Patent Application
20230194537
This invention relates to the field of glycan analysis, for example, in glycoproteins, and further, to the analysis of glycans by fluorescence and mass spectrometry techniques.
Proteins produced in cells may be modified after translation by the addition of covalently-linked, linear or branched chains of carbohydrates. These protein-carbohydrate conjugates are referred to as glycoproteins; the point at which the carbohydrate is attached is referred to as a glycosylation site. Attached polysaccharides or oligosaccharides are referred to as glycans. A wide range of glycans are found on the different glycosylation sites of particular glycoproteins. The particular pattern of glycans on a particular glycoprotein is determined by the specific cell line that produces the glycoprotein and the conditions under which the cells are grown.
Since glycans conjugated to a protein can affect characteristics critical to its function, including pharmacokinetics, stability, bioactivity, or immunogenicity, it is important in many uses to determine which glycans are present. Thus, as glycans are released from a glycoprotein during analysis, it is useful to label them. N-glycans are typically labeled at their free-reducing terminus and are analyzed by methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE), carbohydrate gel electrophoresis, fluorescence analysis, mass spectrometry (MS) and others. U.S. Pat. Nos. 8,124,792 and 8.445,292 describe some methods of glycan analysis, and are hereby incorporated by reference.
There is a need in the art for additional labels and methods that provide rapid labeling of N-glycans under mild conditions without causing the degradation of labile components, and that can provide high sensitivity of detection by both fluorescence and by MS or by HPLC/CE).
The present disclosure provides compounds comprising (a) at least one fluorescent group such as an aromatic or substituted aromatic ring; (b) at least one group capable of binding to an aldehyde or ketone present on an analyte of interest, for e.g.; to a glycoprotein; and (c) at least one negatively charged group that can be detected by negative ion mass spectrometry. These compounds can be used to label and detect the glycans. Therefore, the present disclosure provides methods for labeling or binding aldehyde, ketone-, glycosylamine-containing molecules or analytes, for e.g., glycans.
The present disclosure also provides methods for comparative quantitation of glycans using the 12C/13C labeled isotopologues of the compounds described herein (including for e.g., sulfanilic acid, its derivatives or salts thereof), thereby providing indicating structural characterization and quantitation of glycan alterations.
Accordingly, the present disclosure provides fluorescent, MS active compounds that comprise three functional components: (a) a molecular scaffold comprising an aromatic group (for fluorescence), including but not limited to benzene, (N- or S-) substituted benzene, naphthalene, (N- or S-) substituted naphthalene, anthracene, (N- or S-) substituted anthracene, pyrene, (N- or S-) substituted pyrene, derivatives or salts thereof of any of the preceding compounds; (b) a group for coupling to glycan aldehydes, or glycan ketones, or a glycosylamine, for e.g., an amine, hydrazine or substituted hydrazine, hydrazide or substituted hydrazide, hydrazone or substituted hydrazone, oxime or substituted oxime, an activated carboxylic group such as a succinimidyl or N-hydroxysuccinimidyl (NHS) ester or equivalents (including but not limited to cyanates, thiocyanate, isothiocyanates, isocyanates), carbamates, or derivatives and salts thereof of any of the preceding compounds; and (c) a negatively charged group designed to enhance detection in negative ion mass spectrometry (MS), including but not limited to, sulfonic acid and their derivatives or salts thereof, phosphoric acid and their derivatives or salts thereof, carboxylic acid and their derivatives or salts thereof.
The present disclosure provides methods for detecting the resultant labeled glycans that may be highly fluorescent, thereby providing an excellent method for detection via fluorescence; and that may be highly charged thereby enhancing the detection in negative ion mass spectrometry (MS). The labeled glycans can also be analyzed using capillary electrophoresis (CE), liquid chromatography (LC, UPLC, UHPLC, etc.) or similar techniques. The present disclosure provides methods for combining the above techniques (twoplexed/duplexed/multiplexed) for analysis, for e.g., UPLC-fluorescence—MS analysis, CE-LC, LC-MS, CE-MS, MS-MS, etc. These techniques or their combinations can also be set up for automation.
Certain embodiments provide a method of labeling one or more glycans in a glycoprotein sample, the method comprising:
In certain embodiments, the fluorescent target product is an N-labeled or an 0-labeled glycan.
In certain embodiments,
A is benzene, (N- or S-) substituted benzene, naphthalene, (N- or S-) substituted naphthalene, anthracene, (N- or S-) substituted anthracene, pyrene, (N- or S-) substituted pyrene, or derivatives or salts thereof;
X is an amine, hydrazine or substituted hydrazine, hydrazide or substituted hydrazide, hydrazone or substituted hydrazone, oxime or substituted oxime, an activated carboxylic group such as a succinimidyl or N-hydroxysuccinimidyl (NHS) ester or equivalents (including but not limited to cyanates, thiocyanate, isothiocyanates, isocyanates), carbamates, or derivatives or salts thereof; and,
W is sulfonic acid and their derivatives or salts thereof, phosphoric acid and their derivatives or salts thereof, carboxylic acid and their derivatives or salts thereof.
