U.S. patent application number 14/238183 was filed with the patent office on 2014-07-17 for apparatuses, methods, computer program products, and kits for hi-throughput glycan analysis.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. The applicant listed for this patent is Peter Slade. Invention is credited to Peter Slade.
Application Number | 20140200148 14/238183 |
Document ID | / |
Family ID | 47715394 |
Filed Date | 2014-07-17 |
United States Patent
Application |
20140200148 |
Kind Code |
A1 |
Slade; Peter |
July 17, 2014 |
Apparatuses, Methods, Computer Program Products, And Kits for
Hi-Throughput Glycan Analysis
Abstract
An apparatus for glycan analysis is disclosed. The apparatus
includes a plurality of loading wells adapted to receive a
plurality of samples; a plurality of capillaries arranged in
correspondence with the loading wells, each of the capillaries
including a first portion including a stacking gel and a second
portion including a resolving gel; and a plurality of eluting wells
arranged in correspondence with the capillaries and adapted to
receive a portion of the samples having traversed the
capillaries.
Inventors: |
Slade; Peter; (Seattle,
WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Slade; Peter |
Seattle |
WA |
US |
|
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
47715394 |
Appl. No.: |
14/238183 |
Filed: |
August 10, 2012 |
PCT Filed: |
August 10, 2012 |
PCT NO: |
PCT/US2012/050391 |
371 Date: |
March 25, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61523184 |
Aug 12, 2011 |
|
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|
Current U.S.
Class: |
506/8 ;
264/297.8; 506/12; 506/18; 506/19; 506/39 |
Current CPC
Class: |
G01N 2400/00 20130101;
G01N 27/44791 20130101; C12Q 1/34 20130101; G01N 27/44782 20130101;
G01N 2400/38 20130101; G01N 33/6803 20130101; G01N 2333/924
20130101; G01N 33/6842 20130101; G01N 27/44726 20130101 |
Class at
Publication: |
506/8 ; 506/19;
506/18; 506/39; 506/12; 264/297.8 |
International
Class: |
G01N 27/447 20060101
G01N027/447; C12Q 1/34 20060101 C12Q001/34; G01N 33/68 20060101
G01N033/68 |
Claims
1. An apparatus for glycan analysis, comprising: a plurality of
loading wells adapted to receive a plurality of samples; a
plurality of capillaries arranged in correspondence with the
loading wells, each of the capillaries including a first portion
including a stacking gel and a second portion including a resolving
gel; and a plurality of eluting wells arranged in correspondence
with the capillaries and adapted to receive a portion of the
samples having traversed the capillaries.
2. The apparatus of claim 1, wherein the stacking gel is a
pre-poured stacking gel.
3. The apparatus of claim 1, wherein the stacking gel includes
between about 4% and about 8% acrylamide.
4. The apparatus of claim 1, wherein the stacking gel includes
about 6% acrylamide.
5. The apparatus of claim 1, wherein the resolving gel is a
pre-poured resolving gel.
6. The apparatus of claim 1, wherein the resolving gel includes
between about 25% acrylamide and about 35% acrylamide.
7. The apparatus of claim 1, wherein the resolving gel includes
about 30% acrylamide.
8. The apparatus of claim 1, wherein the stacking gel includes
between about 4% and about 8% acrylamide and the resolving gel
includes between about 25% acrylamide and about 35% acrylamide.
9. The apparatus of claim 1, wherein the stacking gel includes
about 6% acrylamide and the resolving gel includes about 30%
acrylamide.
10. The apparatus of claim 1, wherein a total length of the first
and second portions of each capillary is between about 5 cm and
about 30 cm.
11. The apparatus of claim 1, wherein a total length of the first
and second portions of each capillary is about 10 cm.
12. The apparatus of claim 1, wherein the plurality of capillaries
comprise at least five capillaries arranged substantially parallel
to one another, each of the capillaries including a pre-poured
stacking gel arranged in the first portion of the capillary and a
pre-poured resolving gel arranged in the second portion of the
capillary, the capillaries further comprising first and second
support structures arranged at opposite sides so as to form a
single capillary array unit.
13. The apparatus of claim 1, wherein the capillaries have an
internal diameter of between about 150 micrometers and about 250
micrometers.
14. The apparatus of claim 1, wherein the capillaries have an
internal diameter of between about 0.1 millimeter and about 2.5
millimeters.
15. The apparatus of claim 1, further comprising an ion permeable
membrane arranged between the loading wells and the
capillaries.
16. The apparatus of claim 1, further comprising at least two
electrodes arranged on opposite sides of the capillaries.
17. The apparatus of claim 16, wherein the at least two electrodes
are platinum electrodes.
18. The apparatus of claim 16, wherein the at least two electrodes
include a positive electrode arranged between the capillaries and
the eluting wells and a negative electrode arranged between the
capillaries and the loading wells.
19. The apparatus of claim 16, further comprising a power source
connected to the at least two electrodes and configured to subject
at least part of the capillaries to an electric field.
20. The apparatus of claim 19, wherein the electric field has an
intensity of between about 200 V/cm and about 400 V/cm.
21. The apparatus of claim 19, wherein the electric field has an
intensity of between about 250 V/cm and about 350 V/cm.
22. The apparatus of claim 1, further comprising a light source
configured to subject the capillaries to electromagnetic
radiation.
23. The apparatus of claim 22, wherein the light source is a diode
laser.
24. The apparatus of claim 22, wherein the light source is a blue
Argon ion laser.
25. The apparatus of claim 22, wherein the light source is a yellow
Krypton ion laser.
26. The apparatus of claim 22, wherein the electromagnetic
radiation is radiation having a wavelength in the range of about
400-500 nm or in the range of about 500-600 nm.
27. The apparatus of claim 22, further comprising a fluorescence
detector configured to detect fluorescence emitted from the
capillaries.
28. The apparatus of claim 27, wherein the fluorescence detector is
a CCD camera.
29. The apparatus of claim 28, further comprising a bandpass filter
arranged between the capillaries and the CCD camera and configured
to allow radiation having a wavelength of about 510 nm to pass.
30. The apparatus of claim 1, wherein a largest width, depth, or
height of the apparatus does not exceed about twelve inches.
31. The apparatus of claim 27, further comprising a signal
processor configured to process a signal related to fluorescence
detected by the fluorescence detector.
32. The apparatus of claim 31, wherein the signal processor is
configured to generate an electrophoretogram showing peaks
representing individual glycans having migrated through the
capillaries so as to reveal a time point at which each glycan
passed across the fluorescence detector before eluting off the end
of the capillary.
33. The apparatus of claim 27, further comprising a computer in
communication with the fluorescence detector, the computer being
configured to process a signal related to fluorescence detected by
the fluorescence detector, wherein the computer is configured to
generate an electrophoretogram showing peaks representing
individual glycans having migrated through the capillaries so as to
reveal a time point at which each glycan passed across the
fluorescence detector before eluting off the end of the
capillary.
34. The apparatus of claim 33, wherein the computer includes or is
configured to access an empirically-derived database of glycan
migration times.
35. The apparatus of claim 34, wherein the computer includes or is
configured to access and run a computer program product configured
to consult the empirically-derived database of glycan migration
times to compare migration times obtained by running an experiment
with the apparatus to identify individual glycans having migrated
through the capillaries during the experiment.
36. An array of capillaries for glycan analysis, comprising: at
least five capillaries arranged substantially parallel to one
another, each of the capillaries including a pre-poured stacking
gel arranged in a first section of the capillary and a pre-poured
resolving gel arranged in a second section of the capillary, and
first and second support structures arranged at opposite sides of
the at least five capillaries such that the at least five
capillaries form a single unit.
37. The array of claim 36, wherein the stacking gel includes
between about 4% and about 8% acrylamide.
38. The array of claim 36, wherein the stacking gel includes about
6% acrylamide.
39. The array of claim 36, wherein the resolving gel includes
between about 25% acrylamide and about 35% acrylamide.
40. The array of claim 36, wherein the resolving gel includes about
30% acrylamide.
41. The array of claim 36, wherein the stacking gel includes
between about 4% and about 8% acrylamide and the resolving gel
includes between about 25% acrylamide and about 35% acrylamide.
42. The array of claim 36, wherein the stacking gel includes about
6% acrylamide and the resolving gel includes about 30%
acrylamide.
43. The array of claim 36, wherein a length of the first section
and the second section of each capillary is between about 5 cm and
about 15 cm.
44. The array of claim 36, wherein a total length of the first and
second sections of each capillary is between about 10 cm and about
30 cm.
45. The array of claim 36, wherein the capillaries have an internal
diameter of between about 100 micrometers and about 300
micrometers.
46. The array of claim 36, wherein the capillaries have an internal
diameter of between about 50 micrometers and about 100
micrometers.
47. The array of claim 36, wherein the capillaries have an internal
diameter of between about 0.1 millimeter and about 2.5
millimeters.
48. The array of claim 36, further comprising an ion permeable
membrane arranged on at least one extremity of each of the
capillaries.
