The conjugation of proteins with polyethylene glycol and other polymers: Altering properties of proteins to enhance their therapeutic potential

doi:10.1016/0169-409X(93)90005-O

Advanced Drug Delivery Reviews

Volume 10, Issue 1, January–April 1993, Pages 91-114

https://doi.org/10.1016/0169-409X(93)90005-O Get rights and content

Abstract

The development of protein drugs for therapeutic purposes using recombinant DNA technology has generated a great deal of interest in methods of enhancing their delivery. One method of improving the delivery of proteins, and thereby increasing their therapeutic index, is covalent conjugation to polymers. This paper discusses polymers which are suitable for conjugation, methods of conjugation, the properties of polymer-protein conjugates and possibilities for designing drug delivery systems using these conjugates. The properties of protein-polymer conjugates include a dramatic increase in bioavailability, decreased immunogenicity, and enhanced solubility and stability. These altered properties improve the delivery and efficacy of proteins and impart a flexibility to their clinical usage.

References (124)

C.P. Carpenter et al.
Response of dogs to repeated intravenous injection of polyethylene glycol 4000
Toxicol. Appl. Pharmacol.
(1971)
Y.N. Kwee et al.
Anaphylaxis to polyethylene glycol (PEG) in a multivitamin tablet
J. Allergy Clin. Immun.
(1982)
C.O. Beauchamp et al.
A new procedure for the synthesis of polyethylene glycol-protein adducts
Anal. Biochem.
(1983)
M.J. Knauf et al.
Relationship of effective molecular size to systemic clearance in rats of recombinant interleukin-2 chemically modified with water-soluble polymers
J. Biol. Chem.
(1988)
L. Aldwin et al.
A water-soluble monitorable peptide and protein crosslinking agent
Anal. Biochem.
(1987)
S. Zalipsky et al.
Attachment of drugs to polyethylene glycols
Eur. Polymer J.
(1983)
A.J. Garman et al.
The preparation and properties of novel reversible polymer-protein conjugates
FEBS Lett.
(1987)
J. Maxfield et al.
Conformation of poly(ethylene oxide) in the solid state, melt and solution measured by Raman scattering
Polymer
(1975)
E. Boccu et al.
Pharmacokinetic properties of polyethylene glycol derivatized superoxide dismutase
Pharmacol. Res. Commun.
(1982)
A. Koide et al.
Modification of amino groups in porcine pancreatic elastase with polyethylene glycol in relation to binding ability towards anti-serum and to enzymic activity
Biochem. Biophys. Res. Commun.
(1983)

C-J. Jackson et al.

Synthesis, isolation and characterization of conjugates of ovalbumin with monomethoxypolyethylene glycol

Anal. Biochem.

(1987)

M. Kunitani et al.

On-line characterization of polyethylene glycol-modified proteins

J. Chromatogr.

(1991)

Y. Takakura et al.

Control of pharmaceutical properties of soybean trypsin inhibitor by conjugation with dextran: synthesis and characterization

J. Pharm. Sci.

(1989)

Y. Takakura et al.

Biopharmaceutical and pharmacological properties

J. Pharm. Sci.

(1989)

G.W. Hart et al.

Primary structural requirements for the enzymatic formation of the N-glycosidic bond in glycoproteins; studies with natural and synthetic peptides

J. Biol. Chem.

(1979)

C. Ronin et al.

Enzymatic N-glycosylation of synthetic Asn-X-Thr containing peptides

FEBS Lett.

(1978)

T. Suzuki et al.

Physiochemical and biological properties of polyethylene glycol-coupled immunoglobulin G

Biochim. Biophys. Acta.

(1984)

K.J. Wieder et al.

Some properties of polyethylene glycol: phenylalanine ammonia-lyase adducts

J. Biol. Chem.

(1979)

F.H. Brucato et al.

Catabolism of streptokinase and polyethylene glycol-streptokinase: evidence for transport of intact forms through the biliary system in the mouse

Blood

(1990)

R.M. Jackson et al.

Polyethylene glycol-conjugated superoxide dismutase in unilateral lung injury due to re-expansion (re-oxygenation)

Am. J. Med. Sci.

(1990)

M.C. Carter et al.

Instability of Succinyl ester linkages in O′-monosuccinyl cyclic AMP-protein conjugates at neutral pH

J. Immunol. Methods

(1985)

M. Leonard et al.

Acylation of human hemoglobin with polyoxyethylene derivatives

Biochim. Biophys. Acta.

(1984)

K. Kitamura et al.

Polyethylene glycol modification of the monoclonal antibody A7 enhances its tumor localization

Biochem. Biophys. Res. Commun.

