PEGylation of Proteins: A Structural Approach
The type of reactive moiety controls the site and stability of the covalent link and also the total number of PEGylation sites on a given protein.
With the completion of the Human Genome Project, molecular biologists are continuously discovering new classes of proteins and new therapeutic uses for known proteins. The ~30,000 genes defined by the Human Genome Project translate into 300,000 to one million proteins.1 Converting these proteins into effective biopharmaceuticals, however, may require challenging formulation development, because most recombinant proteins have limited chemical or physical stability in liquid state. For this reason, a renewed interest in conjugating proteins with polyethylene glycol (PEG) can be expected, with the final goal of bringing PEGylated protein drugs to market.
A. Sorina Morar
PROTEIN PROPERTIES AND PEGYLATION USE
Depending on their ionic charge, size, and structure, proteins vary in their thermal stability, solubility, and susceptibility to proteolysis. The intermolecular packing and surface chemistry of proteins determine many of these biopharmaceutical properties, and many stress factors can cause protein unfolding and degradation, ultimately leading to loss of biological activity.2 Slight changes in pH, ionic strength, or temperature, for example, all can reduce biological activity in vivo.3 Other potential stress factors include proteases and oxidation. In vivo, these protein properties can translate into a high clearance rate of a therapeutic protein from the body. In addition, short plasma half-life and reactions with the immune system complicate effective delivery of therapeutic proteins in humans.4 Bypassing these problems can be accomplished by either stabilizing the proteins or increasing their solubility, thus allowing for low dosage volumes and longer circulation times.
To achieve the desired stability and solubility, proteins can be modified using methods such as crosslinking,5 fusion to other proteins, changing the oligomerization state, glycosylation, mutations of cysteine residues, or polymer attachment.6 Currently, one of the current most successful methods for stabilizing proteins and increasing their solubility is to use polymer therapeutics, i.e., to link an active molecule to a polymer molecule such as polyethylene glycol (PEG). Polymer therapeutics includes polymer drugs, polymer conjugates, and polymeric micelles.7 In general, conjugating a protein to a polymer can accomplish several desirable objectives: a longer in vivo half-life; reduced immunogenicity, toxicity, and clearance rate through the kidneys; successful transportation across a cell membrane; protection against proteolysis; modification of electro-osmotic flow; increased pH and thermal stability; a low volume of distribution and sustained adsorption from the injection site; and improved formulation properties of the protein. These superior properties can increase effective potency8 , improve response to the drug, increase patient tolerance and reduce side effects, reduce overall dosage, decrease office visits, and lower the cost to the patient. A better drug profile and an improved quality of life are thus achieved.9 In the biotechnology industry, better biophysical characterization and understanding of PEGylated protein properties would allow for better control of the conjugation reaction and an improved drug product.
POLYETHYLENE GLYCOL'S PROPERTIES
PEG is FDA approved for human administration by mouth, injection, or dermal application. It has been used for many years in various capacities. Early work used PEG in crystallography for crystal growth, purification in two-phase systems, incorporation into liposomes for an increased serum lifetime, or attachment to surfaces to reduce protein adsorption.10 In addition, PEG has been used to promote correct protein folding.
Many of the benefits of PEGylated proteins lie in the properties of PEG. PEG is inert, non-toxic, and non-immunogenic.11 The polymer is easily cleared from the body, through the kidney for molecules with molecular weight below 20 kDa, or through the liver for molecules with molecular weight above 20 kDa.12 PEG is a viscous liquid at molecular weights lower than 1000 and solid at higher molecular weights. PEG is typically prepared by anionic polymerization, providing a variety of molecular sizes. It contains two OH- groups that can be activated, 13 and the polymerization can be controlled such that the molecular weight distribution is narrow. Currently, there are two main types of commercially available PEG: linear and branched.
PEG -(CH2-CH2-O)n -, is a highly soluble, amphiphilic polyether diol. Its solutions are neutral. X-ray structural analysis shows that PEG chains can assume two extreme structural conformations: a zigzag, random coil structure for shorter chains, or a winding, helical structure for longer chains.14 These conformations are reversible in water and dependent on solution conditions.15 The presence of the ether oxygen atom allows for the hydration of the highly mobile chains through the formation of the oxonium ion. Thus, in aqueous solutions PEG can adopt a structure with helical elements in which three water bridges are formed per monomer unit.16 Consequently, the highly mobile PEG chains are heavily hydrated, and have a large exclusion volume that can inhibit the approach of another molecule. For this reason, PEG's hydration properties determine the overall hydrodynamic properties of PEG bioconjugates.
