycated haemoglobin(s) proteins in particular, they are preferably analyzed at a wave comprising glucose, more precisely one or several glycated length of 415 nm. haemoglobin(s) each comprising, on one or several globin In the context of the invention, the results of the electro chain(s) (for example on the beta chains), one or several phoretic separation may be presented as illustrated in the glucose residue(s) bound to the amino acid in the N-terminal examples, in the form of electrophoretic profiles generated 10 position, from other proteins, in particular from at least one from a detection signal proportional to the quantity of hae other haemoglobin, and preferably from the other haemoglo moglobin detected. Thus, the method of the invention gener bins present in a biological sample and, if appropriate, to ally further comprises a step for generating an electrophero assay said glycated haemoglobin(s). gram from the detection signal. The present invention also pertains to a buffer composition As indicated above, when it is present in the analyzed 15 Suitable for capillary electrophoretic analysis of a biological biological sample, a haemoglobin of interest may be quanti sample comprising haemoglobins and in particular one or fied. To this end, the Surface area of the peak corresponding to several glycated haemoglobin(s), more particularly one or said haemoglobin is determined. several glycated haemoglobin(s) comprising one or several Thus, said method generally also comprises a step for glucoses on one or several globin chains (for example on the determining, in particular from an electropherogram, the beta chains). Said buffer composition comprises at least one quantity of one or several haemoglobin(s) present in the bio compound that is capable of specifically complexing glucose logical sample and/or the proportion of one or several hae residues of glycated haemoglobins and also of providing said moglobin(s) present in the biological sample with respect to haemoglobin(s) with several negative electric charges at an the total quantity of proteins, to the total quantity of haemo alkaline pH. globin and/or to the quantity of certain haemoglobins (for 25 Said buffer composition is in particular that used to carry example with respect to the quantity of HbA or HbA) present out the method for capillary electrophoresis of a biological in the biological sample. In particular, the proportion of gly sample presented in the present application. cated haemoglobin A, S, and/or C present in the biologi In particular, according to a particular embodiment of the cal sample with respect to the total quantity of all of the invention, the buffer composition comprises a polycarboxylic haemoglobins or with respect to the quantity of certain hae 30 boronic acid, in particular a dicarboxylic acid or a tricarboxy moglobin(s) present in the biological sample, for example lic acid, for example 3.5-dicarboxyphenylboronic acid, as a with respect to the total quantity of A, Sand/or Chaemoglo compound which can specifically complex glucose residues bin respectively, can be determined. and provide negative electric charges at an alkaline pH. In one particular embodiment of the invention, this assay According to another particular embodiment of the inven may be obtained directly from the electrophoretic profile. 35 tion, the buffer composition comprises, in addition to the According to a particular embodiment of the invention, the compound which is capable of specifically complexing glu analysis method also comprises a step for quantification of cose residues and of providing negative electric charges at an one or several haemoglobin(s) present in the analyzed bio alkaline pH, logical sample with respect to one or several Standardized a flow retardant; and/or calibrator(s) (for example one or several reference biological 40 a buffer compound with a pKa in the range 8.0 to 11.0: samples); this enables standardization of the results. and/or Thus, in order to obtain a high level of precision during a base; and/or assay, each capillary is generally calibrated every time the a salt (in particular a sodium salt, for example sodium electrophoresis apparatus is started up, using reference bio chloride); and/or logical samples (for example standard samples containing 45 an appropriate diluting Solution, for example water. known concentrations of different natural and/or synthetic These various constituents may be as defined in the present fractions of glycated haemoglobin, purified if necessary). As application. an example, in order to assay HbA, at least two calibrators According to a particular embodiment of the invention, the are generally used, for example a first calibrator comprising a buffer composition has a pH of 9.0 or more, preferably a pH known high concentration of HbA and a second calibrator 50 in the range 9.0 to 11.0 and more preferably a pH in the range comprising a low known concentration of HbA. Then, with 9.0 to 10.0, for example a pH in the range 9.3 to 9.5, and still each capillary of the electrophoresis apparatus, the quantity more preferably a pH of 9.4. Such a pH may in particular be of HbA present in each