The activated carboxylic group is selected from: a succinimidyl, N-hydroxysuccinimidyl (NHS) ester, N-hydroxysuccinimidyl (NHS) cyanate, N-hydroxysuccinimidyl (NHS) thiocyanate, N-hydroxysuccinimidyl (NHS) isothiocyanate, and N-hydroxysuccinimidyl (NHS) isocyanate or a salt thereof.
In certain embodiments, the fluorescent reagent of Formula I is a benzenesulfonic acid derivative or a salt thereof.
In certain embodiments, fluorescent reagent is a compound of Formula II, III or IV, its derivatives or salt thereof:
In certain embodiments, R1, R2, R3 and R4 are independently H, methyl, ethyl, C3-C6 alkyl, C1-C6 alkoxy or halogen.
In certain embodiments, the fluorescent reagent is a compound of Formula V, VI or VII, its derivatives or salt thereof:
wherein, X is selected from an amine, hydrazine or substituted hydrazine, hydrazide or substituted hydrazide, hydrazone or substituted hydrazone, oxime or substituted oxime, an activated carboxylic group such as a succinimidyl or N-hydroxysuccinimidyl (NHS) ester or equivalents (including but not limited to cyanates, thiocyanate, isothiocyanates, isocyanates), carbamates, or derivatives and salts thereof.
In certain embodiments, the fluorescent reagent is a compound of Formula IX, X or XI, its derivatives or salt thereof:
In certain embodiments, the fluorescent reagent is any of Compounds 1, 2 or 3, their derivatives or salts thereof:
In certain embodiments, the fluorescent reagent is Compound 9 or Compound 10, their derivatives or salts thereof:
In certain embodiments, the fluorescent reagent is Compound 11, 12 or 13, their derivatives or salts thereof:
In certain embodiments, the fluorescent reagent is Compound 6, 7 or 8, their derivatives or salts thereof:
In certain embodiments, the aromatic ring carbocycle is labeled with 13C, and wherein the carbocyle contains between one and seven 13C atoms.
In certain embodiments, the glycoprotein is either in a solution or is immobilized on a support selected from the group consisting of a resin, a bead, a membrane, an array, a surface, a cartridge, a solid, a plate and a well.
In certain embodiments, the deglycosylation is done either by a physical method, a chemical method or by an enzyme.
In certain embodiments, the enzyme is a glycosidase.
In certain embodiments, the glycosidase is PNGase F.
Certain embodiments provide a method of detecting glycans in a sample, comprising:
In certain embodiments for detecting glycans,
A is benzene, (N- or S-) substituted benzene, naphthalene, (N- or S-) substituted naphthalene, anthracene, (N- or S-) substituted anthracene, pyrene, (N- or S-) substituted pyrene, or derivatives or salts thereof;
X is an amine, hydrazine or substituted hydrazine, hydrazide or substituted hydrazide, hydrazone or substituted hydrazone, oxime or substituted oxime, an activated carboxylic group such as a succinimidyl or N-hydroxysuccinimidyl (NHS) ester or equivalents (including but not limited to cyanates, thiocyanate, isothiocyanates, isocyanates), carbamates, or derivatives or salts thereof; and,
W is sulfonic acid and their derivatives or salts thereof, phosphoric acid and their derivatives or salts thereof, carboxylic acid and their derivatives or salts thereof.
In certain embodiments for detecting glycans, the activated carboxylic group is selected from: a succinimidyl, N-hydroxysuccinimidyl (NHS) ester, N-hydroxysuccinimidyl (NHS) cyanate, N-hydroxysuccinimidyl (NHS) thiocyanate, N-hydroxysuccinimidyl (NHS) isothiocyanate, and N-hydroxysuccinimidyl (NHS) isocyanate.
In certain embodiments for detecting glycans, the fluorescent reagent of Formula I is a benzenesulfonic acid derivative, or salts thereof.
In certain embodiments for detecting glycans, the fluorescent reagent is a compound of Formula II, III or IV, their derivatives or salts thereof:
In certain embodiments for detecting glycans, R1, R2, R3 and R4 are independently H, methyl, ethyl, C3-C6 alkyl, C1-C6 alkoxy or halogen.
In certain embodiments for detecting glycans, the fluorescent reagent is a compound of Formula V, VI or VII, their derivatives or salts thereof:
wherein, X is selected from an amine, hydrazine or substituted hydrazine, hydrazide or substituted hydrazide, hydrazone or substituted hydrazone, oxime or substituted oxime, an activated carboxylic group such as a succinimidyl or N-hydroxysuccinimidyl (NHS) ester or equivalents (including but not limited to cyanates, thiocyanate, isothiocyanates, isocyanates), carbamates, or derivatives and salts thereof.
In certain embodiments for detecting glycans, the fluorescent reagent is a compound of Formula IX, X or XI, their derivatives or salts thereof:
In certain embodiments for detecting glycans, the fluorescent reagent is Compound 1, 2 or 3, its derivatives or salt thereof:
In certain embodiments for detecting glycans, the fluorescent reagent is Compound 9 or Compound 10, its derivatives or salt thereof:
In certain embodiments for detecting glycans, the fluorescent reagent is Compound 11, 12 or 13, their derivatives or salts thereof:
In certain embodiments for detecting glycans, the fluorescent reagent is Compound 6, 7 or 8, their derivatives or salts thereof:
In certain embodiments for detecting glycans, the aromatic ring carbocycle of any one of the compounds described above is labeled with either 12C or 13C, and contains between one and seven 12C/13C atoms respectively, thereby generating 12C/13C labeled isotopalogues.
