U.S. patent application number 11/018077 was filed with the patent office on 2005-08-25 for method for analyzing a glycomolecule.
Invention is credited to Alergand, Tal, Amor, Yehudit, Bangio, Haim, Gulko, Mirit Kolog, Kasuto, Idil Kelson, Kleinman, Fredi, Markman, Ofer, Maya, Ruth, Rebe, Sabina, Rosenfeld, Rakefet, Samokovlisky, Albena.
Application Number | 20050186645 11/018077 |
Document ID | / |
Family ID | 34700181 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186645 |
Kind Code |
A1 |
Amor, Yehudit ; et
al. |
August 25, 2005 |
Method for analyzing a glycomolecule
Abstract
The invention relates generally the structural analysis of
glycomolecule-containing macromolecules, such as those that occur
either attached to proteins (proteoglycans, glycoproteins), lipids,
or as free saccharides.
Inventors: |
Amor, Yehudit; (Jerusalem,
IL) ; Markman, Ofer; (Rehovot, IL) ; Gulko,
Mirit Kolog; (Rishon-Lezion, IL) ; Samokovlisky,
Albena; (Ashdod, IL) ; Kleinman, Fredi;
(Rishon Le Zion, IL) ; Alergand, Tal; (Gedera,
IL) ; Rosenfeld, Rakefet; (Maccabim, IL) ;
Maya, Ruth; (Rinatia, IL) ; Rebe, Sabina; (Tel
Aviv, IL) ; Kasuto, Idil Kelson; (Tel Aviv, IL)
; Bangio, Haim; (Petach Tiqva, IL) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY
AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
34700181 |
Appl. No.: |
11/018077 |
Filed: |
December 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60531578 |
Dec 18, 2003 |
|
|
|
Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
G01N 33/5308 20130101;
G01N 2333/42 20130101; G01N 2400/02 20130101; G01N 2333/924
20130101 |
Class at
Publication: |
435/007.92 |
International
Class: |
G01N 033/53; G01N
033/537; G01N 033/543 |
Claims
What is claimed is:
1. A method for determining a glycomolecule fingerprint for a
glycomolecule, the method comprising: providing a glycomolecule,
wherein said glycomolecule has been modified by desialylation or
treatment with N-Glycosidase F (PNGaseF); adding said glycomolecule
to a substrate comprising a plurality of saccharide-binding agents;
detecting glycomolecule bound to saccharide-binding agents in the
plurality; and obtaining a fingerprint for the glycomolecule based
on the binding of the glycomolecule to the saccharide-binding
agents.
2. The method of claim 1, wherein said glycomolecule been modified
by desialylation.
3. The method of claim 2, wherein substantially all of the sialic
acid residues have been removed from said glycomolecule.
4. The method of claim 2, wherein the desialylation is effected by
reacting said glycomolecule with a sialidase.
5. The method of claim 4, wherein said glycomolecule is reacted
with said sialidase in the presence of a protease inhibitor.
6. The method of claim 1, wherein said glycomolecule been modified
by treatment with PNGaseF.
7. The method of claim 6, wherein substantially all of
Asn-acetylglucosamine bonds have been cleaved in said glycomolecule
by said PNGaseF.
8. The method of claim 1, wherein said glycomolecule been modified
by desialylation and treatment with PNGaseF.
9. The method of claim 1, further comprising reacting said
glycomolecule with a reducing agent and an alkylating agent prior
to obtaining said fingerprint.
10. The method of claim 9, wherein said glycomolecule been modified
by desialylation.
11. The method of claim 10, wherein said method comprises reacting
said glycomolecule with said reducing agent and alkylating agent
following desialylation.
12. The method of claim 9, wherein said reducing agent is selected
from the group consisting of .beta.-mercaptoethanol,
dithiothreitol, and mercaptethylamine.
13. The method of claim 9, wherein said alkylating agent selected
from the group consisting of iodoacetamide and iodoacetic acid.
14. The method of claim 1, wherein all steps of said method are
performed in a single container.
15. The method of claim 1, glycomolecule is detected with a label
associated with said glycomolecule.
16. The method of claim 15, wherein said label is a fluorescent
label.
17. The method of claim 16, wherein said fluorescent label is
selected from the group consisting of fluorescein isothiocyanate
(FITC), rhodamine, Texas Red, and Cy5.
18. The method of claim 15, wherein said label is added to said
glycomolecule prior to adding said glycomolecule to said
substrate.
19. The method of claim 15, wherein said label is added to
glycomolecule after adding said glycomolecule to said
substrate.
20. The method of claim 15, wherein said label is added to
glycomolecule while adding said glycomolecule to said
substrate.
21. The method of claim 15, wherein said label is associated
directly with said glycomolecule.
22. The method of claim 15, wherein said label is associated with a
second saccharide-binding agent that binds to said
glycomolecule.
23. The method of claim 22, wherein said second saccharide-binding
agent is a lectin.
24. The method of claim 22, wherein said second saccharide-binding
agent is an antibody.
25. The method of claim 1, further comprising purifying said
glycomolecule prior to adding said glycomolecule to said
substrate.
26. The method of claim 25, wherein said purification is by column
chromatography.
27. The method of claim 1, wherein said glycomolecule is a
glycoprotein.
28. The method of claim 27, wherein said glycoprotein is from a
cell culture medium.
29. The method of claim 27, wherein said glycoprotein includes at
least a portion of an immunoglobulin polypeptide.
30. The method of claim 29, wherein said immunoglobulin in IgG
isotype.
31. The method of claim 29, wherein said portion comprises an Fc
molecule.
32. The method of claim 1, wherein said method comprsies treating
said glycomolecule with a detergent prior to obtaining said
fingerprint.
33. The method of claim 32, wherein said detergent is an ionic
detergent.
34. The method of claim 33, wherein said detergent is sodium
docecyl sulfate (SDS).
35. The method of claim 1, wherein said substrate is a
microsphere.
36. The method of claim 35, wherein said substrate comprises a
plurality of micropsheres.
37. The method of claim 35, wherein no more than one type of
saccharide-binding agent is present on said microsphere.
38. The method of claim 35, wherein more than one type of
saccharide-binding agent is present on said microsphere.
39. A method for determining a glycomolecule fingerprint for a
glycomolecule, the method comprising: adding a glycomolecule to a
substrate comprising a plurality of saccharide-binding agents;
detecting glycomolecule bound to saccharide-binding agents in the
plurality; and obtaining a fingerprint for the glycomolecule based
on the binding of the glycomolecule to the saccharide-binding
agents.
40. The method of claim 15, wherein said glycomolecule is
associated with a label, and bound glycomolecules are detected by
identifying bound label on said substrate.
41. The method of claim 40, label is a fluorescent label.
42. The method of claim 41, wherein said fluorescent label is
selected from the group consisting of fluorescein isothiocyanate
(FITC), rhodamine, Texas Red, and Cy5.
43. The method of claim 40, wherein said label is added to said
glycomolecule prior to adding said glycomolecule to said
substrate.
44. The method of claim 40, wherein said label is added to
glycomolecule after adding said glycomolecule to said
substrate.
45. The method of claim 40, wherein said label is added to
glycomolecule while adding said glycomolecule to said
substrate.
46. The method of claim 40, wherein said label is associated
directly with said glycomolecule.
47. The method of claim 40, wherein said label is associated with a
second saccharide-binding agent that binds to said
glycomolecule.
48. The method of claim 47, wherein said second saccharide-binding
agent is a lectin.
49. The method of claim 47, wherein said second saccharide-binding
agent is an antibody.
50. The method of claim 40, wherein said label is associated with
an agent that binds to a non-carbohydrate molecule on said
glycomolecule.
51. The method of claim 50, wherein said glycomolecule is a
glycoprotein and said agent binds to a peptide epitope on said
glycoprotein.
52. The method of claim 49, wherien said substrate is substantially
planar.
53. The method of claim 40, wherien said substrate is a
microsphere.
54. The method of claim 53, wherein said substrate comprises a
plurality of micropsheres.