calibrator is measured. This enables obtained by providing a Sufficient quantity of a base as to establish a regression curve (over at least two points) for defined above. each capillary, and thus the measurements carried out using 55 The buffer compositions of the invention are prepared in the various capillaries can be normalized. This also enables the usual manner for analytical buffer compositions, namely standardization of the results obtained with respect to a ref by providing the constituents in the liquid form or in the solid erence method, by using values for HbA determined by this form to be diluted, to an acceptable support. Usually, the method for the calibrators. support is water, distilled or demineralized. According to a particular embodiment of the invention, the 60 The present invention also concerns a kit comprising a capillary electrophoresis carried out is a free solution capil buffer composition of the invention and, if appropriate, lary electrophoresis, in particularis a naked capillary(ies) free instructions for use, to carry out electrophoretic analysis. In Solution capillary electrophoresis. other words, the kit of the invention may comprise or consist From the point of view of the materials used for the capil of a buffer composition of the invention and a packaging laries, those that are normal for capillary electrophoresis are 65 material and, if appropriate, instructions for use. employed. The specialist will be able to adapt the nature of the Thus, the kit of the invention in particular comprises a capillary and its size to the requirements of the analysis. compound which is capable of specifically complexing glu US 9,222,913 B2 19 20 cose residues of glycated haemoglobin and of providing The term “diabetes’ as used in the present application negative charges at an alkaline pH as defined in the present designates type 1 and/or type 2 diabetes. application. When, in addition to said compound, said kit The present invention also concerns the use of a buffer comprises other compounds, in particular one or several com composition of the invention, of a kit of the invention and/or pound(s) selected from: a buffer compound with a pKa in the of a compound which is capable of specifically complexing range 8.0 to 11.0, a flow retardant, a base, a salt (in particular glucose residues of glycated haemoglobins and of providing a sodium salt, for example sodium chloride), an appropriate negative charges at an alkaline pH as defined in the present diluting solution (for example water) and mixtures thereof, application, for the manufacture of a diagnostic kit. Said the various compounds of said kit may be packaged for diagnostic kit may in particular be used for the diagnosis of extemporaneous mixing, or, in contrast, may be packaged 10 diabetes in a human or a non-human mammal and/or to moni together, in particular in the same composition, in the form of torthe glycaemic balance of a human or non-human mammal, in particular a diabetic subject. Said kit may thus allow the a mixture. Alternatively, certain compounds of said kit may efficacy of a treatment against diabetes in a human or a non be packaged separately while others may be packaged human mammal Suffering from diabetes to be evaluated and/ together, in particular in the form of a mixture. 15 or allow a treatment against diabetes in a diabetic Subject to be According to a particular embodiment of the invention, the adapted. buffer composition of the invention is provided in one or The HbA level is generally in the range 4% to 6% (i.e. 20 several parts, to be reconstituted by the consumer before the to 42 mmoles of HbA per mole of haemoglobin in the analysis. Hence, problems with stability which might arise blood) in a human non-diabetic, and more than 7% in a for example when all of the components or Some components diabetic patient (in the absence of treatment). of said composition are packaged in the form of a mixture can In the case of HbA levels in the range 6% to 7% (i.e. a be overcome. concentration of 42 to 53 mmoles of HbA per mole of The kit of the invention may also comprise one or several haemoglobin in the blood), it is recommended that an anti Solution(s) for washing the capillaries and/or one or several diabetic treatment be commenced. dilution segment(s) and/or one or several Solution(s) suitable 25 Beyond a threshold of 8%, which is equivalent to a con for diluting the biological sample to be analyzed (for example centration of 64 mmoles of HbA per mole of haemoglobin a haemolyzing Solution, in particular a haemolyzing Solution in the blood (Panteghini and John, 2007), the patient runs an as defined in the present application) and/or one or several increased risk of developing one of the complications of reference biological sample(s) (for example natural and/or diabetes (microangiopathy, macroangiopathy, etc). It is then synthetic glycated fractions, purified if necessary) which 30 recommended that the patient’s anti-diabetic treatment be enable(s) calibrating each capillary. modified. The present invention also pertains to the use of a buffer Other characteristics and advantages of the invention will composition