U.S. patent application number 14/725614 was filed with the patent office on 2015-12-03 for deglycosylation reagents and methods.
This patent application is currently assigned to NEW ENGLAND BIOLABS, INC.. The applicant listed for this patent is New England Biolabs, Inc.. Invention is credited to John Buswell, Ellen Guthrie, Paula Magnelli, Christopher H. Taron, Ming-Qun Xu.
Application Number | 20150346194 14/725614 |
Document ID | / |
Family ID | 53887174 |
Filed Date | 2015-12-03 |
United States Patent
Application |
20150346194 |
Kind Code |
A1 |
Magnelli; Paula ; et
al. |
December 3, 2015 |
Deglycosylation Reagents and Methods
Abstract
Compositions and methods are provided for efficiently preparing
a completely deglycosylated antibody where efficiency is measured
in relative amounts of reagents in soluble or lyophilized form, and
time and temperature of the reaction. Compositions and methods are
also provided for separating substantially all N-linked glycans
from a glycosylated antibody and for preserving functionality of
the antibody. The methods are compatible with glycan labeling and
protease digestion without the need for prior purification
steps.
Inventors: |
Magnelli; Paula;
(Somerville, MA) ; Guthrie; Ellen; (Andover,
MA) ; Taron; Christopher H.; (Essex, MA) ; Xu;
Ming-Qun; (Hamilton, MA) ; Buswell; John;
(Byfield, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
New England Biolabs, Inc. |
Ipswich |
MA |
US |
|
|
Assignee: |
NEW ENGLAND BIOLABS, INC.
Ipswich
MA
|
Family ID: |
53887174 |
Appl. No.: |
14/725614 |
Filed: |
May 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62080480 |
Nov 17, 2014 |
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62040745 |
Aug 22, 2014 |
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62021936 |
Jul 8, 2014 |
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62018074 |
Jun 27, 2014 |
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62005559 |
May 30, 2014 |
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Current U.S.
Class: |
435/7.92 ;
435/174; 435/188; 435/68.1; 436/501; 436/547 |
Current CPC
Class: |
C07K 2319/30 20130101;
C12N 9/80 20130101; C07K 1/12 20130101; G01N 33/531 20130101; C12Y
201/01063 20130101; C12Y 304/21004 20130101; G01N 33/535 20130101;
G01N 33/543 20130101; C07K 16/00 20130101; G01N 33/5306 20130101;
G01N 2440/38 20130101; C07K 16/2851 20130101; C07K 2317/41
20130101; C07K 2319/60 20130101; C07K 16/2863 20130101; C07K
16/2887 20130101; G01N 33/6854 20130101; C07K 16/241 20130101; G01N
2400/00 20130101; C07K 2317/24 20130101; C12N 9/96 20130101; C12Y
305/01052 20130101; C12N 9/1007 20130101; C07K 14/525 20130101;
C12P 21/005 20130101; C12N 9/6427 20130101; C07K 2317/40
20130101 |
International
Class: |
G01N 33/531 20060101
G01N033/531; C12N 9/96 20060101 C12N009/96; C07K 14/525 20060101
C07K014/525; C07K 16/00 20060101 C07K016/00; C07K 16/28 20060101
C07K016/28; G01N 33/53 20060101 G01N033/53; C12P 21/00 20060101
C12P021/00 |
Claims
1. A composition, comprising: (i) a bile salt or a detergent not
including sodium dodecyl sulfate (SDS); (ii) one or more
glycosidases; (iii) a completely deglycosylated antibody as
determined by electrophoresis or by mass spectrometry; and (iv)
glycan cleavage products.
2. A composition according to claim 1, wherein the detergent is a
dialyzable non-cleavable carboxylated anionic surfactant.
3. A composition according to claim 1, wherein the completely
deglycosylated antibody has antigen binding activity.
4. A composition according to claim 3, wherein the deglycosylated
antibody is selected from deglycosylated human IgG1, human IgG2,
human IgG3, human IgG4, human IgM, human IgA1, human IgA2, human
IgE, murine IgG1, murine IgG2a and murine IgA.
5. A composition according to claim 3, wherein the completely
deglycosylated antibody is suitable for use in immunoassays.
6. A composition according to claim 1, wherein the glycan cleavage
products and/or the antibody are labeled with a fluorescent label,
a radioisotope, methyl acetyl, an antibody or a combination
thereof.
7. A composition according to claim 1, further comprising a
protease.
8. A composition according to claim 7, wherein the protease is
trypsin.
9. A composition according to claim 7, wherein the protease is
selected from trypsin, GluC, AspN, proteinase K, Factor Xa,
Enterokinase, LysC, Arg-C, LysN, IdeS, V-8 Protease, Papain,
Alpha-Lytic Protease, Pyroglutamate Aminopeptidas, Leucine
Aminopeptidase, Methionine Aminopeptidase, Aminopeptidase I,
Aminopeptidase A, Carboxypeptidases (A, B, G, Y), pepsin,
Cathepsins (B, C, D), .alpha.-Chymotrypsin, TEV, thrombin, IdeZ and
IdeE.
10. A composition according to claim 1, wherein one or more of the
glycosidases is a fusion protein.
11. A composition according to claim 10, wherein the glycosidase
fusion protein is immobilized on a matrix.
12. A composition according to claim 10, wherein the fusion protein
comprises a mutant O6-alkylguanine-DNA-alkyltransferase (AGT) and
optionally is immobilized through affinity binding of the AGT to a
matrix.
13. A composition according to claim 1, wherein the composition
comprises an aqueous buffer.
14. A composition, comprising: (a) a bile salt or a dialyzable
non-cleavable carboxylate anionic surfactant; (b) one or more
glycosidases; (c) a completely deglycosylated biologically active
protein as determined by electrophoresis or by mass spectrometry;
and (d) glycan cleavage products.
15. A composition according to claim 14, wherein the one or more
glycosidase are a plurality of glycosidases.
16. A composition according to claim 15, wherein the plurality of
glycosidases comprise exoglycosidases and/or endoglycosidases.
17. A composition according to claim 14, further comprising a
protease.
18. A composition according to claim 17, wherein the protease is
trypsin
19. A method, comprising (a) incubating a composition comprising a
bile salt or a dialyzable non-cleavable carboxylate surfactant, not
including sodium dodecyl sulfate (SDS); and one or more
glycosidases with a glycosylated antibody for less than 60 minutes;
and (b) completely cleaving glycans from the glycosylated antibody
to form a deglycosylated antibody and glycan cleavage products.
20. The method according to claim 19, further comprising isolating
the deglycosylated antibody or cleaved glycan products.
21. The method according to claim 19, further comprising
characterizing the antibody and/or the glycan cleavage
products.
22. The method according to claim 19, further comprising
determining the antigen binding activity of the deglycosylated
antibody.
23. The method according to claim 22, comprising determining an
activity of the antibody by an antibody-antigen binding assay
selected from a radioimmune assay, an ELISA, an affinity binding
assay, or an immunoprecipitation assay.
24. The method according to claim 19, further comprising (c)
obtaining deglycosylated antibody for therapeutic use.
25. The method according to claim 19, further comprising (c)
obtaining deglycosylated antibody for diagnostic use.
26. The method according to claim 19, wherein the composition
further comprises a protease for forming a mixture of peptide
fragments and glycan cleavage products.
27. The method according to claim 19, wherein the composition is
lyophilized prior to incubating with glycosylated antibody wherein
the glycosylated antibody is in an aqueous buffer.
28. The method according to claim 19, wherein the glycosidase is
immobilized on a matrix.
29. The method according to claim 19, wherein the incubating is at
a temperature of about 20.degree. C.-60.degree. C.
30. A kit comprising a lyophilized glycosidase, an immobilized
glycosidase, or a lyophilized buffer, or a combination thereof,
wherein the lyophilized buffer comprises: a bile acid; or a
dialyzable non-cleavable carboxylate anionic surfactant, excluding
SDS; or a combination thereof.
31. A kit according to claim 30, further comprising a protease.
Description
CROSS REFERENCE
[0001] This application claims right of priority to U.S.
Provisional Application Ser. Nos. 62/005,559, filed May 30, 2014,
62/018,074, filed Jun. 27, 2014, 62/021,936 filed Jul. 8, 2014,
62/040,745 filed Aug. 22, 2014 and 62/080,480 filed Nov. 17,
2014.
BACKGROUND
[0002] It is estimated that over 50% of human proteins are
glycosylated. Many health and disease biomarkers are glycosylated
proteins and specific glycoforms may be correlated with health or
disease state. This includes glycosylated proteins linked to
cancer, diabetes, inflammation and other medical conditions, as
well as development. Furthermore, glycosylated proteins are not
just limited to human or mammals, but are found in all eukaryotic
systems, as well as prokaryotes, Archaea and plants.
[0003] Antibodies are glycoproteins. The glycans on an antibody can
be structurally heterogeneous and can vary significantly in health
and disease. During antibody production in vitro, the glycans
attached to an antibody are affected not only by the cell type used
for its production, but also by the cell culture conditions.
Changes in nutrient availability, pH, cell density and CO2 levels
can markedly alter the antibody glycoforms produced by the cells.
For therapeutic antibodies, this can affect tissue distribution,
serum half-life, resistance to proteolysis, complement activation,
antibody-dependent cytotoxicity (ADCC), and inflammation.
Consequently, it is desirable to manufacture therapeutic antibodies
within specific regulatory-approved limits for glycoform
variation.
[0004] Glycan profiling of glycoproteins such as antibodies
requires methods for testing and identifying glycoforms in an
antibody sample. Analysis of glycan structure is an unstandardized,
time-consuming and low-throughput process with deglycosylation
incubation times of as much as 16 hours under conditions that not
only result in partial deglycosylation but also risk damage to the
proteins. (Jenkins, et al., Mol Biotechnol. 39(2):113-8 (2008);
Liu, et al., J Pharm Sci. 97(7):2426-47 (2008). Attempts to shorten
the incubation period have resulted in the use of detergents that
affect the protein integrity and hence functionality and interfere
with mass spectrometry. Antibodies can be difficult to
deglycosylate because glycans can be buried within the molecule's
structural fold. Standard methods denature the antibody structure
to expose the glycans using such methods as extreme heat and harsh
denaturants like sodium dodecyl sulfate (SDS). However, these
methods have several drawbacks: for example SDS can denature the
enzymes used to remove glycans, SDS is difficult to remove, and
even trace amounts of SDS can interfere with sample analysis
methods like mass spectrometry. These methods are also
time-consuming. Alternative methods attempt to increase the rate of
deglycosylation by using a high concentration of
Peptide-N-Glycosidase F (PNGase F). However, this approach does not
overcome the presumed and undesirable bias associated with partial
deglycosylation. Partial deglycosylation may result in certain
glycoforms being preferentially released from the protein over
others. This approach is also costly and is not readily scalable as
it requires significant amounts of PNGase F.
[0005] Current approaches to improve deglycosylation for antibody
characterization continue to result in ever more complex methods,
require additional time to process, are inefficient, and
incompletely remove glycans. Furthermore, the production of
deglycosylated antibodies is currently limited to site-mutagenesis
or bacterial expression systems. Unfortunately, these systems have
difficulties in producing properly assembled and folded antibodies
in any meaningful quantity.
SUMMARY
[0006] In general, preparations including compositions and methods
of use are described for completely deglycosylating proteins such
as antibodies resulting in unbiased removal of glycans that are the
substrates of the glycosidase or mixture of glycosidases used in
the deglycosylation reaction. Also described are methods and
reagents for separating glycans from the protein for further
analysis of the glycans which may be optionally labeled. Where this
involves peptide cleavage by means of a protease, surprisingly it
has been shown that peptide cleavage and deglycosylation can be
achieved in a single step. For ease of use the glycosidase and/or
buffer compositions may be lyophilized and added to the
glycosylated protein to effect deglycosylation.
[0007] In general in one aspect, an artificial in vitro preparation
or composition that cannot occur in nature is provided that
includes (i) a bile salt or a detergent not including sodium
dodecyl sulfate (SDS); (ii) one or more glycosidases; (iii) a
completely deglycosylated antibody as determined by electrophoresis
or by mass spectrometry; and (iv) glycan cleavage products. In one
aspect, the detergent is a dialyzable non-cleavable carboxylate
anionic surfactant. In general, in another aspect, an artificial in
vitro preparation or composition that cannot occur in nature is
provided that includes (a) a bile salt or a dialyzable
non-cleavable carboxylate anionic surfactant; (b) one or more
glycosidases; (c) a completely deglycosylated biologically active
protein as determined by electrophoresis or by mass spectrometry;
and (d) glycan cleavage products.
[0008] In another aspect, the completely deglycosylated antibody
has antigen binding activity and may be used in immunoassays. In
another aspect, the glycan cleavage products and/or the antibody
are labeled with a fluorescent label, a radioisotope, methyl
acetyl, an antibody or a combination thereof. This may facilitate
analysis of the glycan type as well as determining glycan binding
sites on the protein. Examples of a deglycosylated protein include
antibodies. Examples of representative antibodies include
deglycosylated human IgG1, human IgG2, human IgG3, human IgG4,
human IgM, human IgA1, human IgA2, human IgE, murine IgG1, murine
IgG2a and murine IgA.
[0009] In another aspect, a protease may be included in the
composition, wherein the protease may be, for example, trypsin.
Other examples of proteases may include GluC, AspN, proteinase K,
Factor Xa, Enterokinase, LysC, Arg-C, LysN, IdeS, V-8 Protease,
Papain, Alpha-Lytic Protease, Pyroglutamate Aminopeptidas, Leucine
Aminopeptidase, Methionine Aminopeptidase, Aminopeptidase I,
Aminopeptidase A, Carboxypeptidases (A, B, G, Y), pepsin,
Cathepsins (B, C, D), .alpha.-Chymotrypsin, TEV, thrombin, IdeZ and
IdeE.
