Note: Descriptions are shown in the official language in which they were submitted.
CA 02758323 2011-11-08
Use of Amino-Oxy Functional Groups in the Preparation of Vaccines
This application claims benefit of priority of U.S. Provisional Application
Nos. 60/539,573 filed January 29, 2004, and 60/589,019, filed July 20, 2004.
Background of the invention
The present invention relates to a process of covalently linking proteins and
polysaccharides to form conjugate vaccines comprising a reaction between
carbonyl-containing groups and amino-oxy functional groups.
In the process of vaccination, medical science uses the body's innate ability
to protect itself against invading agents by immunizing the body with antigens
that
will not cause the disease but will stimulate the formation of antibodies that
will
protect against the disease. For example, dead organisms are injected to
protect
against bacterial diseases such as typhoid fever and whooping cough, toxoids
are
injected to protect against tetanus and diptheria, and attenuated organisms
are
injected to protect against viral diseases such as poliomyelitis and measles.
It is not always possible, however, to stimulate antibody formation merely
by injecting the foreign agent. The vaccine preparation must be immunogenic,
that is, it must be able to induce an immune response. Certain agents such as
tetanus toxoid can innately trigger the immune response, and may be
administered in vaccines without modification. Other important agents are not
immunogenic, however, and must be converted into immunogenic molecules or
constructs before they can induce the immune response.
The immune response is a complex series of reactions that can generally
be described as follows: (1) the antigen enters the body and encounters
antigen-
presenting cells that process the antigen and retain fragments of the antigen
on
their surfaces; (2) the antigen fragments retained on the antigen-presenting
cells
are recognized by T cells that provide help to B cells; and (3) the B cells
are
CA 02758323 2011-11-08
stimulated to proliferate and divide into antibody forming cells that secrete
antibody against the antigen.
Most antigens only elicit antibodies with assistance from the T cells and,
hence, are known as T-dependent (TD). Examples of such T-dependent antigens
are tetanus and diphtheria toxoids.
Some antigens, such as polysaccharides, cannot be properly processed by
antigen presenting cells and are not recognized by T cells. These antigens do
not
require T cell assistance to elicit antibody formation but can activate B
cells
directly and, hence, are known as T-independent antigens (TI). Such T-
independent antigens include H. influenzae type b polyribosyl-ribitol-
phosphate
(PRP) and pneumococcal capsular polysaccharides.
There are other differences between T-independent and T-dependent
antigens.
A) T-dependent antigens, but not T-independent antigens, can prime an
immune response so that a memory response results on secondary challenge
with the same antigen.
B) The affinity of the antibody for antigen increases with time after
immunization with T-dependent, but not T-independent antigens.
C) T-dependent antigens stimulate an immature or neonatal immune
system more effectively than T-independent antigens.
D) T-dependent antigens usually stimulate IgM, IgG1, IgG2a, and IgE
antibodies, while 1-independent antigens stimulate IgM, IgG1, IgG2b, and IgG3
antibodies.
T-dependent antigens can stimulate primary and secondary responses,
which are long-lived in both adult and in neonatal immune systems, but must
frequently be administered with adjuvants (substances that enhance the immune
2
CA 02758323 2011-11-08
response). Very small proteins, such as peptides, are rarely immunogenic, even
when administered with adjuvants.
T-independent antigens, such as polysaccharides, are able to stimulate
immune responses in the absence of adjuvants, but cannot stimulate high level
or
prolonged antibody responses. They are also unable to stimulate an immature or
B dell defective immune system (Mond, J. J., Immunological Reviews, 64:99
= (1982); Mosier, D. E. et al., J. Immunol., 119:1874 (1977)).
For T-independent antigens, it is desirable to provide protective immunity
against such antigens to children, especially against capsular polysaccharides
found on organisms such as H. influenzae, S. pneumoniae, and Neisseria
meningiditis.
One approach to enhance the immune response to T-independent antigens
involves conjugating polysaccharides such as H. influenzae PRP (Cruse, J. M.,
Lewis, R. E. Jr., eds., Conjugate Vaccines in Contributions to Microbiology
and
Immunology, Vol. 10, (.1989)), or oligosaccharide antigens (Anderson, P. W. et
al.,
J. Immunol., 142:2464, (1989)) to a T-dependent antigen such as tetanus or
diphtheria toxoid. Recruitment of T cell help in this way has been shown to
provide enhanced immunity to many infants that have been immunized.
Protein-polysaccharide conjugate vaccines stimulate an anti-
polysaccharide antibody response in infants who are otherwise unable to
respond
to the polysaccharide alone.
Conjugation of a protein and a polysaccharide may provide other
advantageous results. For example, Applicant has found that a
protein/polysaccharide conjugate may enhance the antibody response not only to
the polysaccharide component, but also to the protein component. This effect
is
described, for example, in U.S. Patent No. 5,955,079. This effect also is
described in A. Lees, et at., Vaccine, 12(13):1160 (1994).
3
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Techniques have been developed to facilitate coupling of proteins and
polysaccharides. See, for example, Dick, W. E. et al., "Glyconjugates of
Bacterial
Carbohydrate Antigens: A Survey and Consideration of Design and Preparation
Factors," Conjugate Vaccines (Eds. Cruse, et al.), p. 48 (1989). Many
techniques
for activation of carbohydrates, however, are not suitable for use in aqueous
media because the activating or functional reagents are not stable in water.
For
example, N,N'-carbonyldiimidazole, as described in Marburg et al., U.S. Patent
No. 4,695,624, must be used in organic media..
Homofunctional and heterofunctional vinylsulfone reagents have been used
to activate polysaccharides. The activated polysaccharides are reacted with a
protein, peptide, or hapten, under appropriate reaction conditions, to produce
the
conjugate. This is described in more detail in U.S. Patent No. 6,309,646.
Another
method for producing conjugate vaccines comprises mixing a uronium 'salt
reagent with a soluble first moiety, such as a polysaccharide or carbohydrate,
and
combining therewith a second moiety, such as a protein, peptide, or
carbohydrate,
to form the conjugate vaccine. This method is described in U.S. Patent No.
6,299,881.
Most carbohydrates must be activated before conjugation, and cyanogen
bromide (CNBr) is frequently the activating agent of choice. See, e.g., Chu et
al.,
Inf. & Imm., 40:245 (1983). The first licensed conjugate vaccine was prepared
with CNBr to activate HI B PRP, which was then derivatized with adipic
dihydrazide and coupled to tetanus toxoid using a water-soluble carbodiimide.
The use of 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate, also
called "CDAP," has been described for use in aqueous media to activate
polysaccharides. These activated polysaccharides may be directly or indirectly
coupled to proteins. The use of CDAP is described in, for example, U.S. Patent
No. 5,849,301 and in Lees, et at., "Activation of Soluble Polysaccharides with
1-
4
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Cyano-4-Dimethylamino Pyridinium Tetrafluoroborate For Use in Protein-
Polysaccharide Conjugate Vaccines and Immunological Reagents," Vaccine,
14(3):190 (1996).
To briefly summarize the CNBr-activation method, CNBr is reacted with the
carbohydrate at a high pH, typically a pH of 10 to 12. At this high pH,
cyanate
esters are formed with the hydroxyl groups of the carbohydrate. These, in
turn,
are reacted with a bifunctional reagent, commonly a diamine or a dihydrazide.
These derivatized carbohydrates may then be conjugated via the bifunctional
group. In certain limited cases, the cyanate esters may also be directly
reacted to
protein.
The high pH is necessary to ionize the hydroxyl group because the reaction
requires the nucleophilic attack of the hydroxyl ion on the cyanate ion (CN-).
As a
result, CNBr produces many side reactions, some of which add neo-antigens to
the polysaccharides. Wilcheck, M. et al., Affinity Chromatography. Meth.
Enzymol., 104:3-55 (1984). More importantly, many carbohydrates or moieties
such as Hib, PRP, and capsular polysaccharides from and pneumococcal type 6
and Neisseria meningitis A can be hydrolyzed or damaged by the high pH
necessary to perform the cyanogen bromide activation.
Another problem with the CNBr activation method is that the cyanate ester
formed is unstable at high pH and rapidly hydrolyzes, reducing the yield of
derivatized carbohydrate and, hence, the overall yield of carbohydrate
conjugated
to protein. Many other nonproductive side reactions, such as those producing
carbamates and linear imidocarbonates, are promoted by the high pH. This
effect
is described in Kohn et al., Anal. Biochem, 115:375 (1981). Moreover, CNBr
itself
is highly unstable and spontaneously hydrolyzes at high pH, further reducing
the
overall yield.
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CA 02758323 2011-11-08
Protein-polysaccharide conjugate vaccines may also be formed via
reductive amination. In this method, aldehydes on the polysaccharide are
reacted
with amines on the protein to form a reversible Schiff base. The Schiff base
is
subsequently reduced to form a stable linkage between the amine and the
aldehyde. This process is beset by a number of problems. The formation of the
Schiff base is slow and inefficient, and the overall reaction is further
impeded by
the large size of the two components (i.e., the polysaccharide and protein),
which
need to be in close proximity with each other in order to react. In order to
overcome this problem, the polysaccharide is often broken down into
oligosaccharides prior to coupling.
The use of dimethylsulfoxide (DMSO) promotes the formation of the Schiff
base, but this organic solvent can harm the protein. Sometimes a multistep
protocol is used, in which a spacer group (e.g., hexane dia mine or adipic
dihydrazide) is added to the polysaccharide via reductive amination, and this
spacer is subsequently ligated to the protein. Using a high concentration of
the
spacer helps to force the reaction and increase the yield. Elevated
temperatures
and prolonged reaction times are also used to promote the reaction. However,
these can also be detrimental to the protein and the polysaccharide.
Furthermore,
as amines must be deprotonated to react with aldehydes, the Schiff base
formation usually requires the use of alkaline solutions, i.e., solutions at a
pH 8.
Prolonged reactions at elevated temperature and pH can be detrimental to both
the protein and the polysaccharide. Furthermore, the reductive step, which
usually involves the use of cyanoborohydride or pyridine-boranes, can be
inefficient and deleterious to the protein. Also, these reagents can be
hazardous
to work with in large quantities. A further limitation of the reductive
amination
method is the highly random nature of the linkage sites between the protein
and
the polysaccharide.
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CA 02758323 2011-11-08
Accordingly, there remains a need in the art for an efficient and effective
process for preparing conjugate vaccines_
Summary of the Invention
One embodiment includes a process for preparing a conjugate vaccine,
comprising:
(a) reacting a first moiety containing at least one carbonyl-containing group
with at least one amino-oxy reagent to form at least one pendent functional
group
on the first moiety, wherein the first moiety is chosen from polysaccharides,
oligosaccharides, carbohydrates, and carbohydrate-containing molecules;
(b) reacting the first moiety containing at least one pendent functional group
with a second moiety to form a composition comprising a conjugate, wherein the
second moiety is chosen from proteins, peptides, and haptens; and
(c) combining the conjugate with a pharmaceutically acceptable delivery
vehicle to form a conjugate vaccine. =
Another embodiment includes a process for preparing a conjugate vaccine,
comprising:
(a) reacting a first moiety containing at least one pendent amino-oxy group
with a second moiety to form a composition comprising a conjugate,
(b) wherein the first moiety is chosen from polysaccharides,
oligosaccharides, carbohydrates, and carbohydrate-containing molecules, and
the
second moiety is chosen from proteins, peptides, and haptens; and
(c) combining the conjugate with a pharmaceutically acceptable delivery
vehicle to form a conjugate vaccine.
Another embodiment includes a process for preparing a conjugate vaccine,
comprising:
(a) reacting a first moiety chosen from polysaccharides, oligosaccharides,
carbohydrates, and carbohydrate-containing molecules, with
7
=
CA 02758323 2011-11-08
(b) a second moiety reacted with at least one amino-oxy reagent, wherein
the second moiety is chosen from proteins, peptides, and haptens, to form a
composition comprising a conjugate; and
(c) combining the conjugate with a pharmaceutically acceptable delivery
vehicle to form a conjugate vaccine.