as defined in the present application and/or to a become apparent from the following examples and figures kit of the invention for the analysis, by capillary electrophore which illustrate the invention. sis, of (one or several) glycated haemoglobins comprising 35 one or several glucose residue(s), in particular the A, Sand DESCRIPTION OF THE FIGURES Chaemoglobins contained in a biological sample compris ing one or several haemoglobin(s). FIG. 1: electropherogram obtained on CE Agilent from a The present invention also pertains to the use of a buffer normal human blood sample using the analysis buffer composition as defined in the present application and/or a kit 40 described in patent U.S. Pat. No. 5,599.433, which contains of the invention and/or a compound which is capable of 100 mM of CAPS and 300 mM of boric acid (pH: 11.00; specifically complexing glucose residues (one or several) of temperature: 24°C.; Voltage: 6.1 kV, i.e. 190V/cm; injection: glycated haemoglobins (one or several glycated haemo 50 mbars 20s). globin(s)) and of providing this or these glycated haemoglo FIG. 2: standard electrophoretic profiles for A, and A. bin (s) with several negative electric charges at an alkaline pH 45 obtained on CE Agilent from reference samples containing to separate, by capillary electrophoresis, one or several gly different purified fractions of glycated haemoglobin using the cated haemoglobin(s) comprising a glucose residue bound to analysis buffer described in patent U.S. Pat. No. 5,599.433, the amino acid in the N-terminal position on at least one which contains 100 mM of CAPS and 300 mM of boric acid globin chain (for example on the beta chains), and more (pH: 11; temperature: 24°C.; Voltage: 6.1 kV, i.e. 190V/cm; particularly to separate, by capillary electrophoresis, haemo 50 injection: 50 mbars 20s). globin A. from other proteins, in particular from at least one FIG. 3: Standard electrophoretic profiles for A, and A. other haemoglobin and preferably from the other haemoglo obtained on CE Agilent from reference samples containing bins present in a biological sample and, if appropriate, to different purified fractions of glycated haemoglobin, using a assay said haemoglobin(s) separated thereby. buffer composition containing 200 mM of CAPSO and 10 In particular, the buffer composition of the invention, the 55 mM of putrescine but no borate compound, nor boronate kit of the invention and/or a compound which is capable of compound (pH: 10.20; temperature: 24°C.; Voltage: 6.1 kV. specifically complexing glucose residues of glycated haemo i.e. 190V/cm; injection: 50 mbars 20s). globins and of providing negative electric charges at an alka FIG. 4: Standard electrophoretic profiles for A, and A. line pH as defined in the present application are appropriate obtained on CE Agilent from reference samples containing for use, for example in the context of a method of the inven 60 different purified fractions of glycated haemoglobin, using a tion for diagnosing diabetes in a human or non-human mam buffer composition containing 200 mM of CAPSO, 10 mM of mal and/or for monitoring the glycaemic balance of a human putrescine and 50 mM deborate (pH: 10.20; temperature: 24° or non-human mammal (in particular a diabetic Subject), in C.; Voltage: 6.1 kV, i.e. 190V/cm; injection: 50 mbars 20s). particular to evaluate the efficacy of a treatment against dia FIG. 5: Standard electrophoretic profiles for A, and A. betes and/or to adapt a diabetes treatment in a diabetic sub 65 obtained on CE Agilent from reference samples containing ject. The biological sample(s) analyzed then originate from different purified fractions of glycated haemoglobin, using a said human or non-human mammal. buffer composition containing 200 mM of CAPSO, 10 mM of US 9,222,913 B2 21 22 putrescine and 50 mM of 3-carboxyphenylboronic acid (pH: putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, 10.20; temperature: 24°C.; Voltage: 6.1 kV, i.e. 190 V/cm; at a pH of 9.40, and a fused silica capillary, uncoated (tem injection: 50 mbars 20s). perature: 34° C.; Voltage: 9.4 kV, i.e. 520V/cm; injection 8 FIG. 6: Standard electrophoretic profiles for A, and A. mbars 6 s). obtained on CE Agilent from reference samples containing FIG. 15: electropherogram obtained on Capillaries(R (Se different purified fractions of glycated haemoglobin, using a bia) from a pool of normal blood comprising F and Bart's buffer composition containing 200 mM of CAPSO, 10 mM of variants, diluted to /6" in the haemolyzing solution (water+1 putrescine and 50 mM of 3,5-dicarboxyphenylboronic acid g/L de Triton X100), using a buffer composition containing (pH: 10.20; temperature: 24° C.; voltage: 6.1 kV, i.e. 190 200 mM of CHES buffer, 20 mM of putrescine and 30 mMof V/cm; injection: 50 mbars 20s). 10 FIG. 7: Standard electrophoretic profiles for A, and A. 