WO1999017120A1 - Preparing conjugates using polyethylene glycol linkers - Google Patents

Preparing conjugates using polyethylene glycol linkers Download PDF

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Publication number
WO1999017120A1
WO1999017120A1 PCT/US1998/019716 US9819716W WO9917120A1 WO 1999017120 A1 WO1999017120 A1 WO 1999017120A1 US 9819716 W US9819716 W US 9819716W WO 9917120 A1 WO9917120 A1 WO 9917120A1
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WIPO (PCT)
Prior art keywords
substituent
polyethylene glycol
solid surface
bead
peg
Prior art date
Application number
PCT/US1998/019716
Other languages
French (fr)
Inventor
Kenneth A. Davis
James E. Bishop
Original Assignee
Becton, Dickinson And Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Becton, Dickinson And Company filed Critical Becton, Dickinson And Company
Priority to AU94021/98A priority Critical patent/AU9402198A/en
Publication of WO1999017120A1 publication Critical patent/WO1999017120A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6943—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a pill, a tablet, a lozenge or a capsule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface

Definitions

  • the predominant recognition methodology involves the use of molecular species which recognize the target.
  • protein molecules that recognize a specific target can be generated as antibodies. Such antibodies can then be used to recognize the target and bind to it.
  • labeling techniques By use of appropriate labeling techniques, the bound target can be identified and/or separated from the remaining population.
  • flow cytometry is uniquely suited for such applications due to its capability to perform simultaneous multiparameter analysis and to separate (or sort) unique cell populations from heterogeneous mixtures of cells.
  • the critical parameter is the recognition of the antigen or target. This is strongly affected by the conformation of the target, and, in general, it is essential to have the target in a confirmation resembling its native conformation.
  • the instant invention presents a rapid simple method for preparing solid phases, preferably beads, with antigens or other substituents presented on the surface in such a manner that the antigens/substituents retain their original functionality and conformation, as well as much of their native structure, to permit their use in a wide array of applications.
  • the substituent is attached to the surface of the solid phase by using a bifunctional derivative of polyethylene glycol.
  • the polyethylene glycol (PEG) acts not only to facilitate the attachment of the substituent to the solid surface, but also acts as a buffer to prevent or reduce any interaction of the solid surface with the attached substituent or, indeed, with any other biological compounds to which it may become exposed during the use of the solid surface conjugates.
  • the polyethylene glycol has a dual function, the first being that of a linking group facilitating the attachment of the substituent to the solid phase, and the second being that of maintaining, to the greatest extent possible, the native conformation of the substituent. While not wishing to be bound by theory, it is believed that this is the result of three properties conveyed by the PEG, namely: additional flexibility of the attached components due to the linking group; a reduction in the conformational distortion of the substituent due at least partially to a reduction in the "sticking" of the other portions of the substituent to the solid surface; and a reduction in the non-specific binding of other proteins and similar materials to the solid surface.
  • the complexes of the instant invention are formed by attaching the substituent(s) to the solid phase by the use of the PEG as a bifunctional derivative to link the substituent to the solid phase.
  • the solid phase must have a surface functional group available to facilitate the bonding of the PEG.
  • the solid phase is a bead which possesses amine (NH2) functionalities.
  • NH2 amine
  • a preferred bead of this type is a 6 micron polymethylmethacrylate (PMMA-NH2) bead.
  • PMMA-NH2 polymethylmethacrylate
  • the choice of the solid phase will be governed by the specific applications intended. A variety of substituents can, thus , be attached to the solid phase by this methodology.
  • substituents include, but are not limited to, fiuorophores (such as phycoeryhtrin), streptavidin (which can be used to bind biotinylated ligands), oligonucleotides, and biological compounds such as proteins and nucleic acids. Indeed, so long as the substituent will bind, or can be chemically modified to permit it to bind, to the PEG, the methodology can be utilized.