[0010] In another aspect, the glycosidase (one or more
glycosidases) may be a plurality of glycosidase and may include for
example, one or more exoglycosidases and/or one or more
endoglycosidases. One or more of the glycosidases may be a fusion
protein where for example, the fusion protein may be immobilized on
a matrix. An example of a fusion protein includes a mutant
O6-alkylguanine-DNA-alkyltransferase (AGT) which can optionally be
immobilized through affinity binding of the AGT to a matrix. The
glycosidase or one or more glycosidases may include one or more
N-glycan glycosidases, one or more O-glycosidases and/or one or
more endoglycosidases.
[0011] In another aspect, the composition may include an aqueous
buffer.
[0012] In general in one aspect, a method is provided that includes
(a) incubating a composition comprising a dialyzable non-cleavable
carboxylate surfactant, not including sodium dodecyl sulfate (SDS),
and/or a bile salt; and one or more glycosidases with a
glycosylated antibody for less than 60 minutes; and (b) completely
cleaving glycans from the glycosylated antibody to form a
deglycosylated antibody and glycan cleavage products.
[0013] In one aspect of the method the deglycosylated antibody
and/or cleaved glycan products are isolated and optionally
purified. The antigen binding activity of the deglycosylated
antibody may be characterized. The antigen binding properties of
the antibody when tested were found to be preserved. Binding of
deglycosylated antibody to antigen could be quantified using
antibody-antigen binding assay selected from, for example, a
radioimmune assay, an ELISA, an affinity binding assay, or an
immunoprecipitation assay. The deglycosylated antibodies are
suitable for therapeutic and/or diagnostic use.
[0014] In one aspect of the method, a protease such as trypsin is
contained within the composition containing one or more
glycosidases for cleaving protein into deglycosylated peptide
fragments and glycan cleavage products.
[0015] In one aspect, the composition is lyophilized prior to
incubating with glycosylated antibody wherein the glycosylated
antibody is in an aqueous buffer. In another aspect, the
glycosidase is immobilized on a matrix. In one aspect, the
incubating is at a temperature of about 20.degree. C.-60.degree. C.
In another aspect, incubating is for a time of 5 minutes or
less.
[0016] In general in one aspect, a kit is described containing one
or more lyophilized glycosidases, and/or one or more immobilized
glycosidases, or one or more glycosidases in solution and a
lyophilized buffer or non-lyophilized buffer, or a combination
thereof, wherein the lyophilized or non-lyophilized buffer
comprises: a bile acid; or a dialyzable non-cleavable carboxylate
anionic surfactant, excluding SDS; or a combination thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The figures and drawings are intended to illustrate one or
more versions of the compositions and/or methods described herein.
Unless stated otherwise, these are not intended to be limiting for
the purpose of interpreting the scope of any claims.
[0018] FIGS. 1A-1D show a cartoons of glycosylated antibodies and
the mass distribution of glycosylated and deglycosylated antibody
heavy chains determined by mass spectrometry. Partial
deglycosylation is demonstrated resulting from standard heat
pre-treatment prior to the addition of PNGase F (New England
Biolabs, Ipswich, Mass.) (also see Example 1). In the absence of
surfactants.
[0019] FIG. 1A shows a diagram of an antibody composed of two heavy
chains (H, white); two light chains (L, grey) and glycan molecules
(G) attached to the heavy chain. Preferably, there are no glycans
in a completely deglycosylated antibody molecule.
[0020] FIG. 1B is a control ("no pre-treatment" and "no PNGase F"
treatment) showing the profile of glycoforms present in the
starting monoclonal IgG antibody sample. The cartoons illustrate
the glycoform for each observed major peak.
[0021] FIG. 1C shows the profile of antibody pre-treated by heat
and incubated with PNGase F for 1 hour. The four major peaks (mass
50191, 50353, 50516, and 50823) represent fully glycosylated
glycoproteins. Under these conditions, less than approximately 10%
of the antibody heavy chain is deglycosylated (see cartoon of an
antibody heavy chain without an attached glycan at far left peak,
near mass 48747).
[0022] FIG. 1D shows that heat combined with an extended 16 hour
PNGase F incubation does not result in a substantially
deglycosylated antibody. Increasing the PNGase F incubation time to
16 hours increased the amount of deglycosylated heavy chain
fraction slightly, but a substantial proportion of the antibody is
still glycosylated as evidenced by the major glycosylated
glycoprotein peaks at 50191, 50353, 50516, and 50823.
[0023] FIGS. 2A-2C show the mass distribution of an antibody heavy
chain in mass spectrometry and demonstrate that a stronger
pre-treatment of combined heat denaturation and a reducing agent
prior to PNGase F treatment is insufficient to produce a
substantially deglycosylated antibody (see also Example 2).
[0024] FIG. 2A is the control showing the profile of glycoproteins
present in the starting monoclonal IgG antibody sample (no
pre-treatment and no PNGase F treatment).
[0025] FIG. 2B is the profile of antibody pre-treated by heat
denaturation in combination with a reducing agent dithiothreitol
(DTT) and incubated with PNGase F for 1 hour. The combination
pre-treatment did not improve deglycosylation as compared to heat
denaturation alone (compare FIG. 1B and FIG. 2B). The antibody
heavy chain remains substantially glycosylated, with less than 10%
deglycosylated (see peak near mass size 48750).
[0026] FIG. 2C shows that the same pre-treatment with an extended
16 hour PNGase F incubation also failed to substantially
deglycosylate the antibody heavy chain. Only partial
deglycosylation was achieved as evidenced by the three remaining
major peaks for the shown glycoforms. Bias in the deglycosylation
rate of certain glycoforms is evidenced by the absence of the
fourth glycoform (indicated by the arrow). This demonstrates that
that different glycoforms are deglycosylated at different rates
under these reaction conditions.
[0027] FIGS. 3A-3B demonstrate that an antibody is not
substantially deglycosylated after pre-treatment of the antibody
using a commercially available reagent, RapiGest.TM. (Waters,
Milford, Mass.) ahead of PNGase F digestion. As described in
Example 3, a glycosylated antibody was pre-treated with
manufacturer's suggested amount of RapiGest. The pre-treated
antibody was incubated with PNGase F for 1 hour before ESI-TOF MS
analysis.
[0028] FIG. 3A is a control (no PNGase F) showing the antibody
heavy chain glycoforms present in the original sample (mass 50192,
50354, and 50823).
[0029] FIG. 3B demonstrates that substantially all heavy chain
glycoforms (indicated by the arrow) were still present after using
RapiGest. Only a small fraction of antibody heavy chain was
deglycosylated (mass 48748).
[0030] FIGS. 4A-4C show the wide range of conditions in which a
carboxylated surfactant lauroylsarcosine (LS), DTT and PNGaseF can
produce a substantially deglycosylated antibody in 5 minutes at
50.degree. C. (see also Example 4). Shown are the SDS-PAGE mobility
shifts of a mouse monoclonal IgG antibody (New England Biolabs,
Ipswich, Mass.) (described in Example 1) after deglycosylation with
PNGase F in the presence of varying amounts of LS in combination
with varying amounts of DTT. The arrows indicate lanes where
complete deglycosylation of the substrate is detected by a shift in
the gel position corresponding to a reduction in molecular
weight.
[0031] FIG. 4A compares reaction conditions using 0.2 mM, 1 mM, 4
mM and 20 mM DTT in combination with 0%, 0.05%, 0.1%, 0.5% LS.
[0032] FIG. 4B compares reaction conditions using 20 mM, 40 mM and
80 mM DTT in combination with 0%, 0.2%, 0.4%, 0.5% LS.
[0033] FIG. 4C compares reaction conditions using 4 mM and 20 mM
DTT in combination with 0%, 0.5%, 2%, 4% and 5% LS.
[0034] FIG. 5 shows that low concentrations of DTT enhance
deglycosylation of antibodies in the presence of 0.5% LS and
PNGaseF for an incubation period of 5 minutes at 50.degree. C. (see
also Example 5). Mouse monoclonal IgG antibody (described in
Example 1) treated with PNGase F in 0.5% LS in combination with
0.02 mM, 0.05 mM or 0.1 mM DTT is shown by SDS-PAGE mobility
shifts. Deglycosylation of the substrate was observed by an
increase in sample mobility on SDS-PAGE corresponding to a
reduction of molecular weight resulting from loss of glycans (see
"X").
[0035] FIG. 6 shows that DTT can be substituted for
tris(2-carboxyethyl)phosphine (TCEP) to enhance deglycosylation of
antibody under similar conditions to those described in FIG. 5 (see
also Example 6).
[0036] FIGS. 7A and 7B show that a range of temperatures can be
used for complete deglycosylation of a mouse monoclonal IgG
antibody by PNGaseF using a buffer containing a bile salt or
carboxylated surfactant in combination with a reducing agent (0.5%
LS and 20 mM DTT or 2% deoxycholate (SDC) 4 mM DTT) detected by a
SDS-PAGE mobility shift (see arrows). Additional details for the
one-step and two-step reactions are provided in Example 7.
[0037] FIG. 7A: A one-step reaction of 5 minutes incubation with
PNGaseF performed over different temperatures. The results with two
different buffers are shown.
[0038] FIG. 7B: A two-step reaction with a pretreatment incubation
of 5 minutes at 50.degree. C. and a subsequent 5 minute incubation
with PNGase F performed over different temperatures. The results
with two different buffers are shown.
[0039] FIG. 8 demonstrates deglycosylation of murine anti-maltose
binding protein (anti-MBP) at 22.degree. C. comparing immobilized
PNGase (Benzyl-Guanine-mutant AGT (BG-HS) PNGase F) and soluble
(Free) PNGaseF with a 5 minute, 10 minute and 15 minute incubation.
The results in FIG. 8 shows complete antibody deglycosylation in a
two-step reaction, after incubation for 5 minutes at room
temperature with immobilized PNGase F as well as with soluble
PNGaseF (see also Example 8).
[0040] FIG. 9 shows that complete deglycosylation of an antibody
using a one-step reaction (also described in Example 9) can be
achieved within 3 minutes. A mouse monoclonal IgG antibody was
treated with PNGase F in the presence of buffer containing a
carboxylated surfactant and reducing agent for 1 to 5 minutes at
50.degree. C. Arrows show samples where complete mobility shift is
observed, indicating complete deglycosylation.
[0041] FIGS. 10A-10B graphically represents composition and
characterization of N-glycans released from an antibody (see also
Example 10).
[0042] FIG. 10A shows the glycans (A to I) released from the
glycosylated antibody when incubated with PNGase F and a reducing
agent (DTT) in the presence of the bile salt, SDC (hatched bars);
or PNGase F and DTT only (white). A minimum ten-fold increase in
glycan yield was observed when DTT plus SDC was used compared with
DTT only in peak fractions. The ten-fold increase is indicative of
the efficient and substantially complete removal of all glycans
from the antibody by this method.
[0043] FIG. 10B provides pie charts showing the percentage
composition of IgG N-glycans after deglycosylation using:
[0044] (i) DTT and SDC; the precise composition of the N-glycans
present in the monoclonal IgG sample is provided showing an
unbiased representation of substantially all nine types of
glycans.
[0045] (ii) DTT only: in contrast to (i), not all glycans are
removed where the major N-glycans (D and B) are underrepresented
and minor glycans species (A, H, E and I) are overrepresented
compared to deglycosylation in the presence of DTT and SDC and
hence showing a bias in the removal of N-glycans by PNGase F in the
presence of a reducing agent alone.
[0046] FIGS. 11A-11B show the chromatographic profiles of
fluorescently labeled N-glycans released from an antibody and
labeled in a one pot method FIG. 11A or a two pot method FIG. 11B
(see also Example 11). The glycans were cleaved from a monoclonal
IgG antibody using PNGase F in the presence of DTT and SDC. The
glycans cleaved from the antibody were either labeled directly in
the deglycosylation mixture in a one pot method, or first isolated
from the deglycosylation mixture before labeling in a two pot
method. Whereas the overall profile in FIGS. 11A and 11B were
similar, the absolute quantities of glycans were significantly
increased in the one pot method (compare fluorescent counts on
y-axes of FIGS. 11A and 11B) indicating the efficiency of the one
pot method.
[0047] FIG. 11A shows the overall profile of labeled glycans using
a one pot method where the glycans were not isolated prior to
labeling.
[0048] FIG. 11B shows the overall profile of labeled glycans using
a two pot method where the glycans were first isolated prior to
labeling.
[0049] FIGS. 12A-12C show the chromatographic profiles of
fluorescently labeled N-glycans released from three different
antibodies in 5 minutes and labeled in a one pot method. The
N-glycans were cleaved from each antibody using PNGase F in the
present of LS and DTT before labeling (see also Example 14).
[0050] FIG. 12A is the N-glycan profile of rituximab.
[0051] FIG. 12B is the N-glycan profile of cetuximab.
[0052] FIG. 12C is the N-glycan profile of etanercept.
[0053] FIGS. 13A-13C demonstrate that antibodies deglycosylated by
PNGase F using a combination of DTT, bile salt and mild heat are
functional and retain the ability to specifically recognize and
bind to its cognate antigen.
[0054] FIG. 13A is an SDS-PAGE of each treated sample. The
deglycosylated heavy chain of IgG runs slightly faster (lower) than
the glycosylated heavy chain (as indicated by arrows with cartoons
of the glycosylated and deglycosylated heavy chain). (+) is an
anti-MBP antibody treated with PNGase F in the presence of SDC and
a reducing agent; (-) is a parallel sample but with no PNGase F
treatment.
[0055] FIG. 13B is ESI-TOF MS analysis for the sample treated with
PNGase F in the presence of a surfactant and a reducing agent shows
substantial deglycosylation of the antibody. The negative control
shows the antibody glycoforms present in the original sample when
not treated with PNGase F.
[0056] FIG. 13C confirms that the deglycosylated antibody of the
positive sample is functional and retains its ability to recognize
and bind to its antigen similar to the glycosylated antibody. A
western blot was performed on an MBP-fusion protein. The white
triangles above the gel indicate the decreasing amounts of the
cognate antigen from 4.2 ng to 0.5 ng loaded on the gel that was
blotted.