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, and a fused obtained on CE Agilent from reference samples containing silica capillary, uncoated (temperature: 34° C.; Voltage: 9.4 different purified fractions of glycated haemoglobin, using a kV, i.e. 520V/cm; injection 8 mbars 6 s). buffer composition containing 200 mM of CAPSO, 15 mM of FIG. 16: electropherogram obtained on Capillaries(R (Se putrescine and 100 mM of 3,5-dicarboxyphenylboronic acid 15 bia) from a normal human blood sample comprising a HbD (pH: 10.20; temperature: 30° C.; voltage: 10 kV, i.e. 310 variant, diluted to /6" in the haemolyzing solution (water+1 V/cm; injection: 50 mbars 20s). g/L de Triton X100), using a buffer composition containing FIG. 8: Standard electrophoretic profiles Ao, A., and A. 200 mM of CHES buffer, 20 mM of putrescine and 30 mMof obtained on CE Agilent from reference samples containing 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, and a fused HbA and/or different purified fractions of glycated haemo silica capillary, uncoated (temperature: 34° C.; Voltage: 9.4 globin, using a buffer composition containing 200 mM of kV, i.e. 520V/cm; injection 8 mbars 6 s). CHES, 20 mM of putrescine and 30 mM of 3,5-dicarbox FIG. 17: comparison of the results obtained by the inven yphenylboronic acid at a pH of 9.40. Fused silica capillary, tors by capillary electrophoresis using the Capillaries(R) (Se uncoated (temperature: 34° C.; Voltage: 17.3 kV i.e. 520 bia) analyzer with the results obtained by HPLC with the V/cm; injection 50 mbars 20s). 25 Variant II Turbo(R) (Bio-Rad) analyzer. FIG. 9: Standard electrophoretic profiles Ao, A., and A. FIG. 18A: agarose gel of HbA, carried out on Hydrasys(R) obtained on CE Agilent from reference samples containing (Sebia) automated machine. Tracks 1 and 2: weak (5.0%) A HbA and/or different purified fractions of glycated haemo calibrator and strong (10.8%) A calibrator. Tracks 3 to 9: globin, using a buffer composition containing 200 mM of normal whole blood incubated for 3 hat 37°C. with glucose CHES, 20 mM of putrescine and 30 mM of 3,4-dicarbox 30 at a concentration of 0 g/L (reference; track 3), 1 g/L (track 4), yphenylboronic acid at a pH of 9.40. Fused silica capillary, 5 g/L (track 5), 10 g/L (track 6), 20 g/L (track 7), 30 g/L (track uncoated (temperature: 34° C.; voltage: 17.3 kV i.e. 520 8) and 50 g/L (track 9). V/cm; injection 50 mbars 20s). FIG. 18B: capillary electrophoresis profiles obtained with FIG.10: standard electrophoretic profiles for A, and A. the Capillaries(R) (Sebia) analyzer with the samples of FIG. obtained on Capillaries(R (Sebia) from reference samples 35 18A. The analysis was not, however, carried out for the containing different purified fractions of glycated haemoglo sample containing 1 g/L of glucose as there was no visible bin, using a buffer composition containing 200 mM of CHES difference in the gel (FIG. 18A). The samples were diluted to buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphe /6" in the haemolyzing solution (water+1 g/L de Triton nylboronic acid, at a pH of 9.40. Fused silica capillary, X100), using a buffer composition containing 200 mM of uncoated (temperature: 34°C.; Voltage: 9.4 kV, i.e. 520V/cm; 40 CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicar injection 20 mbars 6 s). boxyphenylboronic acid, at a pH of 9.40, and a fused silica FIG. 11: electropherogram obtained on Capillaries(R (Se capillary, uncoated (temperature: 34°C.; Voltage: 9.4 kV, i.e. bia) from a normal human blood sample diluted to /6th in the 520V/cm; injection 8 mbars 6 s). haemolyzing Solution (water+1 g/L de Triton X100), using a FIG. 18C: table summarizing values for HbA obtained on buffer composition containing (A) 200 mM of CAPSO buffer, 45 agarose gel and with the Capillaries(R) (Sebia) analyzer for the 10 mM of putrescine and 50 mM of 3,5-dicarboxyphenylbo analyses of the blood presented in FIGS. 18A and 18B. ronic acid at a pH of 10.2, or (B) 200 mM of CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic EXAMPLES acid, at a pH of 9.40. Fused silica capillary, uncoated (tem perature: 34° C.; Voltage: 9.4 kV, i.e. 520V/cm; injection 8 50 A. Apparatus and Methods mbars 6 s). Capillary Electrophoresis FIG. 12: study of the influence of the concentration of The principle of separation is free Solution capillary elec 3,5-dicarboxyphenylboronic acid in the buffer composition trophoresis at an alkaline pH (pH>9), in order to obtain nega on the separation between A and A. haemoglobins (tem tively charged haemoglobin fractions (the isoelectric point of perature: 30° C.; voltage: 10 kV, i.e. 310 V/cm). 55 haemoglobins is in the range 6.05 to 7.63). FIG. 13: electropherogram obtained on Capillaries(R (Se The capillary electrophoresis of biological samples was bia) from a normal human blood sample comprising a variant carried out on a capillary electrophoresis apparatus provided HbE, diluted to /6" in the haemolyzing solution (water+1 g/L with 8 fused silica capillaries with an internal diameter of 25 de Triton X100), using a buffer composition containing 200 microns, a useful length of 16 cm and a total length of 18 cm mM of CHES buffer, 20 mM of putrescine and 30 mM of 60 (Capillaries(R (Sebia) capillary electrophoresis system) or on 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, and a fused capillary electrophoresis apparatus provided with one fused silica capillary, uncoated (temperature: 34° C.; Voltage: 9.4 silica capillary with an