49. A library of information elements stored in a medium readable
by a computer, comprising: a plurality of empirically-derived
capillary migration times corresponding to a plurality of
individual charged, fluorescently-labeled glycans having migrated
through a capillary including a first portion including a stacking
gel and a second portion including a resolving gel upon subjection
of the capillary to an electric field; and a migration time
corresponding to a dextran ladder.
50. The library of elements of claim 49, wherein the dextran ladder
includes oligomers having an increasing number of glucose molecule,
the increasing number going from one glucose molecule to about
twenty glucose molecules.
51. The library of elements of claim 49, wherein the dextran ladder
includes a linear oligomer having a plurality of synthesized
maltoses.
52. The library of elements of claim 49, wherein the
empirically-derived migration times corresponding to a plurality of
individual glycans include empirically-derived migration times
corresponding to a plurality of polysaccharides.
53. The library of elements of claim 49, wherein the
empirically-derived migration times corresponding to a plurality of
individual glycans include empirically-derived migration times
corresponding to a plurality of oligosaccharides.
54. The library of elements of claim 49, wherein the
empirically-derived migration times corresponding to a plurality of
individual glycans include empirically-derived migration times
corresponding to a plurality of proteoglycans.
55. The library of elements of claim 49, wherein the
empirically-derived migration times corresponding to a plurality of
individual glycans include empirically-derived migration times
corresponding to a plurality of glycoproteins.
56. The library of elements of claim 49, wherein the
empirically-derived migration times corresponding to a plurality of
individual glycans include empirically-derived migration times
corresponding to a plurality of glycolipids.
57. The library of elements of claim 49, wherein the
empirically-derived migration times corresponding to a plurality of
individual glycans include empirically-derived migration times
corresponding to a plurality of O-linked glycans.
58. The library of elements of claim 49, wherein the
empirically-derived migration times corresponding to a plurality of
individual glycans include empirically-derived migration times
corresponding to a plurality of N-linked glycans.
59. The library of elements of claim 49, further comprising an
empirically-derived electrophoretogram showing peaks including at
least one peak corresponding to a dextran ladder.
60. A method for high throughput glycan analysis, comprising:
loading a plurality of glycoprotein samples in a plurality of
loading wells; denaturing the glycoprotein samples in the loading
wells using a denaturing solution; cleaving a glycan from each of
the denatured glycoprotein samples in the loading wells using a
glycan cleaving enzyme; labeling the cleaved glycans with a charged
fluorescent label; applying an electric field configured to migrate
the labeled glycans from the loading wells across an ion permeable
membrane and into and along one of a plurality of capillaries
arranged in correspondence with the loading wells, each of the
capillaries including a first portion including a stacking gel and
a second portion including a resolving gel; exciting the labeled
glycans migrating along the capillaries with a light source adapted
to cause the labeled glycans to emit fluorescent radiation;
detecting fluorescent radiation emitted by the labeled glycans; and
analyzing the labeled glycans based on the detected fluorescent
radiation.
61. The method of claim 60, wherein denaturing the glycoprotein
samples in the loading wells includes denaturing the glycoprotein
samples using SDS.
62. The method of claim 60, further comprising mixing each of the
glycoprotein samples with a TBE buffer solution.
63. The method of claim 60, wherein cleaving a glycan from each of
the denatured glycoprotein samples includes cleaving the glycans
using PNGase F.
64. The method of claim 60, wherein cleaving a glycan from each of
the denatured glycoprotein samples includes cleaving the glycans
using endoglycosidase-H.
65. The method of claim 60, wherein cleaving a glycan from each of
the denatured glycoprotein samples includes cleaving the glycans
using one or more of Endo D, Endo F1, Endo F2, and Endo F3.
66. The method of claim 60, wherein cleaving a glycan from each of
the denatured glycoprotein samples includes cleaving the glycans
using one or more of ABS (arthrobacter ureafaciens sialidase), NAN
1 (recombinant sialidase), AMF (almond meal alpha-fucosidase), BKF
(bovine kidney alpha-fucosidase), BTG (bovine testes
beta-galactosidase), SPG (streptococcus peneumoniae
beta-galactosidase), GUH (streptococcus pneumonia hexosaminidase,
recombinant in E. coli), and JBM (jack bean mannosidase).
67. The method of claim 60, wherein cleaving a glycan from each of
the denatured glycoprotein samples includes cleaving the glycans
using peptide-N-(N-acetyl-.beta.-glucosaminyl)asparagine
amidase.
68. The method of claim 60, wherein cleaving the glycan using
peptide-N-(N-acetyl-.beta.-glucosaminyl)asparagine amidase includes
cleaving N-linked glycans.
69. The method of claim 60, wherein labeling the cleaved glycans
with a charged fluorescent label includes labeling the cleaved
glycans at a reducing end of the glycans with disodium
8-aminonaphtalene-1,3,6-trisulphonate.
70. The method of claim 60, wherein labeling the cleaved glycans
with a charged fluorescent label includes labeling the cleaved
glycans at a reducing end of the glycans with potassium
7-amino-1,3-naphtalene disulfonate.
71. The method of claim 60, wherein labeling the cleaved glycans
with a charged fluorescent label includes labeling the cleaved
glycans at a reducing end of the glycans with sodium
4-amino-naphtalene sulfonate.
72. The method of claim 60, wherein labeling the cleaved glycans
with a charged fluorescent label includes labeling the cleaved
glycans at a reducing end of the glycans with a charged fluorescent
label comprising a hydrazide functional group.
73. The method of claim 60, wherein labeling the cleaved glycans
with a charged fluorescent label includes labeling the cleaved
glycans at a reducing end of the glycans with a charged fluorescent
label comprising one or more of ALEXA FLUOR 350 hydrazide, ALEXA
FLUOR 488 hydrazide, ALEXA FLUOR 647 hydrazide, ALEXA FLUOR 594
hydrazide, and ALEXA FLUOR 555 hydrazide.
74. The method of claim 60, wherein labeling the cleaved glycans
with a charged fluorescent label includes labeling the cleaved
glycans at a reducing end of the glycans with a charged fluorescent
label comprising a hydroxylamine functional group contained in one
or more of ALEXA FLUOR 350 hydroxylamine, ALEXA FLUOR 488
hydroxylamine, and ALEXA FLUOR 647 hydroxylamine.
75. The method of claim 60, wherein labeling the cleaved glycans
with a charged fluorescent label includes labeling the cleaved
glycans at a reducing end of the glycans with a charged fluorescent
label comprising a hydrazide functional group contained in
8-hydrazide-pyene-3,6,8-trisulfonate.
76. The method of claim 60, wherein labeling the cleaved glycans
with a charged fluorescent label includes labeling the cleaved
glycans at a reducing end of the glycans with a charged fluorescent
label comprising a hydroxylamine functional group contained in
8-hydroxylamine-pyene-3,6,8-trisulfonate.
77. The method of claim 60, wherein labeling the cleaved glycans
with a charged fluorescent label includes labeling the cleaved
glycans at a reducing end of the glycans using one or more of APTS,
ANTS, ANDA, and ANSA.
78. The method of claim 60, wherein the charged fluorescent label
comprises a sulfonic acid.
79. The method of claim 60, wherein labeling the cleaved glycans
with a charged fluorescent label further includes forming a
hydrazone between the hydrazide functional group and a sugar
carbonyl of the glycans.
80. The method of claim 79, wherein labeling the cleaved glycans
with a charged fluorescent label further includes stabilizing the
hydrazone by reduction with sodium cyanoborohydride.
81. The method of claim 60, wherein applying an electric field
includes applying an electric field having an intensity of between
about 200 V/cm and about 400 V/cm.
82. The method of claim 60, wherein applying an electric field
includes applying an electric field having an intensity of between
about 250 V/cm and about 350 V/cm.
83. The method of claim 60, wherein exciting the labeled glycans
includes exciting the labeled glycans with a light source.
84. The method of claim 83, wherein the light source is a laser
diode.
85. The method of claim 83, wherein the light source has a
wavelength in the range of about 400-500 nm or in the range of
about 500-600 nm.
86. The method of claim 60, wherein detecting fluorescent radiation
includes detecting fluorescent radiation using a fluorescence
detector.
87. The method of claim 86, wherein detecting fluorescent radiation
includes filtering fluorescent radiation directed to the
fluorescence detector using a bandpass filter.
88. The method of claim 87, wherein the band pass filter is a 510
nm bandpass filter.
89. The method of claim 86, wherein analyzing the labeled glycans
includes analyzing the labeled glycans based on an
electrophoretogram generated by the fluorescence detector or by a
signal processor or computer configured to process one or more
signals obtained from the fluorescence detector, the
electrophoretogram showing peaks representing individual glycans as
they migrate along the capillaries and are detected by the
fluorescence detector.
90. The method of claim 89, wherein analyzing the labeled glycans
includes comparing measured migration times to that of a
fluorescently labeled dextran standard ladder and to known
migration times for specific glycan structures and molecular
weights previously recorded in an empirically-derived database.
91. The method of claim 90, wherein loading the glycoprotein
samples in the loading wells further includes loading a dextran
standard ladder, and wherein analyzing the labeled glycans is
dependent upon their migration time relative to the dextran
standard ladder.