Consequently, the most important property of a PEGylated protein is the increased molecular size resulting from the large hydrodynamic volume of the PEG. The effective volume of PEG has a more significant effect on proteins that are smaller than 70 kDa,6 as described in the following explanation.
A protein's size may be approximated by estimating the hydrodynamic radius RH using two equations derived for proteins from pulsed-field gradient nuclear magnetic resonance (NMR) studies.17 In the equation for a folded protein,
RH native = 4.75 N0.29 [1]
N represents the number of protein residues. According to Equation 1, the RH value of myoglobin, a protein of medium size is about 20 Å. A value of 17 Å for the radius of gyration, Rg, has been determined by other methods.18 The calculated RH is in agreement with the value of 22 Å measured by a diffusion experiment for lactalbumin, a protein similar to myoglobin in terms of structure, molecular weight, and number of amino acids.
For polymer chains in solvents, the size description is based on statistical conformations of the chain in two dimensions. The dynamic scaling is described by the gyration radius Rg as a function of its monomer unit number N0 :19
Rg ~ N03/4 [2]
For a 20-kDa PEG chain (454 monomer units), Equation 2 yields a Rg value of 98 Å. A value of 70 Å for the Rg gyration radius of a 20-kDa PEG has been previously reported.20 Using a random coil configuration for PEG, the Flory radius estimates that a 20-kDa PEG occupies a sphere with a radius of 76.5 Å.21 When comparing a 20-kDa PEG with a native protein of medium molecular weight (~150 amino acids), a ratio of ~5 for the polymer gyration radius to protein hydrodynamic radius is obtained. Because of the polymer's hydration, the three-dimensional hydrophilic environment that PEG creates around the protein is likely much larger. Clearly, at the macromolecular level the PEG 's properties become dominant.
A recent study shows that the viscosity radius of the PEGylated protein depends only on the molecular weight of the native protein and the total weight of grafted PEG and suggests that PEG forms a dynamic layer over the surface of the protein.22
Not only does the protein size change upon PEGylation, but the conformation and electrostatic binding properties of a PEG-conjugated protein might be different from those of the unmodified protein. When a PEG moiety is added to a protein, the resulting species is a polymer–protein hybrid, that does not necessarily retain the physical properties of the protein. The conjugate's properties are dominated by sterical hindrance at and away from the active site, conformational changes of both PEG and protein, altered binding properties resulting from changes in local pI, pKa, hydrophobicity and hydrophilicity of the protein. Little is known about the effect of free or bound PEG on protein structure. Most studies point to an unchanged protein secondary structure.23-25 As a result, most of the protein's biological activity is preserved.
In general, the pharmacokinetics and pharmacodynamic properties of a PEG-protein conjugate depends on the site at which the PEG is attached, the molecular weight of the PEG used, the number of PEG molecules attached to a protein, and the stability of the protein-PEG bond.26 For example, the size and structure of PEG largely determine the rate of clearance. The type of reactive moiety controls the site and stability of the covalent link and also the number of PEGylation sites on a given protein. In addition, the PEG's size and structure likely affect the maximum number of PEGylation sites that can be "accessed." Not surprisingly, the location of the site attachment has been correlated with the pharmokinetic activity9 and stability of the PEGylated species in vivo as demonstrated by resistance to proteolytic degradation.27
THE PEGYLATION REACTION
The PEGylation reaction is defined as the covalent attachment of one or more PEG molecules to a biologically active protein.28 To optimize a PEGylation reaction, a formulation scientist must consider many factors, including the goal of PEGylation. Typically, a minimal number of PEGylation sites is desired to reduce loss in bioactivity. PEG structure and size are just two factors that can limit the degree of PEGylation. For example, branched PEGs increase the molecular weight of the mono-PEGylated protein, and also can limit the steric availability of PEGylation sites. With a linear PEG, one expects to see a large gyration radius as a result of increased chain length versus branched PEG of a similar molecular weight. Also, the interaction of a branched PEG with protein could be different than that of a linear PEG. Thus, a variety of species can be synthesized.