[0057] FIGS. 14A-14B demonstrate that rapid deglycosylation is
effective using lyophilized PNGase F (lyo PNGase) and buffer
containing a bile salt or surfactant and DTT. PNGase F and the
buffer can be lyophilized either together (lyo master mix) or
separately (lyo buffer) without any detrimental effect in activity
(see also Example 14).
[0058] FIG. 14A shows the SDS-PAGE of a monoclonal IgG completely
deglycosylated with lyophilized PNGase F and buffer (dried together
or separately). The arrows indicate that the shift in migration
corresponds to full deglycosylation of the IgG, comparable with the
positive control ("fresh PNGase F"), which used PNGase F and buffer
that had not been lyophilized (shown on right). PNGase F was absent
from the negative controls ("C").
[0059] FIG. 14B is the ESI-TOF MS analysis for the samples
described in FIG. 14A ((i) complete deglycosylation using a
lyophilized master mix (PNGase F-buffer); (ii) complete
deglycosylation using lyophilized PNGase F lyophilized buffer);
(iii) negative control (no PNGase F)). The analysis confirms that
the lyophilized and rehydrated buffer and enzyme substantially
deglycosylates the antibody in 5 minutes at 50.degree. C.
[0060] FIGS. 15A-15B illustrate that a buffer containing a bile
salt or carboxylated anionic detergent and DTT can be used to
efficiently deglycosylate a variety of antibody isotypes (see also
Example 15). The SDS-PAGE gels show the controls (no enzyme (-))
containing buffer and the isotype antibody, but no PNGase F. (+)
refers to the deglycosylation reaction using PNGase F in the
presence of 0.5% LS and 20 mM DTT. The isotype and source (human,
"h"; mouse "m") of the antibody are indicated. The PNGase F band is
indicated on the side of each gel as well as the light chain (LC)
of the isotype. Each experimental reaction showed a mobility shift
of the heavy chain (the location of which is indicated by a "HC" on
the side) compared with the negative control indicating successful
deglycosylation.
[0061] FIG. 15A Antibody isotypes: hIgG1, hIgG2, and hIgG4.
[0062] FIG. 15B Antibody isotypes: hIgM, hIgA1, hIgE, mIgG1,
mIgG2A.
[0063] FIGS. 16A-16B shows SDS-PAGE gels in which the amount of
PNGaseF required for complete deglycosylation is substantially
reduced in the presence of LS and a reducing agent compared to the
reducing agent alone under the otherwise same one-step reaction
conditions (for experimental conditions see Example 17). The ratio
of amounts (.mu.g/.mu.l) of PNGaseF: IgG was shown to be greater
than 20 fold, greater than 50 fold, greater than 75 fold, greater
than 90 fold more PNGase F in the absence of LS thus showing that
complete deglycosylation can be achieved with significantly lower
amounts of PNGase F in the presence of LS then would be the case
without LS.
[0064] FIG. 16A shows complete deglycosylation of a monoclonal IgG
using 62.5 Units of PNGase F and where the reaction buffer contains
0.5% LS (see black arrow).
[0065] FIG. 16B shows that, when LS is absent, even the most
concentrated stock available (4000 units) is not sufficient for
complete deglycosylation (indicated by an X).
[0066] FIG. 17A-17B shows SDS-PAGE gels in which the amount of
PNGaseF required for complete deglycosylation is substantially
reduced in the presence of LS compared to the buffer alone under
the otherwise same two-step reaction conditions. (For experimental
conditions see Example 18). The ratio of PNGaseF: IgG was shown to
be greater than 20 fold, greater than 50 fold, greater than 75
fold, greater than 90 fold more PNGase F in the absence of LS.
[0067] FIG. 17A shows complete deglycosylation of a monoclonal IgG
using 62.5 Units of PNGase F and where the reaction buffer contains
0.5% (see black arrow).
[0068] FIG. 17B shows that, when LS is absent, even the most
concentrated stock available (4000 units) is not sufficient for
complete deglycosylation (indicated by an X).
[0069] FIG. 18A-18C shows that the conditions for PNGase F
deglycosylation (carboxylic detergent, heat) under non-reducing
conditions preserve the functional integrity of the antibody
molecule. Mouse monoclonal IgG, rituximab, and etanercept were
pre-treated under various conditions in the absence of DTT, to
determine the amount of LS and the temperature and incubation time
needed to promote effective deglycosylation. The deglycosylated,
intact, monoclonal antibody retains its antigen binding properties
(see also Example 19).
[0070] FIG. 18A shows that rapid deglycosylation of antibodies is
effective in the absence of a reducing agent with pretreatment for
2 to 15 minutes at temperatures ranging from 80.degree. C. to
55.degree. C. The SDS-PAGE shows a shift in size (indicated by the
arrows), corresponding to complete deglycosylation.
[0071] FIG. 18B shows that rapid deglycosylation will preserve the
structure of the antibody in the absence of a reducing agent. To
detect the presence of multimeric proteins (i.e. IgG
hetero-tetramers) the samples were also run on SDS PAGE without DTT
in the sample buffer. It is evident that the multimeric structure
has been preserved, notice the lack of monomeric structures running
under the 80KD marker.
[0072] FIG. 18C shows the functional activity for a deglycosylated,
intact, monoclonal anti-MBP, by it recognition of the corresponding
antigen (MBP-tagged protein) with equivalent potency compared with
the pre-incubated antibody, or with fresh antibody (both of which
are still glycosylated).
[0073] FIG. 19 shows that rapid deglycosylation is effective using
carboxylic surfactants such as LS or sodium laurate (LAU) (see
example 20). The SDS-PAGE shift in migration indicates complete
deglycosylation (arrows) can be compared with the control ("C").
The carboxylic acid is effective in the presence or absence of a
reducing agent. The figure also shows that a carboxylic acid is
effective for rapid deglycosylation in 5 minutes at 50.degree. C.
in a one-step or two-step reaction.
[0074] FIG. 20A-20B shows the results of simultaneous Rapid PNGase
F (New England Biolabs, Ipswich, Mass.) and Trypsin reactions.
Rapid deglycosylation occurs in the presence of a proteolytic
enzyme such as Trypsin (see example 21). Mouse monoclonal IgG was
simultaneously deglycosylated with PNGase F and cleaved by trypsin
to yield deglycosylated, tryptic peptides for mass spectrometry
analysis.
[0075] FIG. 20A shows the total ions observed from the PNGase
F/Trypsin digestion. The reaction performed on Tris HCl buffer
(optimal for Trypsin activity) showed the same peptide coverage in
the absence of LS as predicted in the presence of LS.
[0076] FIG. 20B shows the fragmentation pattern of the isolated
EDYnSTIR peptide, which corresponds to the unique glycosylation
site of mouse IgG. The peptide is only found as the aspartic acid
form, indicating that deglycosylation was complete.
DETAILED DESCRIPTION
[0077] Embodiments of the invention provide improved methods and
compositions for analyzing glycoproteins such as antibodies by
complete and optionally rapid removal of glycans permitting the
separate analysis without bias of the deglycosylated proteins and
the released glycans and/or the preparation of functional
deglycosylated antibodies.
[0078] The advantages of present embodiments include at least one
of the following: (i) deglycosylation conditions that minimize
oxidation, deamidation, and other unwanted chemical modifications
of the protein or the glycans; (ii) deglycosylation conditions that
do not interfere with downstream analysis using mass spectrometry;
(iii) complete deglycosylation that results in elimination of bias
towards certain species of glycans; (iv) rapid reaction conditions
that are convenient and cost effective; (v) preservation of
function of the deglycosylated proteins; (vi) degradation of
protein into peptides with protease and deglycosylation with a
glycosidase in a single step; (vii) use of reduced amounts of
glycosidase in a reaction and (viii) availability of lyophilized
reagents for deglycosylation suitable for adding to the
glycosylated antibody. These advantages accrue as a result of
certain features of embodiments of the invention. These include one
or more of the following: a lyophilized or solubilized preparation
of a glycosidase in a dialyzable non-cleavable carboxylated anionic
surfactant and/or a bile salt in a buffer. The preparation may
additionally contain a reducing agent. The preparation may
additionally contain a protease such as trypsin. The preparation
may be a solubilized or lyophilized preparation. When combined with
a glycosylated antibody, the preparation can achieve complete
deglycosylation of a glycosylated antibody in less than 60 minutes
at a temperature of less than 70.degree. C. and optionally within a
single step reaction or in two-steps involving a pretreatment or in
more than two-steps as desired.
[0079] The high sensitivity of embodiments of the methods can be
used for determining all or substantially all antibody glycoforms
present in a sample in a simple, effective, comprehensive and rapid
manner. Since substantially all glycans can be removed from a
glycoprotein, the inherent bias found in other methods is avoided.
The sensitivity also means that less sample can be used. The
methods can avoid sample loss arising from multiple handling steps
(e.g., heat denaturation, alkylation, SDS treatment where the SDS
cannot readily be removed, or the inactivation of heat-labile or
acid-labile reagents) normally found in other procedures.
Consequently the methods are suitable for low volume or low
concentration of samples. Additionally, since embodiments of the
methods utilize relatively small amounts of glycosidases, large
sample volumes of sample and/or multiple samples (either
simultaneously or sequentially) can be readily deglycosylated.
[0080] The methods are readily combined with downstream analyses,
such as chromatography (e.g., HPLC, HP anion-exchange
chromatography with pulsed amperometric detection (HPAE-PAD), gel
electrophoresis, mass spectrometry (e.g., MALDI-TOF MS, ESI-TOF
MS), and capillary electrophoresis. The methods can be used, for
example, to determine the molecular weights, charge state,
oxidation, clipping, and deamidation of the deglycosylated proteins
(e.g. antibodies), confirm protein deglycosylation (antibody
deglycosylation), determine protein function (e.g. antibody
function), determine glycan composition, determine glycan
structure, and identify glycoprofile information. This facilitates
a rapid workflow that has practical implications in the industry.
The methods and reagents described herein can be used in
diagnostics, quality control, management and process optimization
of antibody production (such a therapeutic antibodies), glycomics
profiling, and high throughput analyses.
[0081] In one embodiments, the substrate glycoprotein is an
antibody. In one embodiment, the glycans are N-glycans removable by
a PNGase glycosidase.
[0082] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the pertinent art. The scope of certain terms is
provided below. Embodiments described herein may include one or
more ranges of values (e.g., size, concentration, time,
temperature). A range of values will be understood to include all
values within the range, including subset(s) of values in the
recited range, to a tenth of the unit of the lower limit unless the
context clearly dictates otherwise.
[0083] As used herein, the articles "a", "an", and "the" relate
equivalently to a meaning as singular or plural unless the context
dictates otherwise.
[0084] The term "antibody" is used interchangeably with the term
immunoglobulin and include whole antibodies and fragments which
when functional, are capable of binding antigen. An antibody may be
natural or recombinant, polyclonal, monoclonal, humanized,
mammalian (including human, primate, camel, porcine, equine,
rodent, bovid), avian, reptilian, fish, (e.g., hagfish, lampreys,
bony and cartilaginous fish, including elasmobranchii). The
antibody may be derived from an in vivo sample, such as a serum,
plasma r blood sample or other body fluid, secreted, excreted or
otherwise removed from the host. Alternatively, an antibody may be
from an in vitro cell culture. An antibody (e.g., a therapeutic
antibody) may be produced in a bioreactor, or in a multicellular
host organism (e.g. a cultured crop). An antibody may be a single
chain antibody, for example.
[0085] The term "antibody" includes all classes and isotypes of
antibody, and include heavy chain Ig, IgA, IgD, IgE, IgG1, IgG2,
IgG3, IgG4, IgM, IgNAR, IgW, and IgY and may further include
chimeric, multi specific (e.g. bi-specific, tri-specific,
tetra-specific), derivatized and fusion proteins thereof. Also
included in the definition are recombinant antibodies such as
minibody, diabody, triabody, tetrabody, scFv, bi-specific scFv,
tri-specific Fab3, single domain antibodies (sdAb), and
dual-variable domain antibodies. An antibody fragment that includes
Fab, Fab', F(ab')2, Fv, Fc, Fd, single domains (e.g., VhH, V.sub.L,
V.sub.H, V.sub.NAR), and portions thereof, are included in the term
"antibody".
[0086] The antibody may be soluble. The antibody may be in an
aqueous state or may be precipitated, dried or immobilized for
example on a solid surface, such as a bead a column or matrix.
[0087] Antibodies including fragments produced by cells are
typically glycosylated. Generally, antibodies are glycosylated with
N-linked glycans, but they can also contain O-glycans.
Glycosylation may occur on any region of an antibody. A particular
glycoform of an antibody can vary, depending on a variety of
conditions, such as health, disease state or culture conditions.
Such variations can impact the activity and half-life of
antibodies. An antibody that is deglycosylated may have reduced or
no variation in antibody activity or half-life.
[0088] In certain embodiments, the affinity between an antibody and
an antigen when they are specifically bound in a capture
agent/analyte complex is characterized by a KD (dissociation
constant) of less than 10.sup.-6M, less than 10.sup.-7 M, less than
10.sup.-8 M, less than 10.sup.-9 M, less than 10.sup.-9 M, less
than 10.sup.-11 M, or less than about 10.sup.-12 M or less.
[0089] The term "glycan" refers to any sugar, in free form or
attached to another molecule and includes O-glycans and N-glycans.
An N-linked glycan is a glycan covalently linked to an asparagine
residue of a polypeptide chain in the consensus sequence:
-Asn-X-Ser/Thr, where X is any amino acid except proline. An
O-linked glycan is a glycan linked via a glycosidic bond to the
hydroxyl group of serine or threonine. The glycan may be a
monosaccharide, oligosaccharide or polysaccharide, linear, branched
or a mixture of linear and branched chains and composed of a single
type of sugar or multiple types of sugars. The term "glycoforms"
refers to the different molecular forms of a glycoprotein,
resulting from variable glycan structure and/or glycan attachment
site occupancy. The term "glycoprofile" refers to the properties or
characteristics of glycans on one or more glycomolecules. The
profile may include the identity, structure, composition and/or
quantity of any one or more glycans, the glycosylation site(s) or
location(s) on a glycomolecule, and/or glycan occupancy on a
glycomolecule. "Glycoprofile" can be used interchangeably with
"glycosylation profile."