55. The method of claim 53, wherein each microsphere comprises one
type of saccharide binding agent.
56. A kit for analyzing a glycomolecule, the kit comprising a
glycomolecule modification agent selected from the group consisting
of a desialidase and a PNGase F; and a labeling agent for labeling
a glycomolecule.
57. The kit of claim 56, further comprising a substrate comprising
a plurality of saccharide-binding agents.
58. The kit of claim 56, further comprising a reducing agent and an
alkylating agent.
59. The kit of claim 56, wherein said labeling agent binds directly
to a glycomolecule.
60. The kit of claim 56, wherein said labeling agent comprises a
second saccharide-binding agent and a label that associates with
said second saccharide-binding agent.
61. The kit of claim 56, further comprising a substrate holder.
62. A kit for analyzing a glycomolecule, the kit comprising an
antibody specifically binds to a glycoprotein; and a labeling agent
for labeling a glycomolecule.
63. The kit of claim 62, further comprising a glycomolecule
modification agent selected from the group consisting of a
desialidase and a PNGase F.
64. The kit of claim 62, further comprising a substrate comprising
a plurality of saccharide-binding agents.
65. The kit of claim 62, further comprising a reducing agent and an
alkylating agent.
66. The kit of claim 62, wherein said labeling agent binds directly
to a glycomolecule.
67. The kit of claim 62, wherein said labeling agent comprises a
second saccharide-binding agent and a label that associates with
said second saccharide-binding agent.
68. The kit of claim 62, further comprising a substrate holder.
69. The kit of claim 62, wherein said antibody binds to a
polypeptide epitope on said glycoprotein.
70. The kit of claim 62, wherein said antibody binds to a
polysaccharide epitope on said glycoprotein.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of, and priority to,
U.S. Ser. No. 60/531,578, filed Dec. 18, 2003. The contents of this
application are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The invention relates generally to the structural analysis
of glycomolecules, which are molecules that contain carbohydrates
and include carbohydrates attached to proteins (proteoglycans,
glycoproteins), to lipids, or carbohydrates present as free
polysaccharides.
BACKGROUND OF THE INVENTION
[0003] Mammalian glycoprotein oligosaccharides are commonly built
from a limited number of monosaccharides. Nevertheless, structural
diversity is vast, mainly due to complex branching patterns.
Glycosylation sites on glycoproteins commonly display
microheterogeneity in that they can be fully or partially occupied
by structurally diverse oligosaccharides. Consequently, a
glycoprotein is not typically isolated as a single structural
entity, but rather as a set of glycosylation variants known as
glycoforms.
[0004] There is evidence that both the in vivo and in vitro
properties of glycoproteins are affected by changes in occupancy
and/or the precise oligosaccharide attached to a particular site.
Distinct biological properties have been correlated with the
presence of particular glycoforms.
[0005] A method for determining the composition and sequence of
polysaccharides in a carbohydrate-containing molecule
("glycomolecule") has been described (see, e.g., WO00/668688,
WO01/84147, WO02/37106, and WO02/44714). In this method, termed
UC-FINGERPRINT.TM. analysis (also known as GMID.TM. analysis), a
carbohydrate-containing molecule is added to a substrate containing
an array of saccharide-binding agents (typically antibodies or
lectins). Saccharide-binding agents bound to the glycomolecule are
identified, and the binding information is used to obtain
composition and sequence information of the monosaccharide subunits
in the polysaccharide.
SUMMARY OF THE INVENTION
[0006] The invention is based in part on the discovery of methods
that facilitate the preparation of glycomolecules for subsequent
analysis in UC-FINGERPRINT.TM. technology. The analysis provides
glycan composition and sequence information, which is often
referred to as a fingerprint of the glycomolecule.
[0007] Among the advantages of the method is simplified sample
preparation and processing. The methods described herein eliminate
the need for multiple pretreatment, treatment, purification, and
buffer changing steps. In addition, the methods facilitate access
to glycomolecules that are otherwise difficult to analyze. These
glycans include, e.g., glycans in inter-subunit clefts or
intra-subunit clefts of glycoproteins.
[0008] In one aspect, the invention provides a method for
determining a glycomolecule fingerprint for a glycomolecule. In
some embodiments, the glycomolecule is one whose native glycan
structure has been modified. The method includes adding the
glycomolecule to a substrate that includes a plurality of
saccharide-binding agents. Glycomolecules bound to
saccharide-binding agents on the substrate are detected. A
fingerprint is obtained for the glycomolecule based on the binding
of the glycomolecule to the saccharide-binding agents.
[0009] In some embodiments, the glycomolecule has been modified by
desialylation. The extent of the desialylation can be modulated, so
that in some embodiments, substantially all of the sialic acid
residues have been removed from the glycomolecule. In other
embodiments, less than all of the sialic acids have been removed
from the glycomolecule.
[0010] A suitable method for desialylating the glycomolecule is by
reacting the glycomolecule with a sialidase. The glycomolecule can
optionally be reacted with the sialidase in the presence of a
protease inhibitor.
[0011] The glycomolecule can alternatively, or in addition, be
modified by treatment with PNGaseF. The extent of treatment with
PNGaseF can be modulated, so that in some embodiments,
substantially all of the bonds between the innermost GlcNAc and
asparagine residues of high mannose, hybrid and complex
oligosaccharides of the glycoprotein have been cleaved. In other
embodiments, less than all of the N-Acetyl Glucosamine acid
residues have been cleaved.
[0012] In some embodiments, the method includes reacting the
glycomolecule with a reducing agent and, preferably, an alkylating
agent prior to obtaining the fingerprint. Examples of suitable
reducing agents include mercaptoethanol, dithiothreitol, and
mercaptethylamine. Examples of suitable alkylating agents
iodoacetamide and iodoacetic acid.
[0013] In some embodiments, all steps of the method are performed
in a single container.
[0014] In some embodiments, the glycomolecule is detected with a
label associated with the glycomolecule. Examples of suitable
labels include, e.g., a fluorescent label. The fluorescent label
can be, e.g., fluorescein isothiocyanate (FITC), rhodamine, Texas
Red, and Cy5.
[0015] The label can be added to the glycomolecule prior to, after,
or while adding the glycomolecule to the substrate.
[0016] In some embodiments, the label is associated directly with
the glycomolecule. In other embodiments, the label is associated
with a second saccharide-binding agent that binds specifically to
the glycomolecule. The second saccharide-binding agent can be,
e.g., a lectin or an antibody. In some embodiments, the label is
associated with an agent (such as an antibody) that binds
specifically to a non-carbohydrate region of the glycomolecule.
[0017] In some embodiments, the method further includes purifying
the glycomolecule prior to adding the glycomolecule to the
substrate. The purification can be, e.g., by column chromatography
ordialysis with a molecular cut off of a defined mass, e.g., 5000
kD The glycomolecule can be any saccharide-containing molecule.
Examples include glycoprotein, polysaccharide, or glycolipid.
[0018] In some embodiments, the glycoprotein is obtained from a
cell culture medium. In some embodiments, the glycoprotein is
purified and/or concentrated prior to being used. In other
embodiments the glycoprotein is obtained from the medium and used
without purification or concentration.
[0019] In some embodiments, the method includes treating the
glycomolecule with a detergent prior to obtaining the fingerprint.
The detergent can be, e.g., a non-ionic detergent or anionic
detergent. Examples of a suitable detergent include, e.g. sodium
docecyl sulfate (SDS), Triton, and Tween80.
[0020] Examples of suitable glycoproteins include, eg.,
immunuglobuin molecules (including IgA, IgD, or IgG, or IgM
isotypes) or fragments of immunoglobulin molecules. For example,
the fragment can include an Fc region of an immunoglubulin.
[0021] In another aspect, the invention provides a kit that
includes a glycomolecule modification agent selected that is a
desialidase and/or a PNGase F, a labeling agent for labeling a
glycomolecule, a container and, optionally instructions for using
the kit to modify the glycomolecule The directions can be provided
on a kit label or as a kit insert, which describe how to manipulate
a glycomolecule using the methods described herein.