In a typical reaction, an activated monofunctional PEG is reacted with one or more accessible lysine residues or the N-terminal amino group. PEGylation at other nucleophilic sites such as cysteine, histidine, arginine or tyrosine also are possible.29-32 Depending on the activated form of PEG, an ester (PEG succinimidyl succinate) or urethane (PEG succinimidyl carbonate) covalent bond is created between the polymer and the protein.33
In a PEGylation reaction, the protein solution is mixed with activated PEG. MonoPEGylated protein molecules with the most reactive site are formed first, and then less reactive sites form diPEGylated species, and so on (Figure 1). These reactions are consecutive pseudo first order reactions (under conditions of excess PEG), as described below:
Figure 1. Protein PEGylation results in a complex mixture of mono-, di-, and multi-PEGylated species
PEG + P → PEG–P
PEG-P + PEG → (PEG)2 –P
(PEG)2 -P + nPEG → (PEG)n+2 –P
A typical reaction set-up places all the PEGylation components in the same solution phase. The PEG-to-protein ratio usually ranges from 1:1 to 5:1, at a pH dictated by the desired specificity of the reaction.
When optimizing a PEGylation reaction toward a high yield and purity for the desired species, the formulator must consider the PEGylation reagent used, reaction conditions, and purification issues.34 The most important factors that affect the PEGylation reaction include, but are not limited to, protein concentration, PEG-to-protein ratio, reaction pH and temperature, reaction time, protein characteristics (molecular weight, surface area, polarity, local amino acid conditions at the PEGylation site, such as lysine pKa, and site accessibility).30 Other aspects of the reaction that could be critical to the reaction outcome are mixing and the PEG addition rate, and the presence of hydrophobic or hydrophilic co-solutes and buffer components. Cosolvents can alter solution properties such as ionic strength, viscosity, and dielectric properties, or can perturb the conformational distribution of PEGylated species. Examples of such co-solutes are unreactive PEGs or dextrans, sugars, salt, alcohol, or detergents. In theory, by fine tuning of these parameters the reaction can be optimized to achieve a high yield of the desired PEG conjugate.
STRUCTURAL CONFORMATION OF PEGYLATED PROTEIN SPECIES
Although not much is known about the structural properties of the protein conjugate, it is generally agreed that the dominant properties of PEG play a large role in the conjugate's overall properties. Because PEG can adopt various conformations dependent upon solution conditions,14 multiple conformations of the PEG-protein hybrid can exist, most likely stabilized by an intricate H-bond lattice. In one conformation of the protein-polymer conjugate, water solvates hydrophilic regions around the protein while hydrophobic PEG clusters interact with corresponding protein patches. These reactions create a shell-like structure (PEGshell-Protein in Figure 2) in which PEG is wrapped around the surface of the protein. Physiologically, this structure translates into a higher stability and reduces the immune system's recognition of the protein.3,35 There is the alternate model, however, in which there is no PEG-protein interaction. The conjugate forms a worm-like helical structure (PEGworm-Protein in Figure 2) in which PEG fluctuates freely in solution. The first model could explain how, at the macromolecular level, PEG coupling to protein can effectively mask the protein surface from proteolytic cleavage. In both models the physical properties of PEG dominate, generating the non-immunogenic benefits in the polymer-protein hybrid.
Figure 2. The PEGylation reaction for a model protein and PEG. The equilibrium between worm- and shell- like conformations would include typical random fluctuations of an unstructured polymer loop until it finds either a surface or its other end. In the spherical conformation, some degree of loop formation in surrounding water is acceptable. While covalently bound, the PEG may also interact non-specifically with the protein.
PEG's interaction with the protein surface has been observed in several instances. Speculation about the behavior of an unstructured random coil PEG when encountering a surface can be based on PEG's flexibility14 and previous work with macromolecules such as DNA,19 where for steric reasons, random coils orient preferentially parallel to a solid surface. Supporting evidence for a resulting shell-like conformation also comes from a study on chemically modified cytochrome c with PEG, where the overall hydrophobicity and thermostability of the protein is increased upon coupling.35 These data agrees with the previous observation that PEG modification enhances protein stability by decreasing electrostatic repulsion between surface charges.5 In addition, PEG's affinity for tryptophan was established and used to explain protein partitioning between phases.36 Work with PEG and Bovine serum albumin suggests the existence of an attractive interaction between protein and polymer.37 Another study found that covalently-bound PEG to protein reduces the interaction with free PEG.38 These observations strongly support non-specific interactions between PEG and protein, interactions that ultimately affect the overall properties of the molecule.