[0090] The term "glycosylation" refers to the covalent attachment
of a carbohydrate to a polypeptide, lipid, polynucleotide or
another carbohydrate. The terms "glycosylated peptide"
"glycosylated polypeptide" or "glycosylated protein" can be used
interchangeably with "glycopeptide," "glycopolypeptide" or
"glycoprotein."
[0091] The term "deglycosylation" refers to the removal of glycans
from a glycan-containing molecule. Deglycosylation can be done
enzymatically or chemically. Single glycosidases or mixtures of
glysidases may be used to deglycosylate a protein. Cleaving
"substantially all" glycans results in a completely deglycosylated
protein where "Complete deglycosylation" refers to >70%,
>80%, >90%, >92%, >94%, >96%, >98% or >99%
deglycosylation by a glycosidase as determined by SDS-PAGE or by
mass spectrometry. For example, complete deglycosylation by an
N-glycan or an O-glycan glycosidase refers to >70%, >80%,
>90%, >92%, >94%, >96%, >98% or >99% N- or
O-glycan deglycosylation. If complete deglycosylation is achieved
by a mixture of N-glycan and O-glycan glycosidases then this refers
to >70%, >80%, >90%, >92%, >94%, >96%, >98% or
>99% N-glycan and O-glycan deglycosylation. An example of
complete deglycosylation is 90%-100% deglycosylation.
[0092] The term "glycosidase" refers to an enzyme that can cleave a
glycan and includes endoglycosidases, exoglycosidases, and
amidases. In one embodiment, the glycosidase is an endoglycosidase
and/or an exoglycosidase. In one embodiment, the glycosidase is an
amidase. In some embodiments, the glycosidase cleaves N-linked
glycans. In some embodiments, the glycosidase cleaves O-linked
glycans. In some embodiments, the glycosidase is selected from the
group consisting of deglycosylated peptide-N-glycosidase from
almonds (PNGase A) (see below) or from rice (PNGase Ar) (see
below), peptide-N-glycosidase F (PNGase F), PNGase Y (Swiss Prot:
Q6CAX5.1 YALI0C23562g YALI0C23562p [Yarrowia lipolytica CLIB122]
Gene ID: 2909617 NCBI Reference Sequence: NC.sub.--006069.1.
O-glycosidase (New England Biolabs, Ipswich Mass.), endoglycosidase
D (Endo D) (New England Biolabs, Ipswich Mass.), endoglycosidase F
(Endo F) (QAbio, Palm Desert, Calif.), endoglycosidase F1 (Endo F1)
(QAbio, Palm Desert, Calif.), endoglycosidase F2 (Endo F2) (QAbio,
Palm Desert, Calif.), endoglycosidase F3 (Endo F3) (QAbio, Palm
Desert, Calif.), endoglycosidase H (Endo H) (New England Biolabs,
Ipswich Mass.), endoglycosidase M (Endo M) (TCI America),
endoglycosidase S (Endo S) (New England Biolabs, Ipswich Mass.),
beta1-3 galactosidase (New England Biolabs, Ipswich Mass.), beta1-4
galactosidase (New England Biolabs, Ipswich Mass.), alpha1-3,6
galactosidase (New England Biolabs, Ipswich Mass.),
beta-N-acetylglucosaminidase (New England Biolabs, Ipswich Mass.),
alpha-N-acetylgalactosamindiase (New England Biolabs, Ipswich
Mass.), beta-N-acetylhexosaminidase (New England Biolabs, Ipswich
Mass.), alpha1-2,3 mannosidase (New England Biolabs, Ipswich
Mass.), alpha1-6 mannosidase (New England Biolabs, Ipswich Mass.),
neuraminidase (New England Biolabs, Ipswich Mass.), alpha2-3
neuraminidase (New England Biolabs, Ipswich Mass.), alpha1-2
fucosidase (New England Biolabs, Ipswich Mass.), or a combination
thereof. In some embodiments, the glycosidase is a fusion protein.
In some embodiments, the glycosidase is PNGase F. PNGase F is a
commercially available enzyme (e.g., New England Biolabs, Ipswich
Mass., Cat. #P0704 or #P0710). In some embodiments, the PNGase F is
a fusion protein. For example, the PNGase F may be PNGase F tagged
with a chitin binding domain (CBD) or a PNGase F-SNAP fusion
protein (see example 16). In some embodiments, the glycosidase is
lyophilized. In some embodiments, the glycosidase is a lyophilized
PNGase F. In some embodiments, the glycosidase is substantially
free of animal-derived reagents.
[0093] As described herein, the glycosidase (which includes a
single glycosidase or a mixture of glycosidases) may be immobilized
on a solid, semi-solid or porous surface. Suitable surfaces for
immobilization of glycosidases (such as PNGaseF) include beads,
resins, columns, wells, plates, microchips, microfluidic devices,
filters and the like. In one embodiment, the glycosidase, such as
PNGase F, is immobilized on an agarose or magnetic bead.
Immobilization may be achieved by means of a fusion protein (e.g. a
PNGaseF fusion protein) where the fusion protein has an affinity
for a specific molecule such as CBD for chitin or maltose binding
domain for maltose. In one embodiment, the fusion protein comprises
a mutant AGT.
[0094] In one embodiment, one unit of glycosidase is the amount of
enzyme required to remove >95% glycans from 5 .mu.g of mouse
monoclonal IgG, for 5 minutes at 50.degree. C. in a 10 .mu.l
reaction volume.
[0095] In another embodiment, one unit of PNGase F is the amount of
enzyme required for complete deglycosylation from 10 .mu.g of
denatured RNase B in 1 hour at 37.degree. C. in a total reaction
volume of 10 .mu.l.
[0096] The term "bile acid" refers to a family of molecules
composed of a steroid structure with four rings, a (five to eight
atom) carbon side chain terminating in a carboxylic acid, and one
or more hydroxyl groups. The four rings are labeled from left to
right (as commonly drawn) A, B, C, and D, with the D-ring being
smaller by one carbon than the other three. The hydroxyl groups can
be in either of two positions, up (or out), termed beta (.beta.;
often drawn by convention as a solid line), or down, termed alpha
(a; seen as a dashed line). All bile acids have a 3-hydroxyl group,
derived from the parent molecule, cholesterol. Bile acids are
reviewed in Hofmann, et al., J. Lipid Res. 51: 226-46 (2010) and
Russell, Annu. Rev. Biochem. 72: 137-74 (2003) and include cholic
acid, glycocholic acid, taurocholic acid, deoxycholic acid,
chenodeoxycholic acid, glycochenodeoxycholic acid,
taurochenodeoxycholic acid and lithocholic acid, although many
others, including synthetically-made variants of naturally
occurring bile acids, are known. A bile acid may be employed as a
salt, i.e., as a bile salt.
[0097] The term "anionic surfactant" refers to a surfactant that
has a negatively charged head, such as a sulfate, sulfonate,
phosphate or caboxylate.
[0098] The term "non-cleavable carboxylated anionic surfactant"
which may be referred to as "detergent" refers to an anionic
surfactant that has a carboxylated head, examples of which include,
but are not limited to, the alkyl carboxylates (soaps), such as
sodium stearate, and LS as well as fluorosurfactants such as
perfluorononanoate, perfluorooctanoate (PFOA or PFO). Other
carboxylated anionic surfactants are known and further examples are
described below. The surfactant/detergent may be rendered inactive
by cleavage under mildly acidic conditions where other chemical
bonds (i.e. peptide bonds, glycosidic bonds etc. are not
affected).
[0099] The term "dialyzable" refers to molecules that are able to
pass through a dialysis membrane (i.e., a semi-semi-permeable
membrane) having a molecular-weight cutoff (MWCO) of 25KD, 50KD, or
preferably in the range of 75KD-100KD, thereby retaining >90% of
a protein having a molecular mass of at least 10 kDa but not the
surfactant.
[0100] The term "non-naturally occurring" refers to a composition
that does not exist in nature. In the context of a protein, the
term "non-naturally occurring" refers to a protein that has an
amino acid sequence and/or a post-translational modification
pattern that is different to the protein in its natural state. For
example, a non-naturally occurring protein may have one or more
amino acid substitutions, deletions or insertions at the
N-terminus, the C-terminus and/or between the N- and C-termini of
the protein. A "non-naturally occurring" protein may have an amino
acid sequence that is different to a naturally occurring amino acid
sequence but that that is at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98% or at least 99% identical
to a naturally occurring amino acid sequence. In certain cases, a
non-naturally occurring protein may contain an N-terminal
methionine or may lack one or more post-translational modifications
(e.g., glycosylation, phosphorylation, etc.) if it is produced by a
different (e.g., bacterial) cell.
[0101] In the context of a preparation, the term "non-naturally
occurring" refers to: a) a combination of components that are not
combined by nature, e.g., because they are at different locations,
in different cells or different cell compartments; b) a combination
of components that have relative concentrations that are not found
in nature; c) a combination that lacks something that is usually
associated with one of the components in nature; e) a combination
that is in a form that not found in nature, e.g., dried, freeze
dried, crystalline, aqueous; and/or d) a combination that contains
a component that is not found in nature. For example, a preparation
may contain a buffering agent (e.g., Tris, HEPES, TAPS, MOPS,
tricine or MES), a detergent, a dye, a reaction enhancer or
inhibitor, an oxidizing agent, a reducing agent, a solvent or a
preservative that is not found in nature. In present embodiments,
two-step reactions and one-step reactions are described for
deglycosylation under conditions that (a) preserve the binding
properties of an antibody for its antigen; (b) preserve the
characteristics of the protein or glycan for further analysis;
and/or (c) preserve the multimeric structure of the protein if such
exists. In some circumstances, a one pot, two-step reaction is
sufficient. However, a one-step reaction can facilitate high
through-put analysis of glycoproteins and can minimize handling
error. Present aspects of the method are effective for nanogram to
microgram amounts of glycosylated protein. The successful
application of aspects of the method to relatively large amount of
substrate protein has utility in detecting rare species of
glycans.
[0102] A "two-step" procedure includes a first step (the
pretreatment step) that, without wishing to be limited by theory,
is believed to cause the substrate glycoprotein to become relaxed
under conditions that avoid aggregation and/or coagulation of the
glycoprotein. The second step (the deglycosylation reaction
step/reaction step) utilizes the same or similar buffers as in the
first step for convenience and maximum activity of the glycosidase.
Here a glycosidase reagent is added for cleavage of the glycans. In
contrast, the "one-step" procedure introduces the glycosidase in a
master mix at the outset. The one-step procedure preferably
utilizes a reducing agent such as DTT that is optional and can be
omitted in the two-step procedure. For example, see Example 7 which
describes one pot methods using a single step to deglycosylate a
protein where an antibody was incubated with a reducing agent, a
buffer comprising SDC or LS, and PNGase F. Incubation of the
mixture for 5 minutes at 50.degree. C. produced a substantially
deglycosylated antibody (see FIG. 7A). Deglycosylation was observed
in less than 3 minutes (see Example 9, and FIG. 9).
[0103] The pH conditions for the two-step and the one-step methods
are similar and are in the range of 4 to 10 for optimal glycosidase
activity. For example, the pH may be about 4.0, 4.5, 5.0, 5.5, 6.0,
6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 or any range thereof.
[0104] The detergent, SDS was found to be unsuitable in the present
embodiments as it denatures the glycoprotein so that it loses any
structure or function it might otherwise have. Moreover, SDS cannot
be readily removed from the reaction mix and it associates
intimately with the protein, and thereafter interferes with mass
spectrometry of the glycans and proteins. In the examples, it was
found that SDS negatively impacts the activity of glycosidases such
as PNGaseF. Therefore SDS was excluded from the present
embodiments.
[0105] Here it has been found that surfactants such as dialyzable
non-cleavable carboxylated anionic surfactants and/or bile acid or
salts thereof are suitable for use in the present methods. Without
being limited by theory it is believed that non-cleavable,
dialyzable carboxylated anionic surfactants or bile acid serve to
relax the protein structure and facilitate enzyme mediated
deglycosylation. In addition, these reagents can be removed so as
not to unduly inhibiting mass spectrometry analysis and may further
permit the protein (such as the antibody) to retain the function as
demonstrated herein.
[0106] Examples of dialyzable, non-cleavable carboxylated anionic
surfactant for use in present embodiments include LS, lauric acid,
stearic acid, palmitic acid, a combination thereof, and salts
thereof. In some embodiments, the carboxylated anionic surfactant
salt is LS. The carboxylated anionic surfactant may be lyophilized
and/or may be substantially free of animal-derived reagents.
[0107] Whereas it is possible to use a range of concentrations of
the dialyzable, non-cleavable carboxylated anionic surfactant such
as sodium LS or SDC, it is preferable to use a concentration of at
least 0.5%, e.g., a concentration in the range of 0.5% to 8% or a
concentration in the range of 1% to 5%.
[0108] Examples of bile acid for use in present embodiments include
cholic acid, chenodeoxycholic acid, lithocholic acid, deoxycholic
acid, ursodeoxycholic acid or salts thereof or a combinations of
the foregoing. In some embodiments, the bile acid is deoxycholic
acid. In one embodiment, the bile acid salt is SDC. The bile acid
may be lyophilized. The bile acid may be substantially free of
animal-derived reagents. Whereas it is possible to use a range of
concentrations of a bile salt such as SDC, it is preferable to use
a concentration of at least 2% such as 3% or 5%. In one embodiment
SDC is used at a concentration of about 2%.