In certain embodiments for detecting glycans, the 12C/13C labeled isotopalogues are used to label glycans, and resultant 12C/13C labeled glycans are used for detection.
In certain embodiments for detecting glycans, the detecting step is determining the mass-based differences in sialic acid linkage isomers after DMT-MM derivatization.
In certain embodiments for detecting glycans, the detection is performed by MS (mass spectrometry), LC-MS, CE-MS, MS-MS, HPLC, HILIC, UPLC/UHPLC, UPLC/UHPLC-CE.
In certain embodiments for detecting glycans, the fluorescent reagent is a compound selected from the following compounds, their derivatives or salts thereof:
Certain embodiments for relatively quantitating glycans provide methods comprising:
a) generating 12C/13C labeled isotope analogues of any one of the fluorescent compounds of claim 35, or any one of the fluorescent compounds described in the methods claims 1-13, or the method claims 19-31 thereby resulting in a 12C labeled isotopologue and a 13C labeled isotopalogue of the selected compound;
b) generating released glycans using a glycosidase enzyme, in the first glycan sample and the second glycan sample;
c) labeling the first glycan sample with the 12C labeled isotopalogue of step (a) to generate 12C labeled glycans, and labeling the second glycan sample with the 13C labeled isotopalogue of step (a) to generate 13C labeled glycans;
d) mixing the 12C labeled and 13C labeled glycans and purifying the 12C/13C labeled glycans by removing the excess fluorescent labels;
e) coinjecting the purified 12C/13C labeled glycans into liquid chromatography or CE, and quantitating the glycans by fluorescence; and,
d) performing tandem mass spectrometry to quantitate the 12C/13C labeled glycans by mass-based differences.
In certain embodiments for relative quantitation of glycans, the method further provides
e) determining the mass-based differences in sialic acid linkage isomers after DMT-MM derivatization.
In certain embodiments for relative quantitation of glycans, the glycans are N-glycans or O-glycans.
In certain embodiments for relative quantitation of glycans the liquid chromatography is done by HILIC, UPLC/UHPLC or HPLC.
In certain embodiments for relative quantitation of glycans the mass spectrometry detection is done on a quadrupole time of flight instrument (UPLC-FLr-QToF-MS/MS).
In certain embodiments for relative quantitation of glycans the first glycan sample is an innovator biomolecule and the second glycan sample is a biosimilar biomolecule.
In certain embodiments for relative quantitation of glycans the first glycan sample is a Fc portion of an antibody and the second glycan sample is a Fab portion of the same antibody.
In certain embodiments for relative quantitation of glycans the first glycan sample is obtained after a first treatment, and the second glycan sample is obtained after a second treatment.
In certain embodiments for relative quantitation of glycans the first glycan sample is obtained before a treatment, and the second glycan sample is obtained after the treatment.
In certain embodiments for relative quantitation of glycans the first and second glycans are samples from bioreactor runs.
In certain embodiments for relative quantitation of glycans the first and second glycans are disease samples.
In certain embodiments for relative quantitation of glycans, the samples are antibodies.
Certain embodiments provide a kit for detecting an analyte in a sample, wherein the kit comprises:
a) a compound selected from any one of the fluorescent compounds of claim 35, or any one of the fluorescent compounds described in the methods claims 1-13, or the method claims 19-31;
(b) instructions for covalently labeling the selected compound of a) to the analyte.
In certain embodiments of the kit, the analyte is a glycoprotein, a biological sample comprising a glycoprotein or part of a glycoprotein, a fusion protein or a fragment thereof, a biosimilar or a fragment thereof, an antibody or a fragment thereof, a cell or part of a cell, a virus or part of a viral protein, protein therapeutic or a fragment thereof.
In certain embodiments the kit further comprises one or more of the following: a buffering agent, a purification medium, a vial comprising the analyte, an organic solvent, one or more reagents for releasing the glycan from a biomolecule, or optionally, one or more reagents to purify the released glycan from the reaction mixture.
In certain embodiments of the kit, the reagent for releasing the glycan from a biomolecule is selected from a physical method, a chemical or an enzyme.
In certain embodiments of the kit, the enzyme is a glycosidase.
In certain embodiments of the kit, the glycosidase is PNGase F.
In certain embodiments of the kit, the instructions for detecting the analyte may also be provided.
In certain embodiments of the kit, the purification medium is selected from the group consisting of a resin, a bead, a membrane, an array, a surface, a cartridge, a solid support, a plate and a well.
In certain embodiments of the kit, the bead is a magnetic bead.
In certain embodiments the kit may further comprise instructions for labeling glycans in a sample for preparation of glycan analysis, the method comprising:
(i) treating the sample with a release reagent, such as PNGase F enzyme, with an appropriate buffer under conditions suitable for the release of the glycan from the biomolecule, thereby forming a reaction mixture;
(ii) adding beads and buffer to the reaction mixture;
(iii) separating the supernatant from the beads;
(iv) washing the beads with wash buffer;
(v) eluting the glycans from the beads with elution buffer;
(vi) performing labeling of the glycans using one or more compounds according to claim 35, thereby forming a glycan-containing solution;
(vii) optionally, removing excess compound using fresh beads; washing beads, separating the beads from excess compound/label/wash solution; and eluting glycans from the beads; and
(viii) collecting the glycan-containing solution.