Yet another embodiment includes a process for preparing a conjugate
vaccine, comprising:
(a) reacting a first moiety with a second moiety containing at least one
pendent amino-oxy group to form a composition comprising a conjugate,
wherein the first moiety is chosen from polysaccharides, oligosaccharides,
carbohydrates, and carbohydrate-containing molecules, and the second moiety is
chosen from proteins, peptides, and haptens; and
(b) combining the conjugate with a pharmaceutically acceptable delivery
vehicle to form a conjugate vaccine_
A further embodiment includes a process for preparing a conjugate
vaccine, comprising:
(a) providing a first moiety chosen from polysaccharides, oligosaccharides,
carbohydrates, and carbohydrate-containing molecules;
(b) providing a second moiety chosen from N-terminal 1,2-aminoalcohols-
which can be oxidized to contain at least one aldehyde group;
(c) functionalizing said second moiety with at least one amino-oxy reagent;
(d) reacting said first moiety with the functionalized second moiety to form
a composition comprising a conjugate; and
(e) combining the conjugate with a pharmaceutically acceptable delivery
vehicle to form a conjugate vaccine.
A further embodiment includes a process for preparing a conjugate
vaccine, comprising:
CA 02758323 2011-11-08
(a) reacting a first moiety containing at least one pendent amino-oxy
group, wherein the first moiety is chosen from polysaccharides,
oligosaccharides,
carbohydrates, and carbohydrate-containing molecules;
(b) reacting the first moiety with a second moiety to form a composition
comprising a conjugate, wherein the second moiety is chosen from glycoproteins
containing at least one carbonyl group; and
(c) combining the conjugate with a pharmaceutically acceptable delivery
vehicle to form a composition comprising an conjugate vaccine.
Still another embodiment includes a process for preparing a conjugate
vaccine, comprising:
(a) reacting a first moiety chosen from polysaccharides, oligosaccharides,
carbohydrates, and carbohydrate-containing molecules with a second moiety
chosen from proteins, peptides, and haptens to form a composition comprising a
conjugate,
(b) wherein the first moiety contains at least one reducing end derivatized
with an amino-oxy reagent, and
(c) combining the conjugate with a pharmaceutically acceptable delivery
vehicle to form a conjugate vaccine.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an SDS-page chromatogram showing a high degree of protein-
polysaccharide conjugation.
Figure 2 is an SDS-page chromatogram showing BSA-polysaccharide
conjugation.
Figure 3 shows the results of a resorcinol assay for protein and
carbohydrate of fractions eluting from an S-400HR TM (Pharmacia) gel
filtration
column.
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Figure 4 shows an SDS-PAGE chromatogram indicating the occurrence of
protein-polysaccharide conjugation.
Figures 5A-5D indicate the presence of higher molecular weight conjugates
of fractions eluting from an S-400HRTM (Pharmacia) gel filtration column.
Figure 6 is an SOS-PAGE chromatogram showing the presence of
conjugate fractions.
Figure 7 is a chromatogram comparing a conjugate with its unconjugated
components.
Figure 8 illustrates the results of an opsonic assay.
Definitions
Amino-oxy reagent refers to a reagent with the structure NH2-0-R. R can
be any group capable of bonding to the amino-oxy nitrogen. According to one
aspect of the disclosure, R is a functional group, e.g., an amine, thiol, or
other
chemical group facilitating coupling to, e.g., a protein.
Conjugate means to chemically link or join together.
Functionalize means to add at least one group that facilitates further
reaction. Typical functional groups include amino-oxy, thiol, maleimide,
halogen,
haloacyl, aldehyde, hydrazide, hydrazine, and carboxyl. Other functional
groups
- would be well known to the person of ordinary skill in the art and can be
found
discussed in Hermanson, Bioconjugation Techniques.
Hapten refers to a small molecule such as a chemical entity that by itself is
not able to elict an antibody response, but can elicit an antibody response
once it
is coupled to a carrier.
Homofunctional, when discussing an amino-oxy reagent, refers to a
reagent that has at least two amino-oxy functional groups. The homofunctional
agent may be homobifunctional or homomultifunctional, i.e., having two, three,
four or more amino-oxy functional groups.
CA 02758323 2011-11-08
Heterofunctional, when discussing an amino-oxy reagent, refers to a
reagent that has at least one amino-oxy functional group and at least one
other
non-amino-oxy functional group. The heterofunctional agent may be
heterobifunctional or heteromultifunctional, i.e., having two, three, four or
more
amino-oxy functional groups. It may also have more than one other non-amino-
oxy functional group, such as two, three, or four or more, of either the same
type
or different types.
Moiety refers to oneof the parts of a conjugate.
Pendent functional group refers to a functional group that is exists on or
is exposed on a molecule.
Spacer refers to an additional molecule that is used to indirectly couple the
first moiety to the second moiety.
Detailed Description of the Invention
A. Strategy for Conjugation
The present invention provides an alternative to prior art processes for
preparing conjugate vaccines. Specifically, the invention provides for new
methods of conjugating a first moiety to a second moiety, where the first
moiety is
chosen from polysaccharides, oligosaccharides, carbohydrates, and
carbohydrate-containing molecules and the second moiety is chosen from
proteins, peptides, and haptens, and the conjugation proceeds using at least
one
amino-oxy functional group.
There are a number of ways of reacting the first and second moiety within
the scope of the invention and each of these methods rely on using at least
one
amino-oxy group in the process.
At least one amino-oxy reagent with one amino-oxy group may be reacted
with the first moiety to form a composition with at least one non-amino-oxy
pendent functional group.
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CA 02758323 2011-11-08
At least one amino-oxy reagent with more than one amino-oxy group may
be reacted with the first moiety to form a composition with at least one amino-
oxy
pendent functional group. In this embodiment, there may optionally
additionally
be present at least one non-amino-oxy pendent functional group.
At least one amino-oxy reagent with one amino-oxy group may be reacted
with the second moiety to form a composition with at least one non-amino-oxy
pendent functional group.
At least one amino-oxy reagent with more than one amino-oxy group may
be reacted with the second moiety to form a composition with at least one
amino-
oxy pendent functional group. In this embodiment, there may optionally
additionally be present at least one non-amino-oxy pendent functional group.
Thus, in this invention, at least one of the first moiety and the second
moiety will be reacted with an amino-oxy reagent, and will result in a
composition
with at least one pendent functional group (at least one of an amino-oxy or
non-
amino-oxy pendent functional group). It is possible to functionalize both the
first
moiety and the second moiety according to any combination of strategies 1 or 2
(first moiety) and 3 or 4 (second moiety), as set forth immediately above. In
another embodiment, either the first moiety or the second moiety may be
functionalized.
The first moiety and the second moiety may then be conjugated together.
This conjugation may proceed directly, by linking the pendent functional group
on
the first moiety directly to the second moiety. Alternatively, this
conjugation may
proceed indirectly, by linking the pendent functional group on the first
moiety to an
additional agent called a spacer, which is then linked to the second moiety.
Certainly, a similar strategy may be followed with a pendent functional group
on
the second moiety, simply by reversing the positions of the first and second
moiety. =
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B. The First Moiety: Polysaccharide, Oligosaccharide,
Carbohydrate,
and Carbohydrate-Containing Molecules
As used herein, "carbohydrate" means any soluble monosaccharide,
disaccharide, oligosaccharide, or polysaccharide. Examples of suitable
polysaccharides for use in the process of the invention include bacterial,
fungal,
and viral polysaccharides. Soluble polysaccharides (i.e., polysaccharides
present
in solution), such as water-soluble polysaccharides, are suitable for use in
accordance with the present invention. Specific examples of suitable
polysaccharides include Salmonella typhi Vi antigen; Neisseria meningiditis
polysaccharide C; and Pneumococcal polysaccharides, such as Pneumococcal
polysaccharide type 14
According to certain embodiments of the present invention, the
carbohydrate is naturally occurring, a semisynthetic, or a totally synthetic
large
molecular weight molecule. According to one embodiment, at least one
carbohydrate-containing moiety is selected from E. coli polysaccharides, S.
aureus polysaccharides, dextran, carboxymethyl cellulose, agarose,
Pneumococcal polysaccharides (Pn), Ficoll, Cryptococcus neoforrnans,
Haemophilus influenzae PRP, P. aeroginosa, S. pneumoniae, Group A and B
streptococcus, N. meningitidis, and combinations thereof.
According to one embodiment, the carbohydrate-containing moiety is a
dextran. As used herein, "dextran" (dex) refers to a polysaccharide composed
of
a single sugar, which may be obtained from any number of sources (e.g.,
Pharmacia). Another preferred carbohydrate-containing moiety is Ficoll, which
is
an inert, semisynthetic, non-ionized, high molecular weight polymer.
Additional
non-limiting examples of moieties that may be used in accordance with the
present invention include lipopolysaccharides ("[PS"),
lipooligopolysaccharides
("LOS"), lipotechoic acid ("LTA"), deaceylated LPS, deaceyiated LTA,
delipidated
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CA 02758323 2011-11-08
LPS, delipidated LTA, and related molecules. Generally, a carbohydrate-
containing molecule that has been coupled using reductive amination requires
the
formation of an aldehyde moiety. In those instances, for example, these
aldehydes may also be coupled using amino-oxy chemistry described herein.
Reductive amination has been used to couple LPS and LOS, both of which
can be coupled using amino-oxy chemistry. Examples of coupling of LPS and
LOS using reductive amination chemistry may be found in Mieszala et al.,
Carbohydrate Research, 338:167 (2003); Jennings et al., Inf. & Immun., 43:407
(1984); and U.S. Patent No. 4,663,160.
C. The Second Moiety: Proteins, Peptides and Haptens
In accordance with the present invention, various different proteins can be
coupled to various different polysaccharides. The following list includes
examples
of suitable proteins that may be used in accordance with the invention: viral
proteins, bacterial proteins, fungal proteins, parasitic proteins, animal
proteins.
Glycoproteins from any of the above sources may also be used to form a
conjugate with the first moiety. Lipids, glycolipids, peptides, and haptens
are also
suitable for use as a second moiety in this invention. Haptenated proteins,
i.e.,
proteins derivatized with haptens, are also suitable for use as a second
moiety in
this invention.
Specific proteins include tetanus toxoid (TT), pertussis toxoid (PT), bovine
serum albumin (BSA), lipoproteins, diptheria toxoid (DT), heat shock protein,
T-
cell superantigens, protein D, CRM197, and bacterial outer-membrane protein.
All
of these protein starting materials may be obtained commercially from
biochemical
or pharmaceutical supply companies (e.g., American Tissue Type Collection in
Rockville, MD or Berna Laboratories of Florida) or may be prepared by standard
methodologies, such as those described in J. M. Cruse and R. E. Lewis (Eds.),
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CA 02758323 2011-11-08
"Conjugate Vaccines in Contributions to Microbiology and Immunology", Vol. 10
(1989).
D. Methods for Functionalizing the First or Second Moiety with an
Amino-Oxy Group
The amino-oxy (also referred to as oxy-amine, amino-oxy, aminooxy, and
amino-oxy) functional group. NH2-0-R, has a lower pKa than the amines found on
proteins, and is nucleophilic at much lower pH. Amino-oxy groups react well
with
carbonyl-containing groups, e.g., aldehydes and ketones, to form highly stable
oximes. The optimum pH for the reaction can range from 4 to 8, for example
from
5 to 7. According to one aspect of the invention, the optimum pH is around 5.
Since oximes are stable, the reductive step in the reductive amination
process,
discussed above, is optional. The high efficiency of the reaction may result
in
shorter reaction times. Furthermore, it is possible to exert some control over
the
reaction sites between the complementary reagents. By contrast, the reaction
of
hydrazides and amines with groups such as, for example, ketones, is slower and
far less efficient.
The protein and polysaccharide are functionalized with complementary
oxime-forming groups, and reacted to form oxime-linked protein-polysaccharide
conjugate vaccines. According to one aspect of the invention, the protein is
directly linked to the polysaccharide.
According to one embodiment, there is provided a process comprising
combining an amino-oxy homofunctional or heterofunctional reagent with an
entity
chosen from polysaccharides, oligosaccharides, carbohydrates, and
carbohydrate-containing molecules containing at least one carbonyl group, to
form
a polysaccharide, oligosaccharide, carbohydrate, or carbohydrate-containing
molecule functionalized via at least one oxime linkage. Functionalized means
to
add a group which facilitates further reaction, for example, thiol, carboxy,
amino-
CA 02758323 2011-11-08
oxy, halogen, aldehydes, and the like. This embodiment may be illustrated by
the
following non-limiting illustration ("Ps" denotes a polysaccharide):
ps--(oFor, Ps-(-CH)õ Ps. (CH = N - OR)õ
NalOx R-ONH2
1-10 mM Oxidized Amino-oxy reagent Oxime
PBS PS
R is a functional group, e.g., an amino-oxy, amine, thiol, or other chemical
group,
such as those listed below, for facilitating coupling to the protein:
NH2-0(CE-12)x¨CH2¨ Bis amino-oxy reagent
(x.>0)
Dithiopyridyl amino-oxy reagent
(x > 0)
(Ci-I1 C
NI-12-0CH H S S rni-1 (-1-4
2, - .2,x- - Bis amino-
oxy reagent with disulfide
(x>0)
R'-NH-C¨(CH2)õ-CH2----
(x >0)
0
HO ¨C ¨(CH2)õ -CH2¨ For x = 0, amino-oxy acetic acid (AoAc)
The at least one pendent functional group is then reacted directly or
indirectly with the protein moiety to yield a protein-polysaccharide
conjugate.