We propose that upon changing the solution conditions the monocovalently bound polymer chain can be driven from the helical or random worm-like conformation when in contact with the solvent, to contact the protein surface in a compact, spherical conformation (Figure 2). The resulting shell-like structure of the monoPEGylated species could inhibit a further PEGylation reaction. The model of such a transition would include typical random fluctuations of unstructured polymer loop until it finds either a surface or its other end. In the spherical conformation, some degree of loop formation in surrounding water is acceptable.
MANUFACTURING CHALLENGES
When optimizing a process for the manufacturing of a protein drug, there are three main considerations: high product quality (purity, stability, and activity), process robustness, and low cost.33 Purifying and characterizing a particular positional isomer are recognized challenges associated with manufacturing of a PEGylated biomolecule. To lower analytical and downstream processing hurdles, it is preferred that the selectivity of the PEGylation reaction be optimized for a certain PEGylation degree and site. For a large-scale production of a PEGylated protein drug, a higher yield of the target product coupled with a lower yield of secondary PEGylation products is extremely advantageous. However, because the progression of the PEGylation reaction depends on several variables, the stoichiometry and the attachment site are hard to control. Ideally, the reaction parameters can be fine-tuned to achieve the desired stoichiometry of PEG conjugates, to produce predominantly mono-, di-, or other target PEG conjugates.39
Various methods have been previously used to force the reaction toward a unique, site-specific, active product that has the suitable degree of PEGylation. Such approaches include the use of protective agents, various types (branched or linear) and sizes of the PEG, mutations of reactive residues, or taking advantage of a specific amino acid reactivity under various solution pH conditions. 13,31,35,36,40-43 In most cases, a mixture of PEGylated isomers is obtained, and the most active isomer is then selectively separated by chromatographic methods.44,45 A combined control of the PEGylation reaction with simultaneous size-exclusion separation of the products has been described.46 The scaling-up of size exclusion chromatography, however, is a significant challenge in the biopharmaceutical industry because of its low throughput and high cost. In most of the cited cases, not only has the PEGylation yield been low, but additional downstream purification of the desired product also has been needed. Besides adding to the production cost, further separation of the PEGylated mixture is extremely difficult because the mixture is complex and the physicochemical characteristics of the species are very similar. Although ion exchange chromatography is routinely used, the dominant PEG properties affect the capacity and resolution of the method. These challenges in developing PEGylated protein drugs still have not been resolved.
APPROVED AND LATE STAGE PEGYLATED PRODUCTS
Despite the manufacturing complexity, PEGylated drug conjugates are an attractive delivery system for therapeutic proteins.47 A series of therapeutic PEGylated proteins, ranging from marketed products to pre-clinical studies, is summarized in Table 1. Among the most recent clinically approved PEGylated drugs in the US are PEG-aspargase 48 and PEG-visomant.49 The field is still expanding, with more and more proteins being conjugated with PEG to improve the molecule's properties (Table 2). PEGylated drugs encompass a large range of medical treatments, from enzyme replacement50 to blood substitute, 51 protein and peptide anticancer drugs52,53 , antibody fragments54-56 , cytokines57 , and adenovirus and adenovirus.58
Table 1. Examples of PEGylated protein drugs*
Cancer drugs are a main target for therapeutics today.24,59 Various anti-cancer agents have a suboptimal pharmacokinetics profile that necessitates prolonged or repetitive administration. PEGylation of these agents may help overcome these shortcomings without compromising the agents' efficacy. Several such PEGylated agents have been developed and evaluated in patients with oncology-related disorders. Attempts have been made to use PEGylated antibodies54 or enzymes60,61 to treat various types of cancer. Special attention is given to targeting the drug directly to the tumor, with the aim of avoiding the undesirable side effects associated with large amounts of chemotherapeutics, implantable devices, or inter veinous lines directed to the affected area.52,53 Initial studies performed with a PEGylated antibody showed enhanced tumor localization of the drug.54,61
Table 2. Various attempts to PEGylate proteins
Interferons, the standard treatment for hepatitis C, are an example of the clinical challenges involved in protein therapy.62 Serum concentrations of interferons decay quickly during the initial 24 hours, requiring multiple (three times a week) doses that can contribute to severe side effects. PEGASYS is a PEGylated form of interferon -α2a in which a 40-kDa branched PEG is attached to the protein.9 The result is a structure that lasts in vivo for more than 168 hours (elimination half-life of 77 hours versus 9 hours for the native interferon), and allows a once-a-week subcutaneous injection regimen at a lower effective dose. A Phase 3 study with 271 cirrhotic patients showed 43% of the patients receiving PEGASYS had viral clearance at the end of the 48 week treatment. For inpatients with chronic hepatitis C and cirrhosis, 180 μg PEG-interferon α-2a administered once weekly was shown to be more effective than three million units of standard interferon α-2a administered three times weekly.9 PEGASYS has received regulatory approval for treatment of hepatitis C, illustrating the value of pursuing PEGylation as a methodology for drug delivery.