92. The method of claim 91, wherein the dextran ladder standard
includes a fluorescently labeled linear polysaccharide made of
glucose molecules, including polysaccharide chains having a number
of glucose molecules varying by unity increment from one glucose
molecule to about twenty-three glucose molecules.
93. The method of claim 92, wherein the dextran ladder standard is
fluorescently labeled at the reducing end of the sugar chain with
the charged fluorescent label comprising a hydroxylamine functional
group contained in the fluorophores ALEXA FLUOR 350
hydroxylamine.
94. The method of claim 92, wherein the dextran ladder standard is
fluorescently labeled at the reducing end of the sugar chain with
the charged fluorescent label comprising a hydroxylamine functional
group contained in the fluorophores ALEXA FLUOR 647
hydroxylamine.
95. The method of claim 94, further comprising, after having
denatured, cleaved, and labeled the glycans, subjecting the glycans
to an exoglycosidase enzyme.
96. A method of making a capillary array for high throughput glycan
analysis, comprising: providing a plurality of capillaries;
pre-pouring, into each of the capillaries, a stacking gel in a
first portion and a resolving gel in a second portion; and
connecting the capillaries structurally on opposite sides such that
the capillaries are arranged substantially in parallel to one
another and form a single unit.
97. The method of claim 96, wherein pre-pouring the stacking gel
includes pre-pouring a stacking gel including between about 4% and
about 8% acrylamide.
98. The method of claim 97, wherein pre-pouring the stacking gel
includes pre-pouring a stacking gel including about 6%
acrylamide.
99. The method of claim 98, wherein pre-pouring the resolving gel
includes pre-pouring a resolving gel including between about 25%
acrylamide and about 35% acrylamide.
100. The method of claim 97, wherein pre-pouring the resolving gel
includes pre-pouring a resolving gel including about 30%
acrylamide.
101. The method of claim 96, wherein a length of the first portion
of each capillary is between about 5 cm and about 15 cm.
102. The method of claim 96, wherein a length of the second portion
of each capillary is between about 5 cm and about 15 cm.
103. The method of claim 96, wherein the capillaries have an
internal diameter of between about 100 micrometers and about 300
micrometers.
104. The method of claim 96, wherein the capillaries have an
internal diameter of between about 0.1 millimeter and about 2.5
millimeters.
105. A method for generating a glycan database, comprising:
empirically obtaining a plurality of empirically-derived migration
times corresponding to a plurality of individual charged,
fluorescently-labeled glycans having migrated through a capillary
including a first portion including a stacking gel and a second
portion including a resolving gel upon subjection of the capillary
to an electric field; and arranging the collected plurality of
empirically-derived migration times in correspondence with a
identification information of each of the plurality of individual
charged, fluorescently-labeled glycans having migrated through a
capillary into a database configured to be accessible by a
computer.
106. A method for identifying a plurality of glycans, comprising:
labeling the glycans with a charged fluorescent label; migrating
the labeled glycans along a plurality of capillaries oriented along
a substantially parallel direction into an electric field, each of
the capillaries including a first portion including a stacking gel
and a second portion including a resolving gel; determining a
migration time relative to a fluorescently labeled dextran standard
ladder for each of the labeled glycans based on detected
fluorescent radiation emitted by the labeled glycans; and comparing
the relative migration time with a database of empirically-derived
migration times corresponding to a plurality of individual charged,
fluorescently-labeled glycans having migrated through a capillary
including a first portion including a stacking gel and a second
portion including a resolving gel upon subjection of the capillary
to an electric field.
107. A kit for glycan analysis, comprising: an array of capillaries
for glycan analysis, including at least five capillaries arranged
substantially parallel to one another, each of the capillaries
including a pre-poured stacking gel arranged in a first section of
the capillary and a pre-poured resolving gel arranged in a second
section of the capillary, and first and second support structures
arranged at opposite sides of the at least five capillaries such
that the at least five capillaries form a single unit; a denaturing
solution adapted for denaturing glycoproteins; a glycan cleaving
enzyme solution adapted for cleaving glycans; and a fluorescent
labeling solution adapted for labeling cleaved glycans.
108. A kit for glycan analysis, comprising: a denaturing solution
adapted for denaturing glycoproteins; a glycan cleaving enzyme
solution adapted for cleaving glycans; and a fluorescent labeling
solution adapted for labeling cleaved glycans.
109. The kit of claim 108, wherein the denaturing solution includes
SDS.
110. The kit of claim 108, wherein the glycan cleaving enzyme
solution includes one or more of PNGase F and
endoglycosidase-H.
111. The kit of claim 108, wherein the glycan cleaving enzyme
solution includes one or more of Endo D, Endo F1, Endo F2, Endo F3,
ABS (arthrobacter ureafaciens sialidase), NAN 1 (recombinant
sialidase), AMF (almond meal alpha-fucosidase), BKF (bovine kidney
alpha-fucosidase), BTG (bovine testes beta-galactosidase), SPG
(streptococcus peneumoniae beta-galactosidase), GUH (streptococcus
pneumoniae hexosaminidase, recombinant in E. coli), and JBM (jack
bean mannosidase).
112. The kit of claim 108, wherein the fluorescent labeling
solution includes one or more of disodium
8-aminonaphtalene-1,3,6-trisulphonate, potassium
7-amino-1,3-naphtalene disulfonate, sodium 4-amino-naphtalene
sulfonate, a charged fluorescent label comprising a hydrazide
functional group, ALEXA FLUOR 350 hydrazide, ALEXA FLUOR 488
hydrazide, ALEXA FLUOR 647 hydrazide, ALEXA FLUOR 594 hydrazide,
ALEXA FLUOR 555 hydrazide, ALEXA FLUOR 350 hydroxylamine, ALEXA
FLUOR 488 hydroxylamine, ALEXA FLUOR. 647 hydroxylamine,
8-hydrazide-pyene-3,6,8-trisulfonate,
8-hydroxylamine-pyene-3,6,8-trisulfonate, APTS, ANTS, ANDA, and
ANSA.
113. The method of claim 60, wherein one or more of the plurality
of glycoprotein samples are recombinant proteins.
114. The method of claim 113, wherein one or more of the plurality
of glycoprotein samples are antibodies.
115. The method of claim 113, wherein one or more of the plurality
of glycoprotein samples are intracellular proteins, membrane
associated proteins or secreted proteins.
116. The method of claim 114, wherein one or more of the
glycoprotein samples are analyzed for reduced core fucosylation
117. The method of claim 116, wherein an antibody-producing cell is
grown in a culture medium treated with an agent that alters core
fucosylation.
118. The method of claim 117, wherein the core fucosylation levels
of one or more of the glycoprotein samples are compared before and
after treatment.
119. The apparatus of claim 1, which identifies a core fucosylated
glycoprotein from a non-fucosylated glycoprotein.
120. The apparatus of claim 1, which differentiates between a 5% to
0.1% fucosylated glycoprotein.
121. The method of claim 60, wherein labeling the cleaved glycans
with a charged fluorescent label further includes forming a oxime
between the hydroxylamine functional group and a sugar carbonyl of
the glycans.
Description
BACKGROUND
[0001] 1. Field
[0002] The present application generally relates to apparatuses,
methods, and computer program products for hi-throughput glycan
analysis.
[0003] 2. Background
[0004] Carbohydrates or glycans linked to the surface of proteins
play an important role for ensuring correct cellular and protein
function and mediating protein folding, signaling, and other
important cellular systems. The analysis of glycans is challenging,
however, and involves time consuming sample preparation and
complex, low-throughput analytical techniques. There is a need for
new and improved apparatuses, methods, and computer program
products that efficiently and simply allow the performance of
hi-throughput analysis of glycans while retaining sufficient
resolution and sensitivity. Such a need is especially applicable in
numerous fields, including in academic and industrial research and
in bioproduction and pharmaceutical industries, for example, where
large numbers of glycans need to be analyzed rapidly and
efficiently.
SUMMARY
[0005] Apparatuses, systems, methods and computer program products
for high-throughput glycan analysis are provided.
[0006] In one aspect, an apparatus for glycan analysis is provided.
The apparatus including: (1) a plurality of loading wells adapted
to receive a plurality of samples; (2) a plurality of capillaries
arranged in correspondence with the loading wells, each of the
capillaries including a first portion including a stacking gel and
a second portion including a resolving gel; and (3) a plurality of
eluting wells arranged in correspondence with the capillaries and
adapted to receive a portion of the samples having traversed the
capillaries.
[0007] In one aspect, an array of capillaries for glycan analysis
is provided. The array of capillaries including: (1) at least five
capillaries arranged substantially parallel to one another, each of
the capillaries including a pre-poured stacking gel arranged in a
first section of the capillary and a pre-poured resolving gel
arranged in a second section of the capillary, and (2) first and
second support structures arranged at opposite sides of the at
least five capillaries such that the at least five capillaries form
a single unit.
[0008] In one aspect, a library of information elements stored in a
medium readable by a computer is provided. The library of
information including: (1) a plurality of empirically-derived
capillary migration times corresponding to a plurality of
individual charged, fluorescently-labeled glycans having migrated
through a capillary including a first portion including a stacking
gel and a second portion including a resolving gel upon subjection
of the capillary to an electric field; and (2) a migration time
corresponding to a dextran ladder.