According to another embodiment, the protein is functionalized with at least
one pendent amino-oxy group, which is subsequently reacted with a carbonyl
group on a polysaccharide, oligosaccharide, carbohydrate, or carbohydrate-
containing moiety. The carbonyl group is formed with, for example, sodium
periodate. For example, in the case of a polysaccharide, the functionalized
protein is reacted with the polysaccharide to form a protein-polysaccharide
conjugate. The following scheme illustrates a non-limiting aspect of this
process:
16
CA 02758323 2011-11-08
EDC PBS
Protein ¨ CO2H Protein ¨ (0144n
Bis AO reagent
0 1 or 2 Steps
Ps ______________ Ps ¨ __ H It may not be necessary
NaI04 to remove excess Na104 or to quench
excess reagent
\I
PS ¨ CH --=---- N --0¨ Protein
quench reaction with NH2OH
Methods for functionalizing a protein with an amino-oxy group are known to
those of ordinary skill in the art. The protein can be functionalized with
amino-oxy
groups chemically, enzymatically or by genetic engineering. Described herein
are
methods for functionalizing the protein on either amines or carboxyl groups,
and
for controlling the number of amino-oxy groups on the protein.
In yet another embodiment, the polysaccharide is functionalized with
pendent amino-oxy groups and subsequently reacted with a glycoprotein
containing carbonyl groups. These may be present, for example, by oxidizing
the
carbohydrate on the glycoprotein. Aldehydes may be created by selective
oxidation of N-terminal serine or threonine.
In accordance with the present invention, for example when the
polysaccharide, oligosaccharide, carbohydrate, or carbohydrate-containing
moiety
is functionalized with an amino-oxy group, the protein advantageously contains
at
least one carbonyl group in the form of, e.g., a ketone or aldehyde moiety.
Aldehydes may be created on proteins containing an N-terminal serine or
threonine, and the resulting protein can be reacted with an amino-oxy reagent,
17
CA 02758323 2011-11-08
thus uniquely functionalizing the N-terminal. This monovalently-functionalized
protein can then be reacted directly, for example, with a carbonyl-containing
polysaccharide, if the amino-oxy reagent is homofunctional or indirectly,
using
spacers. N-terminal serine or threonine can occur naturally, or be engineered
into
a protein.
In the instances where the protein is functionalized with at least one amino-
oxy group, the polysaccharide, oligosaccharide, or carbohydrate contains at
least
one carbonyl group. The carbonyl groups may be a natural part of the
polysaccharide structure, e.g., the reducing end of the polymer, or created,
for
example, by oxidation. Reductive amination has been widely used to produce
protein-polysaccharide conjugates. As a result, means to produce carbonyl-
containing polysaccharides are well-known to those versed in the art.
Some polysaccharides contain a reducing sugar on their end, e.g., Hib
PRP and Neisseria PsC. These contain aldehydes as hemiacetals and can be
reacted with amino-oxy reagents_ Additional aldehydes may be created by
specific degradation of the polysaccharide. General procedures are described
in,
for example, Lindberg et al. "Specific Degradation of Polysaccharides - Adv in
Carbohydrate Chemistry and Biochemistry," Tipson et al., eds. Vol 31, pp. 185-
240 (Academic Press, 1975). For example, when PRP is oxidized with sodium
periodate, the polysaccharide chain is cleaved so as to produce
oligosaccharides
with an aldehyde on each end.
Many other methods for creating aldehydes are known to those versed in
the art. For example Jennings et al., U.S. Patent No. 4,356,170 entitled
"Immunogenic Polysaccharide-Protein Conjugates"; Tai et al., U.S. Patent No.
5,425,946 entitled "Vaccines against Group C Neisseria Meningitidis"; Porro,
U.S.
Patent No. 5,306,492 entitled "Oligosaccharide Conjugate Vaccines"; Yang et
al.,
U.S. Patent No. 5,681,570 entitled "Immunogenic conjugate molecules";
18
CA 02758323 2011-11-08
Constantino et al., "Development and phase 1 clinical testing of a conjugate
vaccine against meningococcus A and C," Vaccine 10:691 (1992); Laferriere et
al., "The synthesis of Streptococcus pneumoniae polysaccharide-tetanus toxoid
conjugates and the effect of chain length on immunogenicity," Vaccine, 15:179
(1997).
In accordance with the present invention, it may be desirable to add
aldehyde moieties to proteins and/or polysaccharides. Those of ordinary skill
in
the art will appreciate that there are many acceptable methods for doing so.
Suitable non-limiting examples of methods to add aldehydes to proteins and
polysaccharides include the following:
1. Hydroxyl groups are reacted with chlorohexanol dimethyl acetal in a
base, and the masked aldehyde is subsequently revealed by mild acid
hydrolysis.
Dick et al., Conjugate Vaccines (Eds. Cruse, et al.), pp. 91-93 (1989).
Scheme A
v4E-0 tb1^1 CI
_____________________________________________________ = 0--tOi.A^CHkOtAte),
r
'so
Protein ¨0t--"ir
.toca4
1---0--tcty5-042-4-Cil- Protein
2. Glucouronic lactone and sodium cyanoborohydride are used to
reductively aminate protein amines. Saponification is used to open the
lactone.
The sugar is then oxidized to an aldehydes using sodium periodate.
Scheme B
cn,cH
CH-2 H
ti
0
NaBH,CN
OH
F NH I
E
HOc H 1
Na C=0 11
Croi-1
kCI)
HO _________________________________________________ (
19
_
CA 02758323 2011-11-08
3. A carboxylated carbohydrate, for example, glucuronic acid, galactaric
acid, glyceric acid, or tartaric acid is added to protein amines using a
carbodiimide
reagent. The glycosylated protein is then oxidized to create aldehyde moieties
using sodium periodate.
= 0 =
HO-C
-0
HO-C-H
HO- 6-11
27,163-2
0
MR 9R1-7
Galactaric acid Glucuronic acid
4. Aldehydes can also be created via enzymatic oxidation, using suitable
oxidizing enzymes such as, for example, glucose oxidase, galactose oxidase,
and
neurominidase. For example, neurominidase may be used to remove terminal
sialic acid, followed by galactose oxidase. (Hermanson, Bioconjugation
Techniques, p. 116-117).
5. Chemical addition of aldehydes to amines on proteins or
polysaccharides can be effected using succinimidyl-p-formyl benzoate or
succinimidyl-p-formylphenoxyacetate. These NHS esters of aldehydes react with
amines and result in the addition of an aldehyde.
0
0
0.1
0
SFB
Succinimidyl-p-formyl benzoate
MW 247
0
0 111 0
0
SFPA
Succinimidyl-p-formylphenoxyacetate
CA 02758323 2011-11-08
6. Still another method uses the reaction of a bis-aldehyde (e.g.,
gluteraldehyde) with an amine. (Hermanson, Bioconjugation Techniques, p. 119-
120).
Scheme C
A-NH, + NaCNBH,
0
Glutaratdehydo Reductive Amination
to Secondary Amino Linkage
with Terminal Forntyt Group
7. Another suitable process is the addition of glyceraldehydes to protein
amines using reductive amination, followed by oxidation with sodium periodate
to
create aldehydes.
Optionally, if the conjugate contains residual free amino-oxy groups or
aldehydes, and if it is desired to quench these groups, an additional step may
be
taken. One of the methods for quenching a conjugate having an aldehyde is by
reduction, e.g., using sodium borohydride_ Alternatively, residual carbonyls
may
be quenched with a mono amino-oxy reagent, e.g., amino-oxy-acetate. Residual
amino-oxy groups can be quenched with a monofunctionalcarbonyl, e.g.,
glyceraldehyde, acetone or succinic semialdehyde.
E. Amino-Oxy Reagents
The preparation of conjugate vaccines may be accomplished by the use of
various amino-oxy reagents. A variety of useful honnofunctional and
heterofunctional amino-oxy reagents may be prepared by one skilled in the art,
and may also be obtained from Solulink, lnc.TM, 9853 Pacific Heights Blvd.,
Suite
H, San Diego, California 92121, and still others are described in the
literature.
Many more can be conceived of and easily synthesized. Toyokuni et al.,
"Synthesis of a new heterofunctional linker, N-[4-(amino-oxy)butyl]rnaleimide
for
21
CA 02758323 2011-11-08
facile access to a thiol-reactive 18F-labeling agent." Bioconjugate Chem.
14:1253
(2003).
Suitable non-limiting examples of reagents that may be used in accordance
with the present invention include those prepared by SolulinkTM (San Diego,
California). For example, bis(amino-oxy)cystamine is a homofunctional amino-
oxy-reagent that can be converted to a heterofunctional thiol-amino-oxy
reagent.
"Boc" is the art-recognized acronym for the t-butoxy carbonyl protecting
group.
Boc-amino-oxy acetate can be used to synthesize a number of suitable amino-oxy
reagents according to, for example, the following scheme:
0
HOC AOAc HOG¨NH¨ 0 ¨CH2 ¨c ¨NH ¨R"
EDC
-NH2 De pro te c t
= -CONHNHI
-0-NH2 0
I I
Ni-120¨clia¨c ¨NH __ R"
The ligands identified by R" are suitable, non-limiting examples of
nucleophilic ligands that may be used in accordance with the present
invention.
The above reagents are based on 2-(Boc-amino-oxy) acetic acid, available
from Bachem (Prod. No. A4605.005). Other useful starting reagents for making
amino-oxy reagents include N-Boc-hydroxylamine and N-Fmoc-hydroxylamine.
These reagents are available from Aldrich Chemical. N-Boc-Hydroxylamine can
be used to prepare a useful amino-oxy reagent as follows:
BOG-NH-OH BOC-NH-OC H2R"
X CH2R"
Deprotect
X= halide, e.g., Br, Cl, I
R" is a functional group
NH2O-CH2R"
22
CA 02758323 2011-11-08
Homofunctional annino-oxy reagents may be used in accordance with the
present invention. Suitable homofunctional amino-oxy reagents that may be used
include, for example, bis(amino-oxy)ethylene diamine, bis(amino-oxy) butane,
and
bis(amino-oxy)tetraethylene glycol, all of which are known and can be prepared
by
art-recognized methods. For example, bis(amino-oxy)butane may be prepared as
follows:
Br-(CI2CH2)2-Br ________________ BocNHO-(CH2CH2)2-ONHBoc
Excess
BOCNH-OH Deprotect
NH20-(CH2CH2)2-ONH2
Synthesis of various useful heterofunctional amino-oxy reagents have been
described in the literature, for example Mikolajczyk et al., Bioconjugate
Chem.
5:636 (1994) (a mateimide-amino-oxy reagent); Mikola & Hanninen Bioconjugate
Chem. 3:182 (1992) (amino-oxy alklyamines); Webb & Kaneko Bioconjugate
Chem. 1:96 (1990) (amino-oxy- dithionitropyridyl reagents). Jones et al.
describe
the synthesis of amino-oxy ethers from N-Boc hydroxylamine and alkyl iodides
and bromides, which provide another route to useful amino-oxy reagents. Dixon
&
Weiss, J. Org Chem. 49:4487 (1984), describe bis-amino-oxy reagents that may
be used in accordance with the present invention.
Ketones may be added to amines using, for example, reagents like NHS
levulate (from SolulinkTm). Carbohydrate groups on a protein, e.g.,
glycoproteins,
can be oxidized to carbonyls with, for example, sodium periodate. In addition,
reverse proteolysis may be used to add carbonyls or amino-oxy groups as
described in Rose et al., "Preparation of well-defined protein conjugates
using
enzyme-assisted reverse proteolysis," Bioconjugate Chem. 2:154 (1991). N-
23
CA 02758323 2011-11-08
terminal threonines or serines on proteins may be selectively oxidized to
aldehydes.