  • the PEG utilized must be of a sufficient size to convey the desired properties, but not so large as to significantly hinder the kinetics of derivitization, attachment of the substituent, or the functionality of the attached substituents.
  • a molecular weight of 1,000-5,000, more preferably 2,000-3,000, and even more preferably 2,000 would be used, but it is o be understood that PEG polymers of even higher and/or lower molecular weights can be used depending on the particular application.
  • succimidyl propionate-PEG-2000- orthopyridyl-disulfide OPSS-PEG2000-SPA
  • Shearwater Polymers Huntsville, AL
  • other functional moieties on the PEG termini may be used as the particular application dictates.
  • the benefits of the OPPS-PEG2000-SPA are two-fold, namely, to activate the solid surface in order to facilitate the attachment of the substituent, and to provide a "barrier layer" between the solid phase and the substituent(s) on the surface or components in the surrounding medium.
  • the functional groups on the PEG reagent will be selected based on the chemistry of the solid surface and the desired functionality for substituent attachment.
  • the solid phase will have a primary amine group (preferably a PMMA-NH2) and the one end of the PEG molecule will have a succinimidyl group, thereby facilitating formation of an amino ester (peptide) linkage between the solid surface and the PEG molecule.
  • the PEG molecule has a protected sulfhydryl group (preferably orthopyridyl-disulfide)which can be deprotected by reduction with DTT and, thus, be available to form a thioether link with a maleimide derivatized substituent or, which could react, intact, with a sulfhydryl derivatized substituent by disulfide interchange.
  • a protected sulfhydryl group preferably orthopyridyl-disulfide
  • PEG attachment methodology to provide a PEG spacer between the solid phase and the substituent also will facilitate the binding of ligands or molecules to the substituent, help to retain the native properties of the bound substituents; and enhance mobility, and thus accessibility, of the attached substituents. Further, the specificity of bonding to the substituent(s) is enhanced, because the PEG linker provides a "barrier" to reduce the non-specific binding of other species to the solid surface.
  • Polymethacrylate amino beads (6.1 microns PMMA-NH2), at a concentration of 1.5% w/v, were admixed with OPSS-PEG2000-SPA, at a concentration of 5 mM, in an aqueous buffer containing 50 mM sodium phosphate/ 150 mM sodium chloride/ 1 mM EDTA 0.01% w/v Tween 20, adjusted to a pH of 7.7, and reacted at room temperature for 2 hours.
  • the beads were subsequently washed with reaction buffer, reduced with 25mM dithiothreitol, and subsequently washed with 50 mM MES (2-(N- morpholino)ethanesulfonic acid) /150mM sodium chloride/1 mM EDTA/0.01 %w/v Tween 20 adjusted to a pH of 6.0.
  • PE phycoerythrin
  • BSA-maleimide both PE and BSA maleimide were synthesized using succinimidyl 4-(N-maleimido-methyl)-cyclohexane-l-carboxylate (SMCC) (Pierce Biochemical Corp) to arrive at a protein to bead ratio of 5 micrograms per square centimeter of bead surface area.
  • SCC succinimidyl 4-(N-maleimido-methyl)-cyclohexane-l-carboxylate
  • the ratio of maleimide PE to maleimide BSA was adjusted to achieve the desired number of PE molecules per bead. Residual -SH groups were capped by reacting with 0.5 mM N-ethylmaleimide for 15 minutes at room temperature.
  • the resultant beads were washed into PBS/0.1% sodium azide/0.2% gelatin/0.01% w/v Tween 20.
  • the beads prepared by this method exhibited more uniform fluorescence and unaltered emission characteristics (essentially indistinguishable from that of PE- conjugated antibodies)and greater stability than PE beads made with shorter linkers.
  • Example 2 Preparation of Streptavidin Beads with a PEG2000 Spacer The beads were prepared by the same synthetic procedure as described in Example
  • biotin-PerCP as did conventional beads with a shorter spacer. While not wishing to be bound by theory, it is postulated that this is due to the greater accessibility of streptavidin on the PEG spacer, versus streptavidin held onto the bead surface with a shorter spacer.
  • non-specific binding e.g. IgGl to PerCP or PE
  • Example 3 Synthesis of oligonucleotide beads with a PEG2000 spacer
  • the beads were prepared by the same synthetic procedure as described in Example 1, except that the OPSS-PEG2000-SPA labeled, reduced, and washed beads were reacted with 120 pmole/cm2 of 5'-maleimido oligonucleotide, which was previously synthesized by the reaction of the 5' NH2-22 mer oligonucleotide with SMCC.
  • the beads prepared by this method bound FITC labeled complimentary oligonucleotide with a low CV and low non-specific binding.