[0109] Further examples of detergents suitable for use as described
herein may be obtained from Sigma Life Sciences (see Biofiles
(2008) Vol. 3 No. 3 "Detergents and Solubilization reagents"),
G-Biosciences "Detergents: A handbook and Selection Guide to
Detergents and Detergent Removal".
[0110] In addition to the anionic carboxylic surfactants and/or
bile salts, it has been found to be advantageous but not essential
to include a reducing agent to the deglycosylation pretreatment or
reaction mixture.
[0111] In some embodiments, the reducing agent is DTT,
.beta.-mercaptoethanol, or TCEP. The final concentration of the
reducing agent can be determined by those of skill in the art. In
some embodiments the reducing agent is DTT. For example, the final
concentration of DTT may be between about 0.2 mM to about 100 mM.
For example, the final concentration may be about 0.2 mM, 0.4 mM,
0.6 mM, 0.8 mM, 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30
mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM,
80 mM, 85 mM, 90 mM, 95 mM, 100 mM, DTT, or any range thereof (see
Example 4 and 5). In some embodiments, the reducing agent is TCEP.
In some embodiments, the final concentration of TCEP is between
about 0.2 mM to about 10 mM. For example, about 0.2 mM, 0.4 mM, 0.6
mM, 0.8 mM, 1 mM, 2 mM, 5 mM, 10 mM TCEP, or any range thereof (see
Example 6).
[0112] In one embodiment, a glycoprotein can be optionally
pre-treated before deglycosylation where the entire deglycosylation
reaction may be completed in less than 60 minutes such as less than
30 minutes. In some embodiments, the deglycosylation reaction
mixture (either with or without a heat pre-treatment) is incubated
at a temperature of about 25.degree. C. to as high as 70.degree. C.
(e.g., about 25.degree. C., 30.degree. C., 37.degree. C.,
40.degree. C., 43.degree. C., 45.degree. C., 48.degree. C.,
50.degree. C., 53.degree. C., 55.degree. C., 58.degree. C.,
60.degree. C., 63.degree. C., 65.degree. C., 70.degree. C. or a
range thereof) for about 2 minutes, 3 minutes, 4 minutes, 5
minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 5
hours, 10 hours, or any range thereof. See for example, Example 7.
In one embodiment, the mixture is incubated at about 37.degree. C.,
45.degree. C., or 50.degree. C. for about 30 minutes, 15 minutes,
10 minutes, 5 minutes or for about 3 minutes or less. In some
embodiments, the reaction may be at least 90% complete in 2 minutes
to 60 minutes at a temperature in the range of 30.degree. C. to
50.degree. C. The reaction conditions, e.g., the temperature, may
vary depending on the enzyme used.
[0113] The concentration of the antibody in the reaction may be in
the range of 10 ng/.mu.l to 10 .mu.g/.mu.l, e.g., 100 ng/.mu.l to 5
.mu.g/.mu.l, although concentrations outside of this range are
envisioned.
[0114] In certain embodiments, a mixture that contains antibodies
that are pre-treated with surfactants without glycosidase at a
temperature of about 45.degree. C. to about 95.degree. C. for 1
minute to 60 minutes, e.g., about 1 minute, 2 minutes, 3 minutes,
or less than about 5 minutes, 10 minutes, 15 minutes, or 30
minutes, before adding the glycosidase results in substantially
complete deglycosylation after addition of the glycosidase. This is
shown in, for example, Examples 7, and 17. In one embodiment, the
pre-treatment incubation is about 15 minutes at about 55.degree. C.
In another embodiment, the pre-treatment incubation is about 2
minutes at about 80.degree. C. (see for example, FIGS. 7A-7B, FIG.
8, Example 7 and Example 8 demonstrating various ranges of suitable
temperatures for deglycosylation from ambient (room) temperature to
about 63.degree. C.). In other examples, a glycosylated antibody
can be optionally pre-treated with a reducing agent such as DTT and
a buffer comprising a dialyzable, non-cleavable carboxylated
anionic detergent such as LS or a bile acid such as SDC before
incubation with the glycosidase.
[0115] The Examples demonstrate deglycosylation of antibodies in
less than 60 minutes, 30 minutes, 15 minutes, 5 minutes, 4 minutes
and 3 minutes. Complete deglycosylation can be achieved at a
temperature in the range of room temperature to about 55.degree. C.
for 5 minutes. By way of an example, a temperature of 55.degree. C.
may be used for 10 minutes or a temperature of 37.degree. C. may be
used for 15 minutes which in combination with the a dialyzable,
non-soluble carboxylated anionic detergent or a bile acid results
in sufficient opening of the substrate glycoprotein to permit
access to the glycans for cleavage.
[0116] In some embodiments, it is desirable to deglycosylate an
antibody at ambient temperatures (about 22.degree. C.). It was an
unexpected finding that deglycosylation of an antibody in the
mixture could still be rapid. In one embodiment, the mixture is
incubated at an ambient temperature for about 2 minutes, 3 minutes,
4 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2
hours, 5 hours, 10 hours, or a range thereof. Example 8 and FIG. 8,
show rapid deglycosylation in 15 minutes, 10 minutes, 5 minutes or
less using an immobilized PNGase F or free PNGase F (either with or
without a pre-treatment) at ambient temperature. In one embodiment,
the ambient temperature is about 18.degree. C. to about 25.degree.
C. (e.g., about 18.degree. C., 19.degree. C., 20.degree. C.,
21.degree. C., 22.degree. C., 23.degree. C., 24.degree. C.,
25.degree. C., or a range thereof). Immobilized PNGase F may be
preferably used at ambient temperatures for reasons other than
enzyme activity. However the immobilized PNGaseF may also be used
at any of the higher temperatures described herein.
[0117] Determining which glycoforms are present in an antibody
sample is useful in determining whether the antibody will have an
expected activity and/or half-life depending on the specific
glycoform that is present. The present methods are effective for
deglycosylation of glycoforms.
[0118] Deglycosylation at ambient temperatures (as shown herein
(see Example 16) is particularly desirable for in-line production
or analytical processes. Ambient temperature deglycosylation is one
particular application of using immobilized PNGase F in the methods
described herein.
[0119] In one embodiment, the deglycosylated antibody is separated
(isolated, purified) from the cleaved glycans. For example, the
separation methods can include protein A affinity chromatography,
protein G affinity chromatography, protein L affinity
chromatography, etc. Additional separation methods include use of a
universal N-glycan binding reagent, such as described in U.S.
Provisional Ser. No. 62/020,335, filed Jul. 2, 2014, the teachings
of which are incorporated herein by reference in their
entirety.
[0120] The glycoforms can be determined using any standard
techniques, such as chromatography, electrophoresis, spectrometry
or mass spectrometry. In Example 14, the glycoforms were determined
by mass spectrometry.
[0121] In one embodiment, the deglycosylated antibody or
deglycosylated fragment thereof, and/or the cleaved glycans are
labeled. Labels are art-standard and a non-exhaustive list includes
fluorescent label, radioisoptope, methyl acetyl, an antibody, or a
combination thereof. In one embodiment, cleaved glycans are labeled
with 2-aminobenzamide (2-AB). Alternatively, or in combination with
2-AB, cleaved glycans can be labeled with 2-aminobenzoic acid
(anthranilic acid; 2-AA). Advantageously, the methods described
above are compatible with direct labeling of the products without
the need for prior purification of the products from the mixture.
Illustrative examples of this embodiment are described in Examples
11 and 12. Furthermore, labeling of the glycans can be achieved
under high aqueous conditions. High aqueous conditions refers to a
solution having at least or about 60%, 65%, 70%, 75%, 80% or 85%
(vol/vol) water. In one embodiment, the glycans are labeled in the
presence of at least 80% water.
[0122] In one embodiment, methods further comprise incubating the
mixture with a labeling reagent at a temperature of about
55.degree. C. to about 75.degree. C. for about 30 minutes to about
3 hours. In one embodiment, the mixture is incubated with a
labeling reagent for about 30 minutes, 45 minutes, 1 hour, 1.5
hours, 2 hours, or 3 hours. In one embodiment the mixture is
incubated with a labeling reagent at about 55.degree. C.,
65.degree. C., 65.degree. C., 70.degree. C., or 75.degree. C. In
one embodiment, the mixture is incubated with a labeling reagent at
about 65.degree. C. for about 2 hours. Examples of labeling agents
include fluorimetric dyes such as fluorescein isothiocyanate,
tetramethylrohodamine isothiocyante, lissamine rhodamine B,
naphthaline-5-sulfonic acid, Alexa Fluor.RTM. dyes (488, 546, 555,
568, 594, 647, 350, 532), Qdot.RTM. fluorophores, Pacific Blue.TM.,
Pacific Orange.TM., and Oregon Green.RTM. (all commercially
available from Life Technologies, Carlsbad, Calif.).
[0123] Substantially all glycans can be removed from a glycoprotein
in an unbiased manner. By way of example, N-linked glycans may be
cleaved from an antibody, isolated and labeled for analysis. In
Example 16, samples were analyzed by LC/ESI-MS. The released
glycans may be labeled with or without prior isolation of the
released glycans (e.g. as illustrated in Example 16). As further
described in Example 11, substantially all N-linked glycans may be
cleaved and either isolated before labeling in a two-pot method
(illustrated in FIG. 11B), or directly labeled without isolation in
a one pot method (illustrated in FIG. 11A). The overall profile of
labeled glycans was identical whether the glycans were first
isolated or not. However, the absolute quantities of glycans was
significantly increased in the one pot method. In these
embodiments, the glycans may be cleaved from a glycosylated
antibody (such as monoclonal IgG antibody) using a glycosidase
(such as PNGase F) in the presence of DTT and SDC. Similar
deglycosylation results were also demonstrated using a dialyzable,
non-cleavable carboxylated anionic surfactant, exemplified by LS,
in a one-pot deglycosylation and labeling method (see Example 12
and FIGS. 12A-12C). This demonstrates the unexpected compatibility
of a glycosidase reaction comprising bile salts or carboxylated
surfactants with direct labeling of the glycans which minimize
antibody precipitation during glycan labeling
[0124] In one embodiment, the deglycosylated antibody retains at
least 75%, 80%, 85%, 90%, 95%, 99% or 100% epitope binding avidity
and/or affinity compared to the epitope binding avidity and/or
affinity of the corresponding glycosylated antibody tested under
identical reaction conditions. Epitope binding avidity and/or
affinity can be confirmed using art standard techniques, such as
western blot, ELISA, Biacore, etc. For example, an antibody was
completely deglycosylated and the its activity was confirmed by a
western blot to retain epitope binding (see Example 13 and 19,
FIGS. 13C and 18C). Embodiments of the methods can be applied to
any glycosylated antibody class or isotype. As shown in Examples 13
and 15, all antibodies can be successfully deglycosylated to a
substantially deglycosylated form (see also FIGS. 13A-13C, and
FIGS. 15A-15B).
[0125] In one embodiment, a protease such as trypsin can be added
to a reaction mixture containing a glycosidase such as PNGaseF
without substantial loss of activity of the glycosidase. The
protease can cleave the antibody into peptide fragments while the
glycosidase removes the glycans in a single reaction. Examples of
suitable proteases include: Trypsin, GluC, AspN, proteinase K,
Factor Xa, Enterokinase (New England Biolabs, Ipswich, Mass.),
LysC, Arg-C, (Promega, Madison, Wis.), LysN (Life Technologies,
Carlsbad, Calif.), IdeS (Genovis, Cambridge, Mass.), V-8 Protease,
Papain, Alpha-Lytic Protease, Pyroglutamate Aminopeptidas, Leucine
Aminopeptidase, Methionine Aminopeptidase, Aminopeptidase I,
Aminopeptidase A, Carboxypeptidases (A, B, G, Y), pepsin,
Cathepsins (B, C, D), .alpha.-Chymotrypsin (Sigma-Aldrich, St.
Louis, Mo.), TEV, Thrombin, IdeZ and IdeE (New England Biolabs,
Ipswich, Mass.).
[0126] In one embodiment, reactants may be lyophilized before use
without negatively affecting deglycosylation.
[0127] Also provided is a kit. In one embodiment, the kit comprises
one or more lyophilized reagents. In one embodiment, the
lyophilized reagent is selected from the group consisting of:
lyophilized glycosidase such as lyophilized PNGase F; a lyophilized
buffer, wherein the buffer comprises a bile acid, or a salt
thereof, a carboxylated anionic surfactant, or a salt thereof, or a
combination thereof; a lyophilized reducing agent; and a
combination thereof. Bile salts, dialyzable, non-cleavable
carboxylate anionic surfactants, and salts thereof are described
above. Additionally or alternatively, the kit comprises an
immobilized glycosidase such as PNGase F. Illustrative lyophilized
reagents, immobilized PNGase F, and methods of using them are
described in the Examples (see Examples 8 and 14). In one
embodiment, the kit further comprises one or more components
selected from the group consisting of a glycan labeling reagent, a
control standard, and instructions for use. In one embodiment, one
or more reagents are substantially free of animal-derived products.
In another embodiment a protease may be included in the kit, for
example trypsin where the protease may be lyophilized with the
glycosidase and/or the reaction buffer or separately.
[0128] Examples of reagents and methods for preparing a
deglycosylated protein illustrated herein by an antibody or
deglycosylated fragment thereof are not intended to be limiting but
rather illustrate embodiments of the invention. Embodiments are
applicable to any glycosylated protein not limited to antibodies
including antibody fragments. Examples of reagents and methods for
obtaining substantially all linked glycans from a glycoprotein are
the result of in vitro analysis and do not constitute nor can be
construed nor intended as naturally occurring mixtures or events
such as might occur in vivo. Embodiments of the deglycosylation
methods are compatible with the direct labeling of the glycans
cleaved from proteins such as antibodies including fragments
thereof without prior purification of the glycans from a mixture
comprising the antibody or fragments thereof and the glycan.