[0022] The kit may additionally contain a plurality of
saccharide-binding agents, a reducing agent, a detergent and an
alkylating agent. The labeling agent may bind directly to the
glycomolecule. Alternatively, the labeling agent is present with,
or capable of being associated with, a second saccharide-binding
agent that binds to the glycomolecule. The second
saccharide-binding agent can be, e.g., a lectin or an antibody.
[0023] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention,
suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
These include, but are not limited to, WO00/68688, WO01/84147,
WO02/37106, and WO02/44714. In the case of conflict, the present
Specification, including definitions, will control. In addition,
the materials, methods, and examples are illustrative only and not
intended to be limiting.
[0024] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1A is a schematic diagram showing the representative
bi-antennary glycans of human milk lactoferrin (hmLF). The various
glycans differ in the presence of the (2,6) linked sialic acid
residues and the (1,3) linked antennary fucose.
[0026] FIGS. 1B and 1C are fingerprints obtained by using a labeled
anti-lactoferrin antibody as a probe. Twenty-four array-bound
lectins were used in these experiments, and are grouped by their
specificities on the abscissa. The group of complex N-linked
glycans contains lectins that do not bind to monosaccharides, but
rather require a complex N-linked glycan containing at least 3
antennae; Results are shown for three independent experiments.
Signals were corrected for differences in scanning parameters
(laser power and PMT gain) for each slide, if applicable, and for
differences in levels of probe fluorescence if these differ between
experiments. FIG. 1C shows fingerprints of the same hmLF sample
following gradual enzymatic trimming of the glycans. Cross-hatched,
native hmLF; dark shading, hmLF following de-sialylalation; dark
shading, hmLF following removal of terminal galactose residues;
open, hmLF following removal of terminal GlcNAc.
[0027] FIG. 2A-2C are fingerprints of a Bows melanoma cell-line
derived tissue plasminogen activator (tPA). The fingerprint
obtained in FIG. 2A was obtained using direct labeling of the
sample. The fingerprint obtained in FIG. 2B was obtained using a
glucose/mannose recognizing probe that recognizes both high-mannose
and complex bi-antennary glycans. The fingerprint shown in FIG. 2C
was obtained using a glucose/mannose-recognizing probe that
recognizes only high mannose type glycans. Since each of the
fingerprints was obtained using a different probe, signals were
corrected for the variation in fluorescence of the labeled probes
(or sample for FIG. 2A), and for the variability in scanning
parameters. Deconvolution of fingerprints obtained using lectin
probes requires several fingerprints each obtained with a different
probe, in order to ensure signals from all lectins. Commonly 2-4
different probes, depending on the complexity of the glycosylation
pattern of the sample, are required.
[0028] FIGS. 3A-3C are fingerprints of desialylated bovine fetuin.
The fingerprint in FIG. 3A was obtained using a terminal
galactose-recognizing probe. The fingerprint in FIG. 3B was
obtained using a complex N-linked glycan-recognizing probe, and the
fingerprint in FIG. 3C was obtained using a Gal/GalNAc recognizing
probe that preferentially recognized O-linked glycans. Signal
correction is same as for FIGS. 2A-2C. The high correlation between
the fingerprints of panels FIG. 3A and FIG. 3B demonstrate the
nearly uniform distribution of complex N-linked glycans at the
three N-linked glycosylation sites; the low signals in FIG. 3C
correlate to the low levels of O-linked glycans in the sample.
[0029] FIG. 4 shows comparisons of fingerprints of tPA from
conditioned media with fingerprints from purified tPA. Fingerprints
were obtained using a glucose/mannose-recognizing probe, which
recognizes both the high-mannose and the bi-antennary type glycans.
The fingerprints are corrected as described for FIGS. 2A-2C.
cross-hatches, purified tPA; dark shading, tPA spiked into DMEM
with 2% FCS collected after 48 hours of culture growth; light
shading, tPA spiked into DMEM with 2% FCS after 1 week of culture
growth; open, tPA spiked into DMEM with 10% FCS after 1 week of
culture growth.
[0030] FIG. 5 shows comparisons of fingerprints of variable
concentrations of human polyclonal IgG The reduction in sample
concentration demonstrates that the technology can be applied to
early stages of therapeutic protein development. IgG concentrations
are: 1 .mu.M (cross-hatches); 0.7 .mu.M (dark shading); 0.3 .mu.M
(light shading); and 0.1 .mu.M (open).
[0031] FIG. 6 is a representation of a manual holder for a slide
wash.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The invention provides methods for determining the glycan
composition and sequence of carbohydrate-containing molecules. In
some embodiments, the methods include modifying a glycomolecule.
These methods enhance the information obtained when the
carbohydrate content of the carbohydrate-containing molecule is
analyzed.
[0033] The methods for analyzing the glycomolecule are particularly
suited for analyzing molecules in the UC-FINGERPRINT.TM. method,
which is also referred to as a glycomolecule identification
(GMID.TM.) method. In this method, information about the
carbohydrate content of a glycomolecule is obtained by adding a
glycomolecule to a substrate to which is attached one or more
saccharide-binding agents (also referred to herein as first
saccharide-binding agents). The first saccharide-binding agents
that have bound the glycomolecule are identified, and the resulting
binding information is used to generate a fingerprint of the
glycomolecule.
[0034] For example, one way to perform the method is with a set of
20-30 lectins printed on a membrane-coated glass slide in
replicates of 4-8. A sample of intact glycoprotein is applied to
the array, and its binding pattern is detected by either direct
labeling of the glycoprotein using FITC, or by using an
FITC-labeled probe that is directed at either the protein
moiety--an antibody for example, or a carbohydrate moiety--a
lectin. The resulting fingerprints are highly characteristic of the
glycosylation pattern of the sample. The large number of lectins,
each with its specific recognition pattern, ensures high
sensitivity of the fingerprint to changes in the glycosylation
pattern. Other fluorescent labels such as Cy3 Cy5 can also be used.
In addition, labeling can be effected using biotin-avidin systems
known in the art.
[0035] Modifications of Glycomolecules Prior to Fingerprinting
Analysis
[0036] It has now been unexpectedly found that the modifications
describe herein enhance the type and amount of information that can
be obtained in UC-FINGERPRINT.TM. analysis. One modification is
removing some or all of the sialic acid residues from
glycomolecule, a process know as desialylation, prior to
UC-FINGERPRINT.TM. analysis. Sialic acid residues are negatively
charged residues that cap carbohydrate moieties attached to many
sites on glycoproteins. Sialic acid residues can be removed using
the enzyme sialidase, which is also known as neuraminidase. The
extent of desialylation can be controlled by modulating the extent
of digestion of the glycomolecule with the sialidase. In addition
to sialidase, any other method that reduces the sialic acid content
of a glycomolecule (including a glycoprotein) can be used.
[0037] Another modification that has been found to enhance the
information revealed in a UC-FINGERPRINT.TM. analysis is digestion
with PNGaseF, which is also known as N-glycosidase F. PNGase F
cleaves N-linked glycoproteins between the innermost GlcNAc and
asparagine residues of high mannose, hybrid and complex
oligosaccharides. O-linked glycan residues (such as N-GlcNAc and
O-Fuc) are not affected and are available for subsequent analysis.
This modification is particularly suitable glycoproteins for which
O-linked glycan composition is of interest. An example of such a
protein is erythropoietin (EPO).
[0038] In addition to PNGaseF, a glycomolecule can be modified with
other glycosidase-modifying anzymes known in the art, e.g., Endo
F2, Endo F3, Endo H.
[0039] A further modification for preparing a glycomolecule for
UC-FINGERPRINT.TM. analysis is to expose the glycomolecule to a
reducing agent and, alkylating agent. Exposure to a reducing agent
can disrupt intra- and inter-chain disulfide bonds and make
available for analysis glycans that would not otherwise be
detected. Suitable reducing agents include, e.g.,
.beta.-mercaptoethanol, dithiothreitol, and mercaptethylamine.