internal diameter of 25 microns, with kV, i.e. 520V/cm; injection 8 mbars 6 s). a useful length of 24 cm and a total length of 32 cm (PCE FIG. 14: electropherogram obtained on Capillaries(R (Se capillary electrophoresis system from Agilent Technologies). bia) from a control AFSC diluted to /6" in the haemolyzing 65 Detection was carried out at a wavelength of 425 nm. The solution (water+1 g/L de Triton X100), using a buffer com blood samples were diluted in a haemolyzing solution (Triton position containing 200 mM of CHES buffer, 20 mM of X1001 g/L in water) and injected by hydrodynamic injection. US 9,222,913 B2 23 24 The capillary was washed before each analysis with 0.25 M Example 5 sodium hydroxide, then with the buffer composition. Buffer Composition Capillary electrophoresis was carried out from reference The buffer compositions in which the capillary electro samples (Exocell, USA), containing different purified frac phoresis was carried out comprised water, a buffer compound tions of glycated haemoglobin (fractions A and A or frac with a pKa in the range 8 to 11 (CAPS, CAPSO or CHES tions Ao A, and A1), using a buffer composition containing depending on the case), a base allowing the pH to be adjusted 200 mM of CAPSO, 10 mM of putrescine and 50 mM of to the desired value, an optional flow retardant (putrescine), 3-carboxyphenylboronic acid (pH 10.20). The standard elec and an optional borate compound (boric acid) or boronate. trophoretic profiles for A, and A. obtained are presented in 3,5-dicarboxyphenylboronic acid (3,5-dCPBA) was 10 FIG. 5. The peak corresponding to HbA lies between the obtained from the firms Combi-blocks Inc. (San Diego, USA) peaks corresponding to HbA, and Hb A. and Apollo Scientific Ltd (Cheshire, United Kingdom). 3,4-dicarboxyphenylboronic acid (3,4-dCPBA) was syn Example 6 thesized by the firm BoroChem SAS (Caen, France). 15 B. Results Capillary electrophoresis was carried out from reference samples (Exocell, USA), containing different purified frac Example 1 tions of glycated haemoglobin (fractions Ao and A or frac tions Ao A, and A1), using a buffer composition containing Capillary electrophoresis was carried out from normal 200 mM of CAPSO, 10 mM de DAB and 50 mM of 3,5- human blood (comprising haemoglobins Hb Ao, HbA and dicarboxyphenylboronic acid (pH 10.20). The standard elec HbA) diluted to /6th in a haemolyzing solution (1 g/L de trophoretic profiles for A, and A. obtained are presented in Triton X 100 dissolved in demineralized water), using the FIG. 6. The peak corresponding to HbAllies after the peaks analysis buffer described in patent U.S. Pat. No. 5,599.433, corresponding to HbA, and Hb A. which contained 100 mM of CAPS and 300 mM of boric acid, 25 pH 11. The electropherogram obtained is presented in FIG.1. Example 7 The separation between the peaks of haemoglobins is poor. Capillary electrophoresis was carried out from reference Example 2 samples (Exocell, USA) containing different purified frac 30 tions of glycated haemoglobin (fractions Ao and A or frac Capillary electrophoresis was carried out from reference tions Ao A, and A), using a buffer composition containing samples (Exocell, USA), containing different purified frac 200 mMofCAPSO, 15 mMofflow retardant (putrescine)and tions of glycated haemoglobin (fractions Ao and A or frac 100 mM of 3,5-dicarboxyphenylboronic acid (pH 10.20). The tions A, A, and A), using the analysis buffer described in standard electrophoretic profiles for A, and A obtained patent U.S. Pat. No. 5,599.433, which contained 100 mM of 35 are presented in FIG. 7. The peak corresponding to HbAllies CAPS and 300 mM of boric acid, pH 10.20. The standard after the peaks corresponding to HbA, and Hb A, and is electrophoretic profiles for A, and A obtained are pre distinct from these peaks, the separation between the haemo sented in FIG. 2. The separation between the HbA and globin HbA and the haemoglobins HbA and Hb A, is HbA, haemoglobins is clearly insufficient; the HbA. elec excellent. trophoretic peak overla 40 the HbA1/HbA1, peaks. Example 8 Example 3 Capillary electrophoresis was carried out from reference samples containing Hb Ao and/or different purified fractions Capillary electrophoresis was carried out from reference 45 of glycated haemoglobin (fractions A and A or fractions samples (Exocell, USA), containing different purified frac AoA, and A), using a buffer composition containing 200 tions of glycated haemoglobin (fractions A and A or frac mM of CHES, 20 mM of flow retardant (putrescine) and 30 tions Ao A, and A1), using a buffer composition containing mM of 3,5-dicarboxyphenylboronic acid (at a pH of 9.40). 