Alternatively, deglycosylated proteins such as antibodies including
deglycosylated fragments thereof can be further processed before
proteomic analysis by any suitable means, including dialysis, drop
dialysis, filtration with molecular sieves, or solid phase
extraction. Glycans can also be further processed, if desired, by
for example, solid phase extraction with normal or reverse phase
(C18, graphite carbon, HILIC). Also provided are methods and
reagents that facilitate sample processing at ambient temperatures.
Such methods and reagents are particularly useful for in-line
sample analyses in industrial production applications.
[0129] Below is a description of a novel modified PNGase derived
from almonds which cleaves alpha 1,3 fucose suitable for removing
N-glycans from proteins made recombinantly in plants, insects,
mollusks and helminthes. These glyco-epitopes are absent in humans
as well as other vertebrates and as such have been implicated in
allergenic and immunogenic responses in humans and may be a factor
for rapid clearance of the plant or insect derived recombinant
therapeutic protein (Bardor, et al., Current Opinion Structural
Bio. 16:576-583 (2006)). The almond derived glycosidase is found in
nature as an N-glycosylated heterodimer. In embodiments of the
invention, it is used as a single chain deglycosylated polypeptide.
It was found to be similarly effective as other glycosidases in
preparations described in the examples for PNGaseF.
TABLE-US-00001 TABLE 1 Examples of plant derived glycosidases
(PNGases) Source Genbank Accession # Prunus dulcis (Almond) P81898
Populus trichocarpa (Black cottonwood) XP_002316856, XP_002316193,
XP_002311245 Vitus vinifera (Common grape) XP_002285454, CAN82504,
XP_002283158, CBI20191, CAN82508, CBI25436, XP_002273437, CAN73340
Ricinus communis (Caster oil plant) XP_002524519, XP_002520774
Glycine max (Soybean) XP_003525973, XP_003541051, XP_003545222,
XP_003519424 Solanum lycopersicum (Tomato) NP_001234709 Medicago
truncatula (Barrel Medic) XP_003589332, XP_003616684 Arabidopsis
thaliana NP_188110, NP_568155 Thellungiella halophila BAJ33648
Sorghum bicolor XP_002454968, XP_002454967, XP_002457153,
XP_002454965, XP_002454966, XP_002441054 Oryza sativa Indica Group
EEC79173 Oryza sativa Japonica Group (Rice) NP_001042348, EAZ10975,
NP_001042351, BAB92157, EAZ10974, NP_001042356, EEE54084,
NP_001055461 Arabidopsis lyrata subsp. lyrata XP_002873210
Brachypodium distachyon XP_003565482, XP_003565481, XP_003565312,
(purple false brome) XP_00356851 Hordeum vulgare subsp. vulgare
(Barley) BAK07800, BAJ92852, BAJ99566, BAJ91834, BAJ89900, BAJ93147
Picea sitchensis (Sitka spruce) ABK25189 Zea mays (Corn)
NP_001152324, NP_001142407, ACF88207 Physcomitrella patens subsp.
patens (moss) XP_001785381, XP_001764866, XP_001760904 Selaginella
moellendorffii (spikemoss) XP_002982418, XP_002966580
[0130] In an embodiment of the invention, a PNGase was used as
described above which had at least 85%, 90%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% amino acid sequence homology to the following
sequence:
TABLE-US-00002 (SEQ ID NO: 1)
AAVPHRHRLPSHHLASLKLNASAPPTTYFEVDRPIRPPRGSVGPCST
LLLSNSFGATYGRPPVTAAYAPPSCLAGGGGGGGGASSIALAVLEWS
ADCRGRQFDRIFGVWLSGAELLRSCTAEPRATGIVWSVSRDVTRYAA
LLAEPGEIAVYLGNLVDSTYTGVYHANLTLHLYFHPAPPPPPPPQQA
DLIVPISRSLPLNDGQWFAIQNSTDVQGKRLAIPSNTYRAILEVFVS
FHSNDEFWYTNPPNEYIEANNLSNVPGNGAFREVVVKVNDDIVGAIW
PFTVIYTGGVNPLLWRPITGIGSFNLPTYDIDITPFLGKLLDGKEHD
FGFGVTNALDVWYIDANLHWLDHKSEETTGSLISYEAQGLVLNVDSG
FSGLDGQFVTSASRHISATGLVKSSYGEVTTNFYQRFSYVNSNVYSK
NGSVQVVNQTIDAKSGVFAKDALAVLLSEELHQIFPLYVYTGTSDEE
ADEYTLISHVKLGVNEKETSGGKMGFSYNSLRNAQSAHGSMKVKKNL
VVGGLGETHQAYKYVGADGCYFRDVRSKNYTVLSDHSGDSCTKRNPY
NGAKFSLRNDQSARRKLMVNNL.
[0131] To further enhance properties such as expression levels and
activity of the selected PNGase cloned in non-natural host cells,
mutations may be targeted to dibasic sites in the protein for
example, a basic amino acid could be converted to any other amino
acid particularly those observed at the same location in other
isoforms from the same organism. Selection of amino acids to
substitute for dibasic sites may be made in such a way to avoid
disrupting any secondary structures such as a helixes or .beta.
sheets. These potential structures are identified by computer
programs such as Jpred3 (Cole, et al., Nucleic Acids Research,
36(suppl 2):W197-W201 (2008)).
[0132] The selected PNGase was expressed as a single polypeptide in
non-cognate host cells and when expressed in animal cells or yeast,
baculovirus/insect cells, the recombinant PNGase itself became
modified with N-linked glycans.
[0133] For ease of purification, a host cell such as yeast capable
of secreting PNGase was selected although the PNGase could also be
purified from the lysate of the host cells. Examples of suitable
host cells for expressing the plant PNGase may include yeast such
as Kluyveromyces lactis (K. lactis) or Pichia pastoris (P.
pastoris). Where the PNGase was synthesized in the nonnative-host
cell with a high mannose N-linked glycan, it was preferably
deglycosylated using a suitable high mannose N-linked glycans
cleavage enzyme such as Endo H.
[0134] The activity of the plant PNGase for N-linked glycans
containing .alpha.1,3 fucose can be determined using any substrate
for which the N-linked glycans have been fully described without
the need for first cleaving the protein into peptides. For example,
HRP and pineapple bromelain have N-linked glycans containing
.alpha.1,3 fucose that have been fully characterized. Substantially
all the glycans on these proteins have an GlcNAc linked to a fucose
via an .alpha.1,3 glycosidic bond and also to a second GlcNAc
linked to two mannoses (bromelain) or three mannoses (HRP) where
the first mannose is also linked to a xylose. In contrast, snail
hemocyanin and human IgG has a GlcNAc linked to fucose via an
.alpha.1,6 glycosidic bond. HRP and pineapple bromelain are both
useful glycoprotein substrates for assaying for cleavage of
N-linked glycans containing .alpha.1,3 fucose by glycosidases.
These glycoproteins can be readily conjugated to a label that
produces a colored or fluorimetric signal. Examples of fluorimetric
dyes: fluorescein isothiocyanate, tetramethylrohodamine
isothiocyante, lissamine rhodamine B, naphthaline-5-sulfonic acid,
Alexa Fluor.RTM. dyes (488, 546, 555, 568, 594, 647, 350, 532)
Qdot.RTM. fluorophores, Pacific Blue.TM., Pacific Orange.TM., and
Oregon Green.RTM..
[0135] The specific activities of the PNGase dipeptide purified
from almond and the recombinant monopeptide PNGase Ar cloned from
rice and expressed in either K. lactis or P. pastoris can be as
much as 500,000 to 600,000 units/mg of protein. One unit is defined
as the amount of enzyme required to remove >95% of the
carbohydrate from 10 .mu.g of denatured RNase B in 1 hour at
37.degree. C. in a total reaction volume of 10 .mu.l.
[0136] The ability to deglycosylate N-glycans containing .alpha.1,3
fucose and to characterize these N-glycans has many uses. These
include meeting federal regulation requirements for the
characterization of therapeutic proteins. Additionally, removal of
the N-glycans can reduce unwanted side effects of administering
these therapeutic proteins. Examples of applications are provided
below.
[0137] HRP produces a colored, fluorimetric, or luminescent
derivative of the labeled molecule when incubated with a specific
substrate, allowing it to be detected and quantified. HRP is often
used in conjugates (molecules that have been joined genetically or
chemically) to determine the presence of a molecular target. For
example, an antibody conjugated to HRP may be used to detect a
small amount of a specific protein in a western blot. To avoid
cross-reactivity, antibodies can be prepared against HRP that has
been deglycosylated with PNGase using embodiments of the method
described herein.
[0138] Individualized vaccines have been developed for treatment of
lymphomas using proteins. Deglycosylation of these proteins is
preferable. A vaccine for non-Hodgkin's lymphoma has been produced
in tobacco plants where removal of the N-glycan involves .alpha.1,3
fucose (McCormick, et al., PNAS, 105:10131-10136 (2008)). PNGase Ar
can be used to analyze whether the activity of the intact
deglycosylated protein is equivalent to the glycosylated protein
but with the advantages that accrue from deglycosylation.
[0139] All references cited herein are incorporated by reference
including U.S. Provisional Application Ser. Nos. 62/005,559, filed
May 30, 2014, 62/018,074, filed Jun. 27, 2014, 62/021,936 filed
Jul. 8, 2014, 62/040,745 filed Aug. 22, 2014 and 62/080,480 filed
Nov. 17, 2014.
EXAMPLES
Example 1
Pre-Treatment of an Antibody by Mild Heat Alone Prior to PNGase F
Treatment does not Produce a Completely Deglycosylated Antibody
[0140] Preparations of a concentrated sample of IgG: Aliquots of
Anti-MBP Monoclonal Antibody (New England Biolabs, Ipswich, Mass.)
were lyophilized and resuspended in 250 .mu.L of water (yielding a
final concentration of 4 .mu.g antibody/.mu.L in 200 mM NaCl, 4 mM
EDTA, 40 mM Tris pH 7). Concentrated IgG (36 .mu.g) was diluted
with water to a total volume of 27 .mu.L. Samples were heated to
55.degree. C. for 10 minutes, and then kept at 4.degree. C. To all
samples, 3 .mu.L of detergent free buffer were added. Control
reactions did not contain any deglycosylating enzyme, whereas 1
.mu.L of Rapid PNGase F was added to each deglycosylation
experimental sample. Reactions were incubated for 1 hour or 16
hours at 37.degree. C. then kept at 4.degree. C. until further
analyzed. To determine the intact mass of the IgG by ESI-TOF, the
deglycosylated IgG samples were further treated with 10 mM DTT for
30 minutes at room temperature, and formic acid was added to 0.1%.
Samples were analyzed by reverse phase liquid chromatography (LC)
with PLRP-S (5 .mu.m particles, 1000 A pore size) and electrospray
ionization time-of-flight mass spectrometry (ESI-TOF MS) using
methods known in the art. Results are shown in FIGS. 1A-1C.
Example 2
Pre-Treatment of an Antibody by Heat Denaturation in Combination
with a Reducing Agent
[0141] prior to PNGase F treatment does not produce completely
deglycosylated antibody
[0142] A concentrated IgG sample was prepared as described in
Example 1. The monoclonal IgG (36 .mu.g) was diluted in 40 mM DTT
to a volume of 27 .mu.L. Samples were incubated at 55.degree. C.
for 10 minutes, after which 3 .mu.L of reaction buffer (500 mM
sodium phosphate pH 7.5) were added. Negative controls did not
contain any deglycosylating enzyme whereas 500 U of PNGase F
Glycerol Free was added to each deglycosylation experimental
sample. Reactions were incubated for 1 hour or 16 hours at
37.degree. C. then kept at 4.degree. C. until further analyzed. The
intact mass of the detergent-free IgG sample was analyzed by
ESI-TOF as described in Example 1. Results are shown in FIGS.
2A-2C.
Example 3
Treatment with the Commercial Reagent RapiGest.TM. Prior to PNGase
F Deglycosylation does not Produce Completely Deglycosylated
Antibody
[0143] A concentrated IgG sample was prepared as described in
Example 1. Aliquots of monoclonal IgG (64 .mu.g) were rehydrated in
0.1% RapiGest.TM. in a total volume of 20 .mu.L. Samples were
incubated at 55.degree. C. for 10 minutes, after which, 10 .mu.L of
reaction buffer. Control reactions did not contain any
deglycosylating enzyme, whereas 1 .mu.l of Rapid PNGase F were
added to each deglycosylation experimental sample. Reactions were
incubated for 1 hour at 37.degree. C. The intact mass of the
detergent-free IgG sample was analyzed by ESI-TOF as described in
Example 1. Results are shown in FIGS. 3A-3B.
Example 4
Complete Deglycosylation of an Antibody Using a Carboxylated
Surfactant in a Range of Concentrations
[0144] An anti-MBP mouse monoclonal IgG 36 .mu.g, (New England
Biolabs, Ipswich, Mass.) was mixed with varying amounts of DTT
(final concentration ranging from 0.2 to 80 mM) and varying amounts
of LS (final concentration ranging from 0.05 to 5% w/v), in a total
volume of 20 .mu.L. Control reactions did not contain any
deglycosylating enzyme. 1 .mu.l of Rapid PNGase F was added to all
other samples and were incubated for 5 minutes at 50.degree. C. An
aliquot of each sample was separated via SDS-PAGE and visualized
with SimplyBlue.TM. SafeStain (Life Technologies, Carlsbad,
Calif.).
[0145] Similar experiments were repeated using varying
concentrations of SDC (e.g., ranging between about 0.05% to about
5% w/v) (data not shown). Deglycosylation was achieved in 5 minutes
or less at about 50.degree. C., with or without a heat
pre-treatment of the glycosylated sample. Complete deglycosylation
was demonstrated in a range of about 2% to 5% SDC. Results are
shown in FIGS. 4A-4C.