Small linker molecules may also be used to functionalize proteins and
polysaccharides with amino-oxy groups. See, for example, Vilaseca et at.,
"Protein conjugates of defined structure: synthesis and use of a new carrier
molecule," Bioconj. Chem. 4:515 (1993); and Jones et al., "Synthesis of UP
993,
a multivalent conjugate of the N-terminal domain of b2GPi and suppression of
an
anti-b2GPI immune response," Bioconj. Chem. 12:1012 (2001).
As is known to those of ordinary skill in the art, amino-oxy, aminooxy,
aminoxy, and oxy-amine are all synonymous terms.
F. Indirect Conjugation
As stated above, the conjugation between the first moiety and the second
moiety may proceed either indirectly or directly. In certain instances, the
process
of combining a protein and a polysaccharide may lead to undesirable side
effects.
In some cases, direct coupling can place the protein and the polysaccharide in
very close proximity to one another and encourage the formation of excessive
crosslinks between the protein and the polysaccharide. Under the extreme of
such conditions, the resultant material can become very thick (e.g., in a
gelled
state).
Over-crosslinking also can result in decreased immunogenicity of the
protein and polysaccharide components. In addition, the crosslinking process
can
result in the introduction of foreign epitopes into the conjugate or can
otherwise be
detrimental to production of a useful vaccine. The introduction of excessive
crosslinks exacerbates this problem.
Control of crosslinking between the protein and the polysaccharide can be
controlled by the number of active groups on each, concentration, pH, buffer
24
CA 02758323 2011-11-08
composition, temperature, the use of spacers and/or charge, and other means
well-known to those skilled in the art.
For example, a spacer may be provided between the protein and
polysaccharide in order to control the degree of crosslinking_ The spacer
helps
maintain physical separation between the protein and polysaccharide molecules,
and it can be used to limit the number of crosslinks between the protein and
polysaccharide. As an additional advantage, spacers also can be used to
control
the structure of the resultant conjugate. If a conjugate does not have the
correct
structure, problems can result that can adversely affect the immunogenicity of
the
conjugate material. The speed of coupling, either too fast or too slow, also
can
affect the overall yield, structure, and immunogenicity of the resulting
conjugate
product. Schneerson et al., Journal of Experimental Medicine, 152:361 (1980).
G. Vaccine Compositions
This invention further relates to vaccines and other immunological reagents
that can be prepared from the conjugates produced by the method in accordance
with the invention. For example, to produce a vaccine or other immunological
reagent, the conjugates produced by the method according to the invention may
be combined with a pharmaceutically acceptable medium or delivery vehicle by
conventional techniques known to those skilled in the art. Such vaccines or
immunological reagents will contain an effective therapeutic amount of the
conjugate according to the invention, together With a suitable amount of
vehicle so
as to provide the form for proper administration to the patient. These
vaccines
may include alum or other adjuvants.
Exemplary pharmaceutically acceptable media or vehicles include, for
example, sterile liquids, such as water and oils, including those of
petroleum,
animal, vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil,
sesame oil, and the like. Saline is a preferred vehicle when the
pharmaceutical
CA 02758323 2011-11-08
composition is administered intravenously. Aqueous dextrose and glycerol
solutions can be employed as liquid vehicles, particularly for injectable
solutions.
Suitable pharmaceutical vehicles are well known in the art, such as those
described in E. W. Martin, Remington's Pharmaceutical Sciences.
The vaccines that may be prepared in accordance with the invention
include, but are not limited, to Diphtheria vaccine; Pertussis (subunit)
vaccine;
Tetanus vaccine; H. influenzae type b (polyribose phosphate); S. pneumoniae,
all
serotypes; E. coil, endotoxin or J5 antigen (LPS, Lipid A, and Gentabiose); E.
coli,
0 polysaccharides (serotype specific); Klebsiella, polysaccharides (serotype
specific); S_ aureus, types 5 and 8 (serotype specific and common protective
antigens); S. epidermidis, serotype polysaccharide 1, II, and III (and common
protective antigens); N. meningitidis, serotype specific or protein antigens;
Polio
vaccine; Mumps, measles, rubella vaccine; Respiratory syncytial virus; Rabies;
Hepatitis A, B, C, and others; Human immunodeficiency virus I and II (GP120,
GP41, GP160, p24, others); Herpes simplex types 1 and 2; CMV
(cytomegalovirus); EBV (Epstein-Barr virus); Varicella/Zoster; Malaria;
Tuberculosis; Candida albicans, other candida; Pneumocystis carinii;
Mycoplasma; Influenzae viruses A and 13; Adenovirus; Group A streptococcus,
Group B streptococcus, serotypes, la, lb, II, and III; Pseudomonas aeroginosa
(serotype specific); Rhinovirus; Parainfiuenzae (types 1, 2, and 3);
Coronaviruses;
Salmonella; Shigella; Rotavirus; Enteroviruses; Chlamydia trachomatis and
pneumoniae (TWAR); and Cryptococcus neoformans.
The invention also relates to the treatment of a patient by administering an
immunostimulatory amount of the vaccine. The term "patient" refers to any
subject for whom the treatment may be beneficial and includes mammals,
especially humans, horses, cows, pigs, sheep, deer, dogs, and cats, as well as
other animals, such as chickens. An "immunostirnulatory amount" refers to that
26
CA 02758323 2011-11-08
amount of vaccine that is able to stimulate the immune response of the patient
for
prevention, amelioration, or treatment of diseases. The vaccines of the
invention
may be administered by any suitable route, but they preferably are
administered
by intravenous, intramuscular, intranasal, or subcutaneous injection. For
example, carbohydrate-based vaccines can be used in cancer therapy.
In addition, the vaccines and immunological reagents according to the
invention can be administered for any suitable purpose, such as for
therapeutic,
prophylactic, or diagnostic purposes.
The invention also relates to a method of preparing an immunotherapeutic
agent against infections caused by bacteria, viruses, parasites, fungi, or
chemicals
by immunizing a patient with the vaccine described above so that the donor
produces antibodies directed against the vaccine. Antibodies may be isolated
or
B cells may be obtained to later fuse with myeloma cells to make monoclonal
antibodies. The making of monoclonal antibodies is generally known in the art
(see Kohler et al., Nature, 256:495 (1975)). As used herein,
"immunotherapeutic
agent" refers to a composition of antibodies that are directed against
specific
immunogens for use in passive treatment of patients. A plasma donor is any
subject that is injected with a vaccine for the production of antibodies
against the
immunogens contained in the vaccine.
EXAMPLES
Example 1: Preparation of an Amino-Oxy Functionalized Protein
The following example illustrates the preparation of an amino-oxy
functionalized protein that can be conjugated to a polysaccharide. Bovine
serum
albumin (BSA) was used as a model protein.
Bis(amino-oxy)tetraethylene glycol was linked to carboxyl groups on bovine
serum albumin (BSA) with carbodiimide. Monomer BSA was prepared as
described in (Lees et al., Vaccine 14:190, 1996). Bis(amino-oxy)tetraethylene
27
CA 02758323 2014-12-15
glycol (85 mg) (prepared by SolulinkTM, MW 361) was made up in 850 pl of 0.5 M
HCI. 5 N NaOH was added to adjust to a pH ¨4.5. 1 ml of BSA mono (42.2
mg/ml in saline) was added. The reaction was initiated by the addition of 25
pl of
freshly prepared EDC (1-(3-dimethylamino)propyI)-3-ethylcarbodiimide
hydrochloride, 100 mg/ml in water). After approximately 3 hours, the solution
was
dialyzed overnight against saline at 4 C. The solution was then made up to 4
ml
with saline and concentrated with an Amicon Ultra 4TM centrifugal device (30
kDa
cutoff) to ¨0.5 ml, and was further desalted on a 1x15 cm G-10 column
(Pharmacia) equilibrated with saline. The void volume fraction was then
concentrated to ¨ 1m1 using the Amicon Ultra 4TM device. Using the BCA assay
(Pierce Chemical Co), the protein concentration was estimated to be 34 mg/ml
BSA. Trinitrobenzene sulfonic acid assay gave an intense red/orange,
indicating
the presence of amino-oxy group.
Example 2: Preparation of an Amino-Oxy Derivatized Polysaccharide
The following example illustrates the preparation of any amino-oxy
functionalized polysaccharide that can be conjugated to a protein, peptide, or
hapten.
Pn14 (10 ml at 5 mg/ml in water) was activated by the addition of 40 mg of
CDAP (100 mg/ml stock in acetonitrile), followed by triethylamine to raise the
pH
to 9.4. After approximately 2.5 minutes, 4 ml of 0.5 M hexanediamine (pH 9.4)
was added. The reaction was permitted to proceed for about 2 hours. Excess
reagent was then removed by dialysis against saline to yield amino-Pn14.
Amino-Pn14 was then reacted with excess NHS bromoacetate at pH 8 and
= dialyzed against saline in the dark at 4 C. The bromoacetylated Pn14 was
concentrated by pressure filtration and then dialyzed against water.
Amino-oxy cysteamine was prepared from bis amino-oxy cystamine by
TM
TCEP reduction followed by ion exchange on a Dowex 1X-8 column as follows:
28
CA 02758323 2011-11-08
Bis(amino-oxy)cystamine (obtained from Solulink)was made up in 50%
NMP/water at 0.1 M. TCEP was made up in water at 0.5 M and 3x molar
equivalents of 1 M sodium bicarbonate was added. A 1.5 molar excess of TCEP
was combined with Bis(A0)cystamine, and adjusted to pH ¨7 with sodium
carbonate. After 10 minutes, the mixture was diluted 5-fold into 10 mM bistris
at
pH 5. The reaction mixture was applied to a 1x3 cm Dowex 1-x8 column that had
been washed with 1 M NaC1 and equilibrated with 10 mM bistris, pH 5. The
reduced amino-oxy cysteamine is found in the flow through of the column.
Amino-oxy cysteamine was added to the bromoacetylated Pn14 and
reacted at pH 8 in the dark. The reaction mixture was then concentrated,
diafiltered, and then dialyzed against water.
Pn14 concentration was determined to be 9.1 mg/ml by the
resorcinol/sulfuric acid method. Using the TNBS assay and amino-oxy acetate as
the standard, the amino-oxy concentration was estimated at 0.74 mM, resulting
in
about 8 amino-oxy groups per 100 kDa of polysaccharide.
Example 3: Preparation of a BSA-Dextran Conjugate
The following example illustrates the preparation of a conjugate vaccine
using an amino-oxy functionalized protein and an oxidized polysaccharide.
Specifically, the amino-oxy functionalized BSA prepared in Example 2 was
linked
to oxidized dextran.
Dextran was oxidized using sodium periodate as follows: A 10 mg/ml
solution of T2000 dextran (Pharmacia) was made to 10 mM in sodium acetate, pH
5 and then 10 mM sodium periodate (from a 0.5 M stock in water), and incubated
at room temperature in the dark. At 1, 5, 10 and 15 min, an aliquot was
removed,
quenched by the addition of glycerol, and dialyzed against water in the dark.
The
final concentration of dextran was determined to be about 4.5 mg/ml.
29
CA 02758323 2011-11-08
The protein was conjugated to the polysaccharide as follows: 110 plot
each oxidized dextran preparation (1-15 min oxidation) was combined with 15 pl
BSA- amino-oxy (0.5 mg each). After an overnight reaction in the dark at room
temperature, the samples were analyzed by SDS PAGE (4-12% gradient gel,
NuPAGE, lnvitrogen). With reference to Figure 1, lanes are conjugates prepared
with (A) dex ox 1 min; (B) dex ox 5 min; (C) dex ox 10 min; (D) dex ox 15 min;
BSA- amino-oxy only. It is evident that each of the conjugation reactions
resulted
in high molecular weight material that did not enter the gel. Essentially no
unconjugated protein is evident, indicating a high degree of conjugation.
The four conjugates were pooled & fractionated on a S40QHRTM gel
filtration column (1x60 cm), equilibrated with saline. The void volume
fractions
were pooled and assayed for protein and polysaccharide. It was determined that
the pool contained 0.21 mg/m1 BSA and 0,27 mg/mIdextran. At least 50% of the
initial protein and polysaccharide were recovered. Thus, the amino-oxy-protein
with oxidized polysaccharide yielded soluble conjugate in excellent yield.
Example 4: Preparation of AO-Functionalized U.
The following hypothetical example illustrates the preparation of Tetanus
toxoid derivatized with amino-oxy groups using a two-step method.
