DESC:FIELD OF THE INVENTION
The present invention relates to a method for characterizing various isomeric glycoforms of an erythropoiesis stimulating agent. BACK GROUND OF THE INVENTION Cytokines, such as erythropoietin and darbepoetin alfa are major erythropoiesis 5 stimulating agents (ESA’s) that are known to stimulate production of red blood cells in mammals. Darbepoetin alfa is a second generation ESA, derived from erythropoietin alfa (EPO). It is a recombinant protein, and have a longer serum half -life due to the presence of two additional N-glycosylation sites as opposed to erythropoietin. (US7217689, Macdougall et al JAm Soc Nephrol 1999; 1 10 0:2392-2395). The human EPO molecule is known for its micro heterogeneity due to presence of carbohydrate side chains containing different structures and varying sugar content. Further a negatively charged sialic acid molecule typically caps the end of each arm of a carbohydrate chain. 15 Darbepoetin alfa contains upto 22 sialic acid molecules (upto four sialic acids on each of the five N-linked and upto two sialic acids on one O-linked glycosylation site). Darbepoetin isoforms arise mainly due to highly branched N-glycans and based on degree of branching they are characterized as mono, bi, tri and tetra antennary species. Further, the number of sialic acid moieties present in 20 darbepoetin alfa leads to formation of more complex isoforms. Sialic acid content present on darbepoetin alfa are known to impact in-vivo half-life and its therapeutic activity. Further, distribution of these negatively charged sialic acid moieties may also play an important role in those activities. Hence it becomes imperative to study both the sialic acid content and charge distribution 25 of sialic acid containing isoforms present in darbepoetin alfa.
3
Hydrophilic interaction liquid chromatography (HILIC) is often used for glycan analysis of glycoproteins. These HILIC columns separate glycans mainly by hydrogen bonding between polar functional groups and the stationary phase, resulting in size and polarity based separation. However these columns do not separate glycoproteins based on charge or isoforms distribution. Hence, there 5 exists a challenge to characterize molecules like darbepoetin alfa with complex glycan isoforms, using only HILIC chemistry. Henceforth, the primary objective of the invention is to provide a method for extensive analysis of glycan isoforms present in darbepoetin alfa molecule, wherein the said method is a mixed mode separation chromatographic technique 10 coupled to mass spectrometry. Further objective of the invention is to provide optimized chromatographic conditions wherein the said conditions facilitate separation of isomers based on weak anion exchange and reverse phase properties of the stationary phase. SUMMARY OF THE INVENTION 15
The present invention discloses a method for extensive characterization of isomeric glycans of darbepoetin wherein the said method comprises salt gradient based mixed mode chromatographic separation coupled to mass spectrometry.
The said mixed mode resin surface comprises a combination of both weak anion exchange (WAX) and reverse phase (RP), wherein the said weak anion exchange 20 chromatography separates glycans based on charge and reverse phase chromatography separates glycans based on their branching or linkage isomerism, and size.
In addition, buffer conditions and flow rate are optimized as a time dependent factor during the course of chromatographic technique for separation of various 25 possible isomeric glycan forms of darbepoetin alfa with higher resolution.
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BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Illustrates a representative chromatogram of darbepoetin at 420nm using optimized mixed mode coupled with mass spectrometry. Here, di- (D1-D19), tri (T1-T26) and tetra (Q1-Q20) indicates number of sialic acid moieties present on various isoforms of darbepoetin. The theoretical and observed masses 5 for the major glycoforms, which have an intensity of =5% are tabulated in Table 2, with assignment of possible glycan structures.
DESCRIPTION OF THE INVENTION
The present invention discloses a method for separation and characterization of highly sialylated and branched isoforms distribution in an erythropoetin 10 stimulating agent (ESA) by mixed mode chromatography coupled with mass spectrometry.