200 mM of CAPSO and 10 mM of putrescine (pH10.20) but The standard electrophoretic profiles Ao, A., and A. no borate compound, and also no boronate compound. The 50 obtained are presented in FIG.8. The peak corresponding to standard electrophoretic profiles for A, and A obtained HbAllies after the peaks corresponding to Hb Ao, HbA, and are presented in FIG. 3. The peak corresponding to HbA. HbA, and is clearly distinct from these peaks; the separation co-migrated with that of HbA. between the haemoglobin HbA and the HbA and HbA, haemoglobins is excellent. Example 4 55 Example 9 Capillary electrophoresis was carried out from reference samples (Exocell, USA), containing different purified frac Capillary electrophoresis was carried out from reference tions of glycated haemoglobin (fractions Ao and A or frac samples containing Hb Ao and/or different purified fractions tions Ao A, and A1), using a buffer composition containing 60 of glycated haemoglobin (fractions Ao and A or fractions 200 mM of CAPSO, 10 mM of putrescine and 50 mM de A, A, and A), using a buffer composition containing 200 borate (pH 10.20). The standard electrophoretic profiles for mM of CHES, 20 mM of flow retardant (putrescine) and 30 A, and A obtained are presented in FIG. 4. The peak mM of acid 3,4-dicarboxyphenylboronic (at a pH of 9.40). corresponding to HbA lies between the peaks correspond The standard electrophoretic profiles Ao, A., and A. ing to HbA and HbA/HbA, and is too close to the peak 65 obtained are presented in FIG. 9. It will be seen that the peak corresponding to other Hb As to allow a reliable assay of corresponding to HbA lies after the peaks corresponding to HbA. HbA. HbA, and Hb A, and is clearly distinct from these US 9,222,913 B2 25 26 peaks, the separation between the haemoglobin HbA and the Hb Bart and Hb Afractions. As a consequence, in order the HbA, and Hb A, haemoglobins is excellent. to be able to assay HbA in the presence of Hb Bart, these two By comparing the electrophoretic profiles of examples 8 fractions should be capable of being quantified using a Suit and 9, it will be seen that 3,5-dicarboxyphenylboronic acid able integration method. If this is not possible, it will be can produce a slightly better result in terms of separation of 5 necessary to alert the user to this, in case he observed this type HbA, compared with the other fractions, while 3,4-dicar of profile with a shoulder on the expected peak. boxyphenylboronic acid allowed a slightly better result to be obtained in terms of focussing. Example 14 Example 10 10 The results obtained by capillary electrophoresis by the method of invention using the Capillaries(R) (Sebia) analyzer Capillary electrophoresis was carried out on Capillaries(R) were compared with the results obtained with one of the (Sebia) from reference samples (Exocell, USA) containing reference techniques: HPLC with the Variant II Turbo (R) (Bio different purified fractions of glycated haemoglobin (frac Rad).analyzer. tions Ao and A or fractions A, A, and A), using a buffer 15 Capillary electrophoresis was carried out from whole composition containing 200 mM of CHES buffer, 20 mM of blood diluted to /6th in the haemolyzing solution (water+1 putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, g/L de Triton X100), in a buffer composition containing 200 at a pH of 9.40. The standard electrophoretic profiles for A1, and A obtained are presented in FIG. 10. It will be seen that mM of CHES buffer, 20 mM of putrescine and 30 mM of the peak corresponding to HbA lies after the peaks corre 3,5-dicarboxyphenylboronic acid, at a pH of 9.40. sponding to HbA, and Hb A, and is clearly distinct from FIG. 17 shows the very good correlation of this novel these peaks; the separation between the haemoglobin Hb A. technique for analysis by capillary electrophoresis with the and the HbA, and HbA, haemoglobins is excellent. analysis of HbA by HPLC with the Variant II turbo R from Bio-Rad. After calibration of the EC data using 2 calibrators Example 11 25 (weakAlland strong A1), the values obtained by the method of invention were very close to those obtained by the refer Analyses by capillary electrophoresis were carried out on ence method. normal human blood diluted to "/6th in haemolyzing Solution (water+1 g/L de Triton X100), using a buffer composition Example 15 containing either 200 mM of CAPSO buffer, 10 mM of 30 putrescine and 50 mM of 3,5-dicarboxyphenylboronic acid, Study demonstrating the absence of interference of the at a pH of 10.20, or 200 mM of CHES buffer, 20 mM of labile fraction of HbA, with the assay of HbA, using the putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, method of the invention. at a pH of 9.40. The electropherograms obtained on Capillar Hypothesizing that the compound complexing glucose and ies(R (Sebia) using a fused silica capillary, uncoated, are 35 providing negative charges at an alkaline pH used in the presented in FIGS. 11A and 11B respectively. In both cases, context of the analysis method of the invention would be complete separation of the haemoglobin HbA. from the capable of interacting with blood glucose (this interaction is other haemoglobins forms is observed. hypothetical and has not been demonstrated), a study of any interference of free glucose on the result of the blood of Example 12 40 HbA was carried out as follows: normal blood was incu bated for 3 hours at 37° C. with different concentrations of The influence of concentration of 3,5-dicarboxyphenylbo glucose (0 to 50 g/L) in order to create the labile form of ronic acid in the buffer composition on the separation HbA (form obtained before rearrangement of the molecule between the A and