Alkylating agents include, e.g., iodoacetamide and iodoacetic
acid.
[0040] A still further modification that has been found to enhance
the information obtained by UC-FINGERPRINT.TM. analysis is to
subject the glycomolecule to denaturing conditions. This
modification is suitable for glycomolecules containing glycans that
are hiden or obscured because of, e.g., protein aggregation. For
example, a glycoprotein can be heated in the presence of a
detergent prior to performing UC-FINGERPRINT.TM. analysis. The
optimal conditions (including, e.g., selection of detergent,
temperature, buffer composition and concentration, and pH) can be
chosen based on the glycomolecule of interest, the first
saccharide-binding agents that are immobilized on the substrate,
and/or the labaeling scheme that is used to detect glycomolecules
bound to the saccharide-binding agents on the substrate.
[0041] For example, to detect glycans associated with Fc subunits
for IgG molecules, the glycoprotein can be treated in 0.01 to 075%
SDS, more preferably 0.04-0.06% SDS, and most preferably about
0.05% SDS. The sample is in addition boiled for 5-15 minutes at
95-100.degree. C., e.g., at 10 minutes at 100.degree. C., or the
equivalent.
[0042] The above-described modifications can be performed singly or
in any desired combination. For example, a glycoprotein can be
treated with salidase, and then subjected to reducing conditions
prior to submtiting the modified glycoprotein to UC-FINGERPRINT.TM.
analysis.
[0043] The methods described above facilitate identification of
carbohydrate information for glycoproteins that contain glycans
that are obscured by sialic acid residues, and/or obscured because
the glycoproteins otherwise exist as present as multimers and/or
are present in inter-subunit or intra-subunit clefts.
[0044] It has been further unexpectedly discovered that many of the
manipulations described herein--modification, labeling, and
reducing, for example--can be performed without the need for
changing the buffer. This eliminates the need to subject a
glycomolecule of interest to cumbersome, multi-step processing
treatments. In addition, the method can be performed using less
material, with less material loss and in a shorter time.
[0045] If desired, the modifications described above can be
performed directly on glycomolecules (including glycoproteins)
isolated directly from a culture medium.
[0046] Determining Fingerprints of Modified Glycomolecules
[0047] A "glycomolecule fingerprint" refers to the information
provided by the amount of binding detected to one or more
saccharide-binding agents for a glycomolecule of interest. The
fingerprint can be expressed graphically by presenting as a
histogram the relative binding intensities for multiple
saccharide-binding agents. In some embodiments, the analysis of the
glycomolecule includes determining a map of the glycomolecule.
"Mapping"--means defining a sequential order of certain predefined
patterns on the polysaccharide chain. The predefined patterns can
correspond to of location(s) on the glycomolecule that bind to a
saccharide-binding agent, and/or are substrates for a
glycoside-cleaving enzyme.
[0048] A glycomolecule can include any molecule that includes a
saccharide moiety. For example, a glycomolecule can includes
carbohydrate-containing proteins (glycoproteins) or glycolipids,
and free polysaccharides. Glycoproetins include, e.g., fetuin,
.alpha..sub.1 Acid GP, and tPA.
[0049] The modified glycomolecules are added to a substrate that
includes one or more first saccharide-binding agents. The first
saccharide-binding agent may be immobilized to a substrate using
any art-recognized method. For example, immobilization may utilize
functional groups of the protein, such as amino, carboxy, hydroxyl,
or thiol groups. For instance, a glass support may be
functionalized with an epode group by reaction with silane. The
epode group reacts with amino groups such as the free
.epsilon.-amino groups of lysine residues. Another mechanism
consists in covering a surface with electrophilic materials such as
gold. As such materials form stable conjugates with thiol groups, a
protein may be linked to such materials directly by free thiol
groups of cysteine residues. Alternatively, thiol groups may be
introduced into the protein by conventional chemistry, or by
reaction with a molecule that contains one or more thiol groups and
a group reacting with free amino groups, such as the N-hydroxyl
succinimidyl ester of cysteine. Also thiol-cleavable cross-linkers,
such as dithiobis(succinimidyl propionate) may be reacted with
amino groups of a protein. A reduction with sulfhydryl agent will
then expose free thiol groups of the cross-linker.
[0050] For some applications, it is preferable to design a
substrate that contains a plurality of saccharide-binding agents
known to bind, or suspected of binding, to a particular
glycomolecule of interest. For example, heparin, heparin sulfate,
or fragments (such as those produced by heparanase digestion), as
well as variant forms of these polysaccharides can be screened for
their ability to bind to one or more proteins such as, e.g.,
.alpha.FGF, .beta.FGF, PDGF, VEGF, VEGF-R, HGF, EGF, TGF-beta,
MCP-1, -2 and -3, IL-1, -2, -3, -6, -7. -8, -10, and -12, annexin
IV, V, and VI, MIP-1 alpha, MIP-1 beta, ecotaxin, thrombospondin,
PF-4, IP-10, interferon alpha, interferon gamma, selectin L and
selectin P, antithrombin, plasminogen activator, vitronectin, CD44,
SOD, lipoprotein lipase, ApoE, fibronectin, and laminin. These
putative agents can be attached to a surface (i.e., can be first
saccharide binding agents).
[0051] The substrate can be conveniently provided on a membrane
disposed on a supporting surface. For example, the
saccharide-binding agents can be provided on a nitrocellulose
filter on a glass slide. Alternatively, the substrate can be a
microsphere, or bead. In various embodiments, one or more distinct
saccharide-binding agents are provided on a single microsphere.
[0052] Saccharide-Binding Agents
[0053] A suitable saccharide-binding agent is any agent that binds
specifically to a carbohydrate-portion of a glycomolecule. Suitable
saccharide-binding agents include, e.g., lectins, antibodies that
recognize carbohydrate-containing epitopes, and
carbohydrate-modifying enzymes, such as glycosidases.
[0054] Lectins are proteins isolated from plants that bind
saccharides. For the purpose of this application, the term "lectin"
also encompasses saccharide-binding proteins from animal species
(e. g."mammalian lectins"). Examples of lectins include lectins
isolated from the following plants: Conavalia ensiformis, Anguilla
anguilla, Tritium vulgaris, Datura stramonium, Galnthus nivalis,
Maackia amurensis, Arachis hypogaea, Sambucus nigra, Erythtina
cristagalli, Sambucis nigra, Erythrina cristagalli, Lens culinaris,
Glycine max, Phaseolus vulgaris Allomyrina dichotoma, Dolichos
biflorus, Lotus tetragonolobus, Ulex europaeus, and Ricinus
commurcis. Other biologically active compounds such as cytokines,
chemokines and growth factors also bind glycomolecules, and hence,
for the purposes of the present invention are considered to be
lectins.
[0055] Examples of glycosidases include a-Galactosidase,
(3-Galactosidase, N-acetylhexosaminidase, .alpha.:-mannosidase,
.beta.-mannosidase, and .alpha.-Fucosidase.
[0056] Detecting Bound Glycomolecules
[0057] Glycomolecules that have bound to a saccharide-binding agent
on a substrate can be detected using any method that will result in
detection of the bound glycomolecule. For example, the
glycomolecule can be directly labeled before, during, or after it
is added to the substrate. Examples of direct labeling include,
e.g., FITC labeling.
[0058] Alternatively, the bound glycomolecule can be detected with
a label associated with an agent that specifically recognizes the
bound glycomolecule. The agent can recognize a
carbohydrate-containing region of the molecule. When the agent has
this specificity it is referred to as a second saccharide-binding
agent. The second saccharide-binding agent can be an antibody or a
lectin, including the antibodies and lectins described above.
[0059] In some embodiments, the agent recognizes a non-carbohydrate
portion of the glycomolecule. An example of such an agent is an
antibody that recognizes a peptide epitope in a glycoprotein.