In one embodiment of the invention discloses, a method for analyzing and identifying isomeric distribution of glycoforms of erythropoiesis stimulating agent comprising 15
a) treatment of sample comprising erythropoiesis stimulating agents with PNgase F to separate glycans
b) labeling the obtained glycans from step a) with a chromophore
c) separating the labelled glycans obtained from step b) by subjecting the sample to mixed mode chromatography using a gradient with an 20 optimized flow rate and percentage of solvents
d) analyzing the molecular weight of the eluted molecules in peaks using mass spectrometry and assigning glycan structures to the peaks based on the theoretical mass
wherein the chromatographic technique in step (c) is coupled to mass 25 spectrometry.
5
In another embodiment of the invention, the erythropoietin stimulating agent is selected from first generation ESA such as erythropoietin or second generation ESA such as darbepoetin.
In another embodiment of the invention, glycans are labelled with chromophores such as -amino benzamide (2-AB), anthraanilic acid, pyrilium or the like, wherein 5 the chromophore imparts optical property for detection.
In yet another embodiment of the inventions, alternatively labelling of glycans is performed using MS tagging agents.
In another embodiment of the invention, mixed mode chromatography is a combination of ion exchange and reverse phase chromatography. 10
In yet another embodiment of the invention, ion exchange chromatography is a weak anion exchange chromatography which separates glycans based on charge.
In another embodiment of the invention, reverse phase chromatography is based on principles of reverse phase chromatography which resolves glycans based on size, linkages or branching, and isomeric structure of glycans of a 15 glycoprotein/ESA.
In another embodiment of the invention, salt gradient is obtained by mixing combination of acetonitrile and ammonium formate in Milli Q water.
In yet another embodiment of the invention, optimized flow rate used for separation of various isomeric glycoforms of erythropoietin stimulating agent 20 comprises about 0.2 ml/min for 10 minutes, followed by decreasing flow rate for 10 minutes, and maintaining the same flow rate of 0.2 ml/ min from about 21 to about 60 minutes.
In another embodiment of the invention, the said chromatographic technique is compatible with both mass spectrometric and fluorescence detection methods. 25
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In another embodiment of the invention, flow rate and percentage of solvents is optimized in said mixed mode chromatographic technique to separate maximum isomeric glycoforms of erythropoiesis stimulating agent with good resolution.
The present invention discloses a method for separating various isomeric glycoforms of erythropoiesis stimulating agent based on their charge, size, 5 polarity and isomeric structure using mixed-mode chromatography. Further, the said method is coupled with mass spectrometry which identifies mass of molecules eluted in chromatography and glycan structures for the peaks are assigned based on the theoretical mass.
In another embodiment the invention describes, a method for analyzing and 10 identifying isomeric distribution of glycoforms of erythropoiesis stimulating agent comprising
a) treatment of sample comprising erythropoiesis stimulating agents with PNgase F to separate glycans
b) labelling the obtained glycans from step a) with a chromophore 15
c) separating the labelled glycans obtained from step b)
i. by subjecting the sample obtained to mixed mode chromatography using a salt gradient obtained by mixing combination of acetonitrile and ammonium formate in Milli Q water
ii. using a series of flow rate comprising about 0.2 ml/min for about 20 10 minutes, followed by decreasing flow rate for 10 minutes, followed by maintaining flow rate of 0.2 ml/ min for about 40 minutes
d) analyzing the molecular weight of the eluted molecules in peaks using mass spectrometry and assigning glycan structures based on theoretical 25 mass
wherein the chromatographic technique in step c) is coupled to mass spectrometry.
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Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
Examples 5
Example 1. Glycan preparation, labeling & UPLC based separation
Sample comprising erythropoiesis stimulating agent such as darbepoetin alfa was characterized based on the separation of various isomeric glycan species.