A. haemoglobins was studied. The buffer (Amadori rearrangement)). Once incubation had been carried composition used contained 200 mM of CAPSO, 15 mM of 45 out, the blood samples were centrifuged and the pellets putrescine and 0 to 120 mM of 3,5-dicarboxyphenylboronic obtained were reconstituted in physiological water and the acid. The results are presented in FIG. 12. The A/A sepa haemolyzing solution (15 uIl of pellet--25 uL physiological ration increased with the concentration of 3,5-dicarboxyphe water+160 uL of haemolyzing solution (water+1 g/L Triton nylboronic acid. X100)) was then analyzed in parallel using a Hydrasys(R) 50 automated machine from Sebia (HbA gel) and using the Example 13 Capillaries(R (Sebia) technique, using a buffer composition containing 200 mM of CHES buffer, 20 mM of putrescine and Capillary electrophoreses were carried out on Capillaries(R) 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40. (Sebia) using four different samples diluted by /6th in the The HbA1c gel obtained by the analysis on the Hydrasys(R) haemolyzing solution (water+1 g/L de Triton X100): normal 55 automated machine (see FIG. 18A) confirmed the formation human blood comprising a HbE variant (FIG. 13), a AFSC of labile fraction of the Hb A1c migrating to the same level as control (FIG. 14), a pool of normal blood comprising F and the HbAlc and in an increasing concentration as the concen Bart's variants (FIG. 15) and normal human blood compris tration of glucose increased during incubation with the blood. ing a HbD variant (FIG. 16). Capillary electrophoresis was It should be noted that in gel, under the normal conditions of carried out in a buffer composition containing 200 mM of 60 use defined by Sebia, the labile fraction did not appear, in CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicar particular because of the acid pH of the haemolyzing solution. boxyphenylboronic acid, at a pH of 9.40, using a fused silica In contrast, the analyses carried out on Capillaries(R) (Se capillary, uncoated. bia) with a buffer composition of the invention (200 mM of FIGS. 13-16 show the absence of interference from the CHES buffer, 20 mM of putrescine and 30 mM of 3,5-dicar principal variants of haemoglobin (E. F. S., C, D and Bart) 65 boxyphenylboronic acid, at a pH of 9.40), on the same blood with the HbA fraction. Note, however, that in the case of samples showed that the assay was not perturbed by the Bart's haemoglobin, the resolution is not complete between presence of free glucose, regardless of the concentration of US 9,222,913 B2 27 28 incubated glucose, in the range studied (0 to 50 g/L): the a. introducing a buffer composition and a biological profiles and the values of Hb A1c were unchanged (see FIGS. sample into an electrophoresis capillary, said buffer 18B and 18C). composition comprising at least one compound which specifically complexes the glucose residue(s) bound to REFERENCES an amino acid in the N-terminal position in glycated haemoglobins of the biological sample and provides Abraham, E. C.: Cameron, B. F.; Abraham, A.; Stallings, M., said glycated haemoglobin(s) with several negative "Glycosylated hemoglobins in heterozygotes and homozy electric charges at an alkaline pH.wherein said gotes for hemoglobin C with or without diabetes’, Journal compound which specifically complexes the glucose of Laboratory and Clinical Medicine, 104, 602-609, 1984. 10 Beccaria, L., Chiumello, G., GianaZZa, E., Luppis, B.: residue(s) of glycated haemoglobins of the biological Righetti, P.G., “Hemoglobin A separation by isoelectric sample and provides negatice charges at an alkaline pH focusing”. American Journal of Hematology, 4, 367-374, comprises two or more than two functional groups, at 1978. least one of said functional groups specifically complex Bosisio, A.; Righetti, P., “Determination of glycated haemo 15 ing one or more glucose residue(s), thereby providing globin by isoelectric focusing in non-linear pH gradients'. one negative electric charge per complexed glucose resi Journal of Chromatography, 307, 103-110, 1984. due,and at least one off said functional groups does not Doelman, C: Siebelder, C; Nijhof, W.; Weykamp, W.; Jans complex the glucose residue(s) thereby providing each sens, J.; Penders, T., “Capillary electrophoresis system for glucose residue with one or more additional negative hemoglobin A determinations evaluated. Clinical charges at an alkaline pH and is: Chemistry, 43, 4, 644-648, 1997. (i) a boronate compound with general formulal RB(OH) Hempe, J. M.; Craver, R D., “Quantification of hemoglobin or RB(OH), in which the group R comprises at least variants by capillary isoelectric focusing”.Clinical Chem one arv1 and/or alkyl (linearbranched or cyclic) and/ istry, 40,12,2288-2295, 1994. or an aralkyl and/or other functional groups or het Hempe, J. M.; Granger, JN.; Craver, RD., “Capillary isoelec 25 eroatoms, and/or a combination thereof, and said tric focusing of hemoglobin variants in the pediatric clini group R provides glycated hamoglobins with one or cal laboratory, Electrophoresis, 18, 1785-1795, 1997. several negative electric charge(s) at an alkaline pH Janssens, J., “Capillary