Abstract

The instant invention presents a rapid simple method for preparing solid phases, preferably beads, with antigens or other substituents presented on the surface in such a manner that the antigens/substituents retain their original functionality and conformation, as well as much of their native structure, to permit their use in a wide array of applications. Specifically, the substituent is attached to the surface of the solid phase by using a bifunctional derivative of polyethylene glycol. The polyethylene glycol (PEG), it has been found, acts not only to facilitate the attachment of the substituent to the solid surface, but also acts as a buffer to prevent or reduce any interaction of the solid surface with the attached substituent or, indeed, with any other biological compounds to which it may become exposed during the use of the solid surface conjugates.

Description

PREPARING CONJUGATES USING POLYETHYLENE GLYCOL LINKERS
BACKGROUND OF INVENTION
Specific and high affinity molecular recognition is critical for a variety of diagnostic applications. The predominant recognition methodology involves the use of molecular species which recognize the target. For example, protein molecules that recognize a specific target can be generated as antibodies. Such antibodies can then be used to recognize the target and bind to it. By use of appropriate labeling techniques, the bound target can be identified and/or separated from the remaining population. Among such separation techniques, flow cytometry is uniquely suited for such applications due to its capability to perform simultaneous multiparameter analysis and to separate (or sort) unique cell populations from heterogeneous mixtures of cells.
Other techniques similarly utilize this technology. However, in each, the critical parameter is the recognition of the antigen or target. This is strongly affected by the conformation of the target, and, in general, it is essential to have the target in a confirmation resembling its native conformation.
As conformation is strongly influenced by the environment of a molecule, it becomes extremely difficult to attach a target molecule (or substituent) to a bead or other structure to facilitate its use in a desired application, yet retain a confirmation similar to its native conformation.
In addition, for many applications in flow cytometry, it is useful to utilize beads having other substituents such as fiuorophores attached to the surface. Thus, it would be useful to have an attachment methodology which is also amenable to attaching such other substituents to the surface of the beads in addition to the antibodies. SUMMARY OF INVENTION
The instant invention presents a rapid simple method for preparing solid phases, preferably beads, with antigens or other substituents presented on the surface in such a manner that the antigens/substituents retain their original functionality and conformation, as well as much of their native structure, to permit their use in a wide array of applications. Specifically, the substituent is attached to the surface of the solid phase by using a bifunctional derivative of polyethylene glycol. The polyethylene glycol (PEG),it has been found, acts not only to facilitate the attachment of the substituent to the solid surface, but also acts as a buffer to prevent or reduce any interaction of the solid surface with the attached substituent or, indeed, with any other biological compounds to which it may become exposed during the use of the solid surface conjugates.
Thus, the polyethylene glycol has a dual function, the first being that of a linking group facilitating the attachment of the substituent to the solid phase, and the second being that of maintaining, to the greatest extent possible, the native conformation of the substituent. While not wishing to be bound by theory, it is believed that this is the result of three properties conveyed by the PEG, namely: additional flexibility of the attached components due to the linking group; a reduction in the conformational distortion of the substituent due at least partially to a reduction in the "sticking" of the other portions of the substituent to the solid surface; and a reduction in the non-specific binding of other proteins and similar materials to the solid surface.
DETAILED DESCRIPTION OF THE INVENTION