The sample comprising darbepoetin alfa was treated with glycanase enzyme PNGase F by incubating at 37°C for 16- 18 hours. After incubation the glycans 10 were purified using LudgercleanTM EB10 column and labeled using commercially available LudgerTag TM 2-AB labeling kit and incubated at 65°C for 2±1 hours. The 2-AB labeled glycans were purified using S-cartridge cleaning kit. The released glycans were analyzed on water UPLC using mixed mode column as GlycanPACTM-AXR1 column (1.9 µm-, 2.1x150 mm). Optimized gradient program 15 followed by column cleaning and equilibration for separating glycans using GlycanPAC-AXR1 column provided in Table 1. The chromatogram obtained is depicted in Figure 1 (GlycanPAC-AXR1). Further, the chromatographic technique is coupled with mass spectrometry to calculate mass of eluted species and the possible structure to the peaks are assigned based on the theoretical mass and is 20 given in table 2. A total of 81 glycan species were found and possible glycan structures were assigned by mass. The theoretical and observed masses for the major glycoforms, which have an intensity of =5% are tabulated in Table 2, with assignment of possible glycan structures.
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Time (Min)
Flow rate
Mobile Phase A
(100% Acetonitrile)
Mobile Phase B
(100mM Ammonium formate pH 4.5)
Mobile Phase C
(Milli-Q water)
0-2
0.2 ml
0.0
10.0
90.0
2-13
0.2 ml
4.9
23.8
71.3
13-14
0.1 ml
4.9
23.8
71.3
14-23
0.1 ml
6.0
25.0
69
23-35
0.2 ml
6.8
27.4
65.8
35-50
0.2 ml
8.3
29.5
62.2
50-60
0.2 ml
10.0
40
50
Table 1: Represents gradient flow rate employed for GlycanPACTM-AXR1 for separation of darbepoetin isomeric glycoforms.
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Peak No.a
Theoretical
Mass
(MW)
Observed Mass (MW)
Possible Glycan
DRL_DA RMP Structure
D2, D3 3566.35 3566.0 3566.0
D4, D5, D6 3201.224 3200.9 3200.9
D9, D10 2836.09 2835.87 2835.87
D11, D12 3242.25 3242.93 3242.93
T1, T2, T3, T4 3857.45 3857.09 3857.09
T6, T7 3492.32 3491.99 3491.99
T13 3127.18 3126.91 3126.91
T19 3169.18 3168.91 3168.91
Q1 4190.52 4190.19 4190.19
Q2, Q3 3783.416 3783.03 3783.03
Q6, Q7, Q8,
Q9, Q10, Q11,
Q12
3867.41 3867.03 3867.03
Table 2: List of observed and theoretical mass of major glycans with
assignment of possible glycan structure. Symbols used in figure are: Nacetylglucosamine
- ?, Mannose - ?, Galactose - ?, Sialic acids - ?, Fucose - ?
5 a For peak assignment please refer to ,CLAIMS:We claim:
1. A method for analyzing and identifying isomeric distribution of glycoforms of erythropoiesis stimulating agent comprising
a) treatment of sample comprising erythropoiesis stimulating agents with 5 PNgase F to separate glycans
b) labelling the obtained glycans from step a) with a chromophore
c) separating the labelled glycans obtained from step b)
i. by subjecting the sample obtained to mixed mode chromatography using a salt gradient obtained by mixing combination of 10 acetonitrile and ammonium formate in Milli Q water
ii. using a series of flow rate comprising about 0.2 ml/min for about 10 minutes, followed by decreasing flow rate for 10 minutes, followed by maintaining flow rate of 0.2 ml/ min for about 40 minutes 15
d) analyzing the molecular weight of the eluted molecules in peaks using mass spectrometry and assigning glycan structures based on theoretical mass
wherein the chromatographic technique in step c) is coupled to mass spectrometry. 20
2. The method according to claim1 wherein, the erythropoiesis stimulating agents is selected from first generation erythropoiesis stimulating agent such as erythropoietin or second generation erythropoiesis stimulating agent such as darbepoetin.
3. The method according to claim1 wherein, glycans are labelled with 25 chromophores such as 2-amino benzamide (2-AB), anthraanilic acid, pyrilium or the like, wherein the chromophore imparts optical property for detection.
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4. The method according to claim1 wherein, the glycans are alternatively labelled with MS tagging agents.
5. The method according to claim1 wherein, mixed mode chromatography is a combination of weak anion exchange and reverse phase chromatography.