1 ml tetanus toxoid (10 mg/ml) in 2 M NaCI is made to pH 8 by the addition
of 50 pl 1 M HEPES, pH 8. The protein is bromoacetylated by the addition of 7
I
of 0.1 M NHS bromoacetate. After a 1 hour incubation, 2 pmoles of
aminocysteamine is added. After an overnight reaction, excess reagent is
removed by dialysis against 2 M NaCI.
The protein concentration is determined using the BCA assay (Pierce
Chemical) and the presence of the amino-oxy group confirmed using TNBS.
CA 02758323 2011-11-08
Example 5: Preparation of amino-oxy-derivatized BSA using a two-step
method
Bromoacetylation of BSA:
4.1 ml of monomeric BSA (48.5 mg/m1) was made to pH 8 by the addition
of 400 pl 1 M HEPES, pH 8 and 5.5 ml water. 1 ml of 0.2 M NHS bromoacetate
(ProChem) in NMP was slowly added while vortexing. After an overnight reaction
at room temperature in the dark, the solution was dialyzed against saline for
2
days, centrifuged and filtered. 10.6 ml of BSA at 15.3 mg/ml was obtained.
Preparation of Amino-oxy cysteamine:
51.5 mg of Bisaminoxocystamine was added to a solution of 56 mg TCEP
made up in 1.1 ml 1 M sodium carbonate, 586 pl DMSO, and 586 pl water. After
minutes, the TCEP was removed on a 1x5 cm Dowex lx-8 column,
equilibrated with 10 mM Bistris, pH 6. The DTNB positive flow thru was pooled
15 and found to be 22.6 mM thiol.
6 ml was added to the bromoacetylated BSA and the pH adjusted to 8. The
reaction was allowed to proceed overnight in the dark, and was then dialyzed
for 2
days at 4 C against multiple changes of saline. The amino-oxy BSA was
determined to be about 8.6 mg/ml. Reaction of an aliquot with TNBS at pH .8
gave
an orangish color, indicating the presence of the amino-oxy group.
Example 6: Use of CDAP to Prepare Amino-Oxy Derivatized
Polysaccharide and Amino-oxy Conjugates
This experiment illustrates the use of CDAP to prepare amino-oxy
derivatized polysaccharide and amino-oxy conjugates. It illustrates how
chemistry
other than oxidation can be used to functionalize a polysaccharide with amino-
oxy
groups.
31
CA 02758323 2011-11-08
I. Preparation of an amino-oxy derivatized_polysaccharide using CDAP
chemistry
A solution of bifunctional amino-oxy reagent was prepared by solubilizing
29 mg of bis-amino-oxy acetate (ethylene diamine) (prepared by SolulinkTM) in
200 pl 1 M NaAc, pH 5. Dextran was activated using CDAP chemistry as follows.
To a solution of 0.5 ml T2000 dextran at 10 mg/ml in water, 25 pl of CDAP
(100mg/mlacetonitrile) was added and 30 seconds later the pH was raised by the
addition of 25 pl 0.2 M triethylamine (TEA) and three 5 pl of TEA neat.
At 2.5 minutes, the pH was reduced by the addition of 100 pl 1 M NaAc, pH
5. 200 pl of the BisA0 solution was then added. After ¨30 minutes reaction,
the
solution was desalted on a 1 x 15 cm P6DG column (BioRad) equilibrated with
NaAc buffer (10 mM NaAc, 150 mM NaCI, 5 mM EDTA, pH 5). The desalted
polysaccharide was estimated at 1.7 mg/ml dextran, using the resorcinol assay,
and about 11 amino-oxy groups/100 kDa dex using a TNBS assay.
II. Preparation of oxidized ovalbumin
To a 0.4 ml solution of ovalbumin (14.4 mg) (OVA), 10 pl of 1 M sodium
acetate, pH 5 was added, followed by the addition of 10 pl 0.5 M sodium
periodate
(in water). After a 15 minutes incubation at room temperature in the dark, the
reaction was quenched with the addition of a few drops of 50% glycerol. The
reaction mixture was then dialyzed in the dark against NaAc buffer. By
adsorption
at 280 nm, the concentration of oxidized ovalbumin ("OVA(ox)") was 6.6 mg/ml.
Ill. Preparation of conjugates and controls
The following solutions were prepared and each was incubated overnight at
room temperature in the dark:
A. 500 pl Dex AO (0.85 mg) + 75 pl OVA(ox) + 100 pl 1 M NaAc pH 5.
B. 250 pl Dex AO (0Ø43 mg) + 37.5 pl NaAc buffer + 50 pl 1 M NaAc
C. 250 pl NaAc buffer + 37.5 pl OVA(ox) + 50 pl 1 M NaAc.
32
CA 02758323 2011-11-08
Each was then assayed by SDS PAGE and SEC HPLC. Only sample A
contained high molecular weight (HMVV) material, with - 20% of protein
conjugated, as estimated by SEC HPLC. Neither B nor C indicated any HMW
material by SEC HPLC or SDS PAGE.
Example 7: Use of Cyanogen Bromide to Label Polysaccharide with a Bis-
Amino-Oxy Reagent.
This prophetic example demonstrates the derivatization of a polysaccharide
with an amino-oxy reagent using cyanogen bromide (CNBr).
Polysaccharide (e.g., Pn-14) is made up at 10 mg/ml in water, and is
treated with CNBr at 1 mg per mg of polysaccharide at pH 10.5 for 6 minutes in
a
p1i-stat. The reaction mixture is then reduced to -pH 7 by the addition of 0.5
M
bis-amino-oxy reagent (e.g., bis-A0(EDA). After an overnight reaction, the
solution is dialyzed into water and assayed for amino-oxy groups with TNBS,
and
for carbohydrates with the resorcinol assay. This amino-oxy derivatized
polysaccharide is used for conjugation with a carbonyl-containing protein.
According to another embodiment, the CNBr-activated polysaccharide can
be reacted with amino-oxy acetate. This will result in a polysaccharide
functionalized with carboxyl groups. The carboxyl groups can then be further
functionalized and indirectly or directly linked to proteins (with, for
example,
carbodiimide).
Example 8: Conjugation of Amino-Oxy Derivatized Protein with
Oxidized Polysaccharide
This example illustrates the preparation of amino-oxy derivatized protein
with the functionalization occuring on the amines. This amino-oxy derivatized
protein is then covalently linked to the clinically relevant polysaccharides
Neisseria
meningididis A and C.
=
33
CA 02758323 2011-11-08
I. Functionalization of protein with amino-oxy groups of a protein on its
amines (protein with pendent amino-oxy groups on amines)
Amines on the protein are bromoacetylated and then reacted with a thiol-
amino-oxy reagent to produce a protein with pendent amino-oxy groups.
Bis(amino-oxy acetate)cystamine 2HC1 was prepared by Solulink. TM Monomeric
BSA was at 42.2 mg/ml. NHS bromoacetate was obtained from Prochem and
made up at 0.1 M in NMP (N-methyl-2-pyrrolidone). The amino-oxy protein was
prepared as follows. In each of 2 tubes, a solution of 0.5 ml of BSA (21.1 mg)
and
250 pl H20 + 100 pl 1 M HEPES, pH 8 was prepared. One tube was reacted with
a 30 fold molar excess of NHS bromoacetate (93 pl) and the other at a 10 fold
molar excess (31 pl).
After about 1 hr, each was made up to 15 ml with sodium acetate buffer (10
mM NaAc, 0.15 M NaCI, 5 mM EDTA, pH 5) and concentrated to about 200 pl
using an Amicon Ultra 15TM device (30 kDa cutoff).
Amino-oxy acetate cysteamine was prepared as follows:
To a solution of 9.8 mg of Bis(A0Ac)cystamine (prepared by SolulinkTM) in
114 ill 1 M sodium acetate + 114 pl NMP, 22.8 pi of 0.25 M TCEP in 1 M HEPES,
pH 8 was added as a reducing agent. After 1 hour, the partially reduced amino-
oxy thiol reagent was added to each of the bromoacetylated BSA preparations,
the pH was adjusted to about pH 8 and the reaction allowed to proceed
overnight
in the dark at 4 C.
Each was desalted using the Amicon Ultra 15TM device by making volume
up to 15 ml with NaAc buffer and centrifuging. The desalting process was
repeated four times. The final volume was about 200 pl and was then made up to
about 1 ml with NaAc buffer. This product was termed BSA-S-AO. By
adsorbance at 280 nm, the 30x prep was determined to be 29.8 mg/mi and the
10x prep 24.8 mg/ml.
34
CA 02758323 2014-12-15
11, Preparation of oxidized Neissena meningiditis polysaccbaride A and C
(Neiss PsA and Neiss PsC)
Neiss PsA and PsC were solubilized overnight at room temperature at 10
mg/ml in water and then stored at 4 C. 50 pl of 1 M sodium acetate, pH 5, was
added to 1 ml of each polysaccharide solution, followed by the addition of 25
pl
0.5 M sodium periodate (0.5 M in water). After 10 minutes in the dark at room
temperature, each was dialyzed 4 hours against 4 I water. Each was then made
up to 4 ml with water and further desalted using an Amicon Ultra 41" device
(30
kDa cutoff). Using the resorcinol assay, the oxidized Neiss PsA was determined
to be 12.1 mg/ml and the oxidized Neiss PsC was 17.8 mg/ml.
III. Conjugation of BSA-S-AO with oxidized Neiss PsA and PsC
The following mixtures of BSA-S-AO and oxidized PsA and PsC were
prepared.
Conjugate pl Ps pl BSA 1 M NaAc pH 5
BSA10x-PsA 175 pl (2.1 mg) 134 p1(4 mg) 25 pl
BSA30x-PsA 175 pl (2.1 mg) 161 pl (4 mg) 25 pl
BSA10x-PsC 175 p1(3.1 mg) 134 p1(4 mg) 25 pl
BSA30x-PsC 175 pl (3.1 mg) 161 p1(4 mg) 25 pl
After an overnight reaction at room temperature in the dark, conjugates
TM
were assayed by SDS PAGE using a Phast gel (8-25%)(Pharmacia) under
reducing conditions. With reference to Figure 2, from left to right the lanes
are
BSA30x-PsA, BSA30x-PsC, BSA30x, BSA10x ¨PsA, BSA10x-PsC, BSA10x. It is
seen that there is a significant amount of high molecular weight materials
that did
not enter the gel, indicating that conjugation of the protein to the
polysaccharide
occurred.
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The PsA conjugates were pooled and fractionated by gel filtration on a S-
400HR column (1x60 cm, Pharmacia), equilibrated with saline. Similarly, the
PsC
conjugates were pooled and fractionated. Approximately 1 ml fractions were
collected and assayed for protein (by absorbance) and for carbohydrate using
the
resorcinol assay. The results are provided in Figure 3.
For the PsC conjugate, tubes 18-22 were pooled and for the PsA
conjugate, tubes 19-23 were pooled and examined by SDS PAGE using reducing
conditions.
With reference to Figure 4, the BSA-Neiss PsC conjugate is on the left and
the PsA conjugate is next to it. On the right is the molecular weight
standard. A
small amount of free BSA is observed in each, indicating incomplete separation
of
the conjugated and free protein. Each contains a significant amount of
conjugated
high molecular weight material that did enter the gel.
Example 9: Preparation of (BSA-Levulate)-Amino-Oxy-Pn14 Conjugate
This example illustrates the reaction of an amino-oxy group with a ketone
and shows that this can be used for the formation of conjugates and, more
specifically, the preparation of (BSA-Levulate)-Amino-oxy Pn14.
NHS Levulate was obtained from Solulink and made up by solubilizing 5.1
mg in 100 IA NW'. This was slowly added to a vortexed solution of 200 pl BSA
at
48.5 mg/ml, 200 pl water, and 100 pi 1 M HEPES, pH 8. After an overnight
reaction, the mixture was diafiltered using an Amicon Ultra 15 device, (30 kDa
cutoff). The final volume was 0.5 ml. This product is BSA-LEV
100 pl of BSA-LEV was combined with 300 pl of amino-oxy Pn14 (4.5
mg/ml Pn14) and incubated for several days in the dark. The conjugate and the
individual components were assayed by SEC HPLC using a Superose 6 column
(Pharmacia). The conjugate was then fractionated on an S400HR column.
Protein was assayed using the Bradford dye method, and polysaccharide with the
36
CA 02758323 2011-11-08
resorcinol method. The high molecular weight fraction was found to contain 0.6
mg BSA/mg Pn14.