Example 5
Complete Deglycosylation of an Antibody Using a Range of
Concentration of the Reducing Agent DTT
[0146] An anti-MBP mouse monoclonal IgG (36 .mu.g, New England
Biolabs) was mixed with varying amounts of DTT (final concentration
ranging from 0 to 0.1 mM) and LS (final concentration 0.5% w/v) in
a total volume of 20 .mu.L. Control reactions did not contain any
deglycosylating enzyme. 1 .mu.l Rapid PNGase F was added to all
other samples and were incubated for 5 minutes at 50.degree. C. An
aliquot of each sample was separated via SDS-PAGE and visualized
with SimplyBlue SafeStain. Results are shown in FIGS. 5A-5D.
Example 6
Complete Deglycosylation of an Antibody Using a Range of
Concentrations of Reducing Agent, Tris(2-Carboxyethyl)Phosphine
(TCEP)
[0147] An anti-MBP mouse monoclonal IgG (36 .mu.g) was mixed with
varying amounts of TCEP (final concentration ranging from 0 to 20
mM) and LS (final concentration 0.5% w/v) in a total volume of 20
.mu.L. Control reactions did not contain any deglycosylating
enzyme. 1 .mu.l Rapid PNGase F was added to all other samples and
incubated for 5 minutes at 50.degree. C. An aliquot of each sample
was separated via SDS-PAGE and visualized with SimplyBlue
SafeStain. Results are shown in FIG. 6.
Example 7
Complete Deglycosylation of an Antibody Using a Range of
Temperatures
[0148] Sixteen samples of anti-MBP mouse monoclonal IgG (36 .mu.g)
were mixed with DTT (final concentration 20 mM) and LS (final
concentration 0.5% w/v) in a total volume of 20 .mu.L.
Additionally, a second set of 16 samples of anti-MBP mouse
monoclonal IgG (36 .mu.g) were mixed with DTT (final concentration
4 mM) and SDC (final concentration 2% w/v) in buffer to form a
total of volume of 20 .mu.l. Eight LS-containing samples and 8
SDC-containing samples were treated in a two-step reaction by
pre-incubating the sample at 50.degree. C. for 5 minutes prior to
addition of 1 .mu.l Rapid PNGase F. 1 .mu.l Rapid PNGase F was
added to the remaining 8 LS-containing samples and 8 SDC-containing
samples in a one-step reaction with no pre-incubation. Controls
(separate IgG samples in the corresponding LS or SDC buffer) did
not contain any PNGase F. All samples (pretreated at 50.degree. C.;
or not) were incubated for 5 minutes in a thermocycler programmed
with an 8-point temperature gradient (from 38.degree. C. to
63.degree. C.). An aliquot of each sample was separated via
SDS-PAGE and visualized with SimplyBlue SafeStain. Results are
shown in FIGS. 7A-7B.
Example 8
Complete Deglycosylation of an Antibody at Ambient Temperature
[0149] An antibody can be completely deglycosylated at ambient
temperature in about 5, 10 and 15 minutes using free Rapid PNGase F
or immobilized forms of recombinant PNGase F. Immobilization
utilized a fusion protein of PNGase F in which PNGaseF was fused
via SNAP-Tag.RTM. (New England Biolabs, Ipswich, Mass.) which in
turn was covalently bound to benzylguanine agarose beads. Although
agarose beads were used here any suitable matrix can be used as is
known in the art.
[0150] Samples of anti-MBP mouse monoclonal IgG (100 .mu.g) were
mixed with 0.5% LS and 20 mM DTT, and incubated at 37.degree. C.
for 15 minutes. Samples were loaded to a column containing PNGase F
HS-BG Agarose Beads. Reactions were incubated at about 22.degree.
C. for 5, 10 or 15 minutes. A control reaction using soluble (free)
PNGase F was performed under the same conditions. Negative controls
("Anti-MBP -") were not treated with enzyme.
[0151] Immobilized PNGase F or soluble Rapid PNGase F treatment
resulted in a mobility shift on SDS-PAGE, corresponding to complete
deglycosylation of the heavy chain of IgG. The intact mass of the
control and deglycosylated antibody samples were analyzed by
ESI-TOF as described in Example 1. Results are shown in FIG. 8.
Example 9
Deglycosylation of an Antibody in 3 Minutes or Less to Produce a
Substantially Deglycosylated Antibody
[0152] An anti-MBP mouse monoclonal IgG (36 .mu.g) was mixed with
DTT (final concentration 20 mM) and LS (final concentration 0.5%
w/v) in a total volume of 20 .mu.L with buffer. Control reactions
did not contain any PNGase F. To all other samples, 1 .mu.l of
Rapid PNGase F was added. Samples were incubated for 1 to 5 minutes
at 50.degree. C. An aliquot of each sample was separated via
SDS-PAGE and visualized with SimplyBlue SafeStain (Life
Technologies). The results are shown in FIG. 9. Complete
deglycosylation was observed within an incubation time of three
minutes.
Example 10
Cleaving Substantially all N-Linked Glycans from an Antibody to
Produce an Unbiased N-Glycan Composition
[0153] A concentrated IgG sample was prepared as described in
Example 1. Monoclonal IgG was mixed with 4 mM DTT, in the presence
or absence of 2% SDC, in a total volume of 20 .mu.L. Samples were
heated to 55.degree. C. for 10 minutes in a pretreatment followed
by 1 .mu.l Rapid PNGase (not in controls) and a second incubation
of 4 hours at 37.degree. C. The released sugars were isolated by
solid phase extraction (SPE) with a graphite cartridge, dried, and
labeled with 2-aminobenzamide (2AB). Samples were analyzed by
LC/ESI-MS, glycan abundance was estimated by peak integration in
the fluorescent channel. Glycan structures were manually assigned
based on their retention time (fluorescent trace) and their
corresponding m/z value. The results are shown in FIGS.
10A-10B.
Example 11
Direct Fluorescent Labeling of Glycans Released from an Antibody
without Prior Glycan Purification in the Presence of a Bile
Salt
[0154] Aliquots (14 .mu.g) of Anti-MBP Monoclonal Antibody were
suspended in 2% SDC, and 4 mM DTT in a total volume of 20 .mu.L. 1
.mu.l Rapid PNGase F was added to each deglycosylation sample.
Reactions were incubated for 15 minutes at 50.degree. C. after
which liberated glycans were directly labeled with 2-aminobenzamide
(2AB) in the same reaction vessel (one pot), or were purified by
SPE and dried as described in example 12 (two pot). Fresh labeling
solution (20 .mu.L, containing 2AB, sodium cyanoborohydride and
acetic acid) was added to the dried glycans, or directly to a
deglycosylation reaction (high aqueous labeling conditions). High
aqueous conditions refers to a reaction having at least or about
60%, 65%, 70%, 75%, 80% or 85% (vol/vol) water. The labeling
reaction was incubated at 65.degree. C. for 2 hours after which,
N-glycans were processed to removed unbound label by HILIC SPE and
analyzed by LC-MS, results are shown in FIG. 11A-11B. Similar
results were obtained using a dialyzable non-cleavable carboxylated
anionic surfactant in place of the bile salt (see Example 12).
Example 12
Direct Fluorescent Labeling of Glycans Released from an Antibody
without Prior Glycan Purification in the Presence of a Carboxylated
Anionic Surfactant
[0155] Therapeutic monoclonal IgGs and IgG fusion proteins
(rituximab, cetuximab, etanercept) were deglycosylated with 20 mM
DTT, 0.5% LS and Rapid PNGaseF for about 5 minutes as described in
example 9, for complete deglycosylation. Immediately after, a
labeling mixture (containing the reagents for reductive amination)
was added and incubated for an additional 2 hours at 65.degree. C.
The labeling reaction may be performed under high aqueous
conditions (described above). High aqueous conditions include
>60% preferably at least or about 80% water. Fluorescently
labeled glycans were subjected to HILIC SPE purification, and
analyzed by HPLC-MS. Results are shown in FIGS. 12A-12C.
Example 13
Monoclonal Antibody Activity is Retained after Deglycosylation in
the Presence of Lauroylsarcosine Under Reducing Conditions
[0156] A concentrated IgG sample as described in Example 1 was
used. The monoclonal IgG (36 .mu.g) was mixed with 4 mM DTT and
0.5% LS in a total volume of 20 .mu.L. Control reactions did not
contain any deglycosylating enzyme. 1 .mu.l Rapid PNGase F was
added experimental samples and were incubated for 5 minutes at
50.degree. C. The intact mass of the IgG sample was analyzed by
ESI-TOF.
[0157] To determine the functional activity of the intact,
deglycosylated monoclonal IgG, the anti-MBP-monoclonal antibody was
analyzed on a Western blot against its corresponding antigen
(maltose binding protein, MBP), using MBP-Endo H fusion protein
(Endo Hf, New England Biolabs, Ipswich, Mass.). Western blots of
serial dilutions of Endo Hf were prepared on PVDF membrane
(Immobilon-P Millipore, Billerica, Mass.) and immunoblotted with
either deglycosylated anti-MBP mouse monoclonal antibody (treated
with PNGase F under non-reducing conditions described above) or
glycosylated anti-MBP mouse monoclonal antibody (not treated with
Rapid PNGase F). Results are shown in FIGS. 13A-13B.
Example 14
Buffer and PNGase F can be Lyophilized Separately or in Combination
and Used to Produce a Deglycosylated Antibody
[0158] Rapid PNGase F and a reaction buffer containing 0.5% LS and
20 mM DTT were lyophilized either together ("master mix") or
separately. Prior to testing the activity of the lyophilized master
mix, or the individually lyophilized buffer and Rapid PNGase F,
each component was rehydrated. Each enzyme-buffer mixture was added
to an IgG sample. A negative control reaction contained 36 .mu.g of
antibody in 50 .mu.L of fresh 0.5% LS and 20 mM DTT. A positive
control reaction was identical to the negative control but with 2
.mu.l of Rapid PNGase F. All reactions were incubated at 50.degree.
C. for 5 minutes. An aliquot of each sample was separated via
SDS-PAGE and visualized with SimplyBlue SafeStain. Similar results
can be achieved by substituting LS with a bile salt such as SDC
(e.g. 2% SDC and 8 mM DTT) in a dried master mix or dried reaction
buffer containing a bile salt as described in Examples 4 and 5.
Results are shown in FIGS. 14A-14B.
Example 15
One-Step or Two-Step Complete Deglycosylation is Non-Specific for
Antibody Isotypes
[0159] The general effect of complete deglycosylation was
demonstrated using 11 different isotypes of an antibody. The
Anti-hCD20 isotype collection featuring the variable region of the
therapeutic antibody rituximab was obtained from Invitrogen (San
Diego, Calif.). Purified isotypes consisting of human IgG1, human
IgG2, human IgG3, human IgG4, human IgM, human IgA1, human IgA2,
human IgE, murine IgG1, murine IgG2a and murine IgA were used
prepared at a final concentration of 1 mg/ml. Two deglycosylation
reactions were set up for each isotype. A negative control reaction
consisted of 16 .mu.g of the antibody mixed with a buffer giving a
final concentration of 0.5% LS, and 20 mM DTT in a total of 20
.mu.L. Reactions were identical to the negative control, but with
the addition of 1 .mu.l Rapid PNGase F. All reactions were
incubated at 50.degree. C. for 10 minutes. An aliquot of each
sample was separated via SDS-PAGE and visualized with SimplyBlue
SafeStain (See FIGS. 15A-15B). Similar results have been obtained
with a buffer comprising 2% SDC in place of LS.
[0160] A two-step complete deglycosylation reaction that is isotype
non-specific occurs. The two-step reaction includes a brief heat
pretreatment step before enzymatically cleaving the glycans. The
temperature and time of the heat pre-treatment can be varied, and
generally shorter times may be combined with higher temperatures.
Here, the pre-treatment time is as short as one or two minutes, and
the temperature can be up to about 95.degree. C. In this example,
anti-hCD20 isotype collection antibodies in a buffer containing a
bile salt or a carboxylated anionic surfactant was pre-treated at
80.degree. C. for approximately 2 minutes before adding Rapid
PNGase F and incubating the mixture at 50.degree. C. for about 10
minutes. Complete deglycosylation was confirmed by SDS-PAGE or
other art-standard techniques.
Example 16
Ambient Temperature in-Line Analysis During Antibody Production
[0161] Large scale production of antibodies, such as those used for
commercial or therapeutic antibody production, need analytical
processes that are suitable for in-line analysis. During the
production, the antibodies may need to be analyzed at multiple time
points during cell cultivation to monitor the glycan profile of the
glycosylated antibodies. In this example, a sample of the antibody
culture was aseptically withdrawn from the bioreactor at one or
more time points and loaded onto a device comprising immobilized
PNGase F. The device was a column packed with beads having
immobilized PNGase F. Alternatively a microfluidic device might be
used with PNGase F immobilized on a surface that comes into contact
with the sample. The device can be either pre-loaded or
subsequently loaded with a buffer as described herein. 0.5% LS, 20
mM DTT, or 2% SDC, 20 mM DTT was used in a buffer. The sample in
the device was allowed to react at ambient temperature for 15
minutes or less (e.g., 10 minutes, 5 minutes, or less) before
eluting and analyzing the eluent for aglycosylation of the antibody
and/or glycan composition. FIG. 8 demonstrated that immobilized
PNGaseF was as effective as soluble PNGaseF in the same buffer.
Example 17
PNGase F/IgG Ratios for Complete Deglycosylation with Reducing
Agents, in Five Minutes at 50.degree. C. (See FIGS. 16A-16B)
[0162] To determine an effective ratio of PNGaseF to antibody using
a one-step reaction for five minutes at 50.degree. C., samples of
anti-MBP mouse monoclonal IgG (36 .mu.g) were combined with DTT (20
mM), with or without LS (final concentration 0.5% w/v) in 10 .mu.l
of buffer. Controls did not contain any deglycosylating enzyme.
Two-fold dilutions of stock recombinant PNGase F (8000 units/.mu.l)
were added to samples which were incubated for 5 minutes at
50.degree. C. (one-step, simultaneous mild heating and
deglycosylation). Aliquots from each tube were mixed with sample
buffer, heated, and loaded on a Novex.RTM. 10-20% Tris Glycine gel
(Life Technologies, Carlsbad, Calif.). After electrophoresis, the
gel was stained with SimplyBlue SafeStain to visualize bands (see
FIGS. 16A-16B).