TRENDS AND OPPORTUNITIES
The fact that several PEGylated proteins are approved for use and more PEGylated protein drugs are currently in clinical trials is proof of the advantages provided by this drug delivery route. As new proteins are designated as potential therapeutics, PEGylation will continue to grow as a viable and important option.
Although PEGylation remains an excellent choice for protein stabilization and controlled dosage of protein drugs, however, it still poses a number of challenges. Manufacturing costs of goods, PEG polydispersity, drug clearance from the body, and loss of biological activity resulting from PEGylation are just a few of the areas in need of further investigation. Because of the improvement of PEG purification processes, the commercially available PEGs are nowadays less polydisperse, allowing for larger molecular weight polymers to be used. In addition, the use of branched PEGs and the development of robust site specific PEGylation have expanded and will continue to expand the polymer options available for protein PEGylation. Bigger and better PEGs would require better characterization of the overall structural properties of the PEG and the conjugated complex. A conformational approach to PEGylated species equilibrium offers insight into alternative options for optimization of the PEGylation reaction.
A. Sorina Morar, PhD, is a scientist, Process Development, Diosynth Biotechnology, 3000 Weston Parkway, Carey, NC 25713, 919.388.5649, fax: 919.678.0366, sorina.morar@diosynth-rtp.com
REFERENCES
1. Maggio ET, Ramnarayan K. Recent developments in computational proteomics. Trends Biotechnol. 2001, 19:266-72.
2. Krishnamurthy R, Manning MC. The stability factor: importance in formulation development. Curr. Pharm. Biotechnol. 2002, 3:361-71.
3. Meyer JD, Ho B, Manning MC. Effects of conformation on the chemical stability of pharmaceutically relevant polypeptides. Pharm. Biotechnol. 2002, 13:85-107.
4. Goldberg M, Gomez-Orellana I. Challenges for the oral delivery of macromolecules. Nat. Rev. Drug Discov. 2003, 2:289-95.
5. DeSantis G, Jones JB. Chemical modification of enzymes for enhanced functionality. Curr. Opin. Biotechnol. 1999, 10:324-30.
6. Marshall SA, et al. Rational design and engineering of therapeutic proteins. Drug Discov. Today 2003, 8:212-21.
7. Burnham NL. Polymers for delivering peptides and proteins. Am. J. Hosp. Pharm. 1994, 51:210-18.
8. Harris JM, Chess RB. Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discov. 2003, 2:214-21.
9. Heathcote EJ, et al. Peginterferon α-2a in patients with chronic hepatitis C and cirrhosis. N. Engl. J. Med. 2000, 343:1673-80.
10. Harris JM. Poly(ethylene glycol) chemistry: biotechnical and biomedical applications. New York: Plenum; 1992.
11. ACS Symposium series 680. Poly(ethylene glycol) chemistry and biological applications. 1997 ed. Washington, DC: American Chemical Society; 1997.
12. Yamaoka T, Tabata Y, Ikada Y. Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice. J. Pharm. Sci. 1994, 83:601-06.
13. Zalipsky S. Chemistry of polyethylene glycol conjugates with biologically active molecules. Adv. Drug Deliv. Rev. 1995, 16:157-82.
14. Heymann B, Grubmuller H. Elastic properties of poly(ethylene-glycol) studied by molecular dynamics stretching simulations. Chem. Phys. Lett. 1999, 307:425-32.
15. Tasaki K. Poly(oxyethylene)-water interactions: a molecular dynamics study. J. Am. Chem. Soc. 1996, 118:8459-69.
16. Oesterhelt F, Rief M, Gaub HE. Single molecule force spectroscopy by AFM indicates helical structure of poly(ethylene-glycol) in water. New J. Phys. 1999, 1:6.1-6.11.