[0009] In one aspect, a method for high throughput glycan analysis
is provided. The method including: (1) loading a plurality of
glycoprotein samples in a plurality of loading wells; (2)
denaturing the glycoprotein samples in the loading wells using a
denaturing solution; (3) cleaving a glycan from each of the
denatured glycoprotein samples in the loading wells using a glycan
cleaving enzyme; (4) labeling the cleaved glycans with a charged
fluorescent label; and (5) applying an electric field configured to
migrate the labeled glycans from the loading wells across an ion
permeable membrane and into and along one of a plurality of
capillaries arranged in correspondence with the loading wells, each
of the capillaries including a first portion including a stacking
gel and a second portion including a resolving gel; (6) exciting
the labeled glycans migrating along the capillaries with a light
source adapted to cause the labeled glycans to emit fluorescent
radiation; (7) detecting fluorescent radiation emitted by the
labeled glycans; and (8) analyzing the labeled glycans based on the
detected fluorescent radiation.
[0010] In one aspect, a method of making a capillary array for high
throughput glycan analysis is provided. The method including: (1)
providing a plurality of capillaries; (2) pre-pouring, into each of
the capillaries, a stacking gel in a first portion and a resolving
gel in a second portion; and (3) connecting the capillaries
structurally on opposite sides such that the capillaries are
arranged substantially in parallel to one another and form a single
unit.
[0011] In one aspect a method for generating a glycan database is
provided. The method including: (1) empirically obtaining a
plurality of empirically-derived migration times corresponding to a
plurality of individual charged, fluorescently-labeled glycans
having migrated through a capillary including a first portion
including a stacking gel and a second portion including a resolving
gel upon subjection of the capillary to an electric field; and (2)
arranging the collected plurality of empirically-derived migration
times in correspondence with a identification information of each
of the plurality of individual charged, fluorescently-labeled
glycans having migrated through a capillary into a database
configured to be accessible by a computer.
[0012] In one aspect, a method for identifying a plurality of
glycans is provided. The method including: (1) labeling the glycans
with a charged fluorescent label; (2) migrating the labeled glycans
along a plurality of capillaries oriented along a substantially
parallel direction into an electric field, each of the capillaries
including a first portion including a stacking gel and a second
portion including a resolving gel; (3) determining a migration time
relative to a fluorescently labeled dextran standard ladder for
each of the labeled glycans based on detected fluorescent radiation
emitted by the labeled glycans; and (4) comparing the relative
migration time with a database of empirically-derived migration
times corresponding to a plurality of individual charged,
fluorescently-labeled glycans having migrated through a capillary
including a first portion including a stacking gel and a second
portion including a resolving gel upon subjection of the capillary
to an electric field.
[0013] In one aspect, a kit for glycan analysis is provided. The
kit including: (1) an array of capillaries for glycan analysis,
including at least five capillaries arranged substantially parallel
to one another, each of the capillaries including a pre-poured
stacking gel arranged in a first section of the capillary and a
pre-poured resolving gel arranged in a second section of the
capillary, and first and second support structures arranged at
opposite sides of the at least five capillaries such that the at
least five capillaries form a single unit; (2) a denaturing
solution adapted for denaturing glycoproteins; (3) a glycan
cleaving enzyme solution adapted for cleaving glycans; and (4) a
fluorescent labeling solution adapted for labeling cleaved
glycans.
[0014] In one aspect, a kit for glycan analysis is provided. The
kit including: (1) a denaturing solution adapted for denaturing
glycoproteins; (2) a glycan cleaving enzyme solution adapted for
cleaving glycans; and (3) a fluorescent labeling solution adapted
for labeling cleaved glycans.
[0015] The foregoing general description and the following detailed
description are exemplary only and are not limiting in any way of
the scope of the invention. Other embodiments or variations upon
embodiments specifically discussed herein, including various
combinations of features of embodiments discussed herein, may be
realized from the following detailed description or may be learned
by practice of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The accompanying drawings illustrate various exemplary
embodiments disclosed herein. The drawings are exemplary only and
are not in any way limiting of the scope of the invention.
[0017] FIG. 1 illustrates an exemplary apparatus for glycan
preparation and analysis.
[0018] FIG. 2 illustrates an exemplary system for glycan
preparation and analysis.
[0019] FIG. 3 illustrates an exemplary glycan preparation and
analysis workflow.
[0020] FIGS. 4A-4D illustrate an exemplary capillary array and
related glycan resolution and sensitivity data.
[0021] FIG. 5 illustrates capillary gel electrophoresis and glycan
resolution and sensitivity data for seven carbohydrates.
[0022] FIG. 6 illustrates an exemplary apparatus for glycan
preparation and analysis.
[0023] FIG. 7 illustrates an exemplary system and related glycan
analysis data.
[0024] FIGS. 8-10 illustrate various data and electrophoretograms
regarding the separation of oligo maltose standards and glycans
from IgG and other glycoproteins on a capillary array system;
Applied Biosystems.RTM. 3130 Genetic Analyzer.
DETAILED DESCRIPTION
[0025] As used herein, the term "antibody" refers to (a)
immunoglobulin polypeptides and immunologically active portions of
immunoglobulin polypeptides, i.e., polypeptides of the
immunoglobulin family, or fragments thereof, that contain an
antigen binding site that immunospecifically binds to a specific
antigen (e.g., CD70) and an Fc domain comprising a complex
N-glycoside-linked sugar chain(s), or (b) conservatively
substituted derivatives of such immunoglobulin polypeptides or
fragments that immunospecifically bind to the antigen (e.g., CD70).
Antibodies are generally described in, for example, Harlow &
Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor
Laboratory Press, 1988). Unless otherwise apparent from the
context, reference to an antibody also includes antibody
derivatives as described in more detail below.
[0026] As used herein, an "antibody derivative" means an antibody,
as defined above (including an antibody fragment), or Fc domain or
region of an antibody comprising a complex N-glycoside linked sugar
chain, that is modified by covalent attachment of a heterologous
molecule such as, e.g., by attachment of a heterologous polypeptide
(e.g., a ligand binding domain of heterologous protein), or by
glycosylation (other than core fucosylation), deglycosylation
(other than non-core fucosylation), acetylation, phosphorylation or
other modification not normally associated with the antibody or Fc
domain or region.
[0027] As used herein, the term "monoclonal antibody" refers to an
antibody that is derived from a single cell clone, including any
eukaryotic or prokaryotic cell clone, or a phage clone, and not the
method by which it is produced. Thus, the term "monoclonal
antibody" is not limited to antibodies produced through hybridoma
technology.
[0028] As used herein, the term "Fc region" refers to the constant
region of an antibody, e.g., a C.sub.H1-hinge-C.sub.H2-C.sub.H3
domain, optionally having a C.sub.H4 domain, or a conservatively
substituted derivative of such an Fc region.
[0029] As used herein, the term "Fc domain" refers to the constant
region domain of an antibody, e.g., a C.sub.H1, hinge, C.sub.H2,
C.sub.H3 or C.sub.H4 domain, or a conservatively substituted
derivative of such an Fc domain.
[0030] As used herein, the term "low fucosylation" or "reduced
fucosylation" does not refer to a single glycoprotein molecule
having less fucose residues attached to it. Rather, reference is
made to a `glycoprotein preparation` prepared from cells, or, from
a cell medium preparation comprising the glycoproteins secreted by
the cell. The glycoprotein preparation comprises a population of
individual glycoprotein molecules, with members of the population
having different glycosylation features. For purposes of
illustration and not limitation, for an IgG1 antibody expressed in
a modified CHO cell, "low fucosylation" or "reduced fucosylation"
refers to a smaller number of individual glycoproteins having a
fucose residue on an N-linked GlcNAc residue of a glycan at
position 297 of the Fc. Such "low fucosylation" or "reduced
fucosylation" refers to a relatively low (or reduced) number of the
glycoproteins of the population having fucose residues on them, as
compared to, a population of the same glycoprotein made in a cell
line that lacks a modification, or a cell line grown in a medium
with, say, a fucose analog that reduces fucosylation. By way of
illustration, if a glycoprotein is 1% fucosylated as compared with
the same glycoprotein made by a wild-type cell, or in a cell
culture medium without, say, a fucosylation inhibitor, only 1% of
the molecules of Fc-containing protein are fucosylated as compared
with the amount of fucosylation observed in a corresponding
wild-type cell (arbitrarily set to 100%, whether or not all of the
molecules of Fc-containing protein are fucosylated in the wild-type
cell under the same conditions).