The complexes of the instant invention are formed by attaching the substituent(s) to the solid phase by the use of the PEG as a bifunctional derivative to link the substituent to the solid phase. As such, the solid phase must have a surface functional group available to facilitate the bonding of the PEG. In a preferred embodiment of this invention, the solid phase is a bead which possesses amine (NH2) functionalities. A preferred bead of this type is a 6 micron polymethylmethacrylate (PMMA-NH2) bead. However, the choice of the solid phase will be governed by the specific applications intended. A variety of substituents can, thus , be attached to the solid phase by this methodology. Such substituents include, but are not limited to, fiuorophores (such as phycoeryhtrin), streptavidin (which can be used to bind biotinylated ligands), oligonucleotides, and biological compounds such as proteins and nucleic acids. Indeed, so long as the substituent will bind, or can be chemically modified to permit it to bind, to the PEG, the methodology can be utilized.
The PEG utilized must be of a sufficient size to convey the desired properties, but not so large as to significantly hinder the kinetics of derivitization, attachment of the substituent, or the functionality of the attached substituents. In general, a molecular weight of 1,000-5,000, more preferably 2,000-3,000, and even more preferably 2,000 would be used, but it is o be understood that PEG polymers of even higher and/or lower molecular weights can be used depending on the particular application. While the preferred heterobifuctional reagent of this invention, succimidyl propionate-PEG-2000- orthopyridyl-disulfide (OPSS-PEG2000-SPA) is available commercially (Shearwater Polymers, Huntsville, AL), other functional moieties on the PEG termini may be used as the particular application dictates. The benefits of the OPPS-PEG2000-SPA are two-fold, namely, to activate the solid surface in order to facilitate the attachment of the substituent, and to provide a "barrier layer" between the solid phase and the substituent(s) on the surface or components in the surrounding medium.
Similarly, the functional groups on the PEG reagent will be selected based on the chemistry of the solid surface and the desired functionality for substituent attachment. In a preferred embodiment, the solid phase will have a primary amine group (preferably a PMMA-NH2) and the one end of the PEG molecule will have a succinimidyl group, thereby facilitating formation of an amino ester (peptide) linkage between the solid surface and the PEG molecule. Further, in this preferred embodiment, the PEG molecule has a protected sulfhydryl group (preferably orthopyridyl-disulfide)which can be deprotected by reduction with DTT and, thus, be available to form a thioether link with a maleimide derivatized substituent or, which could react, intact, with a sulfhydryl derivatized substituent by disulfide interchange.
The use of the PEG attachment methodology to provide a PEG spacer between the solid phase and the substituent also will facilitate the binding of ligands or molecules to the substituent, help to retain the native properties of the bound substituents; and enhance mobility, and thus accessibility, of the attached substituents. Further, the specificity of bonding to the substituent(s) is enhanced, because the PEG linker provides a "barrier" to reduce the non-specific binding of other species to the solid surface.
Examples The following examples illustrate certain preferred embodiments of the instant invention, but are not intended to be illustrative of all embodiments.
Example 1 - Preparation of phycoeryhtrin coated beads with a PEG 2000 linker
Polymethacrylate amino beads (6.1 microns PMMA-NH2), at a concentration of 1.5% w/v, were admixed with OPSS-PEG2000-SPA, at a concentration of 5 mM, in an aqueous buffer containing 50 mM sodium phosphate/ 150 mM sodium chloride/ 1 mM EDTA 0.01% w/v Tween 20, adjusted to a pH of 7.7, and reacted at room temperature for 2 hours. The beads were subsequently washed with reaction buffer, reduced with 25mM dithiothreitol, and subsequently washed with 50 mM MES (2-(N- morpholino)ethanesulfonic acid) /150mM sodium chloride/1 mM EDTA/0.01 %w/v Tween 20 adjusted to a pH of 6.0. The resultant beads were then reacted at room temperature fro 2 hours with PE (phycoerythrin)-maleimide and BSA-maleimide (both PE and BSA maleimide were synthesized using succinimidyl 4-(N-maleimido-methyl)-cyclohexane-l-carboxylate (SMCC) (Pierce Biochemical Corp) to arrive at a protein to bead ratio of 5 micrograms per square centimeter of bead surface area. The ratio of maleimide PE to maleimide BSA was adjusted to achieve the desired number of PE molecules per bead. Residual -SH groups were capped by reacting with 0.5 mM N-ethylmaleimide for 15 minutes at room temperature. The resultant beads were washed into PBS/0.1% sodium azide/0.2% gelatin/0.01% w/v Tween 20. The beads prepared by this method exhibited more uniform fluorescence and unaltered emission characteristics (essentially indistinguishable from that of PE- conjugated antibodies)and greater stability than PE beads made with shorter linkers.
