Polysaccharide conjugates of biomolecules

Details Polysaccharide conjugates of biomolecules Polysaccharide conjugates of biomolecules

1999-05-18

2002-12-03

Gitomer, Ralph (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S059000, C514S367000, C536S051000, C524S054000

Reexamination Certificate

active

06489309

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
In the fields of medicine and clinical chemistry, many studies and determinations of physiologically reactive species such as cells, proteins, enzymes, cofactors, nucleic acids, substrates, antigens, antibodies, and so forth are carried out using conjugates involving specific binding pair members or labels or the like. Various assay techniques that involve the binding of specific binding pair members are known. These assay techniques generally also involve a label used in the detection part of the assay.
Polysaccharides, particularly dextran, have been conjugated to specific binding pair members to increase the stability of the specific binding pair member. Conjugation of these members to polysaccharides also increases the bulkiness of these molecules, which can enhance their effectiveness in assays involving specific binding pair members by interfering with binding to complementary specific binding pair members. Additionally, these conjugates, when present on a surface, permit specific binding of a complementary specific binding pair member to the surface with greatly reduced non-specific binding.
Aminodextran or carboxymethyldextran have usually been utilized for forming conjugates to specific binding pair members. Coupling the dextran to a protein, for example, can then be carried out through formation of an amide. However, aminodextrans and carboxymethyldextrans have a charge that often must be neutralized to control non-specific binding. Such neutralization is difficult to do without derivitizing the conjugate biomolecule at the same time.
An alternative method of conjugation is to first partially oxidize the polysaccharide with periodate to introduce aldehyde groups. Coupling to amine containing ligands and receptors can then be carried out by reductive amination. Although dextrans that are partially oxidized are not charged, the oxidation is difficult to precisely control, and the products have substantially reduced stability toward hydrolysis. A procedure for introducing aldehyde groups onto polysaccharides that does not compromise stability, is more readily carried out, and that permits ready conjugation, is therefore needed.
2. Description of the Related Art
Lauritzen, et al., discuss dot immunobinding and immunoblotting or picogram and nanogram quantities of small peptides on activated nitrocellulose in
Journal of Immunological Methods
(1990) 131:257-267.
Effective immunoadsorbents based on agarose-polyaldehyde microsphere beads: synthesis and affinity chromatography are disclosed by Margel, et al.,
Analytical Biochemistry
(1983) 128:342-350.
U.S. Pat. No. 4,264,766 (Fischer) discloses immunological diagnostic reagents.
U.S. Pat. No. 4,801,504 (Burdick, et al.) discusses fluorescent labels having a polysaccharide bound to polymeric particles.
Polysaccharide-modified immunoglobulins having reduced immunogenic potential or improved pharmacokinetics is discussed in European Patent No. 0 315 456 B1.
Wang, et al., describe a facile synthesis of an aldehydic analog of platelet activating factor and its use in the production of specific antibodies in
Chemistry and Physics of Lipids
(1990) 55:265-273.
SUMMARY OF THE INVENTION
In one aspect, the present invention pertains to polymers comprising a sequence of repeating monosaccharide units, each of which is independently selected from monosaccharide units of the formula:
wherein one of the A's is a bond to the C1 glycosidic carbon of another of the units and the other A's are independently selected from the group consisting of H and QL, wherein L is a linking group linking O and Q and Q is C(Z)═D wherein D is O or CR
1
R
2
wherein R
1
and R
2
are independently H, alkyl or aryl and Z is H or C(Y)═O wherein Y is R
3
, OR
3
or NR
3
R
4
wherein R
3
and R
4
are independently H, alkyl or aryl and wherein any of L, R
1
, R
2
, R
3
and R
4
may be taken together to form a ring with the proviso that at least two of the A's in the polymer are QL. Also included are compounds produced by reaction of the above polymer with a biomolecule such as a polypeptide.
Another aspect of the present invention is a compound comprising the structure:
wherein n is 50 to 50,000, A
1
is independently selected from the group consisting of H and Q
1
L
1
, wherein L
1
is a linking group linking O and Q and Q
1
is C(Z)═D wherein D is O or CR
1
R
2
wherein R
1
and R
2
are independently H, alkyl or aryl and Z is H or C(Y)═O wherein Y is R
3
, OR
3
or NR
3
R
4
wherein R
3
and R
4
are independently H, alkyl or aryl and wherein any of L, R
1
, R
2
, R
3
and R
4
may be taken together to form a ring with the proviso that at least two of the A's in the compound are Q
1
L
1
.
Also included are compounds produced by reaction of the above compound with a polypeptide.
Another aspect of the present invention is a compound that is a modified polysaccharide having pendant aldehyde functionalities wherein each of the aldehyde functionalities is attached through a linker to a position corresponding to a hydrogen atom of a different hydroxyl group of unmodified polysaccharide.
Another embodiment of the present invention is a method for introducing an amine-reactive functionality into a dextran. The method comprises reacting the dextran with an alkylating agent having a functionality that reacts with an hydroxyl group of the dextran thereby forming an alkylated dextran wherein the alkylating agent has an olefin group and treating the alkylated dextran to convert the olefin group to an amine-reactive functionality.
Another embodiment of the present invention is a method for conjugating a polysaccharide to a biomolecule such as a polypeptide. The polysaccharide is reacted with an alkylating agent having a functionality that reacts with a hydroxy group of the polysaccharide thereby forming an alkylated polysaccharide wherein the alkylating agent has an olefin group. The alkylated polysaccharide is treated to convert the olefin group to an amine-reactive functionality, which is reacted with an amine functionality on the biomolecule to produce polysaccharide conjugated to the biomolecule.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention provides a simple, inexpensive method for conjugation of a macromolecule to a polysaccharide for use in an immunoassay. Amine reactive functionalities such as an aldehyde are introduced into the polysaccharide for linking to a macromolecule. One advantage of the present invention is that, unlike prior methods, the number of amine reactive functionalities introduced into the polysaccharide can be controlled.
Before proceeding further with the description of the specific embodiments of the present invention, a number of terms will be defined.
Monosaccharide—a carbohydrate that cannot be hydrolyzed into simpler compounds such as an aldehyde alcohol or a ketone alcohol, e.g., a hexose or a pentose.
Polysaccharide—a carbohydrate containing three or more monosaccharide units; the polysaccharide can be hydrolyzed into the simpler monosaccharide units. Examples of polysaccharides by way of illustration and not limitation are dextran, starch, glycogen, polyribose and the like.
Dextran—a polysaccharide consisting of linear 1-6 linked (98%) glucose units; a polymerized glucose.
Alkyl—a monovalent branched or unbranched radical derived from an aliphatic hydrocarbon by removal of one H atom; includes both lower alkyl and upper alkyl.
Lower alkyl—alkyl containing from 1 to 5 carbon atoms such as, e.g., methyl, ethyl, propyl, butyl, isopropyl, isobutyl, pentyl, isopentyl, etc.
Upper alkyl—alkyl containing more than 6 carbon atoms, usually 6 to 20 carbon atoms, such as, e.g., hexyl, heptyl, octyl, etc.
Alkylidene—a divalent organic radical derived from an aliphatic hydrocarbon, such as, for example, ethylidene, in which 2 hydrogen atoms are taken from the same carbon atom.
Aryl—an organic radical derived from an aromatic hydrocarbon by the removal of one atom and containing one or more aromatic rings, usually one to four aromatic rin

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