Conjugates of defined stoichiometry
View Patent ↗The invention relates to a process for the production of a biomolecule-linker conjugate of uniform stochiometry. It especially relates to a conjugate consisting of a biomolecule of a molecular weight between 5 kD and 500 kD and a hydrophilic linker molecule said linker having a molecular weight between 1 and 15 kD and between 4 and 60 charged residues, characterized in that said conjugate comprises at least one biomolecule-linker product of uniform stoichiometry in a pre-selected amount.
1. A process for production of a fraction of conjugates, each conjugate comprising a biomolecule and a linker and having a pre-defined ratio of biomolecule to linker so that the fraction has a pre-determined and uniform stoichiometry with respect to biomolecule and linker,
wherein the biomolecule has at least two reactive sites, is selected from the group consisting of polypeptides, polysaccharides, and lipopolysaccharides, and, has a molecular weight of between 5 kD and 500 kD, and
wherein the linker is a hydrophilic linker having a molecular weight between 1 kD and 15 kD and containing between 4 and 60 charged residues,
the process comprising:
covalently coupling the biomolecule to the linker, thereby generating a mixture of coupling products comprising the conjugates having the pre-defined ratio of biomolecule to linker,
fractionating by chromatography the mixture of coupling products, and
collecting a fraction comprising the conjugates having the pre-defined ratio of biomolecule to linker, wherein said pre-defined ratio of biomolecule to linker is selected from the group consisting of 1:1, 1:2, 1:3, and 1:4.
2. The process of claim 1 wherein the charged residues are negatively charged.
3. The process of claim 1 wherein the charged residues are positively charged.
4. The process of claim 1 wherein the hydrophilic linker comprises 6 to 50 charged residues.
5. The process of claim 1 wherein the linker comprises a peptide backbone.
6. The process of claim 1 wherein the biomolecule is a polypeptide.
7. The process of claim 1 wherein the chromatographic fractionation is based on differences in apparent molecular weight of the coupling products.
8. The process of claim 7 wherein the linker has an apparent molecular weight between 20% and 500% of that of the biomolecule as determined using molecular sieve chromatography.
9. The process of claim 1 wherein the chromatographic fractionation is based on differences in charge of the coupling products.
10. The process of claim 1 wherein the pre-defined ratio of biomolecule to linker is 1:2.
11. The process of claim 1 wherein the pre-defined ratio of biomolecule to linker is 1:1.
12. The process of claim 1 wherein the linker comprises a β-alanine-glutamic acid peptide.
13. The process of claim 1 wherein the conjugate further comprises a detectable label.
14. The process of claim 1 wherein the conjugate further comprises a member of a bioaffinity binding pair.