(1990)

R.H-L. Chen et al.

Properties of two urate oxidases modified by the covalent attachment of polyethylene glycol

Biochim. Biophys. Acta

(1981)

J.S. Beckman et al.

Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance

J. Biol. Chem.

(1988)

A. Abuchowski et al.

Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase

J. Biol. Chem.

(1977)

K.V. Savoca et al.

Preparation of a non-immunogenic arginase by the covalent attachment of polyethylene glycol

Biochim. Biophys. Acta

(1979)

A. Abuchowski et al.

Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol

J. Biol. Chem.

(1977)

I. Wilkinson et al.

Tolerogenic polyethylene glycol derivatives of xenogeneic monoclonal immunoglobulins

Immunol. Lett.

(1987)

W. Regelson

The biologic activity of water-soluble polymers

C.G. Gebelein

Bioactive polymeric systems; an overview

S.R. Sandler et al.

J.M. Harris

Laboratory synthesis of polyethylene glycol derivatives

Rev. Macromol. Chem. Phys.

(1985)

H.F. Symth et al.

Toxicology of polyethylene glycols

J. Am. Pharm. Assoc. Sci.

(1950)

A. Fisher

Immediate and delayed allergic contact reactions to polyethylene glycol

Contact Derma.

(1978)

A. Abuchowski et al.

Soluble polymer-enzyme adducts

K. Savoca et al.

Enzyme-polyethylene glycol adducts: modified enzymes with unique properties

F.F. Davis et al.

Soluble nonantigenic polyethylene glycol-bound enzymes

A. Abuchowski et al.

Cancer therapy with chemically modified enzymes

Cancer Biochem. Biophys.

(1984)

S. Zalipsky et al.

Evaluation of a new reagent for covalent attachment of polyethylene glycol to proteins

Biotech. Appl. Biochem.

(1992)

N.V. Katre et al.

Chemical modification of recombinant interleukin-2 by polyethylene glycol increases its potency in the murine meth a sarcoma model

P.J. Shadle et al.

Conjugation of Polymer to Colony Stimulating Factor-1

(1989)

T.P. King et al.

Preparation of protein conjugates with alkoxypolyethylene glycols

Int. J. Peptide Protein Res.

(1980)

S. Dreborg et al.

Immunotherapy with monomethoxypolyethylene glycol modified allergens

Crit. Rev Ther. Drug Carrier Syst.

(1990)

B.A. Andrews et al.

PEG activation and ligand binding for the affinity partitioning of proteins in aqueous two-phase systems

Biotech. Tech.

(1990)

W.Y. Lee et al.

Suppression of reaginic antibodies

Immunol. Rev.

(1978)

F.M. Veronese et al.

Surface modification of proteins: activation of monomethoxypolyethylene glycols by phenylchloroformates

Appl. Biochem. Biotech.

(1985)

D.E. Nitecki et al.

Preparation of a Polymer/interleukin-2 Conjugate

(1990)

S. Zalipsky et al.

Succinimidyl carbonates of polyethylene glycol: useful reactive polymers for preparation of protein conjugates

Polymer Prepr.

(1990)

E. Boccu et al.

Coupling of monomethoxypolyethylene glycols to proteins via active esters

Z. Naturforsch.

(1983)

Cited by (264)