[0060] If desired, bound glycomolecules can be detected using a
series of agents. For example, desialo darbepoetin alfa
(ARASNEP.TM.) bound to glycans can be detected using anti-human EPO
monoclonal mouse antibody followed by an anti-mouse
IgG-FITC-labeled antibody.
[0061] The label can be any label that is detected, or is capable
of being detected. Examples of suitable labels include, e.g.,
chromogenic label, a radiolabel, a fluorescent label, and a
biotinylated label. Thus, the label can be, e.g., colored lectins,
fluorescent lectins, biotin-labeled lectins, fluorescent labels,
fluorescent antibodies, biotin-labeled antibodies, and
enzyme-labeled antibodies. In preferred embodiments, the label is a
chromogenic label. The term "chromogenic binding agent" includes
all agents that bind to saccharides and which have a distinct color
or otherwise detectable marker, such that following binding to a
saccharide, the saccharide acquires the color or other marker. In
addition to chemical structures having intrinsic,
readily-observable colors in the visible range, other markers used
include fluorescent groups, biotin tags, enzymes (that may be used
in a reaction that results in the formation of a colored product),
magnetic and isotopic markers, and so on. The foregoing list of
detectable markers is for illustrative purposes only, and is in no
way intended to be limiting or exhaustive. In a similar vein, the
term "color" as used herein (e.g. in the context of step (e) of the
above described method) also includes any detectable marker.
[0062] The label may be attached to the agent using methods known
in the art. Labels include any detectable group attached to the
glycomolecule, or detection agent that does not interfere with its
function. Labels may be enzymes, such as peroxidase and
phosphatase. In principle, also enzymes such as glucose oxidase and
.beta.-galactosidase could be used. It must then be taken into
account that the saccharide may be modified if it contains the
monosaccharide units that react with such enzymes. Further labels
that may be used include fluorescent labels, such as Fluorescein,
Texas Red, Lucifer Yellow, Rhodamine, Nile-red,
tetramethyl-rhodamine-5-isothiocyanate,
1,6-diphenyl-1,3,5-hexatriene, cis-Parinaric acid, Phycoerythrin,
Allophycocyanin, 4',6-diamidino-2-phenylindole (DAPI), Hoechst
33258, 2-aminobenzamide, and the like. Further labels include
electron dense metals, such as gold, ligands, haptens, such as
biotin, radioactive labels.
[0063] The agent can additionally be detected using enzymatic
labels. The detection of enzymatic labels is well known in the art.
Examples include, e.g., ELISA and other techniques where enzymatic
detection is routinely used. The enzymes are available
commercially, e.g., from companies such as Pierce.
[0064] In some embodiments, the label is detected using fluorescent
labels. Fluorescent labels require an excitation at a certain
wavelength and detection at a different wavelength. The methods for
fluorescent detection are well known in the art and have been
published in many articles and textbooks. A selection of
publications on this topic can be found at p. O-124 to O-126 in the
1994 catalog of Pierce. Fluorescent labels are commercially
available from Companies such as SIGMA, or the above-noted Pierce
catalog.
[0065] The agent may itself contain a carbohydrate moiety and/or
protein. Coupling labels to proteins and sugars are techniques well
known in the art. For instance, commercial kits for labeling
saccharides with fluorescent or radioactive labels are available
from Oxford Glycosystems, Abingdon, UK, and ProZyme, San Leandro,
Calif. USA). Reagents and instructions for their use for labeling
proteins are available from the above-noted Pierce catalog.
[0066] Coupling is usually carried out by using functional groups,
such as hydroxyl, aldehyde, keto, amino, sulfhydryl, carboxylic
acid, or the like groups. A number of labels, such as fluorescent
labels, are commercially available that react with these groups. In
addition, bifunctional cross-linkers that react with the label on
one side and with the protein or saccharide on the other may be
employed. The use of cross-linkers may be advantageous in order to
avoid loss of function of the protein or saccharide.
[0067] While the labeling has been described with respect to
modified glycomolecules, the invention also encompasses these
labeling methods when used to perform UC-FINGERPRINT.TM. analysis
of unmodified glycomolecules.
[0068] Obtaining a Fingerprint
[0069] The intensity of label associated with bound glycomolecules
can be detected using methods known in the art. Some detection
methods are described in WO 93/22678. Particularly suitable for the
method of the present invention is the CCD detector method. This
method may be used in combination with labels that absorb light at
certain frequencies, and so block the path of a test light source
to the VLSI surface, so that the CCD sensors detect a diminished
light quantity in the area where the labeled agent has bound. The
method may also be used with fluorescent labels, making use of the
fact that such labels absorb light at the excitation frequency.
Alternatively, the CCD sensors may be used to detect the emission
of the fluorescent label, after excitation. Separation of the
emission signal from the excitation light may be achieved either by
using sensors with different sensitivities for the different
wavelengths, or by temporal resolution, or a combination of
both.
[0070] The fingerprint is preferably determined by correcting for
the glucose concentration of the media from which the glycomolecule
is taken.
[0071] The acquired binding information can be used directly, e.g.,
following visual inspection of the binding pattern. Alternatively,
the binding information can be stored, e.g., as a photograph or
digitized image. If desired, the binding information can be stored
in a database. Interpretation of binding information is also
discussed in, e.g., WO00/68688, WO01/84147, WO02/37106, and
WO02/44714, the contents of which are incorporated by reference
herein in their entirety.
[0072] Kits
[0073] The invention additionally provides kits for modifying
glycomolecules and then subjecting them to UC-FINGERPRINT.TM.
analysis. The contents of a kit can include one or more of a
modification agent(s), a labeling reagent for detecting a
glycomolecule that is bound to a saccharide-binding agent, and, if
desired, a substrate that contains or is capable of attaching to
one or more saccharide-binding agents. The substrate can be, e.g.,
a microsphere.
[0074] Each kit preferably includes saccharide-binding agent or
agents. The reagent is preferably supplied in a solid form or
liquid buffer that is suitable for inventory storage, and later for
exchange or addition into the reaction medium when the test is
performed. Suitable packaging is provided. The kit may optionally
provide additional components that are useful in the procedure.
These optional components include buffers, capture reagents,
developing reagents, labels, reacting surfaces, means for
detection, control samples, instructions, and interpretive
information.
[0075] The kit may optionally include a detectable second
saccharide-binding agent and, if desired, reagents of detecting the
second binding agent. The plurality of first saccharide-binding
agents is preferably attached at predetermined location on the
substrate and a detectable second saccharide-binding agent. In
other embodiments, the kit is provided with a substrate and first
saccharide-binding agents that can be attached to the substrate, as
well as second saccharide-binding agents.
[0076] If desired, a slide holder as shown in FIG. 6 may be
included in the kit. The holder is divided to chambers in a size
slightly bigger then a standard microscope slide, with a proper
space to reach with a gloved finger, the slide is held by stoppers
to prevent forward--backward and side movement, and its bottom is
equipped with bumps to avoid the formation of vacuum and laminar
forces between the slide and the chamber bottom. Each chamber can
typically hold 5-10 ml of liquid and is by thus a vessel for slide
wash and incubation. A cover holder with typical feet reach the
holding surface of the slides and by thus allows to flip the
chambers and discard liquids in a more effective way.
[0077] The invention will be further illustrated in the following
non-limiting examples.
EXAMPLE 1
Comparative Glycomolecule Fingerprints of Desialized and
Non-Desialized Glycoproteins
[0078] The glycomolecule fingerprint of human milk lactoferrin
(hmLF) before and after disialyzation was examined. hmLF is a
glycoprotein with a relatively simple glycosylation structure (Spik
et al. Eur J Biochem. 1982;121(2):413-9). The hmLF structure
includes two glycosylation sites that are occupied by any of 5
major glycans, resulting in 25 possible glycoforms. All of these
glycans are of the complex bi-antennary type, containing a core
fucose and differing in their levels of sialylation and the
variable presence of antennary fucose (FIG. 1A). The various
glycans differ in the presence of the (2,6) linked sialic acid
residues and the (1,3) linked antennary fucose.