[0031] Thus, in a "low fucosylation" or "reduced fucosylation"
glycoprotein, fucosylation is reduced about 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% in comparison with a cell that does
not contain the modification or a cell not grown in the presence of
a fucosylation modifier (for e.g., small molecule like a fucose
analog). In a specific embodiment, the reduction is about 99.1%,
99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% in
comparison with a cell that does not contain the modification, or
grown without a fucose inhibitor. In another specific embodiment,
the reduction is about 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%,
98.7%, 98.8%, or 98.9% in comparison with a cell that does not
contain the modification, or grown without a fucose inhibitor. In
another specific embodiment, the reduction is about 97.1%, 97.2%,
97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, or 97.9% in comparison
with a cell that does not contain the modification, or grown
without a fucose inhibitor. In another specific embodiment, the
reduction is about 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%,
96.8%, or 96.9% in comparison with a cell that does not contain the
modification, or grown without a fucose inhibitor. In another
specific embodiment, the reduction is about 95.1%, 95.2%, 95.3%,
95.4%, 95.5%, 95.6%, 95.7%, 95.8%, or 95.9% in comparison with a
cell that does not contain the modification, or grown without a
fucose inhibitor. In another specific embodiment, the reduction is
about 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, or
94.9% in comparison with a cell that does not contain the
modification, or grown without a fucose inhibitor.
[0032] FIG. 1 illustrates an exemplary apparatus for glycan
preparation and analysis, which may simplify glycan analysis by
performing both sample preparation and analysis with a
high-throughput array screening technique and a high resolution
analytical technique for complex glycoforms. The apparatus may
purify individual glycans and may also be used for protein
separation. FIG. 2 illustrates an exemplary system for glycan
preparation and analysis, including a capillary array within an
apparatus in communication with a computer having a glycan database
and identification software.
[0033] According to various exemplary embodiments, N-linked glycans
may be enzymatically cleaved using PNGase, and glycans may be
fluorescently labeled at their reducing end with a modified dye
(e.g., ALEXA FLUOR 448, etc.) containing either a hydrazide or
oxyamine functional group (e.g., a carbonyl reactive group)
on-line, in a 100 microliters sample well. Labeling may involve the
formation of a hydrazone between a sugar carbonyl and the
fluorophore hydrazide or the formation of an oxime between the
sugar carbonyl and the fluorophore hydroxylamine. The labeled
glycans may inherit a negative charge due to sulfonic acids that
may be in the dye and, as a result, they may migrate in an electric
field. The labeled glycans may then be separated by capillary gel
electrophoresis and may be detected by fluorescence using, for
example, a laser diode (e.g., 488 nm) for excitation and a CCD
camera (including, e.g., a 510 nm bandpass filter) for detection,
for example. Detection may generate an electrophoretogram showing
peaks representing individual glycans as they migrate paste the
laser/detector. The ALEXA FLUOR 448 may provide fluorescence at a
sufficient linear range to be used quantitatively.
[0034] As shown in FIG. 1, the glycans may be separated using a
capillary 10 subjected to an electric field (generated by a
positive electrode 3 and negative electrode 1 interfaced to
opposite ends of the capillary 10) of about 200-400 V/cm with a run
time of about 10-15 min. The capillary 10 can include a stacking
capillary gel portion 5 comprised of about 4% to 8% acrylamide (or
an equivalent gel matrix) and a resolving (analytical) capillary
gel portion 6. In various embodiments, the resolving capillary gel
portion 6 can be packed into the capillary 10 after the stacking
capillary gel portion 5. The resolving capillary portion can be
comprised of about 25% to 35% acrylamide (or an equivalent gel
matrix). The total capillary 10 length (stacking portion 5 and
resolving portion 6) can be between about 5 cm to about 15 cm, and
the capillaries may be arranged in an array 12 of up to about
twenty capillaries 10. Each capillary 10 may have an internal
diameter of about 200 micrometers to ensure sufficient resolution
and sensitivity to analyze complex samples. The system may have a
detection limit of between about 1 femtomole and 100 attomoles of
glycan. For greater sensitivity and resolution, capillaries with a
smaller internal diameter of 50-100 micrometers may be used.
[0035] According to various embodiments, the above apparatuses,
systems and procedures may be used to prepare and purify individual
glycans. Specifically, immediately following fluorescent detection,
the glycans may be eluted from the end of the capillary array 12
into a sample removal well, where they may be removed (purified) by
a technician/researcher for further analysis (e.g., mass
spectrometry). This advantageously allows for the development of a
retention time database for glycan identification. For higher
throughput or purification of greater quantities of glycan a larger
capillary array, which may have larger internal diameter of about 1
mm, may advantageously be used.
[0036] According to various embodiments, the glycans may be
identified by comparison of their retention times (on the
electrophoretogram) against a dextran ladder standard (e.g.,
fluorescently labeled carbohydrate oligomers differing by one
glucose molecule). The dextran ladder may be run in parallel with
the glycan samples, and specific glycans may then be identified by
locating the time point at which they elute relative to the dextran
ladder. Known retention times for specific glycan structures and
molecular weights may previously be recorded in an
empirically-derived database, which may then be searched. An
analysis software may then compare retention times (relative to the
dextran ladder) of peaks from an electrophoretogram of unknown
glycans with the retention time database to identify the glycans.
The software may include a database specific for IgG glycans, among
other glycans and glycoforms.
[0037] According to various embodiments, the above systems and
procedures may be used not only for glycan separation and
detection, but also for protein separation/purification using
SDS-PAGE capillaries. In this case, the capillaries may have an
internal diameter of about 1 mm and may contain a 4-12% gradient of
acrylamide.
[0038] According to various embodiments, the above systems and
procedures may be used for easier sample preparation (e.g., on-line
labeling) and to complete analysis from glycoprotein to identified
glycan much more quickly, potentially in hours instead of days and
perhaps even in minutes eventually.
[0039] According to various embodiments, the above systems and
procedures may be used with multiple ALEXA FLUOR fluorophores
(e.g., ALEXA FLUOR 350 hydrazide, ALEXA FLUOR 488 hydrazide, ALEXA
FLUOR 647 hydrazide, ALEXA FLUOR 594 hydrazide, and ALEXA FLUOR 555
hydrazide, etc.). The brightness of the ALEXA FLUOR fluorophores
may allow for greater sensitivity, and the use of multiple colors
of ALEXA FLUOR may allow for the relative quantification of glycans
between different samples.
[0040] According to various embodiments, the above systems and
procedures may be used to simplify sample preparation. The gel
matrix may handle relatively "dirty" samples containing protein and
salt contaminants, which may reduce sample preparation time, and
the hydrazine ALEXA FLUOR may facilitate labeling, on-line and
without acid use. Glycan structure being extremely complex, it can
be impractical to make synthetic standards.
[0041] According to various embodiments, the purification ability
of the above systems and procedures may allow the development of a
database for glycan identification. Such a database of glycan
retention times and glycan identification software advantageously
would allow the average biologist/scientist who is unfamiliar with
mass spectrometry to be able to do high end glycan analysis.
[0042] According to an exemplary embodiment, there is provided an
apparatus for glycan analysis, including: (1) a plurality of
loading wells 2 adapted to receive a plurality of samples; (2) a
plurality of capillaries 10 arranged in correspondence with the
loading wells 2, each of the capillaries 2 including a first
portion including a stacking gel 5 and a second portion including a
resolving gel 6; and (3) a plurality of eluting wells 4 arranged in
correspondence with the capillaries 10 and adapted to receive a
portion of the samples having traversed the capillaries 10.
[0043] In the apparatus, each of the loading wells 2 may be a
conduit leading to one of the capillaries 10. Each of the loading
wells 2 may be a receptacle in fluid communication through an ion
permeable membrane 8 with one of the capillaries 10. Each of the
loading wells 2 may have a volume capacity of between about 10
.mu.l and about 500 .mu.A or of between about 50 .mu.l and about
150 .mu.A for example. Each of the eluting wells 4 may have a
volume capacity of between about 10 .mu.l and about 500 .mu.A or of
between about 50 .mu.l and about 150 .mu.A for example. The
apparatus may further include a reservoir including a buffer
solution, the reservoir 15 being in fluid communication through an
ion permeable membrane 8 with the loading wells 2. The buffer
solution may be TBE. The apparatus may further include a sample
loader configured to load samples in the loading wells.
[0044] The capillaries 10 may be substantially parallel to one
another. The capillaries 10 may have a substantially circular
cross-section, or may have a substantially rectangular
cross-section, for example. The capillaries 10 may be connected
structurally to form a capillary array unit 12 that is removable as
a whole. The capillary array unit 12 may be configured for a single
use and disposable. Each of the capillaries 10 may be configured
for a single use and disposable. The stacking gel portion 5 may be
a pre-poured stacking gel, and may include between about 4% and
about 8% acrylamide, or about 6% acrylamide, for example. The
resolving gel portion 6 may be a pre-poured resolving gel, and may
include between about 25% acrylamide and about 35% acrylamide, or
about 30% acrylamide, for example. In various embodiments, the
stacking gel portion 5 may include about 6% acrylamide while the
resolving gel portion 6 may include about 30% acrylamide.