[0163] Ratios for complete deglycosylation were defined as the
minimal amount of units of PNGase F necessary to completely
deglycosylate 1 .mu.g of IgG in 10 .mu.l of reaction volume at
50.degree. C. for 5 minutes.
Example 18
PNGase F/IgG Ratios for Complete Deglycosylation without Reducing
Agents, in a Two-Step Reaction
[0164] A two-step reaction was utilized to determine an effective
ratio of PNGaseF to antibody in the absence of reducing agent.
Samples of anti-MBP mouse monoclonal IgG were combined with or
without LS (final concentration 0.5% w/v) in 10 .mu.l of buffer.
Samples were incubated at 75.degree. C. for five minutes. Controls
did not contain any deglycosylating enzyme. Two-fold dilutions of
stock recombinant PNGase F (8000 units/up were added to samples
which were incubated for 5 minutes at 50.degree. C. Aliquots from
each tube were mixed with sample buffer, heated, and loaded on a
Novex 10-20% Tris Glycine gel. After electrophoresis, the gel was
stained with SimplyBlue SafeStain to visualize bands (see FIGS.
17A-17B).
[0165] Ratios for complete deglycosylation under non-reducing
conditions were defined as the minimal amount of units of PNGase F
necessary to completely deglycosylate 1 .mu.g of IgG in 10 .mu.l of
reaction volume at 50.degree. C. for 5 minutes. The results in FIG.
16A-16B and FIG. 17A-17B show that the presence of a dialyzable,
non-cleavable carboxylation anionic surfactant significantly
reduced the amount of PNGase F required for complete
deglycosylation in a two-step reaction by >20 fold, >50 fold,
>75 fold or >90 fold PNGase F.
Example 19
Complete Deglycosylation Under Non-Reducing Conditions Preserves
Antibody Binding Function
[0166] Retaining functional structure of a tetrameric antibody
after deglycosylation was established as follows. Anti-MBP mouse
monoclonal IgG (36 .mu.g), rituximab (40 .mu.g, Genentech, San
Francisco, Calif.), and etanercept (25 .mu.g, Amgen, Thousand Oaks,
Calif.) were mixed with LS (final concentration 0.5% w/v) in 20
.mu.l (final volume) of buffer. Reactions were pre-incubated under
various conditions: 80.degree. C. for 2 minutes, 75.degree. C. for
5 minutes, 70.degree. C. for 10 minutes, 65.degree. C. for 10
minutes, 60.degree. C. for 10 minutes, or 55.degree. C. for 15
minutes. Controls did not contain any deglycosylating enzyme, to
all other samples, 1 .mu.l of Rapid PNGase F were added. Reactions
were immediately incubated for 10 minutes at 50.degree. C. Aliquots
were mixed with sample buffer, heated, and loaded on a Novex 10-20%
Tris Glycine gel. Non-reducing gels were also run to demonstrate
structural integrity of dimers and tetramers: aliquots were mixed
with sample buffer in the absence of DTT, heated, and gels were run
as described above. After electrophoresis, gels were stained with
SimplyBlue SafeStain to visualize bands.
[0167] To determine the functional binding activity of the intact,
deglycosylated monoclonal IgG, the anti-MBP-monoclonal antibody was
analyzed on a Western blot against its corresponding antigen
(maltose binding protein, MBP), using MBP-Endo H fusion protein,
Endo Hf. Western blots of serial dilutions of Endo Hf were prepared
on PVDF membrane (Immobilon-P Millipore) and immunoblotted with
either deglycosylated anti-MBP Mouse monoclonal antibody (treated
with PNGase F under non-reducing conditions described above) or
glycosylated anti-MBP mouse monoclonal antibody (not treated with
PNGase F). Controls with fresh anti-MBP were also included. Results
are shown in FIGS. 18A-18C.
Example 20
Complete Deglycosylation of Antibody in a One-Step or Two-Step
Reaction with PNGase F Treatment in the Presence of Two Different
Dialyzable, Non-Cleavable Carboxylated Surfactants
[0168] Aliquots (36 .mu.g) of anti-MBP monoclonal antibody were
suspended in a buffer containing either 2.5% LS or laurate (LAU),
with or without 20 mM DTT, in a 20 .mu.L volume of buffer. Control
reactions did not contain PNGase F. For the one-step reactions, 1
.mu.L of Rapid PNGase F was added and samples were incubated at
50.degree. C. for 5 minutes. For the two-step reactions, samples
were pre-heated at 72.degree. C. for 5 minutes, after cooling 1
.mu.L of Rapid PNGase F was added and samples were incubated at
50.degree. C. for 5 minutes. Aliquots (5.4 .mu.g IgG) of each
sample were separated via SDS-PAGE and visualized with SimplyBlue
SafeStain (see FIG. 19)
Example 21
Simultaneous Digestion of a Protein with PNGase F and Trypsin
[0169] Simultaneous PNGase F/Trypsin digestion was performed to
facilitate peptide mapping (and peptide coverage) of monoclonal
antibodies. This single-step method is advantageous for structural
analysis, such as glycan occupancy determination and compares
favorably with recombinant PNGase F digestion followed by Trypsin
digestion that requires several hours to complete.
[0170] To determine the deglycosylation conditions that were
favorable for protease digestion, 25 .mu.g of anti-MBP mouse
monoclonal IgG were resuspended in a total of 50 .mu.l of buffer.
The antibody was incubated at 95.degree. C. for 5 minutes, and then
6 .mu.l of Rapid PNGase F and 250 ng of Trypsin-ultra.TM., Mass
Spectrometry Grade (New England Biolabs, Ipswich, Mass.) at a ratio
1:100 enzyme:substrate. The samples were incubated at 37.degree. C.
for 30 minutes to 3 hours, or 50.degree. C. for 5 minutes to 30
minutes.
[0171] One microliter of digested sample (simultaneous PNGase
F/trypsin digestion) was injected onto a C18 analytical column and
separated using a 60 minute linear gradient. Multiple-charged
peptide ions were automatically chosen and fragmented by both CID
and ETD. The MS and MS/MS fragmentation data were analyzed,
considering theoretical peptides with a maximum of two missed
cleavages. The analysis allowed variable modifications of
asparagine to account for the conversion to aspartic acid that
occurs after the glycan removal by PNGase F. While the data in
FIGS. 20A-20B shows results for PNGase F/Trypsin in the absence of
LS, the present example provides a method for doing the reaction in
the presence of a carboxylated anionic surfactant.
Example 22
Complete Deglycosylation of an Antibody Containing Both N- and
O-Glycans in a Two-Step Reaction, Involving Pre-Treatment with Heat
in Combination with a Bile Salt or Non-Cleavable Dialyzable
Carboxylated Surfactant Prior to Treatment with a Combination of
Glycosidases
[0172] For antibodies containing O-glycans, it may be desirable to
optionally remove all O-glycans as well as N-glycans. Enzyme
combinations containing an O-glycosidase
(Endo-alpha-N-acetylgalactosaminidase) and 3 exoglycosidases
(neuraminidase, Beta 1-4 galactosidase and Beta N-Acetyl
glucosaminadase (New England Biolabs, Ipswich, Mass.)) for cleaving
O-glycans and N-glycosidases (PNGase F) for cleaving N-glycans were
used together to completely deglycosylate a protein having O- and
N-glycans.
[0173] A therapeutic monoclonal fusion protein (etanercept) was
mixed with 0.5% LS and 20 mM DTT. Samples were pre-incubated at
80.degree. C. for 2 minutes. 5 .mu.l of Protein Deglycosylation Mix
containing all the glycosidases described above (New England
Biolabs, Ipswich, Mass.) were added to samples. Control reactions
included no enzyme; or SDS in place of LS, reactions were incubated
for 30 minutes at 37.degree. C. An aliquot of each sample was
separated via SDS-PAGE and visualized with SimplyBlue
SafeStain.
[0174] Samples treated with Protein Deglycosylation Mix show an
increase in mobility compared with controls corresponding to
complete removal of both O-glycans and N-glycans. This example
underscores problems with the use of SDS which is detrimental to
complete deglycosylation by the enzyme mix.
[0175] For all patents, applications, or other references cited
herein, such as non-patent literature and reference sequence
information (such as database or accession numbers) are
incorporated by reference in its entirety for all purposes as well
as for the proposition that is recited. Where any conflict exits
between a document incorporated by reference and the present
application, this application will control.
[0176] As will be recognized by the person having ordinary skill in
the art following the teachings of the specification, the foregoing
aspects can be claimed by Applicant in any combination or
permutation. To the extent one or more elements and/or features is
later discovered to be described in one or more references known to
the person having ordinary skill in the art, they may be excluded
from the claims by, inter alio, a negative proviso or disclaimer of
the one or more elements and/or features. Headings used in this
application are for convenience only and do not affect the
interpretation of this application or claims.
Sequence CWU 1
1
11586PRTArtificial SequenceSynthetic construct 1Ala Ala Val Pro His
Arg His Arg Leu Pro Ser His His Leu Ala Ser 1 5 10 15 Leu Lys Leu
Asn Ala Ser Ala Pro Pro Thr Thr Tyr Phe Glu Val Asp 20 25 30 Arg
Pro Ile Arg Pro Pro Arg Gly Ser Val Gly Pro Cys Ser Thr Leu 35 40
45 Leu Leu Ser Asn Ser Phe Gly Ala Thr Tyr Gly Arg Pro Pro Val Thr
50 55 60 Ala Ala Tyr Ala Pro Pro Ser Cys Leu Ala Gly Gly Gly Gly
Gly Gly 65 70 75 80 Gly Gly Ala Ser Ser Ile Ala Leu Ala Val Leu Glu
Trp Ser Ala Asp 85 90 95 Cys Arg Gly Arg Gln Phe Asp Arg Ile Phe
Gly Val Trp Leu Ser Gly 100 105 110 Ala Glu Leu Leu Arg Ser Cys Thr
Ala Glu Pro Arg Ala Thr Gly Ile 115 120 125 Val Trp Ser Val Ser Arg
Asp Val Thr Arg Tyr Ala Ala Leu Leu Ala 130 135 140 Glu Pro Gly Glu
Ile Ala Val Tyr Leu Gly Asn Leu Val Asp Ser Thr 145 150 155 160 Tyr
Thr Gly Val Tyr His Ala Asn Leu Thr Leu His Leu Tyr Phe His 165 170
175 Pro Ala Pro Pro Pro Pro Pro Pro Pro Gln Gln Ala Asp Leu Ile Val
180 185 190 Pro Ile Ser Arg Ser Leu Pro Leu Asn Asp Gly Gln Trp Phe
Ala Ile 195 200 205 Gln Asn Ser Thr Asp Val Gln Gly Lys Arg Leu Ala
Ile Pro Ser Asn 210 215 220 Thr Tyr Arg Ala Ile Leu Glu Val Phe Val
Ser Phe His Ser Asn Asp 225 230 235 240 Glu Phe Trp Tyr Thr Asn Pro
Pro Asn Glu Tyr Ile Glu Ala Asn Asn 245 250 255 Leu Ser Asn Val Pro
Gly Asn Gly Ala Phe Arg Glu Val Val Val Lys 260 265 270 Val Asn Asp
Asp Ile Val Gly Ala Ile Trp Pro Phe Thr Val Ile Tyr 275 280 285 Thr
Gly Gly Val Asn Pro Leu Leu Trp Arg Pro Ile Thr Gly Ile Gly 290 295
300 Ser Phe Asn Leu Pro Thr Tyr Asp Ile Asp Ile Thr Pro Phe Leu Gly
305 310 315 320 Lys Leu Leu Asp Gly Lys Glu His Asp Phe Gly Phe Gly
Val Thr Asn 325 330 335 Ala Leu Asp Val Trp Tyr Ile Asp Ala Asn Leu
His Trp Leu Asp His 340 345 350 Lys Ser Glu Glu Thr Thr Gly Ser Leu
Ile Ser Tyr Glu Ala Gln Gly 355 360 365 Leu Val Leu Asn Val Asp Ser
Gly Phe Ser Gly Leu Asp Gly Gln Phe 370 375 380 Val Thr Ser Ala Ser
Arg His Ile Ser Ala Thr Gly Leu Val Lys Ser 385 390 395 400 Ser Tyr
Gly Glu Val Thr Thr Asn Phe Tyr Gln Arg Phe Ser Tyr Val 405 410 415
Asn Ser Asn Val Tyr Ser Lys Asn Gly Ser Val Gln Val Val Asn Gln 420
425 430 Thr Ile Asp Ala Lys Ser Gly Val Phe Ala Lys Asp Ala Leu Ala
Val 435 440 445 Leu Leu Ser Glu Glu Leu His Gln Ile Phe Pro Leu Tyr
Val Tyr Thr 450 455 460 Gly Thr Ser Asp Glu Glu Ala Asp Glu Tyr Thr
Leu Ile Ser His Val 465 470 475 480 Lys Leu Gly Val Asn Glu Lys Glu
Thr Ser Gly Gly Lys Met Gly Phe 485 490 495 Ser Tyr Asn Ser Leu Arg
Asn Ala Gln Ser Ala His Gly Ser Met Lys 500 505 510 Val Lys Lys Asn
Leu Val Val Gly Gly Leu Gly Glu Thr His Gln Ala 515 520 525 Tyr Lys
Tyr Val Gly Ala Asp Gly Cys Tyr Phe Arg Asp Val Arg Ser 530 535 540
Lys Asn Tyr Thr Val Leu Ser Asp His Ser Gly Asp Ser Cys Thr Lys 545
550 555 560 Arg Asn Pro Tyr Asn Gly Ala Lys Phe Ser Leu Arg Asn Asp
Gln Ser 565 570 575 Ala Arg Arg Lys Leu Met Val Asn Asn Leu 580
585
* * * * *