[0079] The fingerprints (FIG. 1B and FIG. 1C) were obtained by
using a labeled anti-lactoferrin antibody as a probe. Twenty-four
array-bound lectins were used and are grouped by their
specificities on the abscissa. The group of complex N-linked
glycans contains lectins that do not bind to monosaccharides, but
rather require a complex N-linked glycan containing at least 3
antennae; the data summarized in FIG. 1B are the result of three
independent experiments. Signals are corrected for differences in
scanning parameters (laser power and PMT gain) for each slide, if
applicable, and for differences in levels of probe fluorescence if
these differ between experiments. Lectins were printed on a
membrane-coated glass slide in replicates of 4-8. Lectins were
purchased from Vector Laboratories (Burlingame, Calif.). The
lectins were dissolved in PBS at pH 7.4 to concentrations of 2-4
mg/ml. Lectins are spotted with a high precision robot for
microarray spotting (MicroGrid, Biorobotics, Cambridge, UK) onto
nitrocellulose coated glass slides (FAST Slides, Schleicher &
Schull, Keene, N.H.), using solid pins of 0.4 mm diameter, at a
center-to-center distance of 0.9 mm. Arrays were blocked with 1%
BSA (Sigma). Samples are incubated with PBS buffer containing 1 mM
CaCl, 1 mM MgCl and 0.1 mM MnCl followed by a wash using the same
buffer. The process was fully automated on the Protein Array
Workstation (Perkin Elmer, Wellesley, Mass.). HmLF was applied to
the array and its binding pattern was detected by scanning with a
confocal laser scanner (ScannArray Express, Perkin Elmer), and data
analyzed using the ArrayPro software package (Media Cybernetics,
Silver Spring, Md.).
[0080] FIG. 1B shows fingerprints of hmLF obtained with a labeled
anti-lactoferrin antibody probe. A polyclonal anti-hmLF antibody
was used to detect hmLF bound to the immobilized lectins. The
polyclonal antibody recognizes all hmLF glycoforms, and thus each
bar in the histogram represents the binding observed on one of the
array-bound lectins, which are grouped by their specificities.
Three independent experiments are depicted, demonstrating the
reproducibility of the platform. A relatively simple fingerprint,
containing few signals, is observed: one major signal arises from a
lectin from the mannose/glucose specificity group, and no signals
are observed in the complex glycan specificity group, which
recognize tri- and higher order antennary structures. These results
indicate that all of the lactoferrin glycans are of the complex
bi-antennary type. Two signals arise from lectins that recognize
the terminal galactose of non-sialylated antennae, one from the Gal
specificity group and another from the Gal/GalNAc group, and
additional signals arise from two lectins that recognize fucose
(both core and antennary), and from a lectin recognizing the sialic
acid.
[0081] 10 mg/ml of hmLactoferrin was desialylated using 50 mU/ml
Neuraminidase from Arthrobacter ureafaciens (Roche cat #269611).
Galactose was removed using 20 mU/ml beta 1,4-Galactosidase from
Streptococcus pneumoniae (Calbiochem, cat #345806).
N-acetylglucosamine was removed with 20 U/ml of beta
1-2,3,4,6-N-Acetylglucosaminidase from Streptococcus pneumoniae
(Calbiochem cat #110116). All cleavage reactions were performed in
the present of 50 mM phosphate buffer at pH 6 containing protease
inhibitors (PI Cocktail Set I -Calbiochem cat #539131) for 19 hours
at 37.degree. C.
[0082] FIG. 1C depicts fingerprints of hmLF following successive
enzymatic trimming of the glycans, and using the same antibody
probe. FIG. 1C shows the fingerprints of the same hmLF sample
following gradual enzymatic trimming of the glycans (KEY--native
hmLF (box with diagonal lines); following de-sialylalation (dark
shaded box); following removal of terminal galactose residues
(light shaded box); following removal of terminal GlcNAc (clear
box)).
[0083] The fingerprint of the native sample is virtually identical
to that observed in the experiments of FIG. 1B. Following
desialylation, signals from the lectin of the glucose/mannose
specificity group, which recognizes the complex bi-antennary core,
and those from the fucose-recognizing lectins remain virtually
unchanged, while the signals from the sialic-acid recognizing
lectin disappear. These outcomes are expected in light of what is
known about the structure of hmLF glycans.
[0084] The galactose-recognizing lectins demonstrate a more complex
behavior. The signals from these lectins increase differentially,
demonstrating the differential sensitivity of these lectins to the
presence of sialic acid: lectin 11 is able to bind the
non-sialylated antenna of a mono-sialylated glycan and thus the
small increase in signal from this lectin following desialylation
indicates a low level of di-sialylated structures in the native
sample. In contrast, the affinity of lectin 15 towards
mono-sialylated glycans is significantly decreased in comparison to
fully desialylated glycans, and thus the large increase in the
signal of this lectin indicates that the native protein contains a
low level of neutral glycans. In addition, the large difference in
the signals observed on these lectins in response to the
desialylated sample demonstrates that their affinity towards
galactose differs significantly.
[0085] The signal from lectin 1 increases following the removal of
the terminal-galactose, demonstrating increased accessibility of
the lectin to the tri-mannosyl core. The signals from the galactose
recognizing lectins disappear, and a signal from a lectin that
recognizes the newly exposed terminal N-acetyl-glucoseamine
(GlcNAc) is evident. The signals from the fucose recognizing
lectins remain unchanged. Following the removal of the GlcNAc only
the signals from lectin 1, recognizing the tri-mannosyl core, and
from the fucose recognizing lectins are observed
[0086] These results clearly demonstrate the sensitivity of the
bound lectins to changes in the glycan structures. The fingerprints
of FIGS. 1B and 1C also demonstrate the complexity of deconvoluting
the fingerprints: signal intensities do not correlate with the
abundance of the recognized epitopes. The abovementioned example of
lectins 11 and 15 having different affinities for the terminal
galactose of the desialylated antennae illustrate this; an
additional example is revealed by comparing the signals of lectins
21 and 22. Lectin 21 recognizes the core fucose, which is present
in all of the lactoferrin glycans, whereas lectin 22, whose signal
is 40% higher than that of lectin 21, recognizes the antennary
fucose present on only approximately 30% of the glycans. Thus
quantification of the glycan epitopes requires a comprehensive
understanding of the lectin glycan recognition.
EXAMPLE 2
Rule-Based Fingerprint Deconvolution
[0087] Deconvolution of the fingerprints is optimally performed by
acquring a detailed understanding of lectin glycan recognition.
This is complicated by the broadness of lectin specificities
towards glycans, and by the fact that the affinities, both within
and between the groups, differ markedly and are unknown.
Measurement of these affinities are hampered by the inability to
obtain a single-glycan-type glycoprotein for each glycan type.
Mathematically, this translates into uncertainties in the
conditional probabilities of observing a signal for a particular
lectin, when the presence of a particular glycan is known. This
limits the use of probabilistic-based algorithms.
[0088] An alternative approach using a rule-based expert system
(Castillo et al., "Expert Systems and Probabilistic Network
Models"--(Monographs in Computer Science) Springer-Verlag, New-York
1997) for fingerprint deconvolution was chosen. The rule base
consists of lectin-glycan recognition rules that were extracted
from the literature and further refined by manual curation of
fingerprints that were run on a large set of well-characterized
glycoproteins. Examples of these rules include:
if(LEC1) then (Tri-antennary, Tetra-antennary) (1)
if(LEC2) then (Hybrid, Tri-antennary, Tetra-antennary) (2)
if(LEC3 I(LEC3 >>LEC2)AND (LEC4, LEC5)) then (High mannose)
(3)
[0089] The rules are written in a natural language form and are
thus easily edited and optimized. In the example above, LEC1-LEC5
represent particular lectins; the first rule reads "if a signal is
observed on LEC1 then there is either a tri-antennary or a
tetra-antennary glycan present in the sample". As emphasized above,
knowledge of the relative probabilities of each of these epitopes
is not available, and thus a straightforward inference from the set
of rules relevant for any particular fingerprint is not
possible.