[0045] A length of the first portion (i.e., stacking gel portion 5)
of each capillary 10 may be between about 5 cm and about 15 cm, and
a length of the second portion (i.e., resolving gel portion 6) of
each capillary 10 may be between about 5 cm and about 15 cm. A
total length of the first and second portions of each capillary 10
may be between about 10 cm and about 30 cm, or may be about 10 cm,
for example. The capillaries 10 may include at least five
capillaries 10, at least ten capillaries 10, or at least twenty
capillaries 10, for example. The plurality of capillaries 10 may
include at least five capillaries 10 arranged substantially
parallel to one another, each of the capillaries 10 including a
pre-poured stacking gel arranged in the first portion of the
capillary and a pre-poured resolving gel arranged in the second
portion of the capillary, the capillaries 10 further including
first and second support structures arranged at opposite sides so
as to form a single capillary array unit. The capillaries may have
an internal diameter of between about 150 micrometers and about 250
micrometers, or between about 50 micrometers and about 100
micrometers, or between about 0.1 millimeter and about 2.5
millimeters, or between about 0.5 millimeter and about 1.5
millimeters, for example.
[0046] The apparatus may further include an ion permeable membrane
8 arranged between the loading wells 2 and the capillaries 10. The
apparatus may further include at least two electrodes arranged on
opposite sides of the capillaries, and the at least two electrodes
may be platinum electrodes and may include a positive electrode 1
arranged between the capillaries 10 and the eluting wells 4 and a
negative electrode 3 arranged between the capillaries and the
loading wells 2. The apparatus may further include a power source
connected to the at least two electrodes and configured to subject
at least part of the capillaries to an electric field. The electric
field may have an intensity of between about 200 V/cm and about 400
V/cm, or of between about 250 V/cm and about 350 V/cm, for example.
The apparatus may further include a light source 19 configured to
subject the capillaries to electromagnetic radiation, and the light
source may be a diode laser, a blue Argon ion laser, or a yellow
Krypton ion laser, for example. The electromagnetic radiation may
be radiation having a wavelength in the range of about 400-500 nm
or in the range of about 500-600 nm, for example. The apparatus may
further include a fluorescence detector 14 configured to detect
fluorescence emitted from the capillaries, and the fluorescence
detector may be a CCD camera or a CMOS camera. The apparatus may
further include a bandpass filter 16 arranged between the
capillaries and the CCD camera and configured to allow radiation
having a wavelength of about 510 nm to pass. The apparatus may be a
bench top apparatus, and may have a largest width, depth, or height
that does not exceed about twelve inches.
[0047] The apparatus may further include a signal processor
configured to process a signal related to fluorescence detected by
the fluorescence detector, and the signal processor may be
configured to generate an electrophoretogram showing peaks
representing individual glycans having migrated through the
capillaries 10 so as to reveal a time point at which each glycan
passed across the fluorescence detector 16 before eluting off the
end of the capillary 10. The apparatus may further include a
computer 20 in communication with the fluorescence detector 14, the
computer 20 being configured to process a signal related to
fluorescence detected by the fluorescence detector 14. The computer
20 may be configured to generate an electrophoretogram showing
peaks representing individual glycans having migrated through the
capillaries 10 so as to reveal a time point at which each glycan
passed across the fluorescence detector 14 before eluting off the
end of the capillary 10. The computer 20 may include or be
configured to access an empirically-derived database of glycan
migration times, and may include or be configured to access and run
a computer program product configured to consult the
empirically-derived database of glycan migration times to compare
migration times obtained by running an experiment with the
apparatus to identify individual glycans having migrated through
the capillaries 10 during the experiment.
[0048] According to an exemplary embodiment, there is provided an
array of capillaries 12 for glycan analysis, including: (1) at
least five capillaries arranged substantially parallel to one
another, each of the capillaries including a pre-poured stacking
gel 5 arranged in a first section of the capillary and a pre-poured
resolving gel 6 arranged in a second section of the capillary, and
(2) first and second support structures arranged at opposite sides
of the at least five capillaries such that the at least five
capillaries form a single unit.
[0049] The capillary array unit 12 may be configured for a single
use and disposable. The stacking gel portion 5 may include between
about 4% and about 8% acrylamide, or about 6% acrylamide, for
example. The resolving gel portion 6 may include between about 25%
acrylamide and about 35% acrylamide, or about 30% acrylamide, for
example. In various embodiments, the stacking gel portion 5 may
include about 6% acrylamide while the resolving gel portion 6 may
include about 30% acrylamide, for example.
[0050] A length of the first portion of each capillary 10 may be
between about 5 cm and about 15 cm, and a length of the second
portion of each capillary 10 may be between about 5 cm and about 15
cm. A total length of the first and second portions of each
capillary 10 may be between about 10 cm and about 30 cm, or may be
about 10 cm, for example. The capillary array 12 may include at
least five capillaries, at least ten capillaries, or at least
twenty capillaries, for example. The array 12 may include at least
ten substantially cylindrical capillaries arranged substantially
parallel to one another, each of the capillaries including a
pre-poured stacking gel 5 arranged in a first section of the
capillary and a pre-poured resolving gel 6 arranged in a second
section of the capillary 10, or at least twenty substantially
cylindrical capillaries arranged substantially parallel to one
another, each of the capillaries including a pre-poured stacking
gel 5 arranged in a first section of the capillary and a pre-poured
resolving gel 6 arranged in a second section of the capillary 10,
for example.
[0051] The capillaries may have an internal diameter of between
about 100 micrometers and about 300 micrometers, or between about
150 micrometers and about 250 micrometers, or between about 50
micrometers and about 100 micrometers, or between about 0.1
millimeter and about 2.5 millimeters, or between about 0.5
millimeter and about 1.5 millimeters, for example. The array 12 may
further include an ion permeable membrane 8 arranged on at least
one extremity of each of the capillaries.
[0052] According to an exemplary embodiment, there is provided a
library of information elements stored in a medium readable by a
computer, including: (1) a plurality of empirically-derived
capillary migration times corresponding to a plurality of
individual charged, fluorescently-labeled glycans having migrated
through a capillary including a first portion including a stacking
gel and a second portion including a resolving gel upon subjection
of the capillary to an electric field; and (2) a migration time
corresponding to a dextran ladder.
[0053] The dextran ladder may include oligomers having an
increasing number of glucose molecules, the increasing number going
from one glucose molecule to about twenty glucose molecules, or may
include a linear oligomer having a plurality of synthesized
maltoses, for example. The dextran ladder may be extracted from
digested starch. The empirically-derived migration times
corresponding to a plurality of individual glycans may include
empirically-derived migration times corresponding to a plurality of
polysaccharides, or a plurality of oligosaccharides, or a plurality
of proteoglycans, or a plurality of glycoproteins, or a plurality
of glycolipids, or a plurality of O-linked glycans, or a plurality
of N-linked glycans, for example. The library may further include a
plurality of empirically-derived electrophoretogram showing peaks
representing individual glycans, and may further include an
empirically-derived electrophoretogram showing peaks including at
least one peak corresponding to a dextran ladder.
[0054] FIG. 3 illustrates an exemplary glycan preparation and
analysis workflow, in accordance with various embodiments. The
method can include the following workflow steps: (Step 32) loading
a plurality of glycoprotein samples in a plurality of loading
wells; (Step 34) denaturing the glycoprotein samples in the loading
wells using a denaturing solution; (Step 36) cleaving a glycan from
each of the denatured glycoprotein samples in the loading wells
using a glycan cleaving enzyme; (Step 38) labeling the cleaved
glycans with a charged fluorescent label; (Step 40) applying an
electric field configured to migrate the labeled glycans from the
loading wells across an ion permeable membrane and into and along
one of a plurality of capillaries arranged in correspondence with
the loading wells, each of the capillaries including a first
portion including a stacking gel and a second portion including a
resolving gel and exciting the labeled glycans migrating along the
capillaries with a light source adapted to cause the labeled
glycans to emit fluorescent radiation; and (Step 42) detecting
fluorescent radiation emitted by the labeled glycans; and analyzing
the labeled glycans based on the detected fluorescent
radiation.
[0055] In various embodiments, an optional (Step 44) can inserted
into the glycan analysis workflow. (Step 44) involves adding
exoglycosidase to the loading wells after the glycans have been
labeled in (Step 38).
[0056] In various embodiments, loading the glycoprotein samples
into the loading wells may include loading each of the glycoprotein
samples into a conduit leading to one of the capillaries. In
various embodiments, denaturing the glycoprotein samples in the
loading wells may include denaturing the glycoprotein samples using
SDS. In various embodiments, loading the glycoprotein samples into
the loading wells may include loading each of the glycoprotein
samples into a receptacle in fluid communication with one of the
capillaries, and may include loading between about 10 .mu.l and
about 500 .mu.l of each glycoprotein sample into one of the loading
wells, or loading between about 50 .mu.l and about 150 of each
glycoprotein sample into one of the loading wells, for example. In
various embodiments, the method may further include mixing each of
the glycoprotein samples with a buffer solution, and the buffer
solution may be TBE.
[0057] In various embodiments, cleaving a glycan from each of the
denatured glycoprotein samples may include cleaving the glycans
using PNGase F, or using endoglycosidase-H, or using one or more of
Endo D, Endo F1, Endo F2, and Endo F3, or using one or more of ABS
(arthrobacter ureafaciens sialidase), NAN 1 (recombinant
sialidase), AMF (almond meal alpha-fucosidase), BKF (bovine kidney
alpha-fucosidase), BTG (bovine testes beta-galactosidase), SPG
(streptococcus pneumoniae beta-galactosidase), GUH (streptococcus
pneumoniae hexosaminidase, recombinant in E. coli), and JBM (jack
bean mannosidase), or using
peptide-N-(N-acetyl-.beta.-glucosaminyl)asparagine amidase, for
example. Cleaving the glycan using
peptide-N-(N-acetyl-.beta.-glucosaminyl)asparagine amidase may
include cleaving N-linked glycans.