A better understanding of the features and advantages of the present invention will be obtained by reference to the following figures that sets forth illustrative embodiments, in which the principles of the invention are utilized.
Glycans conjugated to proteins can affect characteristics critical to their function, including pharmacokinetics, stability, bioactivity, or immunogenicity. Several proteins, including biotherapeutics, are glycoproteins. Glycosylation can also impact drug stability, half-life, clearance in the body, immunogenicity, and other physiochemical properties. Various glycoform modifications can occur during drug design, development and optimization during bioproduction. Therefore it is important to determine the nature and types of glycans present, and analysis especially of N-linked and O-linked glycans are of particularly of interest in biological research, clinical analysis, and pharmaceutical production.
The present disclosure provides compounds that are both fluorescent and MS active for tagging glycans, such as N-linked or O-linked glycans which are useful to label and detect the glycans. Therefore, the present disclosure provides methods for labeling or binding aldehyde-, ketone-, glycosylamine-containing molecules or analytes, for e.g., glycans. The present disclosure also provides methods for comparative quantitation of glycans using the 12C/13C labeled isotopologues of the compounds described herein (including for e.g., sulfanilic acid, its derivatives or salts thereof), thereby providing indicating structural characterization and quantitation of glycan alterations. Such comparative glycomic quantitation has numerous potential applications well documented in literature; the 12C/13C labeled isotopologues of any of the compounds described herein (including for e.g., sulfanilic acid, its derivatives or salts thereof) may be applied in any such glycomic quantitation requiring application, including pharmaceutical, medical and analytical applications.
The terms glycoproteins or glycoconjugates may be used interchangeably, and comprises one or more glycans (the carbohydrate, or the saccharide, or the oligosaccharide portion, or the sugar in the molecule, which terms are meant equivalently through this disclosure, and is so understood and referenced in the art). The glycans can be either N-glycans (linked via N-linkage to the protein) or O-glycans (linked via O-linkage to the protein).
A general method for the analysis of glycans on a glycoprotein is described herein, and generally consists of one or more steps that may include:
(a) deglycosylating any bound glycans from a glycoprotein in a sample so that free glycans are obtained. The glycoprotein may be subjected to deglycosylation by, for example, in the case of N-glycans, enzymatic cleavage of the N-glycans using a glycosidase enzyme such as PNGase-F to result in free N-glycan which can be labeled. The term “bound glycan” as described herein means any glycan that is covalently attached to another molecule, for e.g., when a glycan in a glycoprotein is attached to a protein. Or, in some embodiments, a glycoprotein may be immobilized on a solid support prior to deglycosylation, facilitating separation of released glycans from the protein portion still attached to the solid immobilized support. By solid support is meant anything including a plate, resin, surface, bead, membrane, wall, layer, microarray, or any solid support that is well understood to be useful in the art. Glycans can be also be released from a glycoprotein of interest by chemical means. In general, protocols and workflows for releasing glycans from glycoproteins using deglycosylation enzymes are known in the art and one skilled in the art would be familiar with the ranges of times, temperatures and pHs used in these methods. Exemplary endoglycosidases include endo-alpha-N-acetyl-galactosaminidase, Endoglycosidase F1, Endoglycosidase F2, Endoglycosidase F3, and Endoglycosidase H. In some embodiments of the present invention, the enzyme is the well-known deglycosylation enzyme PNGase F (Peptide-N4-(acetyl-B-glucosaminyl)-asparagine amidase, EC 3.5.1.52), which releases N-glycans from the glycoprotein in the form of glycosylamines, which can then be labeled using the methods described herein. Glycosylamines are known to hydrolyze over time therefore the time between the deglycosylation step and the labeling step should be minimized. For example, N-glycans released from a glycoprotein as glycosylamines preferably may be labeled within 30 min of being released from the glycoprotein, or with shorter periods, such as about 25 min, about 20 min, about 15 min, or about 10 min, being more preferred, in that order, with “about” meaning±3 min. In preferred embodiments, the N-glycans are labeled within about 9, 8, 7, 6, 5, 4, 3, 2 or 1 min after being released from the glycoprotein, with “about” meaning±30 seconds. In some preferred embodiments, the N-glycans are labeled within 30 seconds after release from the glycoprotein.