[0090] The algorithmic solution adopted is an inference engine
based on the Dempster-Shafer theory of evidence (Shafer,
Probability judgment in artificial intelligence. In L. N. Kanal and
J. F. Lemmer, editors, Uncertainty in Artificial Intelligence.
North-Holland, New-York, 1986; Lefevre et al. A Generic Framework
for Resolving the Conflict in the Combination of Belief Structures.
FUSION 2000--3rd International Conference on Information Fusion.
July 2000, Paris, France; Ronald R. Yager (Editor), Janusz Kacprzyk
(Editor), Mario Fedrizzi (Editor). Advances in the Dempster-Shafer
Theory of Evidence. Wiley & Sons 1994). This framework is
powerful in situations where many pieces of evidence (observations)
must be weighted in order to determine a single most probable
model, and there is uncertainty in the system. Here, the lectin
signals are the pieces of evidence, having uncertainties that stem
from the broad specificities of the lectins as well as the
multiplicity of glycans, glycoforms, and lectin recognition
epitopes. The inputs to the inference engine are the lectin binding
signals (the fingerprint) and the set of interpretation rules. The
inference engine translates the rules into "evidence" based on
signal intensities. The three rules shown in the example above are
translated into:
Evidence(Tri-antennary, Tetra-antennary)=Signal(LEC1) (1)
Evidence(Hybrid, Tri-antennary, Tetra-antennary)=Signal(LEC2)
(2)
Evidence(High mannose)={Signal(LEC3) if
Signal(LEC3)>>Signal(LEC2) and (Signal(LEC4)>0 or
Signal(LEC5)>0), 0 otherwise} (3)
[0091] Since each lectin can provide evidence for more than one
glycan, the signals are iteratively processed until the inference
engine converges to the following glycan profile:
Evidence(Tri-antennary)=20%
Evidence(Tetra-antennary)=5 5%,
Evidence(ManHigh7-9)=25%
[0092] Thus, the output is a set of glycan descriptors and
quantitative estimates of the relative abundances of each
descriptor in the analyzed sample. A careful choice of lectins
allows for sufficient data for fingerprint deconvolution. For
example, an analysis of an array of 25 lectins produces 25 signals.
The required output is commonly a set of 5-10 glycan descriptors
(major glycan structures and various additional epitopes).
Mathematically, this indicates a problem whose number of equations
is considerably larger than the number of variables. Thus, as long
as the lectin binding patterns are sufficiently unique, we can
expect the fingerprint to yield a solution.
[0093] Table 1 tabulates the deconvolution of fingerprints of 5
well-characterized glycoproteins: human milk lactoferrin (hmLF),
bovine fetuin, a Bowes melanoma cell line derived tissue
plasminogen activator (tPA), porcine thyroglobulin, and human
al1-acid glycoprotein. The interpretation is based on fingerprints
obtained by direct labeling of the samples. Deconvolution of the
fingerprints is by the Dempster-Shafer rule-based inference
algorithm. The numbers in brackets indicate percentages of each
epitope as reported in the literature and verified by
mass-spectrometry analysis (data not shown). NA indicates not
applicable. * indicates that due to lack of multiple standards with
varying levels of O-linked fucosylation, the quantitation of this
epitope was not parameterized, and thus the output only detects its
existence. ** is in accordance with convention, N-linked and
O-linked glycans distributions were treated independently; the
overall ratio of N/O-linked glycans is directly estimated from the
relative intensities of the signals obtained from N-linked glycan
and O-linked glycan specific lectins. * * * indicates an estimate
of average level of sialylated glycans. A comparison of observed
values to values compiled from the literature and verified by
mass-spectroscopy (data not shown) is also reported.
1TABLE 1 porcine .alpha.1-acid Glycan structures hmLF Bovine fetuin
tPA thyroglobulin glycoprotein N-linked high mannose 0 (0) 0 (0) 46
(47-50) 29 (27) 0 (0) hybrid 0 (0) 0 (0) <1 (4) 0 (0) 0 (0)
complex bi-antennary 100 (100) 20 (14) 48 (40) 43 (41) 40 (40)
tri-antennary 0 (0) 80 (86) 6 (6-9) 28 (32) 44 (42) tetra-antennary
0 (0) 0 (0) <1 (0) 0 (0) 16 (18) O-linked Gal-GalNAc core 0 (0)
100 (100) <1 (0) 0 (0) 0 (0) Gal core 0 (0) 0 (0) 0 (0) 0 (0) 0
(0) Gal branched epitope 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) O-Fucose* no
No yes no no Ration of N/O linked glycans** NA 80/20 NA NA NA
Additional Sialylation*** 75 (80) 100 (100) 52 (50) 60 (58) 100
(100) Epitopes fucose core 100 (100) 0 (0) 54 (50) 71 (73) 1-5 (7)
antennary 30 (33) 0 (0) <1 (0) 0 15 (15) Gal a (1-3) Gal <1
(0) 0 (0) <1 (0) 13 (15) <1 (0)
[0094] Interpretation of the hmLF fingerprints results in the
correct glycan profile (leftmost column of Table 1). Interpretation
of the fingerprints demonstrates the same overall glycan structures
following removal of sialic acid, galactose and GlcNAc, and detects
the removal of sialic acid and appearance of terminal GlcNAc.
Bovine fetuin contains 3 N-linked glycosylation sites, invariably
occupied, and 3 partially occupied O-linked glycosylation sites
(Green et al. J Biol Chem. 1988;263(34):18253-68; Edge and Spiro, J
Biol Chem. 1987;262(33):16135-41; Spiro and Bhoyroo, J Biol Chem.
1974;249(18):5704-17; Yet et al. J Biol Chem. 1988;263(1): 111-7).
Approximately 80% of the glycans are N-linked complex glycans, and
the remaining 20% are O-linked. Interpretation of fetuin
fingerprints results in the correct identification of the
structures of both the N- and O-linked glycans, and their relative
abundance (N- and O-linked glycans each calculated separately in
accordance with convention, and their relative abundance is
estimated from the relative intensities of lectins that
preferentially recognize either N- or O-linked glycans).
[0095] Tissue plasminogen activator (tPA) (Pohl et al.
Biochemistry. 1984;23(16):3701-7; Jaques et al. Biochem J. 1996;316
(Pt 2):427-37; Chan et al. Glycobiology. 1991 Mar;1(2):173-85)
contains 3 N-linked glycosylation sites, 2 of which are fully
occupied, one invariably by a high-mannose glycan and the other by
a complex glycan. The third site shows partial occupancy by an
additional complex glycan. In the sample analyzed (derived from a
Bowes melanoma cell-line) the level of occupancy of the third site
is low, as deduced from the abundance of the high-mannose glycans.
In addition, an O-linked fucose is present. This demonstrates that
the profiling of the tPA glycans is accurate, and includes the
detection of the O-linked fucose, which cannot be readily detected
using standard mass-spectrometry methods. Porcine thyroglobulin
(Ronin et al. J Biol Chem. 1986;261(16):7287-93; de Waard et al. J
Biol Chem. 1991;266(7):4237-43; Spiro and Bhoyroo, J Biol Chem.
1984;259(15):9858-66) contains, in addition to the major structures
detailed in Table 1, low levels of Gal.alpha.(1-3)Gal, an epitope
produced by all mammalians excluding higher primates and man, and
which is highly antigenic in humans. The detection of this epitope
and its quantification are important to manufacturers of biological
therapeutics. The UC-FINGERPRINT.TM. technology correctly detects
the level of this epitope. .alpha.1-acid glycoprotein (Sei et al. J
Chromatogr A. 2002;958(1-2):273-81) contains bi-, tri- and
tetra-antennary glycans. The ability of the fingerprint to resolve
the antennarity of these is evident from the accurate
estimations--all within 10% of those obtained by chromatographic
and mass spectroscopy techniques.