[0058] In various embodiments, labeling the cleaved glycans with a
charged fluorescent label may include labeling the cleaved glycans
at a reducing end of the glycans with disodium
8-aminonaphtalene-1,3,6-trisulphonate, or with potassium
7-amino-1,3-naphtalene disulfonate, or with sodium
4-amino-naphtalene sulfonate, or with a charged fluorescent label
including a hydrazide functional group, or with a charged
fluorescent label including one or more of ALEXA FLUOR 350
hydrazide, ALEXA FLUOR 488 hydrazide, ALEXA FLUOR 647 hydrazide,
ALEXA FLUOR 594 hydrazide, and ALEXA FLUOR 555 hydrazide, or with a
charged fluorescent label including a hydroxylamine functional
group contained in one or more of ALEXA FLUOR 350 hydroxylamine,
ALEXA FLUOR 488 hydroxylamine, and ALEXA FLUOR 647 hydroxylamine,
or with a charged fluorescent label including a hydrazide
functional group contained in 8-hydrazide-pyene-3,6,8-trisulfonate,
or with a charged fluorescent label including a hydroxylamine
functional group contained in
8-hydroxylamine-pyene-3,6,8-trisulfonate, for example. Labeling the
cleaved glycans with a charged fluorescent label may include
labeling the cleaved glycans at a reducing end of the glycans using
one or more of APTS, ANTS, ANDA, and ANSA. The charged fluorescent
label may include a sulfonic acid. Labeling may involve the
formation of a hydrazone between a sugar carbonyl and the
fluorophore hydrazide or the formation of an oxime between the
sugar carbonyl and the fluorophore hydroxylamine.
[0059] In various embodiments, applying an electric field may
include applying an electric field having an intensity of between
about 200 V/cm and about 400 V/cm, or between about 250 V/cm and
about 350 V/cm, for example, and the electric field may be applied
for a period of between about 10 minutes and about 15 minutes, for
example. Exciting the labeled glycans may include exciting the
labeled glycans with a light source, which may be a laser diode and
may have a wavelength in the range of about 400-500 nm or in the
range of about 500-600 nm, for example. Detecting fluorescent
radiation may include detecting fluorescent radiation using a
fluorescence detector, and may includes filtering fluorescent
radiation directed to the fluorescence detector using a bandpass
filter, which may be a 510 nm bandpass filter.
[0060] In various embodiments, analyzing the labeled glycans may
include analyzing the labeled glycans based on an
electrophoretogram generated by the fluorescence detector or by a
signal processor or computer configured to process one or more
signals obtained from the fluorescence detector, the
electrophoretogram showing peaks representing individual glycans as
they migrate along the capillaries and are detected by the
fluorescence detector. In various embodiments, analyzing the
labeled glycans may include comparing measured migration times to
that of a fluorescently labeled dextran standard ladder and to
known migration times for specific glycan structures and molecular
weights previously recorded in an empirically-derived database.
[0061] In various embodiments, loading the glycoprotein samples in
the loading wells may further include loading a dextran standard
ladder, and analyzing the labeled glycans may be dependent upon
their migration time relative to the dextran standard ladder. The
dextran ladder standard may include a fluorescently labeled linear
polysaccharide made of glucose molecules, including polysaccharide
chains having a number of glucose molecules varying by unity
increment from one glucose molecule to about twenty-three glucose
molecules. The dextran ladder standard may be fluorescently labeled
at the reducing end of the sugar chain with the charged fluorescent
label including a hydroxylamine functional group contained in the
fluorophores ALEXA FLUOR 350 hydroxylamine, or including a
hydroxylamine functional group contained in the fluorophores ALEXA
FLUOR 647 hydroxylamine, for example. The method may further
include eluting labeled glycans from the end of each of the
capillaries into a plurality of removal wells arranged in
correspondence with the capillaries, and may further include
purifying the eluted glycans. The method may further include after
having denatured, cleaved, and labeled the glycans, subjecting the
glycans to an exoglycosidase enzyme.
[0062] According to an exemplary embodiment, there is provided a
method of making a capillary array for high throughput glycan
analysis, including: (1) providing a plurality of capillaries; (2)
pre-pouring, into each of the capillaries, a stacking gel in a
first portion and a resolving gel in a second portion; and (3)
connecting the capillaries structurally on opposite sides such that
the capillaries are arranged substantially in parallel to one
another and form a single unit.
[0063] In the method, pre-pouring the stacking gel may include
pre-pouring a stacking gel including between about 4% and about 8%
acrylamide, or about 6% acrylamide, for example. Pre-pouring the
resolving gel may include pre-pouring a resolving gel including
between about 25% acrylamide and about 35% acrylamide, or about 30%
acrylamide, for example. A length of the first portion of each
capillary may be between about 5 cm and about 15 cm, and a length
of the second portion of each capillary may be between about 5 cm
and about 15 cm. A total length of the first and second portions of
each capillary may be between about 10 cm and about 30 cm.
[0064] Providing a plurality of capillaries may further include
providing at least five capillaries, or at least ten capillaries,
or at least twenty capillaries, for example. The capillaries may
have an internal diameter of between about 100 micrometers and
about 300 micrometers, between about 50 micrometers and about 100
micrometers, or between about 0.1 millimeter and about 2.5
millimeters, or between about 0.5 millimeter and about 1.5
millimeters, for example.
[0065] According to an exemplary embodiment, there is provided a
method for generating a glycan database, including: (1) empirically
obtaining a plurality of empirically-derived migration times
corresponding to a plurality of individual charged,
fluorescently-labeled glycans having migrated through a capillary
including a first portion including a stacking gel and a second
portion including a resolving gel upon subjection of the capillary
to an electric field; and (2) arranging the collected plurality of
empirically-derived migration times in correspondence with a
identification information of each of the plurality of individual
charged, fluorescently-labeled glycans having migrated through a
capillary into a database configured to be accessible by a
computer.
[0066] According to an exemplary embodiment, there is provided a
method for identifying a plurality of glycans, including: (1)
labeling the glycans with a charged fluorescent label; (2)
migrating the labeled glycans along a plurality of capillaries
oriented along a substantially parallel direction into an electric
field, each of the capillaries including a first portion including
a stacking gel and a second portion including a resolving gel; (3)
determining a migration time relative to a fluorescently labeled
dextran standard ladder for each of the labeled glycans based on
detected fluorescent radiation emitted by the labeled glycans; and
(4) comparing the relative migration time with a database of
empirically-derived migration times corresponding to a plurality of
individual charged, fluorescently-labeled glycans having migrated
through a capillary including a first portion including a stacking
gel and a second portion including a resolving gel upon subjection
of the capillary to an electric field.
[0067] According to various exemplary embodiments, the apparatus,
array or databases for glycan analysis, may further identify the
fucosylation status of a recombinant protein or glycoprotein (e.g.,
whether it is fucosylated or non-fucosylated), or the %
fucosylation of a glycoprotein.
[0068] Recombinant therapeutic proteins, like antibodies, can
undergo a variety of post-translational modifications, including
glycosylation, and are commercially produced in large bioreactors
in host cells. Antibodies with reduced or low core fucosylation
levels (described below) are desirable in the therapeutic industry
and have been shown to alter Fc effector functions, in particular,
Fc gamma receptor binding and ADCC activity. The glycans attached
to Asn297 on antibodies, are usually referred to as having high
levels of `core fucosylation`. Monoclonal antibodies, such as
IgG1s, have an N-linked glycosylation site at asparagine 297
(Asn297) of each heavy chain. Alternatively, small molecule
inhibitors like catanospermine acting on enzymes in the
glycosylation pathway has resulted in antibodies that lack the
complex N-linked glycan structure, and therefore has low
fucosylation levels. Recently, small molecules, like fucose
analogs, have been used in producing recombinant antibodies and
derivatives that have complex N-linked glycans but have reduced
core fucosylation (see U.S. Publication No. 2009/0317869, whose
disclosure is incorporated by reference in its entirety herein).
Even more recently, genetically-modified cell lines with reduced
ability to fucosylate proteins, but without a knockout of the
fucosylating gene, (that is, the modified cell can conditionally
fucosylate proteins, say, at a different temperature) have been
generated (see U.S. Publication No. 2010/0304436, whose disclosure
is incorporated by reference in its entirety herein). These
observations have lead to interests in identifying whether
antibodies have reduced core fucosylation under certain conditions,
and if so, what is the percentage of reduction in glycosylation or
fucosylation. The apparatus, arrays and methods of the present
invention provide means to identify, characterize and analyze
glycoproteins, such as antibodies, for their fucosylation
status.