(b) The released glycans may then be labeled with a fluorescent reagent. In some embodiments, glycoproteins may be labeled, either after being released from the solid support, or while they are still immobilized. These initial sample prep steps of deglycosylation and labeling may be performed collectively either manually or via automation or in high throughput mode. Allowing enough time for labeling under appropriate conditions to permit a significant reaction to occur between a glycan sample and the fluorescent probe reagent, such as Formula (I) or sulfanilic acids or their derivatives as described in the formulae and compounds shown in
(c) The resultant labeled glycan (labeled for e.g., using the formulae or compounds described in
In an embodiment exemplified in the examples and in the Figures herein, one or more of techniques may be combined for e.g., multiplexing may be performed with UPLC-fluorescence—MS analysis, CE-LC, LC-MS, CE-MS, MS-MS, etc. This is sometimes referred to as twoplexed/duplexed/multiplexed analysis, and is useful for detailed structural elucidation of glycoforms present in a sample. These combinatorial techniques may be set up for automation. In certain embodiments for duplex/multiplex analysis, the formulae or compounds described in
This type of analysis may be useful in studying glycosylation patterns of any biological material, or any medical sample, including but not limited to cancer, metabolic disease samples; for assessing the critical quality attribute (CQA) of a glycosylated protein therapeutics; for quantitative glycomics; for evaluating biological activity, quality/purity and efficacy during bioproduction, including antibodies, protein therapeutics; for comparing innovator and biosimilar glycosylated therapeutic molecules; for selecting therapeutic molecules with a desired glycosylation; to distinguish between Fc and Fab fragments, etc. Nearly 40% of all approved therapeutic proteins are glycosylated, and 8 out of the 10 top selling protein therapeutics contain N-linked oligosaccharides. Glycosylation has an impact on drug stability, half-life, clearance (dose repeats), immunogenicity, and other physiochemical properties. Glycoform modifications can occur during drug design, development and optimization during bioproduction. Critical quality attributes (CQA) may be strictly monitored during bioproduction of biotherapeutics. The consistency of the production process, and changes in glycosylation patterns and levels can be monitored by the glycan profiles.
Following labeling, aqueous media, for example, water, may be added to dilute the reactants to an appropriate range before taking sample analytes to be introduced into an analytical instrument or instruments by which the practitioner intends to analyze the labeled compound or compounds. Analyses, such as measuring fluorescent intensity and performing mass spectrometry are commonly performed in the art and one skilled in the art would be familiar with preparing samples of N-glycans and other compounds for such analysis, including bringing the samples up to pre-determined equal volumes before introducing an aliquot into the instrument. For example, after labeling, volumes of each replicate were brought up to 100 μL before an aliquot was taken and introduced into the analytical instrumentation, but the volume may depend on the quantity of initial reactants and on the particular system on which the labeled compounds are to be analyzed. A dilute acid, such as dilute acetic acid or formic acid, may be used in place of water to bring the volume up to the pre-determined volume.
Compounds
The present disclosure provides compounds that are both fluorescent and MS active. The compounds may be used in labeling or tagging a target analyte, for e.g. a glycan of a glycoprotein and subsequently, in the separation of the labeled glycans and/or detection by a variety of methods. In certain variations, the compounds described herein can be further modified, and the modifications may facilitate rapid labeling, better fluorescence detection, better mass spectrometry detection, and/or enable the translation of compounds into kits described below. In general, the compounds comprise (a) at least one fluorescent group such as an aromatic, substituted aromatic ring, aromatic, a phenyl, a polycyclic aromatic, a heterocycle, (b) at least one group capable of binding to an aldehyde or ketone present on an analyte of interest, for e.g.; a glycoprotein; and (c) at least one negatively charged group that can be detected by negative ion mass spectrometry.
Therefore, the present disclosure provides compounds that are both fluorescent and MS active, having a general formula (I):
wherein,
Accordingly, A, the molecular scaffold can comprise an aromatic group including but not limited to benzene, (N- or S-) substituted benzene, naphthalene, (N- or S-) substituted naphthalene, anthracene, (N- or S-) substituted anthracene, pyrene, (N- or S-) substituted pyrene, derivatives or salts thereof of any of the preceding compounds.
Accordingly, X may be a group for coupling to glycan aldehydes, or glycan ketones, or a glycosylamine, for e.g., an amine, hydrazine or substituted hydrazine, hydrazide or substituted hydrazide, hydrazone or substituted hydrazone, oxime or substituted oxime, an activated carboxylic group such as a succinimidyl or N-hydroxysuccinimidyl (NHS) ester or equivalents (including but not limited to cyanates, thiocyanate, isothiocyanates, isocyanates), carbamates, or derivatives and salts thereof of any of the preceding compounds.
Accordingly, W may be a negatively charged group designed to enhance detection in negative ion mass spectrometry (MS), including but not limited to, sulfonic acid and their derivatives or salts thereof, phosphoric acid and their derivatives or salts thereof, carboxylic acid and their derivatives or salts thereof.
Throughout the present disclosure, by ‘composition(s)’ or ‘compound(s)’ is meant, generally, a compound have the general structure of (I), and in a preferred embodiment, having the structure of sulfanilic acid (SA) and their derivatives or salts thereof. However, by ‘composition(s)’ or ‘compound(s)’ is also meant, any compound/formula as described in Formula I, II, II, IV, V, VI, VII, VIII, IX, X, XI, their derivatives or salts thereof, as shown in
In exemplary embodiments, stable isotope variants of sulfanilic acid—13C6 isotopologue may be generated, which provide superior quantitative and comparative MS/MS based analyses. Similarly, stable 13C6 isotopologues of the compositions/compounds described above (for e.g, any of Formulae I, II, II, IV, V, VI, VII, VIII, IX, X, XI, their derivatives or salts thereof, as shown in
Thus in certain embodiments, described herein are compositions comprising the compounds as shown in
Accordingly, the compounds or compositions disclosed herein are fluorescent, MS active compounds that comprise three functional components: (a) a molecular scaffold comprising an aromatic group (for fluorescence), including but not limited to benzene, (N- or S-) substituted benzene, naphthalene, (N- or S-) substituted naphthalene, anthracene, (N- or S-) substituted anthracene, pyrene, (N- or S-) substituted pyrene, derivatives or salts thereof of any of the preceding compounds; (b) a group for coupling to glycan aldehydes, or glycan ketones, or a glycosylamine, for e.g., an amine, hydrazine or substituted hydrazine, hydrazide or substituted hydrazide, hydrazone or substituted hydrazone, oxime or substituted oxime, an activated carboxylic group such as a succinimidyl or N-hydroxysuccinimidyl (NHS) ester or equivalents (including but not limited to cyanates, thiocyanate, isothiocyanates, isocyanates), carbamates, or derivatives and salts thereof of any of the preceding compounds; (c) a negatively charged group designed to enhance detection in negative ion mass spectrometry (MS), including but not limited to, sulfonic acid and their derivatives or salts thereof, phosphoric acid and their derivatives or salts thereof, carboxylic acid and their derivatives or salts thereof.