EXAMPLE 3
Comparison of Glycomolecule Fingerprints Obtained Using
Direct-Labeled Glycomolecules and Glycomolecule Fingerprints
Obtained Using Labeled Lectins as Second Saccharide-Binding
Agents
[0096] Fingerprint patterns obtained using direct labeling of the
samples were compared to fingerprint patterns obtained using a
labeled lectin to detect glycomolecules bound to a substrate.
[0097] FIGS. 2A-2C shows a fingerprints of a Bows melanoma
cell-line derived tissue plasminogen activator (tPA). Fingerprints
were obtained by direct labeling of the sample (FIG. 2A); labeling
with a glucose/mannose recognizing lectin probe that recognizes
both high-mannose and complex bi-antennary glycans (FIG. 2B); or
with a glucose/mannose recognizing lectin probe that recognizes
only high mannose type glycans (FIG. 2C). Since each of the
fingerprints was obtained using a different probe, signals were
corrected for the variation in fluorescence of the labeled probes
(or sample, for the study shown in FIG. 2A), and for the
variability in scanning parameters. Deconvolution of fingerprints
obtained using lectin probes requires several fingerprints each
obtained with a different probe, in order to ensure correct
assessment of signals from all lectins. Commonly 2-4 different
probes, depending on the complexity of the glycosylation pattern of
the sample, are required.
[0098] The fingerprint in FIG. 2A showed a stronger signal than
those shown in FIGS. 2B and 2C, but lower signal intensities,
demonstrating the increased sensitivity and specificity obtained
with a labeled probe. The fingerprint in FIG. 2A was the input for
the interpretation shown in Table 1, and shows the expected signals
for complex glycans, mainly of the bi-antennary type containing a
core fucose, and high-mannose type glycans. The fingerprint in FIG.
1B shows fewer signals, due mainly to increased specificity. The
fingerprint in FIG. 1C shows even fewer signals: those from lectins
2 and 3 are not observed in this fingerprint.
[0099] These results demonstrate the power of using lectins as
probes, and reveal the existence of a single high-mannose site in
tPA. The fact that no signals are observed from lectins that
recognize the high-mannose type glycans indicates that the
glycoforms bound to these lectins (evident in panels a and b) do
not have an additional high-mannose type lectin available for
interaction with the labeled probe.
[0100] FIGS. 3A-C depicts fingerprints of de-sialylated bovine
fetuin, obtained by using three probes that recognize terminal
galactose (FIG. 3A), tri- and tetra antennary complex N-linked
glycans (FIG. 3B), and N-acetyl-galactoseamine (GalNAc) (FIG. 3C).
This latter probe preferentially recognizes O-linked glycans.
Numerous lectins are sensitive to the presence of sialic acid (Yim
et al. Proc Natl Acad Sci USA. 2001; 98(5): 2222-2225; Tronchin et
al. Infect Immun. 2002; 70(12): 6891-6895) and its removal enables
increased resolution of antennarity. The high correlation between
the fingerprints of panels FIGS. 3A and B suggest that the complex
type N-linked glycans are nearly uniformly distributed at the three
N-linked glycosylation sites, consistent with previous
publications. The fingerprint obtained with the O-linked glycan
recognizing probe (FIG. 3C) shows fewer and lower signals,
consistent with the lower abundance of O-linked glycans on fetuin.
Moreover, the complete absence of signals from the group of
Gal/GalNAc recognizing lectins in this fingerprint suggests that
the majority of the glycoforms have a single O-linked glycosylation
site occupied, consistent with the reported ratio of N/O linked
glycans in fetuin (Yim et al. Proc Natl Acad Sci U S A. 2001;
98(5): 2222-2225; Tronchin et al. Infect Immun. 2002; 70(12):
6891-6895).
EXAMPLE 4
UC-FINGERPRINT.TM. Profiles Determined for glycoproteins Obtained
Directly from Conditioned Medium
[0101] FIG. 4 depicts fingerprints of tPA analyzed directly in
CHO-conditioned media, in comparison with a fingerprint of purified
tPA. CHO cells were grown in DMEM supplemented with 2% or 10% FCS.
Cell culture supernatant was collected after 48 hours or 1 week as
indicated. Human tPA was spiked into the different cell culture
supernatants to a final concentration of 0.7 .mu.M. 150 .mu.l of
the tPA-containing media was collected at various time points and
used for incubation with the lectin array.
[0102] The fingerprints were obtained using a glucose/mannose
recognizing lectin probe. The fingerprints were comparable to the
fingerprint obtained with a purified sample of the protein.
EXAMPLE 5
Labeling of Small Quantities of Glycoproteins for
UC-FINGERPRINT.TM. Profile Determination
[0103] The ability of small quantities of a glycoprotein to be
labeled using UC-FINGERPRINT.TM. technology was examined.
[0104] The results are shown in FIG. 5. Shown are fingerprints of
human polyclonal IgG at concentrations of 0.1 .mu.M (clear box),
0.3 .mu.M (light shading), 0.7 .mu.M (dark shading), and 1 .mu.M
(diagonal lines). Fingerprints are detectable with as little as 0.1
.mu.M of glycoproteins.
[0105] These results demonstrate that UC-FINGERPRINT.TM. technology
can be performed with glycan structures on intact glycoproteins
with minimal sample pretreatment. Thus, the method can provide a
high-throughput solution for accurate analysis of protein
glycosylation. The analysis can additionally be preformed on crude
samples in growth media, obviating the need for time-consuming
purification and degradation steps. Less than 200 .mu.l of sample
volume with protein concentrations of <0.3 .mu.M are sufficient
to produce a quantitative analysis. This renders the technology
applicable to all stages of development of protein therapeutics:
clone selection and optimization, process development, growth
condition monitoring, manufacturing and Quality Control.
Additionally, the methods can be used without purification steps,
which can introduce bias into the resulting glycoform population
(Bond et al. Journal of Immunological Methods, 1993;166:
27-33).
EXAMPLE 6
Fluorescein Labeling and Reduction of Desialylated Glycoprotein
[0106] 0.667 .mu.g/.mu.l of Fc-Chimeric protein was desialized for
about 16.5 hours at 37.degree. C. in 50 mM NaAc pH 4.99, and
protease inhibitor and 100 Units sialidase. The desialylated
protein was then labeled at 25.degree. C. for 2 hours with
agitation in the absence of light in a volume of 500 .mu.l at a
concentration of about 0.667 .mu.g/.mu.l. The reaction included 80
.mu.l 0.2M 2M K.sub.2HPO.sub.4pH 9.18 and 21.5 .mu.g/.mu.l
Flourescein (2 mg/ml in DMSO).
[0107] The desialylated, labeled FC-chimeric protein was made 0.2M
Tris-Cl pH 8.0 and 1 mM DDT and incubated for 10 minutes at
80.degree. C. Iodoacetic acid was then added to a final
concentration of 22 mM.
[0108] Free sialic acid, fluorescein, DDT, and iodoacetic acid were
removed by DG-10 chromatography. Labeling of the protein was
confirmed by measuring absorbance at 280 nm and 495 nm was measured
for various collected fractions.
EXAMPLE 7
PNGase Treatment of Glycoproteins for UC-FINGERPRINT.TM. Profile
Determination
[0109] The UC-FINGERPRINT.TM. profile of PNGase treated native or
denatured erythropoietin (EPO) was determined. Denatured EPO was
prepared using SDS and .beta.-mercaptoethanol and heating to
100.degree. C. for 10 minutes. After cooling, Triton 1% was added
along with PNGaseF (0.5 U/.mu.l).
[0110] UC-FINGERPRINT.TM. profiles on multiple lectins were
prepared for native and denatured EPO, and for PNGaseF-treated
native and denatured EPO. The fingerprints obtained for each were
distinct, demonstrating that denaturing the protein and subjecting
the protein to PNGase F treatment reveals glycan information for
EPO that is not detected when native, denatured EPO is used.
[0111] The descriptions given are intended to exemplify, but not
limit, the scope of the invention. Additional embodiments are
within the claims.
* * * * *