Example 10: Preparation of Amino-oxy-BSA Neisseria PsC Conjugate
Neiss PsC was oxidized to create terminal aldehyde as generally
described in Jennings & Lugowski J. Imm. 127:1011(1981). SEC HPLC indicated
the molecular weight of the PsC was significantly reduced.
After overnight conjugation Of PsC and BSA-AO, analysis was conducted
via SEC HPLC Superose 6 0.5 ml/min. The conjugate was fractionated on a
1x60cm S200HR column, equilibrated 10 mM sodium acetate, 150 mM NaCI, 2
mM EDTA, pH 5. ft was determined by both SEC analysis and gel filtration that
most of BSA was conjugated. The high molecular weight peak was analyzed for
protein and carbohydrate and determined to contain 0.2 mg BSA/mg PsC.
Example 11: Preparation of amino-oxy-BSA ¨ Neisseria PsA conjugate
This example illustrates the preparation of a Neisseria PsA-BSA conjugate
by way of functionalizing the protein with an amino-oxy group.
Neiss PsA was terminally reduced to an alditol with NaBF14 and then
oxidized to create terminal aldehyde as generally described in Jennings &
Lugowski J. Imm. 127:1011 (1981).
Neisseria PsA was solubilized in water at 20 mg/mlfor 15 min. To 1 ml of
the solubilized polysaccharide, 10 mg of sodium borohydricle was added. The pH
was maintained to about 8-9. After 1 hour, 100 pl of 1 M NaAc was added, and
the pH was adjusted to 5. The reduced PsA was desalted on a 1x15 cm G10
column, equilibrated with saline, and the void volume fraction concentrated
with
an Amicon Ultra 4 (10 kDa cutoff device) to about 1 ml. 20 mg of solid sodium
periodate was added, along with 100 pl 1 M sodium acetate at pH 5. After a 15
minute oxidation in the dark at room temperature, the reaction was quenched by
the addition of a drop of glycerol and then desalted on lx15cm G10 column
37
_ .
CA 02758323 2011-11-08
equilibrated with 10 mM NaAC, 150 mM NaCI and 2 mM EDTA, pH 5 (acetate
buffer). The void volume was pooled and found to be positive in the BCA assay,
indicating the presence of reducing sugar. The material was diafiltered and
concentrated with an Ultra 4 device into acetate buffer.
Both the amino-oxy BSA and the PsA(red/ox) were examined by SEC
HPLC. The molecular weight of the PsA was markedly reduced by the
reduction/oxidation process.
Conjugation
In the conjugation step, 150 pi amino-oxy-BSA at 6 mg/ml was combined
with 50 pl PsA (red/ox) and 25 pl 1 M NaAc at pH 5.
After an overnight incubation in the dark at 4 C, the conjugate was
analyzed by SEC HPLC (Superose6, saline, 0.5 ml/min). It was seen that the PsA
contributed very little absorbance and the AO-BSA increased in molecular
weight
on conjugation.
The conjugate was fractionated on a 1x6Ocnn S200HR gel filtration column
=
and the high molecular weight fraction assayed for protein and PsA and was
found to contain 0.4 mg BSA/mg PsA.
Conclusion: The reduction/oxidation method works well to create
aldehydes that can be linked to amino-oxy-protein.. PsA was probably
hydrolyzed
during the Naf3H4 step, which is at elevated pH.
Example 12: Preparation of PRP(ox)-BSA-A0 Conjugate
1. Oxidation of PRP Hib
22.7 mg PRP Hib was made up at 10 mg/ml in water, and combined with
100 pl 1 M NaAc and 46 pl 0.5 M sodium periodate. The reaction proceeded in
the dark and on ice for 15 minutes, and was then quenched with 50% glycerol.
The reaction mixture was diafiltered into water with an Amicon Ultra 4 (10 kDa
cutoff) device, 4 x 4m1, final volume was approximately 1 ml. A resorcinol
assay
38
CA 02758323 2011-11-08
was conducted at 10 mg/m1. The sample was positive in the BCA assay,
indicating the presence of aldehyde.
2. Conjugation amino-oxy BSA
AO-S-BSA was provided at 15 mg/ml. 667 ul BSA-S-AO 10 mg was
combined with 100 ul 1 M sodium acetate, at pH 5, and approximately 1 ml
PRP(ox), and the reaction was permitted to proceed overnight in the dark. It
was
then quenched by the addition of 50 pl 0.25 M amino-oxy acetate.
3. Assay by SEC Superose6 prep grade HR10/30, equilibrated PBS 0.5
ml/min, OD 220.
Here, 0.5 ml conjugate was fractionated on 1x60 cm S200HR, and
equilibrated PBS. All fractions eluted before BSA, indicating higher MW.
Fraction BSA/mg Hib PRP (mg)
9 0.7
10 0.6
11 0.4
12 0.5
Example 13: Preparation of BSA-Pn14 Conjugate via Oxidation of
Glycidic Acid
This example illustrates a protocol whereby glycidic acid was added to
amines on BSA using carbodiimide. The glycidic acid on the protein was then
oxidized and reacted with amino-oxy-Pn14.
I. BSA-Glycidic acid
Monomeric BSA and glycidic acid (obtained from Fluka Chemical) were
combined to a final concentration in water of 12.5 mg/ml and 28 mg/ml,
respectively. The pH was adjusted to about 5 and 220 pl of 100 mg/ml EDC in
water was added. The pH is kept at about 5 for approximately 1.5 hours, and
the
39
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CA 02758323 2011-11-08
= reaction was quenched by the addition of .025 ml 1 M sodium acetate at pH
5.
The reaction mixture is then dialyzed against saline at 4 C overnight.
II. Oxidation of BSA-qlycidic acid
100 pl of 1 M sodium acetate at pH 5 was added to 1 ml of BSA-glycidic
acid (7.8 mg/ml), followed by 25 pl of 0.5 M sodium periodate in water. After
10
minutes in the dark, glycerol was added to quench the reaction and excess
reagent removed using an Amicon Ultra centrifugal device with a 30 kDa cutoff.
The final volume was about 400 ul.
100 pl of the oxidized BSA-glycidic acid was combined with 250 pl of
amino-oxy Pn14 at 9.3 mg/ml along with 50 p11 M sodium acetate at pH 5. An
aliquot was evaluated by SEC HPLC (Superose 6 0.5 ml/min, PBS). After an
overnight reaction, another aliquot was assayed in the same way. It was seen
that a significant portion of the absorbance eluted at the void volume (-15
minutes), indicating that the protein was linked to the high molecular weight
Pn14.
Following gel filtration on an S400HR column (Pharmacia), the high
molecular weight fraction was determined to contain 0.3 mg BSA/mg Pn14. This
ratio is similar to that determined from the percentage of conjugated high
molecular weight protein in the above chromatogram.
Thus, the method of linking glycidic acid to protein using carbodiimide
provides a way to create aldehydes on proteins that can be subsequently linked
to
amino-oxy groups.
Example 14: Use of Amino-Oxy Chemistry to link BSA to Dextran
This example illustrates the coupling of an oligosaccharide via its reducing
end to amino-oxy derivatized protein.
T40 dextran was made up at 100 mg/ml in water. The number of reducing
ends was estimated using the BCA assay with glucose as the standard_ It was
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found that there were 3.5 mM reducing ends/100 mg/ml T40 dextran, so the
average molecular weight was taken to be approximately 28,000 kDa
mg amino-oxy BSA containing -8 amino-oxy/BSA was combined with 2
ratios of T40 dextran at pH 5.
5 (A) 830 pl BSA-AO 15.3 mg/ml was combined with 620 pl T40 dextran at
100 mg/ml and 100 p11 M NaAc at pH 5.
(B) 830 pl BSA-A0 15.3 mg/ml was combined with 3.1 ml 140 dextran at
100 mg/m1 and 500 pl 1 M NaAc at pH 5.
Solutions were reacted at room temperature in the dark for 1 week, and
then assayed by SEC HPLC.
. Both conjugates eluted much earlier than BSA-A0, indicating that their
molecular weight has increased. Conjugates were then fractionated by anion ion
exchange (IEX). Consistent with the SEC profile, the higher the molecular
weight,
the lower ionic strength the conjugate eluted. IEX elution fractions were
analyzed
for the ratio of carbohydrate to protein and plotted on both a weight and mole
ratio
(using 28 kDa WA/ for the T40 dextran)
The peak fraction from each !EX elution was analyzed by SEC HPLC
(Superose 6 1 ml/min). The absence of monomeric BSA and the increasing MW
for high vs. low ratio T4Odex/BSA conjugates. SDS PAGE confirmed the high
molecular weight nature of the conjugate IEX eluants.
Example 15: Use of Amino-Oxy Chemistry to Link Oligosaccharide
and Protein
This example demonstrates the use of amino-oxy chemistry to link an
oligosaccharide indirectly via its reducing end to a protein. A general
description
of the protocol is as follows. The reducing end of T40 dextran (-40 kDa MW)
was
reacted with the amino-oxy group of amino-oxy acetate to create a dextran with
a
single carboxyl group on one end. This carboxy group was then converted to an
41
CA 02758323 2011-11-08
amine by reaction with ethylenediamine and carbodiimide. The amine-tipped
dextran was then thiolated and reacted with maleimide-derivatized BSA, to
create
a conjugate consisting of a protein with "threads" of carbohydrate extending
from
it
I. Addition of amino-oxy acetate to the reducing end of dextran
850 mg of T40 dextran (Pharmacia) was solubilized in 850 pl of water
overnight at room temperature.
235 mg of amino-oxy acetate was solubilized in a mixture of 850 pl DMSO
and 500 pl 1 M sodium acetate, pH 5 and combined with the T40 dextran
solution.
An additional 500 pl of DMSO was added to make the solution approximately 50%
DMSO. After an incubation at about 68 C for about 6 hours, the solution was
extensively dialyzed against water. The product was dextran containing a
single
carboxyl group on its reducing end.
1.8 g of ethylenediamine 2HCI was added to the solution (approximately 22
ml) and the pH adjusted to approximately 5 with 1 N NaOH. 220 mg of EDC (1-(3-
dimethylamino)propy1)-3-ethylcarbodiimide hydrochloride) was added and the pH
maintained at about 5 for 3 hours. The reaction was then quenched by the
addition of 1 M sodium acetate, pH 5, dialyzed against saline, and
concentrated
using an Amicon Ultra j5TM (10 kDa cutoff). It was then further dialyzed
against
saline and then against water.
The product was assayed for amines using TNBS and for carbohydrate
using the resorcinol assay. It was determined that there were approximately
0.45
amines per 40,000 kDa of dextran. This product was dextran containing a single
amine group on its reducing end and was termed NH2-A0Ac-T40 dextran. Using
the resorcinol assay, the solution was determined to have a concentration of
about 119 mg/ml dextran. T40 dextran consists of a distribution of molecular
42
CA 02758323 2011-11-08
weights, which makes it difficult to determine the actual degree of
substitution of
the reducing ends of the polymers.
II. Thiolated dextran and maleimide- BSA
Maleimide-derivatized BSA was prepared as follows: GMBS (40 pl of a 0.1
M stock in NMP) was added to a solution of 200 pl of monomeric BSA (42.2
mg/ml), 50 pl 0.75 M HEPES, 5 mM EDTA at pH 7.3, and 100 pl water. After a 2
hour reaction, the pH was reduced by the addition of 100 pl 1 M sodium
acetate,
at pH 5. The solution was desalted using an Amicon Ultra 4TM (30 kDa cutoff)
ultrafiltration device and 10 mM NaAc, 0.15 M NaCI, 5 mM EDTA, pH 5.
The NH2-A0Ac-T40 dextran was thiolated using SPDP as follows: 0.5 ml
of the NH2-A0Ac-T40 dextran was combined with 100 pl of 1 M HEPES, pH 8 and
100 pl of 0.1 M SPDP were added. After approximately 2 hours, 50 pi of 0.1 M
EDTA pH 5 was added, followed by 100 pi of 1 M sodium acetate, pH 5 and 50 pl
of 0.5 M dithiothreitol in water. After a 1 hour incubation, the solution was
dialyzed into sodium acetate buffer overnight at 4 C.
The thiol tipped T40 dextran and the maleimide derivatized BSA were
combined (a small aliquot of the BSA-maleimide was saved for analysis). After
an
overnight reaction, one half the mixture (about 1 ml) was fractionated by gel
filtration using a 1x60 cm S-400HR column, equilibrated with saline. For
comparison, a mixture of 100 pl BSA monomer (42.2 mg/ml), 300 pl T40 dextran
AOAc, and 0.5 ml saline was similarly fractionated on the same gel filtration
column. Fractions (about 1 ml) were analyzed for protein by absorbance at 280
nm and for dextran using the resorcinol assay.
