3. Poly(ethylene glycol) – the Pioneer in Polymer Therapeutics

PEGs show a spectrum of unique physical and chemical properties, which have been described in literature extensively by the pioneers in PEGylation: Harris, Veronese and recently by Hermanson. Here are summarized the most common known properties.

• PEG fragments can be attached to many different positions in a protein. Amino groups of any solvent accessible lysines as well as the N-termini are the most prominent candidates for conjugation together with thiol functions of available cysteins. The C-terminus or carboxylic groups from aspartic acid and glutamic acid in theory are also possible for conjugation, however, are rarely used.
• PEG can also serve as spacer or cross linker between two moieties.
• PEG provides high solubility and does not contain charged side chains.
• PEG is FDA approved for internal application, is nontoxic, lacks T-cell epitopes, and shows no signs of immunogenicity in animal experiments.
• PEG derivatives are available from pure, monodisperse, discrete molecules with short chain lengths or even one ethylene oxide unit only, to long polydisperse both linear and branched constructs, allowing regio-specific chemical conjugation with small molecules, proteins, peptides and biopharmaceuticals through their broad variety of terminal chemical groups available.

Chemical and Physical Properties of PEGs:

• Good solubility in BOTH hydrophilic AND hydrophobic solvents as: water, toluene, methylene chloride, and many other organic solvents.
• Insoluble in: diethyl ether, hexane, ethylene glycol.
• The solubility is influenced by forming derivatives.
• Highly mobile in water with high exclusion volume; large hydrodynamic radius.
• Form complexes with metal cations.
• Can be used to precipitate proteins and nucleic acids.
• Form two-phase system with aqueous solutions of other polymers.
• Non-toxic, FDA approved for internal consumption. PEGylating Biopharmaceuticals and Small Molecules has the following effects:
• Improves solubility of conjugated molecules.
• Renders proteins non immunogenic and toleragenic.
• Reduces the rate of renal clearance through the kidney and alters pharmacokinetics.
• Renders surface protein rejection.
• Alters electro osmotic flow.
• Moves molecules across cell membranes.

  Table: PEG conjugates in the pharmaceutical market*

  

  * References: 

  • The dawning era of polymer therapeutics; R. Duncan; Nat Rev Drug Discov 2003; 2: 347-360.
  • PEGylation, successful approach to drug delivery; F. M. Veronese and G. Pasut; Drug Discovery Today 2005; 10: 1451-1458. doi:10.1016/S1359- 6446(05)03575-0
  • Poly(ethylene glycol) in Drug Delivery: Pros and Cons as Well as Potential Alternatives; K. Knop, R. Hoogenboom, D. Fischer and U. S. Schubert; Angew Chem. Int. Ed. 2010; 49: 6288-6308. doi:10.1002/anie.200902672
  • Poly(ethylene glycol)-Prodrug Conjugates: Concept, Design, and Applications; S. S. Banerjee, N. Aher, R. Patil and J. Khandare; J Drug Deliv. 2012; 2012: 17. doi:10.1155/2012/103973

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  General References to PEGylation: 