Methacrylated fibrinogen hydrogels for 3D cell culture and delivery
2023, Acta Biomaterialia
Methacrylation was performed on fibrinogen to design a new biomedical hydrogel for 3D cell culture or as a biodegradable delivery matrix for in vivo implantation. The methacrylation of denatured fibrinogen in solution was performed using methacrylic anhydride (MAA). The extent of fibrinogen methacrylation was quantified by proton NMR and controlled using stochiometric quantities of MAA during the reaction. The methacrylated fibrinogen (FibMA) hydrogels were formed by light-activated free-radical polymerization in the presence of macromolecular cross-linking polymers made from acrylated poly(ethylene glycol) (PEG). The biocompatibility and biodegradability of the FibMA hydrogels were characterized by in vitro assays and in vivo implantation experiments using quantitative magnetic resonance imaging (MRI) of the implant volume. The FibMA supported the growth and metabolic activity of human dermal fibroblasts in both 2D and 3D cultures. The methacrylation did not alter important biological attributes of the fibrinogen, including the ability to support cell adhesion and 3D cell culture, as well as to undergo proteolysis. Animal experiments confirmed the biodegradability of the FibMA for potential use as a scaffold in tissue engineering, as a bioink for 3D bioprinting, or as a biodegradable matrix for in vivo sustained delivery of bioactive factors.
This paper describes methacrylated fibrinogen (FibMA) and the formation of a biomedical hydrogel from FibMA for cell culture and other biomedical applications. Inspired from methacrylated gelatin (GelMA), the FibMA is made from blood-derived fibrinogen which is more suitable for clinical use. Sharing similar properties to other hydrogels made from methacrylated proteins, the FibMA has yet to be reported in the literature. In this manuscript, we provide the methodology to produce the FibMA hydrogels, we document the mechanical versatility of this new biomaterial, and we show the biocompatibility using 3D cell culture studies and in vivo implantations.
Main-chain water-soluble polyphosphoesters: Multi-functional polymers as degradable PEG-alternatives for biomedical applications
2020, European Polymer Journal
Polyphosphoesters (PPEs) are a class of (bio)degradable polymers with high chemical versatility and functionality. In particular, water-soluble PPEs with the phosphoester group in the polymer backbone are currently discussed as a potential alternative to poly(ethylene glycol) (PEG). Ring-opening polymerization of typically 5-membered cyclic phosphoesters gives straightforward access to various well-defined PPEs. Several PPE candidates have proven their biocompatibility in vitro in terms of cytocompatibility, antifouling properties, “stealth effect”, degradability (hydrolytic and enzymatic), and some promising in vivo results in drug delivery vehicles. The possibility to control the properties with the appropriate tuning of the lateral chain makes PPEs especially appealing. This review summarizes recent developments of such PPEs for biomedical applications, e.g. in protein-polymer conjugates, hydrogels for tissue engineering, or nanocarriers for drug and gene delivery. We summarize the progress made over the years, highlighting the strengths and the shortcomings of PPEs for these applications to date. We critically evaluate the current state of the art, try to assess their potential and to predict future perspectives, shedding light on the pathway that needs to be followed to translate into clinics.
Conjugation of Amine-Functionalized Polyesters With Dimethylcasein Using Microbial Transglutaminase
2020, Journal of Pharmaceutical Sciences
Protein-polymer conjugates have been used as therapeutics because they exhibit frequently higher stability, prolonged in vivo half-life, and lower immunogenicity compared with native proteins. The first part of this report describes the enzymatic synthesis of poly(glycerol adipate) (PGA(M)) by transesterification between glycerol and dimethyl adipate using lipase B from Candida antarctica. PGA(M) is a hydrophilic, biodegradable but water insoluble polyester. By acylation, PGA(M) is modified with 6-(Fmoc-amino)hexanoic acid and with hydrophilic poly(ethylene glycol) side chains (mPEG12) rendering the polymer highly water soluble. This is followed by the removal of protecting groups, fluorenylmethyloxycarbonyl, to generate polyester with primary amine groups, namely PGA(M)-g-NH₂-g-mPEG12. ¹H NMR spectroscopy, FTIR spectroscopy, and gel permeation chromatography have been used to determine the chemical structure and polydispersity index of PGA(M) before and after modification. In the second part, we discuss the microbial transglutaminase–mediated conjugation of the model protein dimethylcasein with PGA(M)-g-NH₂-g-mPEG12 under mild reaction conditions. SDS-PAGE proves the protein-polyester conjugation.
N-terminal site-specific PEGylation enhances the circulation half-life of Thymosin alpha 1
2019, Journal of Drug Delivery Science and Technology
Covalent attachment of polyethylene glycol (PEG) to peptides and proteins has been used to increase the clinical efficacy and circulation half-life of biopharmaceuticals. The purpose of this study was to enhance the half-life of an important immune-modulating agent, Thymosin alpha 1 (Tα1), using site-specific PEGylation. A novel N-terminal modified Tα1 (Mal-Tα1) was constituted by condensing the N-terminal amino group of Tα1 with 3-maleimidopropionic acid (Mal). Then, Mal-Tα1 was site-specifically conjugated with methoxy polyethylene glycol thiol (mPEG-SH) under mild conditions to form mPEG-Mal-Tα1. The conjugation efficiency was greater than 95%. mPEG-Mal-Tα1 was stable in the presence of 2-mercaptoethanol and exhibited significantly increased immunoactivity compared to Tα1 in mice. The terminal half-life (t_1/2β) of mPEG-Mal-Tα1 was 2.5 times longer than that of mPEG-Cys-Tα1 (PEGylation product of mPEG-Mal conjugated with Cysteine-Tα1) in rats. These results demonstrate that site-specific PEGylation of N-terminal Mal-Tα1 is a promising strategy for enhancing the circulation t_1/2β of Tα1 compared to Cysteine-Tα1.
Degradation versus resorption
2019, Materials for Biomedical Engineering: Absorbable Polymers
The intrinsic ability of pristine natural and synthetic polymers or composite and hybrid macromolecular compounds to provide biomedical-related tunability and functionality enables outstanding effects in modern healthcare applications. Besides the complex implications related to biocompatibility concept, degradation and resorption of biomaterials represent key factors within the personalized therapy aim of modern biomedicine.
Thanks to their compositional and structural peculiarity, as well as to their physicochemical and biofunctional versatility, different polymers proved superior performances in various biomedical applications, including pharmaceutical and curative formulations, systemic disorders and cancer management, and restorative or regenerative tissue therapy.
Within this chapter, important aspects regarding the intricate degradation and resorption mechanisms of biopolymers will be discussed. Moreover, their significant implications in drug delivery and tissue engineering applications will be outlined, together with relevant examples of polymer-based biomaterials and devices for modern biomedicine.
Site-specific polymer-protein conjugates by Cys mutation
2019, Polymer-Protein Conjugates: From Pegylation and Beyond
The attachment of water-soluble polymers, such as polyethylene glycol (PEG), to proteins has been an area of exploration for several decades. With the advent of recombinant technology, it became possible to engineer specific amino acid residues into the amino acid sequence of protein therapeutics for polymer conjugation to improve their efficacy. This chapter will cover the evolution of site-specific conjugation of PEGs to cysteine residues engineered on protein therapeutics. The specific attachment of PEG (PEGylation) to cysteine residues is a very viable method because cysteine residues in proteins are less than 1% of the total amino acid content, and most of them are involved in disulfide bonds. The selection of the site(s) on a protein for cysteine mutation to covalently attach a polymer without the loss of its bioactivity is a critical step in the process of enhancing the therapeutic potential of the protein. The approaches to this selection, the various Cys-directed PEGylation methodologies, and the future of Cys-PEGylation will be discussed.