With reference to Figures 5A-D, it is evident that the protein and dextran
are eluting earlier from the column when in the T40 dextran AOAc-thiol-
maleimide
BSA conjugate than when the components are mixed. This indicates that a
43
CA 02758323 2011-11-08
conjugate of higher molecular weight has been formed. Furthermore, the ratio
of
dextran to protein increased.
The column fractions were further analyzed by SDS PAGE, with the results
provided in Figure 6. From left to right, MW marker, conjugate fractions 18,
20,22,24,26, mixture fractions # 24,26,28, 30, unfractionated conjugate,
starting
BSA-maleimide.
It is evident that the unfractionated conjugate contains only a small
proportion of free protein, indicating that the conjugate was formed in high
yield.
No high molecular weight protein is evident in the mixture fractions. Only
conjugate and essentially no free protein is evident in the conjugate
fractions.
This confirms that a conjugate formed in high yield.
Example 16: Preparation of BSA-Pn14 Conjugates via Glycidic Acid and
Amino-Oxy Derivatized Pn-14.
The following example is illustrative of the preparation of a conjugate using
an aldehyde-substituted protein.
I. In situ synthesis of NHS ester of glycidic acid using TSTU and addition to
BSA
In this step, 7.9 mg of glycidic acid hemi-calcium salt monohydrate (MW
143) was solubilized in 110 pl NMP. This was combined with 200 pl of 0.5 M
TSTU (Novachem) in NMP, and 100 pl of triethylamine, and was added to 1 ml of
24 mg/ml BSA. The pH was adjusted to pH 8. After approximately 2 hours, the
mixture was dialyzed on 2 x 1 liter saline. The number of free amines on BSA
was determined using TNBS. For the control, the number was 33.2 NH2/BSA.
For glycidic acid/TSTU/BSA, the number was 25 NH2/BSA. These results lead to
the conclusion that BSA was labeled with about 8 glycidic acid units /BSA
44
CA 02758323 2011-11-08
Oxidation of Functionalized BSA
A 5 mg aliquot was made up with 25 mM NaAc at pH 5, and 25 mM sodium
periodate. The reaction was allowed to proceed in the dark at room temperature
for 15 minutes, after which a drop of glycerol was added to quench the
reaction.
The mixture was fractionated S200HR, pool main peak & concentrate.
III. Preparation of BSA(ox)-AO-Pn14 Conjugate
In this step, 444 pl of amino-oxy functionalized Pn14 was combined with
0.4 ml BSA(ox) made up at 10.1 mg/ml, and 100 p11 M NaAc at pH 5, and
reacted overnight at room temperature. The Fractionate by gel filtration
S400HR
1x60 cm saline + 0.02% azide SEC HPLC indicated that oxidation of the Glycdic
acidTTSTU/BSA caused polymerization of the BSA.
Also observed was the progressive increase in the high molecular weight
peak, indicating that conjugation was increasing with time. The AO-Pn14 alone
had minimal absorbance.
Example 17: Preparation of Mercaptoglycerol-Bromoacetate BSA
This example illustrates a process for preparing a
BSA(mercaptoglycerol(ox))-AO-dextran conjugate.
Preparation of bromoacetylated BSA
500p1 monomeric BSA (48mg/m1) was combined with 500p1 1 M HEPES, at
pH 8, and 25 pl 0.1 M NHS bromoacetate in NMP. For the control, 250 pl BSA
was combined with 250 p1HEPES and 12 pl NMP
After approximately 1 hour, each was desalted into saline using an Amican
Ultra 4 (30 kDa cutoff) device. The final volume was 450 pl BSA-bromoAc, and
300p1 GSA control
Next, 50 mM mercaptoethanol and 50 mM mercaptoglycerol were prepared
in water.
CA 02758323 2011-11-08
Preparation E: 225 pl BSA-BromoAc was combined with 100 pl 1 M
HEPES at pH 8 and 50 pl of 50 mM mercaptoglycerol.
Preparation F: The BSA control was combined with 100 p11 M HEPES to
pH 8 and 50 pl 50 mM mercaptoglycerol
Preparation G: 225 pl BSA- BromoAc was combined with 100 p11 M
HEPES at pH 8, and 50 pl 50 mM mercaptoethanol.
After 30 minutes, each was desalted with Am icon Ultra using NaAc buffer
(10 mM NaAcetate, 150 mM NaCI, 5 mM EDTA, pH 5). Final volume was 0.5 ml
Each was then made up in 10 mM sodium periodate from a freshly
prepared 0.5 M stock and incubated for 10 minutes at 4 C in the dark, and then
quenched by the addition of glycerol and desalted using the Amicon Ultra
device
and washed into NaAc buffer. By OD 280, each was determined to be about 20
mg/ml BSA.
Preparation E should contain BSA-aldehyde; Preparation F was not labeled
with the bromoacetate, and so it could not react with the mercaptoglycerol.
Thus,
it should not contain aldehydes. Preparation G would have pendent
mercaptoethanol, which does not oxidize, so it should not contain aldehdydes.
315 pl of Amino-oxy dextran, at 15.9 mg/ml, was combined with 250 pl of
each BSA preparation, and incubated overnight at room temperature in the dark.
Each was then fractionated by gel filtration on a S400HR 1x60cm
equilibrated with saline. The high molecular weight fraction was analyzed for
protein and dextran.
The results indicated that only BSA containing the oxidized
mercaptoglycerol formed a conjugate, and this was confirmed by the
protein/dextran ratio of the high molecular weight fraction.
E BSA-mercaptoglycerol(ox) + AO-dex 0.97 mg BSA/mg dex.
F BSA control (ox) + AO-dex 0.1 mg BSA/mg dex.
46
CA 02758323 2011-11-08
G BSA-mercaptoethanol(ox) + AO-dex 0.1 mg BSA/mg dex.
Example 18: Linking of a Protein to a Polysaccharide via Oxime Formation
The following example illustrates the linking of a protein via its N-terminal
group to a polysaccharide via oxime formation.
N-terminal threonine of lysostaphin was oxidized and derivatized with a bis-
amino-oxy.reagent. Oxidation of the protein was performed as generally
described in Gaertner & Offord, "Site-specific attachment of functionalized
poly(ethylene glycol) to the amino terminus of proteins," Bioconjugate Chem.
7:38
(1996). Lysostaphin a 27 kDa protein was produced in lactococcus.
Trial No. 1
The lysostaphin used contained only about 30% free N-terminal threonine.
Conditions of Gaertner & Offord were used for oxidation of the N-terminal
threonine. In more detail, a 50 molar excess of methionine (17.5 pl from a 1M
stock in water) was added to 1 ml of a 10 mg/ml solution of lysostaphin.
Sodium
bicarbonate (1M) was added to adjust the pH to 8_3_ Oxidation was commenced
by the addition of sodium periodate (7 pl from a 0.5 M stock in water). The
reaction mixture was kept in the dark at room temperature for 10 minutes, at
which time 7.1 mg Bis(amino-oxy)tetraethylene glycol (obtained from
SolulinkTM)
prepared as a 50 mg/ml solution in DMSO was added. After 1 hour in the dark,
the solution was dialyzed against saline in the dark at room temperature. The
product is termed lysotaphin AO. The lysostaphin concentration was determined
at OD 280 using 0_49 mg/ml/Absorbance unit.
An aliquot was tested with TNBS at pH 5. It has previously been found that
amino-oxy but not amines reacted with TNBS under these conditions. The assay
was performed as follows: 50 pl of lysostaphin or lysostaphin AO was added to
440 pl of 0.1 M NaAc, pH 5 and then 10 pl of 10 mg/ml TNBS in water added. 5
pl
of 1 mM Amino-oxy acetic acid was used as a standard in the above solution.
47
CA 02758323 2011-11-08
Samples were read at 500 nm after a 6 hour incubation in the dark. The sample
solution was orange, indicating presence of amino-oxy groups. Using the
standard, it was estimated about 30% of lysostaphin was derivatized with the
AO
group. This lysostaphin AO was then reacted with excess oxidized T2000 dextran
in the dark at room temperature to allow conjugation via oxime formation. The
reaction was assayed by SEC HPLC to determine the shift of mass from low
molecular weight (unconjugated protein) to high molecular weight (lysostaphin-
dextran conjugate). A Phenomenex Biosep SEC2000 (300x4.6) equilibrated with
PBS and run at 0.5 ml/min with monitoring at 280 nm was used for SEC HPLC
'10 With reference to Figure 6, the upper chromatogram is the reaction
mixture
at about 1 minute, the middle chromatogram is after an overnight reaction and
the
lower figure is the lysostaphin AO alone. Note the shift to high molecular
weight
material after the reaction was allowed to proceed overnight. This figure
suggests
that the AO group on the lysostaphin linked to the high molecular weight,
oxidized
dextran. About 27% was coupled, based on the percentage of the area of the
high molecular weight peak. This is in the expected percentage since only
about
a third of the lysostaphin contained a free threonine and was derivatized with
AO,
as indicated by TNBS assay.
Example 19: Preparation of DT(ox)-AO-Pn14 Conjugate
This example illustrates the preparation of the DT(ox)-AO-Pn14 conjugate,
and it also demonstrates how reagents can be prepared in as "single pot"
reactions (which may simplify preparation).
I. Mercaptocilycerol-Diptheria toxoid
0.5 ml diphtheria toxoid (-13 mg/ml) was combined with 100 p11 M
HEPES, pH 8 and 10 pl 0.1 M NHS bromoacetate in NMP. It was incubated in the
dark for about 30 minutes, and then 10 pl of 12.3 pl mercaptoglycerol was
added.
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CA 02758323 2011-11-08
Following an overnight reaction, the solution was desalted with an Amicon
Ultra 4
(30kDa cutoff) to a final volume of about 400 pl.
Next, 50 pl of 1 M sodium acetate at pH 5 was added, followed by 9 pl of
0.5 M sodium periodate. Oxidation was allowed to proceed for 10 minutes in the
dark at room temperature. The reaction was then quenched by the addition of
50% glycerol. The low molecular weight components were removed on the same
Amicon Ultra 4 device and diafiltered into saline. The final volume was about
200
pl.
The above protocol eliminated one of the desalting steps by adding excess
mercaptoglycerol to the solution containing bromoacetylated-DT and
bromoacetate.
11. Conjugation
1 ml of amino-oxy Pn14 (-9 mg/ml) was added to the oxidized DT and 100
pl of 1 M sodium acetate at pH 5 added. The reaction was allowed to proceed
overnight at room temperature in the dark, and then fractionated on an S400HR
column (1x60 cm, equilibrated with saline).
The high molecular weight fraction was pooled. Protein was estimated
using 1 mg/m1= 1 OD and the Pn14 concentration determined using the resorcinol
assay. The fraction was found to contain about 1.3 mg DT/mg Pn14.
Example 20: Preparation of a gp350(ox)-A0-S-Pni 4 conjugate.
gp350 is a glycoprotein from Epstein Barr virus that binds to human
complement receptor. It was produced recombinantly in yeast cells by Dr.
Goutam Sen (Uniformed Services University of the Health Sciences, Bethsda,
MD) and purified by hydrophobic interaction chromatography.
The pH of 0.5 ml of gp350 at 8 mg/m1 in PBS was reduced by the addition
of 50 p11 M sodium acetate, pH 4.7, and 11 pl of 0.5 M sodium periodate (in
water) was added. After an 8 minute incubation in the dark, on ice, the
reaction
49
CA 02758323 2011-11-08
was quenched by the addition of 100 pi 50% glycerol. Excess reagent was
removed by diafiltration using an Amicon Ultra 4 (30 kDa cutoff) device. A
total of
four, 4 ml exchanges with PBS were used. The final volume was about 300 pl.
To this solution, 100 pl of 1 M NaAc, pH 5 was added, followed by 400 pl of AO-
S-
Pn14 (9.1 mg/ml).
After an overnight reaction at 4 C in the dark, the reaction was quenched
by the addition of 100 pl of 0.25 M amino-oxy acetate, pH 5. The resulting
conjugate was fractionated by gel filtration on a 1 x 60 cm S400HR column,
equilibrated with saline. The void volume fractions were pooled. The Pn14
concentration was determined by the resorcinol/sulfuric acid method and the
protein from the absorbance at 280 nm, using an extinction coefficient of 1
mg/ml
= 1 absorbance unit. The conjugate contained 0.9 mg gp350/mg Pn14.