  • Poly(ethylene glycol) chemistry biotechnical and biomedical applications J. Milton Harris, Ed; G. Whitesides; Appl Biochem Biotechnol 1993; 41: 233-234. doi:10.1007/bf02916424
  • Peptide and Protein PEGylation III: Advances in Chemistry and Clinical Applications; F. M. Veronese and J. M. Harris; Adv Drug Deliv Rev 2008; 60: 1-88.
  • PEGylation, successful approach to drug delivery; F. M. Veronese and G. Pasut; Drug Discovery Today 2005; 10: 1451-1458. doi:10.1016/ S1359-6446(05)03575-0
  • Introduction and overview of peptide and protein pegylation; F. M. Veronese and G. Pasut; Adv Drug Deliv Rev 2002; 54: 453-456. doi:10.1016/S0169-409X(02)00020-0
  • Bioconjugate Techniques; G. T. Hermanson; 2nd Edition; Elsevier 2008; ISBN 978-0-12-370501-3
  • A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes; V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless; Angew Chem. Int. Ed. 2002; 41: 2596-2599. doi:10.1002/1521- 3773(20020715)41:143.0.co;2-4
  • Click Chemistry: Diverse Chemical Function from a Few Good Reactions; H. C. Kolb, M. G. Finn and K. B. Sharpless; Angew Chem. Int. Ed. 2001; 40: 2004-2021. doi:10.1002/1521- 3773(20010601)40:113.0.co;2-5
  • Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides; C. W. Tornøe, C. Christensen and M. Meldal; J Org Chem 2002; 67: 3057-3064. doi:10.1021/jo011148j
  • The growing impact of click chemistry on drug discovery; H. C. Kolb and K. B. Sharpless; Drug Discovery Today 2003; 8: 1128-1137. doi:10.1016/S1359-6446(03)02933-7
  • CuI-Catalyzed Alkyne–Azide “Click” Cycloadditions from a Mechanistic and Synthetic Perspective; V. D. Bock, H. Hiemstra and J. H. van Maarseveen; Eur J Org Chem 2006; 2006: 51-68. doi:10.1002/ ejoc.200500483
  • A3-type star polymers via click chemistry; O. Altintas, B. Yankul, G. Hizal and U. Tunca; J. Polym. Sci.: Part A: Polym. Chem. 2006; 44: 6458-6465. doi:10.1002/pola.21728
  • Preparation of alumina supported copper nanoparticles and their application in the synthesis of 1,2,3-triazoles; M. L. Kantam, V. S. Jaya, B. Sreedhar, M. M. Rao and B. M. Choudary; J Mol Catal. A: Chem 2006; 256: 273-277. doi:10.1016/j.molcata.2006.04.054
  • A Rapid and Versatile Method to Label Receptor Ligands Using “Click” Chemistry: Validation with the Muscarinic M1 Antagonist Pirenzepine; D. Bonnet, B. Ilien, J.-L. Galzi, S. Riché, C. Antheaune and M. Hibert; Bioconjug Chem 2006; 17: 1618-1623. doi:10.1021/ bc060140j
  • Alkyne-azide click reaction catalyzed by metallic copper under ultrasound; P. Cintas, A. Barge, S. Tagliapietra, L. Boffa and G. Cravotto; Nat Protoc 2010; 5: 607-16. doi:10.1038/nprot.2010.1
  • The origin of pegnology; F. F. Davis; Adv Drug Deliv Rev 2002; 54: 457-458. doi:10.1016/S0169-409X(02)00021-2
  • Non-immunogenic polypeptides; F. F. Davis, T. Van Es and N. C. Palczuk; Patent 1979: US4179337.
  • Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase; A. Abuchowski, J. R. McCoy, N. C. Palczuk, T. van Es and F. F. Davis; J Biol Chem 1977; 252: 3582-6.
  • PEG-proteins: Reaction engineering and separation issues; C. J. Fee and J. M. Van Alstine; Chemical Engineering Science 2006; 61: 924- 939. doi:10.1016/j.ces.2005.04.040
  • Protein conjugates purification and characterization, C.J. Fee, in PEGylated Protein Drugs: Basic Science and Clinical Applications, F.M. Veronese (Ed.). Birkhauser Publishing, Basel, 2009; 113-125.
  • Size-exclusion reaction chromatography (SERC): A new technique for protein PEGylation; C. J. Fee; Biotechnology and Bioengineering 2003; 82: 200-206. doi:10.1002/bit.10561
  • Advances in PEGylation of important biotech molecules: delivery aspects; S. M. Ryan, G. Mantovani, X. Wang, D. M. Haddleton and D. J. Brayden; Expert Opinion on Drug Delivery 2008; 5: 371-383. doi:10.1517/17425247.5.4.371
  • Protein PEGylation: An overview of chemistry and process considerations; V. B. Damodaran and C. J. Fee; European Pharmaceutical Review 2010; 15: 18-26.

  3.1 Branched PEGylating Reagents

  Branched PEGs impart significant water solubility and thus produce compounds with reduced aggregation or surfaces with reduced non-specific binding in diagnostic applications. The PEGylation reagent itself is nonimmunogenic and non-toxic, passing these properties to the PEGylated biopharmaceutical. These PEGs are potentially very useful as drug/protein modifiers to specifically increase the hydrodynamic volume. They are highly methylene chloride soluble - the ideal solvent for carboamide activations.

  Reference:

  • Bioconjugate Techniques; G. T. Hermanson; 2nd Edition; Elsevier 2008; Ch. 18: 711-742; ISBN 978-0-12-370501-3