electrophoresis detection and/or for each glucose residue complexed with the boronate analysis method and unit', EP 0733 900 A2, 1996. group; wherein said boronate compound is a dicaroX Janssens, J.; Chevigne, P.; Louis, P. “Capillary electrophore 30 yphenylboronic acid or sis method using initialized capillary and polyanion-con (ii) a salt of a dicarboxyphenylboronic acid; taining buffer and chemical kit therefore”, U.S. Pat. No. b. performing capillary electrophoresis on the buffered 5,611,903, 1997. biological sample to separate constituents of the biologi Menard, L; Dempsey, M.; Blankstein, L.; Aleyassine, H.; cal sample; and Wacks, M.; Soeldner, J., “Quantitative determination of 35 c. detecting one or several haemoglobin(s) present in the glycosylated hemoglobin A1 by agar gel electrophoresis'. biological sample. Clinical Chemistry, 26, 11, 1598-1602, 1980. 2. A method according to claim 1, in which the concentra Molteni, S.; Frischknecht, H.; Thormann, W., “Application of tion in the buffer composition of the compound which spe dynamic capillary isoelectric focusing to the analysis of cifically complexes glucose residues of glycated haemoglo human hemoglobin variants”, Electrophoresis, 15, 22-30, 40 bin (s) of the biological sample and provides negative charges 1994. at an alkaline pH is in Stoechiometric excess with respect to Panteghini, M. John, W. G., on behalf of the IFCC Scientific the total quantity of proteins, compared with the total quantity Division, “Implementation of haemoglobin A results of all of the haemoglobins present in the biological sample or traceable to the IFCC reference system: the way forward.”. compared with the total quantity of all of the haemoglobins Clin Chem Lab Med., 45 (8), 942-4, 2007. 45 comprising glucose present in the biological sample. Simon, M.: Cuan J., “Hemoglobin Aie by isoelectric focus 3. A method according to claim 1, in which the concentra ing, Clinical Chemistry, 28.1, 9-12, 1982. tion in the buffer composition of the compound which spe Siren, H.; Laitinen, P.; Turpeinen, U.: Karppinen, P., “Direct cifically complexes glucose residues of glycated haemoglo monitoring of glycohemoglobin A in the blood samples bins of the biological sample and provides negative charges at of diabetic patients by capillary electrophoresis. Compari 50 an alkaline pH is in the range 0.10 to 100 mM. son with an immunoassay method'. Journal of Chroma 4. A method according to claim 1, in which the buffer tography A,979, 201-207, 2002. composition further comprises a flow retardant. Stickland, M.; Perkins, C.; Wales, J., “The measurement of 5. A method according to claim 4, in which the concentra haemoglobin A by isolectric focusing in diabetic tion offlow retardant in the buffer composition is in the range patients', Diabetologia, 22,315-317,1982. 55 0.10 to 40 mM. Wilson, D.H., Bogacz, J. P. Forsythe, C.M., Turk, P.J., Lane, 6. A method according to claim 1, in which the buffer T. L., Gates, R.C., Brandt, D. R. “Fully automated assay of composition further comprises: glycohemoglobin with the Abbott IMx analyzer: novel a buffer compound having a pKa in the range 8.0 to 11.0: approaches for separation and detection’. Clin Chem. and/or 39(10), 2090-7, 1993. 60 a base; and/or The invention claimed is: a salt, and/or 1. A method for analysis by capillary electrophoresis of one a diluting solution. or several glycated haemoglobin(s) comprising at least one 7. A method according to claim 6, in Which the buffer globin chain comprising a glucose residuebound to the amino compound is a Zwitterionic compound. acid in the N-terminal position of the beta globin chain, 65 8. A method according to claim 6, in which the concentra contained in a biological sample comprising haemoglobins, tion of buffer compound in the buffer composition is in the said method comprising the following steps range 20 to 500 mM. US 9,222,913 B2 29 30 9. A method according to claim 1, in which the buffer composition has a pH of 9 or more. 10. A method according to claim 1, further comprising a step for generating an electropherogram from a detection signal which is proportional to the quantity of haemo 5 globin(s) detected. 11. A method according to claim 1, further comprising a step for determining the quantity of one or several haemoglo bin (S) present in the biological sample and/or of the propor tion of one or several haemoglobin(s) present in the biological 10 sample with respect to the total quantity of proteins, the total quantity of haemoglobin or the quantity of certain haemoglo bins present in the biological sample. 12. A method according to claim 1, further comprising a step for quantification of one or several haemoglobin(s) 15 present in the biological sample compared with one or several standardized calibrator(s). 13. A method according to claim 1, in which the biological sample is a blood sample. 14. A method according to claim 1, in which the biological sample is diluted in a haemolyzing solution. 15. The method according to claim 1, wherein the boronate compound is a dicarboxphenylboronic acid selected from 3,4-dicarboxyphenylboronic acid and 3,5-dicarboxyphenyl boronic acid. 25