In some embodiments, the compounds or compositions disclosed herein may further comprise one or more linkers which may be optionally placed between the aromatic substituent or component (A) and the negatively charged substituent or component (W); and/or, one or more linkers may be optionally placed between the aromatic substituent or component (A) and the molecular scaffold substituent or component designed to react with a glycan aldehyde or ketone (X) (see Formula (I),
In an illustrative embodiment, sulfanilic acid (SA) or 3-aminobenzene sulfonic acid may be used to label N-glycans released from glycoproteins through standard reductive amination in the presence of reducing agents. As discussed above, similar methods may be applied to any of the compounds shown in
In an illustrative embodiment, the permanent negative charge imparted by the sulfonic acid group can make sulfanilic acid and its derivatives ideal labeling reagents for negative ion mode N-glycan analysis. When compared to the current labeling solutions with permanent positive charges, the use of a permanently negatively charged label and negative ion polarity, such as seen with sulfanilic acid and its derivatives, is highly attractive for its substantially higher analytical benefits to the end user. Some of the reasons for the superior analytical benefits of sulfanilic acid, its derivatives/salts, or its equivalents (described above) are outlined here: (i) the permanently negatively charged label in the negative ion mode generates a cleaner MS and MS/MS spectra due to the lower overall ion flux into the instrument compared to positive ion mode which, although ion counts are generally one order of magnitude lower than positive ion mode, resulting in a more sensitive analyses due to the removal of background ions; (ii) the permanently negatively charged label facilitates the analysis of sialylated N-glycans, since the ionization bias and then instability of sialylated oligosaccharide ions encountered typically during analysis with positive ion polarity is removed. More importantly, increased ionization efficiency of sialylated glycans is achieved, which further increases the overall analytical sensitivity of N-glycans, for e.g, sialylated glycans and associated oligosaccharides; (iii) highly informative MS/MS analysis may be possible with N-glycans labeled with sulphanilic acids and its derivatives. The application of MS/MS with negative ion polarity may result in both glycosidic cleavages and cross ring fragmentation, which may facilitate more informed structural annotation of the N-glycans; (iv) monosaccharide rearrangements, in particular fucose migration, which is typically problematic during MS/MS glycan analysis using positively charged amine containing labels, have not been observed with sulfanilic acids and its derivatives; (v) issues with intramolecular analyte interactions and problems with selectivity on hydrophilic interaction and weak anion exchange phases, especially seen with sialylated N-glycans using the positively charged labels, for e.g., procainamide based labels like the RapiFlour MS reagent, are avoided when permanently negatively charged labels like sulfanilic acid and its derivatives are used. When using permanently negatively charged labels like sulfanilic acid and its derivatives, complications associated with the use of certain exoglycosidase enzymes for monosaccharide and linkage anomericity assessment have also been avoided; (vi) sulfanilic acid and its derivatives can be synthesized as 13C6 isotopologues based on the sulphonation of 13C6 aniline by fuming sulphuric acid to generate a solution for relative MS based quantitation and comparability analysis. Such 13C6 isotopologues generation is not possible using for e.g., RapiFluor MS; (vii) Sulfanilic acid and its derivatives may be compatible with DMT-MM based sialic acid linkage specific derivatization chemistry which may be suitable for both LC-MS and CE-MS based glycan analysis. This method, when combined with the use of 13C6 isotopologue-labeling on the reducing terminus of glycans/saccharides, may enable the differential analysis and/or quantitation of (12C6/13C6) individual glycan/saccharide isomers within a complex mixture of glycans/saccharides; (viii) the additional negative charge imparted on the labeled glycans due to labeling with sulfanilic acid and its derivatives may be beneficial in chromatographic purification methods, for e.g., using mixed mode, ion-exchange, anion exchange, or HIC columns.
Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation.
