Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Binds bacterium or component thereof or substance produced...
Reexamination Certificate
2000-01-21
2001-08-14
Prats, Francisco (Department: 1651)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Binds bacterium or component thereof or substance produced...
C424S164100, C424S178100, C424S243100, C424S244100, C424S234100, C536S123000, C536S123100, C536S124000, C536S018500, C536S063000, C536S054000
Reexamination Certificate
active
06274144
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention is in the general field of methods of preparing saccharide fragments.
Saccharides are important as commodity chemicals and are used often in food and industrial applications. They are also important specialty chemicals in biotechnology, e.g., in the preparation of antibiotics or antibodies, as antigens for vaccines, or as diagnostic reagents.
Saccharides may be obtained from natural sources or synthesized enzymatically or chemically. Synthesis of saccharides having more than about five monosaccharide units often is difficult, especially if one of the units is sialic acid, which is acid labile. Enzymatic synthesis is limited by the available enzymes and substrates and may be relatively expensive.
While natural polysaccharides are sometimes available, in some situations, their use presents problems when they are too large. For example, many food and industrial applications require polysaccharides of specific sizes, and some native polysaccharides may be far too large. In some applications, decreasing the size of the polysaccharide can improve ease of handling and lower production costs. Where a cross-linked saccharide is desired, e.g., to enhance immunogenicity when used in a vaccine, available materials of high molecular weight may form insoluble gels when cross-linked. Reducing the chain length of the starting saccharide can avoid this problem.
Polysaccharides can be cleaved into smaller molecular weight fragments by acid, base, or enzymatic-catalyzed hydrolysis. Acid catalyzed degradation may cleave polysaccharides nonselectively in both carbohydrate and other functional moieties, yielding inconsistent products or non-functional products. For example, sialic acids are found on the carbohydrate moieties of many biologically important polysaccharides and can be determinants of biological functions, including recognition and attachment. They can also be determinants of epitopes for antibody generation and as such should be conserved in attempts to generate saccharide fragments, from polysaccharides. Sialic acids, however, are readily removed by acid.
Enzymatic hydrolysis of polysaccharides can be highly specific but it is usually limited to applications where an enzyme with the desired specificity is readily available. Some saccharide fragments may alternatively be isolated directly from natural sources, but these naturally occurring shorter polysaccharides typically exist in limited quantity. In some cases, saccharide fragments may be chemically and/or enzymatically synthesized. However, even in those cases the enzymes and substrates necessary to conduct the synthesis may be expensive. In general, the synthesis of saccharide fragments of more than five monosaccharide residues can be extremely difficult.
SUMMARY OF THE INVENTION
The invention is based on the discovery that ozone can be used to cleave polysaccharides to yield useful shorter-length saccharide fragments of a desired length, which generally retain structural features of the polysaccharide.
Accordingly, the invention features a method for producing a saccharide fragment by oxidizing a larger polysaccharide having at least one covalent bond between a C1 anomeric carbon of an aldose residue and an oxygen atom of a second monosaccharide residue in a -
D
glycosidic linkage. The method can also be used when the covalent bond is in the form of an -
L
linkage. Typically, the -
D
and -
L
linkages will exhibit similar reactivities. Similarly, the -
D
and -
L
linkages will also exhibit similar reactivities. The method comprises protecting free hydroxyl groups on the larger polysaccharide; reacting the larger polysaccharide with ozone to oxidize the C1 anomeric carbon, thus converting the aldose residue into an aldonic acid ester residue; and cleaving the aldonic acid ester residue to form the saccharide fragment.
In another aspect, the invention features a method of preparing an saccharide fragment by oxidizing a larger polysaccharide having at least one covalent bond between a C1 anomeric carbon of an aldose residue and an oxygen atom of a second monosaccharide residue in an or glycosidic linkage (henceforth, referred to as the “one-step” method).
The glycosidic linkage can be in any form, e.g., -
L
, -
D
, -
L
or -
D
. As is mentioned above, the -
D
and -
L
linkages will typically exhibit similar reactivities, and the -
D
and -
L
linkages will exhibit similar reactivities.
The one-step method comprises reacting the larger polysaccharide with ozone to cleave a bond linking two monosaccharide subunits in the polysaccharide, resulting in the formation of the saccharide fragment.
The larger polysaccharide in the methods described herein can be from any source and can contain labile residues, e.g., sialic acid. Preferably, the larger polysaccharide is substantially pure. The larger polysaccharide is substantially purified when it is separated from those cellular components which accompany it in its natural state. Similarly, the saccharide fragment may optionally be subsequently purified. By a purified saccharide fragment is meant a saccharide fragment separated from the starting polysaccharide.
For purposes of diagnosis and vaccine development, the polysaccharide may be from a bacterial pathogen, e.g., a group B Streptococcus capsular polysaccharide, such as GBS type I, II, III, IV, V, VI, VII, and VIII; the O-antigen of a lipopolysaccharide; a capsular polysaccharide of
Staphylococcus aureus
, e.g., the
Staphylococcus aureus
type 5 or type 8 antigens; the capsular polysaccharide of
Streptococcus pneumonia
; and the capsular polysaccharide of
Bacteroides fragilis.
The ozone can be added in solution, generated in-situ, or be delivered from an external source, e.g., bubbled in.
The aldonic acid ester intermediate can be cleaved by a nucleophile, e.g., a hydroxyl ion, an amine, a thiol, or a carbanion. The aldonic acid ester intermediate may alternatively be cleaved by heating or hydrolysis.
The invention also includes a method for producing antibodies using saccharide fragments produced by ozonolysis. The saccharide fragments can be conjugated to a carrier to create an immunogen, after which the immunogen is injected into a suitable host. Any recognized host is suitable, e.g., rabbit, rat, mouse, goat. Either polyclonal or monoclonal antibodies can be generated.
The invention has many advantages. The methods enable degradation of any polysaccharide containing a glycosidic linkage. The one-step procedure allows ozonolysis to take place in an aqueous solution and without the need for pretreating the starting polysaccharide. In addition, the one-step procedure can be used to depolymerize polysaccharides containing any glycosidic linkage.
If cleavage takes place at the same glycosyl residue, saccharide fragments with the same repeating unit structure can be recovered from abundant, naturally occurring polysaccharides.
Saccharide fragments produced by this method can be easily modified and linked to other molecules (e.g., protein carriers). This can make them useful in drug and vaccine design. The saccharide fragments may also be tagged with chromophores, biotins, peptides, and lipids and thus have diverse potential applications.
A still further advantage of the invention is that it is possible to vary the molecular weight of saccharide fragments generated by varying the ozonolysis conditions. As used herein, “saccharide fragment” is any complex carbohydrate which is formed according to the invention from a starting material which is a “starting polysaccharide”. Thus, while the product is always smaller than the starting material, no particular size limitation is implied on either the starting material or the product. The size of the starting material generally will be dictated by the source of polysaccharide that is readily available. The size of the saccharide fragment will be a function of various factors, such as the desire for a small molecule that is more conveniently adapted to the end use (e.g., solubilized or reacted with labels), consistent with the need to conserve
Hollingsworth Rawle I.
Kasper Dennis L.
Wang Ying
Fish & Richardson P.C.
Prats Francisco
The Brigham and Women's Hospital Inc.
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