Example 2 -Preparation of Streptavidin Beads with a PEG2000 Spacer The beads were prepared by the same synthetic procedure as described in Example
1, except that the OPSS-PEG2000-SPA labeled, reduced, and washed beads were reacted with streptavidin maleimide at 5 micrograms per square centimeter bead surface area. The beads prepared by this method had about twice the ligand binding capacity
(biotin-PerCP) as did conventional beads with a shorter spacer. While not wishing to be bound by theory, it is postulated that this is due to the greater accessibility of streptavidin on the PEG spacer, versus streptavidin held onto the bead surface with a shorter spacer.
Further, non-specific binding (e.g. IgGl to PerCP or PE) was very low; this was attained without the use of blocking buffers or proteins.
Example 3 -Synthesis of oligonucleotide beads with a PEG2000 spacer The beads were prepared by the same synthetic procedure as described in Example 1, except that the OPSS-PEG2000-SPA labeled, reduced, and washed beads were reacted with 120 pmole/cm2 of 5'-maleimido oligonucleotide, which was previously synthesized by the reaction of the 5' NH2-22 mer oligonucleotide with SMCC. The beads prepared by this method bound FITC labeled complimentary oligonucleotide with a low CV and low non-specific binding.
Example 4 - Preparation of Bead Conjugates using other PEG linkers
Bead conjugates were made using the synthetic procedure of Example 1, except that PEG 1000 and PEG 20,000 were substituted for the PEG2000. It was found that the PEG 1000 gave most of the benefits found with the PEG2000, except that the syntheses were not always reproducible, the PEG 20,000 gave acceptable results, but was difficult to synthesize due to the high viscosity of the reaction solution and slower reaction kinetics.
It is apparent that many modifications and variations of this invention as hereinabove set forth may be made without departing from the spirit and scope thereof. The specific embodiments are given by way of example only and the invention is limited only by the terms of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. An improved method for conjugating a substituent to a solid surface comprising attaching a bifunctional derivative of a polyethylene glycol to said solid surface and said substituent such that said bifunctional derivative of polyethylene glycol is a linking agent between said solid surface and said substituent.
2. The method of Claim 1 wherein the solid surface is a bead.
3. The method of Claim 2 wherein the bead possesses amine surface functionalities.
4. The method of Claim 3 wherein the bead is a polymethylmethacrylate bead.
5. The method of Claim 1 wherein the substituent is selected from the group consisting of fiuorophores, streptavidin, and biological compounds.
6. The method of Claim 5 wherein the substituent is a fluorophore selected from the group consisting of phycoerythrin and perdinin chlorophyll protein.
7. The method of Claim 5 wherein the substituent is a biological compound selected from the group consisting of proteins and nucleic acids.
8. The method of Claim 7 wherein the substituent is an oligonucleotide.
9. The method of Claim 7 wherein the substituent is an antibody.
10. The method of Claim 9 wherein the antibody is a monoclonal antibody.
11. The method of Claim 1 wherein the polyethylene glycol has a ? molecular weight of about 1,000-about 5,000.
12. The method of Claim 11 wherein the polyethylene glycol has a ? molecular weight of about 2000.
13. The method of Claim 1 wherein the polyethylene glycol is derivatized with a maleimide compound.
14. The method of Claim 13 wherein the maleimide compound is succinimidyl propionyl dithiopyridine.
PCT/US1998/019716 1997-09-26 1998-09-21 Preparing conjugates using polyethylene glycol linkers WO1999017120A1 (en)

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EP2415486A2 (en) 2001-05-18 2012-02-08 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
US7491805B2 (en) 2001-05-18 2009-02-17 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
US7833992B2 (en) 2001-05-18 2010-11-16 Merck Sharpe & Dohme Conjugates and compositions for cellular delivery
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