Sulfanilic acid (or its derivative/salts, or its equivalent) labeled N-glycans provide high sensitivity for UPLC/UHPLC-fluorescence, and informative negative ion polarity with strong [M-2H]2− pseudomolecular ions suited for LC-MS and CE-MS analysis of complex oligosaccharides. Additionally, a stable isotope variant of sulfanilic acid—13C6 isotopologue may be generated, which provided superior quantitative and comparative MS/MS based analyses. The stable 12/13C6 sulfanilic acid isotopologues are useful for quantitative glycan profiling in a twoplex manner, which generates extracted ion electropherograms (EIE) for 12C6‘light’ and 13C6 ‘heavy’ sulfanilic acid labeled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical materials, for potential biomarker discovery, or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Other glycomic applications include applications such as studying glycosylation patterns of biological or medical samples, or for assessing protein production, protein quality/purity, for comparing innovator and biosimilar glycosylated proteins, or for selecting proteins with the desired glycosylation.
Labeling, Analyzing and Quantitation with Sulfanilic Acid or its Derivatives
In an exemplary embodiment, the labeling of the analyte, for e.g., N-glycan was done with sulfanilic acid or any of its derivatives (described herein in
Sialylation of Glycoproteins
Terminal sialylation of glycosylated polypeptides has been reported to increase serum-half life of therapeutics. In the biotechnological production of therapeutic polypeptides such as immunoglobulins the assessment of oligosaccharide microheterogeniety and its batch-to-batch consistency are important tasks. Monoclonal antibodies (mAbs) are one of the fastest growing classes of protein therapeutics. Antibodies differ significantly from other recombinant polypeptides in their glycosylation pattern. Immunoglobulin G (IgG) for e.g., is a symmetrical, multifunctional glycosylated polypeptide of an approximate molecular mass of 150 kDa consisting of two identical Fab parts responsible for antigen binding and the Fc part for effector functions. Glycosylation tends to be highly conserved in IgG molecules at Asn-297, which is buried between the CH2 domains of the Fc heavy chain, forming extensive contacts with the amino acid residues within CH2 (Sutton and Phillips, Biochem. Soc. Trans. 11 (1983) 130-132). The oligosaccharides bound to the Fc region, do not only effect physicochemical properties (e.g. structural integrity) and abolish or minimize protease resistance but are also essential for effector functions, such as complement binding, binding to macrophage Fc receptors, rapid elimination of antigen-antibody complexes from the circulation, and induction of antibody-dependent cell-mediated cytotoxicity (ADCC) (Cox, K. M., et al., Nature Biotechnol. 24 (2006) 1591-1597; Wright and Morrison, Trends Biotechnol. 15 (1997) 26-32). Because different glycoforms can be associated with different biological properties, the ability to enrich for a specific glycoform may be useful, for example, to elucidate the relationship between a specific glycoform and a specific biological function. Thus, production of glycosylated polypeptide compositions that are enriched for particular glycoforms is highly desirable.
Kits
Conveniently, one or more of the materials required for methods described herein can be provided in kit form either in dried form or in solution. This may be added directly to a sample of the compound analyte which is to be labeled. The kits may further provide instructions for labeling an amine-containing compound or an aldehyde/ketone of interest. For example, the kits may include directions on how to label a N-glycan. The kit may also include reagents, such as PNGase F or one or more other deglycosylation enzymes, to facilitate release and labeling of glycans from a glycoprotein.
In certain embodiments, the kit further comprises instructions for covalently bonding the compound to the analyte. In certain embodiments, the kit further comprises one or more of the following: a buffering agent, a purification medium, a vial comprising the analyte, or an organic solvent, one or more reagents for releasing the glycan from a biomolecule, and optionally, one or more reagents to purify the released glycan from the reaction mixture.
The kit may further comprise instructions for labeling glycans in a sample in preparation of glycan analysis, the method comprising: treating the sample with a release reagent, such as PNGase F enzyme, with an appropriate buffer under conditions suitable for the release of the glycan from the biomolecule, thereby forming a reaction mixture; adding beads and buffer to the reaction mixture; separating the supernatant from the beads; washing the beads with wash buffer; eluting the glycans from the beads with elution buffer; performing dye labeling of the glycans using one or more dye compounds provided herein, thereby forming a glycan-containing solution; optionally, removing excess dye using fresh beads; washing beads, separating the beads from excess dye/wash solution; and eluting glycans from the beads; and collecting the glycan-containing solution. In certain embodiments, the glycan solution may be stored for future use according to instructions provided, or analyzed for its glycan profile using a CE analyzer or UPLC analyzer or a combination thereof, and/or, using UV/visible or fluorescence detection in appropriate excitation and emission wavelengths, and/or, preceding or following the previous techniques with mass spectrometry detection, which may be used for structural elucidation of at least one glycan present in a sample.
The present disclosure provides compounds comprising (a) at least one fluorescent group such as an aromatic or substituted aromatic ring; (b) at least one group capable of binding to an aldehyde or ketone present on an analyte of interest, for e.g.; to a glycoprotein; and (c) at least one negatively charged group that can be detected by negative ion mass spectrometry. The resultant compound, for e.g., the labeled glycan, may be highly fluorescent, thereby providing an excellent method for detection of aldehydes, ketones or glycosylamines in solution, and additionally, may be highly charged thereby enhancing the detection in negative ion mass spectrometry (MS) detection. The compounds can also be analyzed using capillary electrophoresis (CE), liquid chromatography (LC, UPLC, UHPLC, etc.), other similar techniques. The above techniques can also be twoplexed/duplexed/multiplexed for analysis, for e.g., UPLC-fluorescence—MS analysis, or CE-LC, or LC-MS, or CE-MS, or MS-MS, etc. These techniques or their combinations may be set up for automation.