Control gp350 was oxidized and prepared as above but amino-oxy acetate =
was added instead of amino-oxy Pn14.
In competition assays with fluorescently labeled gp350, both the control
and the conjugated gp350 were capable of binding to the complement receptor of
human B cells. (Performed by Goutam Sen USUHS).
Mice were immunized on with the gp350-Pn14 conjugate on days 0 and 10,
and bled on days 10 and 23
Day Anti-Pn14 IgG Titer
10 630
23 10643
The increase in anti-Pn14 IgG on boosting is an indication that the protein
and polysaccharide are covalently linked and acting as a T cell dependent
antigen. As a T cell independent antigen, Pn14 alone does not show an increase
CA 02758323 2011-11-08
in titer.
Example 21: Preparation of a peAcLTA(ox)-AO-SH]-GMBS-BSA Conjugate
LTA was deacylated by incubation for 1 hour in pH 10 sodium bicarbonate
at approximately 75 C. Sample is then dialyzed against water. This is
deacylated
LTA (DeAcLTA).
The sample was then oxidized in 10 mM sodium periodate at pH 5
overnight in the dark at room temperature, dialyzed against water again, and
lyophilized. The sample was taken up in a small volume of water, incubated
overnight with reduced amino-oxy cysteamine and lyophilized. The sample was
taken up in about 1 ml of water and fractionated on an S200HR column,
equilibrated with 10 mM sodium acetate, 150 mM NaCI, and 5 mM EDTA, pH 5.
The low molecular weight fraction containing both Pi and thiol was pooled and
lyophilized and taken up in about 0.75 ml water. This fraction was found to
contain about 1 mM thiol and 350 micromolar phosphate. This material is thiol-
labeled DeAcLTA.
BSA was labeled with a 50 fold molar excess of GMBS (Prochem) at pH
7.2 and desalted in sodium acetate buffer and concentrated using an Amicon
Ultra
4 (30kDa cutoff) device to a final concentration of about 55 mg/ml.
60 pl of the BSA-GMBS was added to the thiol labeled DeAcLTA. As the
reaction proceeded, the concentration of thiols decreased at least 10 fold, as
determined by the DTNB assay.
Conjugation was monitored by SEC HPLC on a Superose 6 column,
(equilibrated with PBS,1 ml min, OD 280). The lower trace indicated the
chromatogram for the GMBS labeled BSA alone and the upper trace indicated the
conjugate, which elutes earlier, indicating an increased molecular weight. By
SDS
PAGE, the MW of the conjugate had clearly increased in a manner consistent
with
the SEC chromatogram. No monomeric BSA is evident in the conjugate_
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CA 02758323 2011-11-08
A western blot was performed to demonstrate the presence of LTA on the
high molecular weight protein. It is seen that the conjugate was reactive for
LTA.
Neither BSA nor LTA alone or the combination indicate any high molecular
weight
LTA.
To further demonstrate the covalent linkage between LTA and BSA, a
double EL1SA was performed. The EL1SA plate was coated with anti-BSA
followed by conjugate or LTA, BSA or the combination_ Anti-LTA is then applied
and the amount bound determined. Thus, only material containing both BSA and
LTA would be detected. Only the conjugate was positive in this assay.
Thus by reaction monitoring, molecular weight, western blotting and double
ELISA all indicated the formation of a covalent link between the protein and
LTA.
Example 22: Preparation of a LTA-TT Conjugate
Reagents were obtained from Aldrich. Aminooxyacetylcystamine was
prepared by Dr. David Schwartz of Solulink Inc. (San Diego, CA). TCEP was
purchased from Pierce.
S. Aureus serotype 5 lab strain (MSSA) was grown by Kemp Biotech
(Frederick, MD) in a 100 liter fermenter. Cells were centrifuged, resuspended
and
centrifuged into aliquots approximating 10 liters of cells_ The cell paste was
stored at -70 C. An aliquot was thawed and resuspended in 0.1 M sodium
citrate,
pH 4.7 and disrupted with a Bead Beater (Biospec Products) using 0.1 m
zirconium beads. The device was ice cooled and run 1 min on and 1 min off for
4
cycles. The liquid was removed and the beads washed with citrate buffer.
Alternatively, cells were treated with 1 mg/ml lysostaphin at pH 5 overnight
at 4 C
and then disrupted using a Microfluidizer Model 110Y (Microfluidizer, Newton,
MA)
with 3 passes at 23,000 psi.
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CA 02758323 2011-11-08
I. LTA extraction
LTA was extracted and purified from cell pellets using either the phenol
extraction method of Fischer et al., Improved preparation of lipoteichoic
acids. Eur
J Biochem, 1983. 133(3): p. 523-30, with minor modifications or using the
butanol
method of Morath et al., Structure-function relationship of cytokine induction
by
lipoteichoic acid from Staphylococcus aureus. J Exp Med, 2001. 193(3): p. 393-
7.
In brief, the disrupted cell suspension was vigorously mixed for 30 min
with an equal volume of n-butanol. The solution was then centrifuged for 20
min
at 13,000 x g. The upper phase (butanol) was removed and the lower, aqueous
phase was lyophilized. Initially the butanol phase was re-extracted and the
new
aqueous phase tested for LTA by ELISA, however an insignificant amount of LTA
was recovered. Pellets were resuspended in 25 ml of citrate buffer and frozen.
Pellets from several extraction runs were combined, and the
disruption/extraction
process repeated.
The solubilized extract was filtered using a Whatman 0.45pm syringe filter
(#6894-2504) and loaded onto a 2.6 x 20 cm Octyl Sepharose' column
(Pharmacia), equilibrated with 0.1M ammonium acetate in 15% n-propanol, pH
4,7, at a flow rate of .5m1/min. The column was then washed with 0.1 M sodium
=
acetate in 15% n-propanof until the absorbance at 280 nm returned to baseline.
The column was then eluted with 25mM sodium acetate in 40% n-propanol at 4
ml/min and fractions of 8 ml collected. The phosphate containing fractions
were
pooled and loaded onto a 5 ml SepharoseTM Q FF column, equilibrated with the
same buffer. When the absorbance returned to baseline, the column was eluted
with buffer + 0.5 M KC!. Phospate containing tubes were pooled, partially
lyophilized to reduce the volume and dialyzed against water to remove salts
and
lyophilized again.
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CA 02758323 2011-11-08
11. Conjugation of LTA to tetanus toxoid
LTA was deacylated by incubating 1 ml (10 mg/m1) in 0.1 M sodium
carbonate + 0.1 M hydroxylamine for 2 hr at 75 C, followed by dialysis against
water using a 3.5 kDa cutoff membrane (Pierce). The solution was then
lyophilized and taken up in 0.5 ml water. The deacylated LTA was oxidized by
the
addition of 100 kill M sodium acetate, pH 5 and 1251.11 0.5 M sodium
metaperiodate. After 2 hrs the reaction was quenched by the addition of 100
ill
50% glycerol and dialyzed overnight in the dark against water. This material
tested positive for aldehydes in the purpald assay (Lee, C.H. and C.E. Frasch,
Quantification of bacterial polysaccharides by the purpald assay: measurement
of
periodate-generated formaldehyde from glycol in the repeating unit. Anal
Biochem, 2001. 296(1): p. 73-82).
(Amino-oxyacetate)cysteamine was prepared by solubilizing 11 mg
Bis(amino-oxyacetate)cystannine in an aqueous solution of 25% NMP. TCEP (17
mg)in 1 M sodium carbonate was added and the solution incubated for 10 min
and then passed over a Dowex lx-8 column (1x3 cm), equilibrated with 10 mM
Bistris, pH 6. The DTNB positive fractions in the flow through were pooled.
The
pooled (amino-oxyacetate)cysteamine was assayed for thiols and determined to
contain 5.2 mole SH. The reagent was combined with the oxidized deacylated
LTA and incubated overnight in the dark at 4 C and then dialyzed against 10 mM
sodium acetate, 0.15 M sodium chloride, 5 mM EDTA, pH 5 to remove excess
reagent. The final solution was 2 ml at 2.5 mM thiol and 25 mM phosphate.
Tetanus toxoid (TT) (obtained from GlaxoSmithKline, Rixensart, Belgium)
was labeled with maleimide by adding a 50-fold molar excess of GMBS (0.1 M
stock in NMP) to a 14.6 mg/mIsolution of TT buffered 1n15 M HEPES, 5 mM
EDTA, pH 7.3. After a 1 hr reaction, the solution was desalted by
diafilitration into
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CA 02758323 2011-11-08
2 M NaCl using an Amicon Ultra 4 (30kDa cutoff) device, concentrating to a
final
volume of 0.4 ml. The retained material was DTNB positive_
The thiolated, deacylated LTA was combined with the maleimide-TT under
a stream of nitrogen and the pH adjusted to 6.5 by the addition of 015 M
HEPES,
pH 7.3. After sealing and incubating overnight at 4 C, the solution was
assayed
and determined to still be 2 mM thiol. An additional 7 mg of IT was labeled
with
maleimide as above, diafiltered and concentrated to 0.5 ml and added to the
reaction mixture. Over 30- min the thiol content steadily decreased and at
that
time the reaction was quenched by the addition of 100 I of 0.5 M
iodoacetamide
+100 I of 1 M sodium carbonate.
The conjugate was purified using size exclusion chromatography on a
Superose" 6(Prep grade)column(1 X 30 cm), equilibrated with saline.
III. Immunization of mice
Groups of 20 Balb/c mice were immunized on days 0, 14 and 28 with 5 pg
of LTA as described above, either mixed with IT or conjugated to TT and with
Ribi
MPL adjuvant and bled 14 days later. Individual sera were assayed for anti-IgG
by ELISA. The results are provided in Fig. 7. M110 (a mouse monoclonal
antibody that binds to LTA) was used as a standard. Anti-LTA levels were
assayed in the sera. Results are shown in Fig. 8. The conjugate induced high
levels, and the mixture induced only very low levels of antibody. To evaluate
the
biological activity of the anti-sera, an opsonophagocytic was performed. Sera
were diluted at 1:25.
IV, Phosphate analysis
Phosphate was determined as described by Chen, P.S., T.Y. Toribara, and
H. Warner, Microdetermination of Phosphorous. Anal Biochem, (1956) 28: p.
1756-1758, with some modifications. In brief, a 100 I sample + 30 I
magnesium
CA 02758323 2014-12-15
nitrate solution in a 13 x 100 mm borosilicate tube were vortexed, dried in a
heating block and flamed with a propane torch until a brown gas was emitted.
300
Al of 0.5 M HCI was added, the tubes capped with glass marbles and heated in a
boiling water bath for 15 min. The tubes were allowed to cool and 700111 of an
ascorbic acid/ammonium molybdate mix added, incubated for 20 min at 45 C and
samples read at 820 nm. The mix was prepared by combining 6 parts of a
solution of ammonium molybdate (0.42 g 2.86 ml sulfuric acid made up to 100
ml with water) and 1 part of 10% ascorbic acid in water. Phosphate standard
was
obtained from Sigma.
Other embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the invention
disclosed herein. It is intended that the specification and examples be
considered
as exemplary only, and the scope of the claims should not be limited by the
preferred embodiments set forth in the examples, but should be given the
broadest interpretation consistent with the description as a whole.
The specification is most thoroughly Understood in light of the teachings of
the references cited within the specification. The embodiments within the
specification provide an illustration of embodiments of the invention and
should
not be construed to limit the scope of the invention. The skilled artisan
readily
recognizes that many other embodiments are encompassed by the invention. Any
of the foregoing process are suitable in accordance with the present
invention.
The above serve only as illustrative examples and are nonlimiting.
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CA 02758323 2011-11-08
Unless otherwise indicated, all numbers expressing quantities of
ingredients, reaction conditions, and so forth used in the specification,
including
claims, are to be understood as being modified in all instances by the term
"about" Accordingly, unless otherwise indicated to the contrary, the numerical
parameters are approximations and may vary depending upon the desired
properties sought to be obtained by the present invention. At the very least,
and
not as an attempt to limit the application of the doctrine of equivalents to
the scope
of the claims, each numerical parameter should be construed in light of the
number of significant digits and ordinary rounding approaches.
Unless otherwise indicated, the term "at least" preceding a series of
elements is to be understood to refer to every element in the series. Those
skilled
in the art will recognize, or be able to ascertain using no more than routine
experimentation, many equivalents to the specific embodiments of the invention
described herein. Such equivalents are intended to be encompassed by the
following claims.
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