[0069] According to various exemplary embodiments, the purification
ability of the systems and procedures presented herein and
described above, may allow the development of a database for
fucosylated or non-fucosylated glycan identification. Such a
database of fucosylated versus non-fucosylated glycan retention
times and fucosylated versus non-fucosylated glycan identification
software advantageously would allow the average biologist/scientist
who is unfamiliar with mass spectrometry to be able to do high end
glycan analysis. The fucosylated or non-fucosylated glycans may be
identified by comparison of their retention times (on the
electrophoretogram) with a dextran ladder standard (e.g.,
fluorescently labeled carbohydrate oligomers differing by one
glucose molecule). The dextran ladder may be run in parallel with
the glycan samples, and specific glycans may then be identified by
locating the time point at which they elute relative to the dextran
ladder. Known retention times for specific fucosylated versus
non-fucosylated glycan structures and molecular weights may
previously be recorded in an empirically-derived database, which
may then be searched. An analysis software may then compare
retention times (relative to the dextran ladder) of peaks from an
electrophoretogram of unknown glycans with the retention time
database to identify the specific fucosylated versus
non-fucosylated glycans. The software may include a database
specific for IgG fucosylated versus non-fucosylated glycans.
[0070] According to an exemplary embodiment, there is provided a
cell or cell line used in expressing a recombinant glycoprotein in
a recombinant cell. In one embodiment, the cell expresses an
Fc-containing protein or glycoprotein. In one embodiment, the
Fc-containing protein or glycoprotein is an antibody. In certain
embodiments, the glycoprotein may be made, either within an
unmodified, or a modified cell. Examples of modified cells would
be, a cell with a FUT gene knockout, or, a cell that conditionally
fucosylates proteins, or, a cell grown in a cell culture medium
treated with a sugar analog, like a fucose analog. By fucose analog
is meant, a small molecule that inhibits fucosylation, as described
below. Exemplary small molecules include, but are not limited to,
fucose alkyne, fucose azides, etc., which are described in U.S.
Publication No. 2009/0317869, and whose disclosure regarding
various small molecule analogs is incorporated by reference in
their entirety herein. Thus the cell is cultured in the culture
medium under conditions sufficient for the cell to express the
protein or glycoprotein. In certain embodiments, the cell itself is
the sample that contains the expressed glycoprotein, and in other
embodiments, the culture medium that contains the expressed and/or
secreted glycoprotein is the sample.
[0071] According to an exemplary embodiment, there is provided an
array of capillaries for glycan analysis which can identify the
fucosylation status of a recombinant protein (whether fucosylated
or non-fucosylated), or the % fucosylation of a glycoprotein.
Further, in a specific exemplary embodiment of the invention, there
is provided a library of information elements stored in a medium
readable by a computer which can identify the fucosylation status
of a protein, or the % fucosylation of a glycoprotein.
[0072] According to another exemplary embodiment, provided herein
are methods for high throughput glycan analysis, to identify
fucosylated versus non-fucosylated proteins, including: (1) loading
a plurality of glycoprotein samples in a plurality of loading
wells; (2) denaturing the glycoprotein samples in the loading wells
using a denaturing solution; (3) cleaving a glycan from each of the
denatured glycoprotein samples in the loading wells using a glycan
cleaving enzyme; (4) labeling the cleaved glycans with a charged
fluorescent label; and (5) applying an electric field configured to
migrate the labeled glycans from the loading wells across an ion
permeable membrane and into and along one of a plurality of
capillaries arranged in correspondence with the loading wells, each
of the capillaries including a first portion including a stacking
gel and a second portion including a resolving gel; (6) exciting
the labeled glycans migrating along the capillaries with a light
source adapted to cause the labeled glycans to emit fluorescent
radiation; (7) detecting fluorescent radiation emitted by the
labeled glycans; and (8) analyzing the labeled glycans based on the
detected fluorescent radiation. In these methods, identification of
fucosylation status or % fucosylation may sometimes further include
the steps of oligosaccharide analysis via HPLC, wherein
fucosyl-containing glycans or oligosaccharides may be quantified by
integration of glycan peak area, and, e.g., protein fucosylation
may be calculated based on glycan peak area. In some embodiments,
the glycoproteins are tagged using click-based sugar analogs that
are commercially available from Invitrogen.
[0073] In some embodiments, the % fucosylation of one or more
glycoproteins may be studied or compared to the % fucosylation of
other glycoprotein(s), using the apparatus, the array or various
exemplary embodiments of the invention. In one embodiment, the
apparatus or the array of capillaries of the invention can identify
an expressed glycoprotein of no more than about 5% fucosylated, or
in other embodiments, no more than about 4%, 3%, 2%, 1%, 0.5% or
0.1% fucosylated glycoproteins. In a specific embodiment, the
percent fucosylation is a mole percent of fucose to glycan. In some
embodiments, the cell glycosylates the Fc-containing protein, but
does not substantially fucosylate the glycosylated Fc-containing
protein. In a specific embodiment, the fucosylation is about no
more than about 10%, 5%, 4%, 3%, 2%, 1%, or 0.1% of the
fucosylation of the glycosylated Fc-containing protein as compared
to a modified cell, or a cell that lacks fucosylation capacities.
In yet another specific embodiment, the percent fucosylation is a
mole percent of fucose to glycoprotein. In a specific embodiment,
the molar ratio of nonfucosylated to fucosylated protein is about
0.90 to 0.10, about 0.91 to 0.09, about 0.92 to 0.08, about 0.93 to
0.07, about 0.94 to 0.06, about 0.95 to 0.05, about 0.96 to 0.04,
about 0.97 to 0.03, about 0.98 to 0.02, or about 0.99 to 0.01.
[0074] According to an exemplary embodiment, there is provided a
kit for glycan analysis, including: (1) an array of capillaries for
glycan analysis, including at least five capillaries arranged
substantially parallel to one another, each of the capillaries
including a pre-poured stacking gel arranged in a first section of
the capillary and a pre-poured resolving gel arranged in a second
section of the capillary, and first and second support structures
arranged at opposite sides of the at least five capillaries such
that the at least five capillaries form a single unit; (2) a
denaturing solution adapted for denaturing glycoproteins; (3) a
glycan cleaving enzyme solution adapted for cleaving glycans; and
(4) a fluorescent labeling solution adapted for labeling cleaved
glycans.
[0075] According to an exemplary embodiment, there is provided a
kit for glycan analysis, including: (1) a denaturing solution
adapted for denaturing glycoproteins; (2) a glycan cleaving enzyme
solution adapted for cleaving glycans; and (3) a fluorescent
labeling solution adapted for labeling cleaved glycans.
[0076] The denaturing solution may include SDS. The glycan cleaving
enzyme solution may include one or more of PNGase F and
endoglycosidase-H, or one or more of Endo D, Endo F1, Endo F2, Endo
F3, ABS (arthrobacter ureafaciens sialidase), NAN 1 (recombinant
sialidase), AMF (almond meal alpha-fucosidase), BKF (bovine kidney
alpha-fucosidase), BTG (bovine testes beta-galactosidase), SPG
(streptococcus peneumoniae beta-galactosidase), GUH (streptococcus
pheumoniae hexosaminidase, recombinant in E. coli), and JBM (jack
bean mannosidase), for example.
[0077] The fluorescent labeling solution may include one or more of
disodium 8-aminonaphtalene-1,3,6-trisulphonate, potassium
7-amino-1,3-naphtalene disulfonate, sodium 4-amino-naphtalene
sulfonate, a charged fluorescent label including a hydrazide
functional group, ALEXA FLUOR 350 hydrazide, ALEXA FLUOR 488
hydrazide, ALEXA FLUOR 647 hydrazide, ALEXA FLUOR 594 hydrazide,
ALEXA FLUOR 555 hydrazide, ALEXA FLUOR 350 hydroxylamine, ALEXA
FLUOR 488 hydroxylamine, ALEXA FLUOR 647 hydroxylamine,
8-hydrazide-pyene-3,6,8-trisulfonate,
8-hydroxylamine-pyene-3,6,8-trisulfonate, APTS, ANTS, ANDA, and
ANSA, for example.
[0078] According to various exemplary embodiments described herein,
one or more aspects of one or more of the foregoing exemplary
embodiments may be performed in whole or in part using a DNA
sequencer such as the APPLIED BIOSYSTEMS 3130 Genetic Analyzer, for
example.
[0079] FIG. 4A illustrates an exemplary capillary array. FIGS.
4B-4D illustrate related glycan resolution and sensitivity data.
FIG. 5 illustrates capillary gel electrophoresis and glycan
resolution and sensitivity data for seven carbohydrates. FIG. 6
illustrates an exemplary apparatus for glycan preparation and
analysis. FIG. 7 illustrates an exemplary system and related glycan
analysis data. FIGS. 8-10 illustrate various data and
electrophorograms regarding the separation of oligo maltose
standards and glycans from IgG and other glycoproteins on a
capillary array system; Applied Biosystems.RTM. 3130 Genetic
Analyzer.
[0080] Other embodiments of the invention will be apparent to one
of ordinary skill in the art having had the benefit of the present
specification and/or having practiced one or more embodiments of
the invention. Further, the present specification including the
drawings are all exemplary and are not in any way limiting of the
scope of the invention, which shall be determined by the following
claims.
* * * * *