Certain embodiments provide the use of such compounds, compositions or kits for labeling an analyte. In certain embodiments, the analyte is a biological molecule; for e.g. glycans or glycoproteins. In specific embodiments, the compounds or compositions described can label glycans.
The kits described herein for labeling glycans comprise the compounds generically described in formula (I) and specifically described in
Synthesis of NHS Ester of Sulfobenzoic Acid. NHS Ester of sulfobenzoic acid can be synthesized essentially by adopting the method of Navotny et al (Journal of Proteome Research, 6 (2007) 124-132). Briefly, to a solution of sulfobenzoic acid in dry DMSO was added 1.5 molar excess of dicyclohexylcarbodiimide. This solution was stirred at room temperature for approximately 10-15 min, after which equimolar amounts of N-hydroxysuccinimide and diisopropylethylamine were added to the reaction mixture. The reaction mixture was stirred at room temperature for 3 h. The precipitated dicyclohexylurea, a side product of the reaction, was filtered and discarded. The remaining solution was then allowed to react for 2 h and was again filtered to remove the side product. After the second filtration, approximately 60 mL of cold acetone (4° C.) was slowly added to precipitate the product. The final product was then washed four times; each washing step involved the addition of approximately 15 mL of cold acetone. After the final washing, the product was dried under vacuum for approximately 2 h to give the desired product.
NHS Conjugation (Reaction Scheme of
Reductive Amination (Reaction Scheme of
Method of Labeling Glycans with sulfanilic acid (for e.g. 3ASA): N-linked glycans were enzymatically removed with PNGase F and glycans are purified using magnetic beads. As glycans are released from a glycoprotein, it is useful to label them. N-glycans are typically labeled at their free-reducing terminus and are analyzed by methods such as high performance liquid chromatography (HPLC), UPLC/UHPLC, HILIC, capillary electrophoresis (CE), carbohydrate gel electrophoresis, fluorescence analysis, mass spectrometry (MS) and others. Glycans were identified by retention time relative to an appropriate size standard ladder. For e.g., glycans can be quantified using maltotriose (M3), maltotetrose (M4) and/or maltopentose (M5) as internal standards (see
CE-MS analysis: A key feature of the CE-MS method is low sample consumption, enabling LC-MS to be performed using the same sample, thereby facilitating quantitative structural analysis using an orthogonal method for verification purposes. CE-MS experiments were performed using an Agilent 7100 capillary electrophoresis system interfaced using an Agilent G1607B orthogonal coaxial sheath sprayer to an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer under the control of Mass Hunter acquisition software, version B.06.01. (Santa Clara, Calif., USA). Samples were injected hydrodynamically (100 mbar, 10 seconds) and electrophoresis separations were performed using an applied voltage of −30 kV. Reagents, sample preparation methodology and conditions used herein for CE-MS was done as described in Varadi et al., Anal. Bioanal. Chem., 2016 December; 408(30):8691-8700, hereby incorporated by reference in its entirety.
Mass spectra were collected using negative ionisation mode over the acquisition range of 500-2000 m/z with 2 GHz digitisation.
LC-MS analysis: LC-MS analysis was also performed using 12C 3ASA labeled N-glycan samples from the different lots of monoclonal antibody, Cetuximab.
Method of Quantitative Glycan Analysis Using 12C and 13C-Sulphanilic Acid Stable Isotope Labeling
The twoplex 12C6/13C6-2D LC-MS platform is ideally suited for differential glycomic analysis of structurally complex glycan pools due to combination and analysis of samples in a single LC-MS injection, and due to minimal technical variation. A distinct advantage of 3ASA labeling is its permanent negative charge and negative ion polarity with strong [M-H]2− pseudomolecular ions well-suited for LC-MS and CE-MS analysis of complex oligosaccharides. The stable isotope variant of sulfanilic acid—13C6 isotopologue was generated which provided superior quantitative and comparative MS/MS based analyses. Quantitative glycan profiling in a twoplex manner generated extracted ion electropherograms (EIE) for 12C6 ‘light’ and 13C6 ‘heavy’ sulfanilic acid labeled glycan isotope clusters.
To evaluate the performance of the twoplex 12C6 and 13C6 3 ASA quantitation using the developed CE-MS method, two different biosimilar drugs were labeled with 12C6 and 13C6 3 ASA, cleaned and analysed by CE-MS. Quantitation was performed based on the peak area of generated EIEs for the isotope clusters of the 12C6 ‘light’ and 13C6 ‘heavy’ 3ASA, respectively. Resulting isotope based quantitation data is shown in
DMT-MM derivatization allows for mass-based differentiation of sialic acid linkage isomers and their simultaneous and differential glycomic analysis. DMT-MM is 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. With HILIC (Hydrophilic Interaction Liquid Chromatography), separation is mediated by changes in their structural polarity. Such methods, when combined with the use of 13C isotopologue-labeling on the reducing terminus of glycans/saccharides, enabled the differential analysis and/or the quantitation of (12C/13C) individual glycan/saccharide isomers within complex mixtures of glycans/saccharides, such as in biological samples, medical samples, etc.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/022988 | 3/16/2018 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62472230 | Mar 2017 | US |