View all citing articles on Scopus

View full text

The conjugation of proteins with polyethylene glycol and other polymers: Altering properties of proteins to enhance their therapeutic potential

Abstract

Toxicol. Appl. Pharmacol.

J. Allergy Clin. Immun.

Anal. Biochem.

J. Biol. Chem.

Anal. Biochem.

Eur. Polymer J.

FEBS Lett.

Polymer

Pharmacol. Res. Commun.

Biochem. Biophys. Res. Commun.

Anal. Biochem.

J. Chromatogr.

J. Pharm. Sci.

J. Pharm. Sci.

J. Biol. Chem.

FEBS Lett.

Biochim. Biophys. Acta.

J. Biol. Chem.

Blood

Am. J. Med. Sci.

J. Immunol. Methods

Biochim. Biophys. Acta.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Immunol. Lett.

The biologic activity of water-soluble polymers

Bioactive polymeric systems; an overview

Laboratory synthesis of polyethylene glycol derivatives

Rev. Macromol. Chem. Phys.

Toxicology of polyethylene glycols

J. Am. Pharm. Assoc. Sci.

Immediate and delayed allergic contact reactions to polyethylene glycol

Contact Derma.

Soluble polymer-enzyme adducts

Enzyme-polyethylene glycol adducts: modified enzymes with unique properties

Soluble nonantigenic polyethylene glycol-bound enzymes

Cancer therapy with chemically modified enzymes

Cancer Biochem. Biophys.

Evaluation of a new reagent for covalent attachment of polyethylene glycol to proteins

Biotech. Appl. Biochem.

Chemical modification of recombinant interleukin-2 by polyethylene glycol increases its potency in the murine meth a sarcoma model

Conjugation of Polymer to Colony Stimulating Factor-1

Preparation of protein conjugates with alkoxypolyethylene glycols

Int. J. Peptide Protein Res.

Immunotherapy with monomethoxypolyethylene glycol modified allergens

Crit. Rev Ther. Drug Carrier Syst.

PEG activation and ligand binding for the affinity partitioning of proteins in aqueous two-phase systems

Biotech. Tech.

Suppression of reaginic antibodies

Immunol. Rev.

Surface modification of proteins: activation of monomethoxypolyethylene glycols by phenylchloroformates

Appl. Biochem. Biotech.

Preparation of a Polymer/interleukin-2 Conjugate

Succinimidyl carbonates of polyethylene glycol: useful reactive polymers for preparation of protein conjugates

Polymer Prepr.

Coupling of monomethoxypolyethylene glycols to proteins via active esters

Z. Naturforsch.