17. Wilkins DK, et al. Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques. Biochemistry 1999, 38:16424-31.
18. Garcia DLT, Huertas ML, Carrasco B. Calculation of hydrodynamic properties of globular proteins from their atomic-level structure. Biophys. J. 2000, 78:719-30.
19. Bae SC, et al. Single isolated macromolecules at surfaces. Curr. opin. Solid State Mater. Sci. 2001, 5:327-32.
20. Devanand K, Selser JC. Asymptotic behavior and long range interactions in aqueous solutions of poly(ethylene oxide). Macromol. 1991, 24:5943-47.
21. Jeppesen C, et al. Impact of polymer tether length on multiple ligand-receptor bond formation. Science 2001, 293:465-68.
22. Fee CJ, Van Alstine JM, Prediction of the viscosity radius and the size exclusion chromatography behavior of PEGylated proteins. Bioconj. Chem. 2004;15:1304-13.
23. Digilio G, et al. NMR structure of two novel polyethylene glycol conjugates of the human growth hormone-releasing factor, hGRF(1-29) -NH2. J. Am. Chem, Soc. 2003;125:3458-70.
24. Greenwald RB. Poly(ethylene glycol) anticancer drug delivery systems. P. R. Health Sci. J. 2002, 21:113-121.
25. Hinds KD, Kim SW. Effects of PEG conjugation on insulin properties. Adv. Drug Deliv. Rev. 2002;54:505-30.
26. Mehvar R. Modulation of the pharmacokinetics and pharmacodynamics of proteins by polyethylene glycol conjugation. J. Pharm. Pharm. Sci. 2000, 3:125-136.
27. Lee KC, et al. Isolation, characterization, and stability of positional isomers of mono-PEGylated salmon calcitonins. Pharm. Res. 1999, 16:813-18.
28. Harris JM, Martin NE, Modi M. Pegylation: a novel process for modifying pharmacokinetics. Clin. Pharmacokinet. 2001, 40:539-51.
29. Morpurgo M, et al. Preparation of characterization of poly(ethylene glycol) vinyl sulfone. Bioconjug. Chem. 1996, 7:363-68.
30. Roberts MJ, Bentley MD, Harris JM. Chemistry for peptide and protein PEGylation. Adv. Drug Deliv. Rev. 2002, 54:459-76.
31. Veronese FM. Peptide and protein PEGylation: a review of problems and solutions. Biomaterials 2001, 22:405-17.
32. Wylie DC, et al. Carboxyalkylated histidine is a pH-dependent product of pegylation with SC-PEG. Pharm. Res. 2001, 18:1354-60.
33. Harris JM. Synthesis of polyethylene glycol derivatives. J. Macromol. Sci. Rev. Macromol. Chem. Phys. 1985, C25:325-73.
34. Wheelwright SM. Protein purification: design and scale up of downstream processing. New York: Whiley;1994.
35. Garcia-Arellano H, et al. High temperature biocatalysis by chemically modified cytochrome c. Bioconjug. Chem. 2002, 13:1336-44.
36. Eiteman MA, Hassinen C, Veide A. A mathematical model to predict the partitioning of peptides and peptide-modified proteins in aqueous two-phase systems. Biotechnol. Prog. 1994, 10:513-19.
37. Abbott NL, Blankschtein D, Hatton TA. Protein partitioning in two-phase aqueous polymer systems. A neutron scattering investigation of the polymer solution structure and protein-polymer interactions. Macromol. 1992, 25:3932-41.
38. Kerwin BA, et al. Interactions between PEG and type I soluble tumor necrosis factor receptor: modulation by pH and by PEGylation at the N terminus. Protein Sci. 2002, 11:1825-33.
39. Bailon P, Berthold W. Polyethylene glycol-conjugated pharmaceutical proteins. Pharm. Sci. Technol. Today 1998, 1:352-56.
40. Roberts MJ, Harris JM. Attachment of degradable poly(ethylene glycol) to proteins has the potential to increase therapeutic efficacy. J. Pharm. Sci. 1998, 87:1440-45.
41. Campbell RM, et al. Pegylated peptides. V. Carboxy-terminal PEGylated analogs of growth hormone-releasing factor (GRF) display enhanced duration of biological activity in vivo. J. Pept. Res. 1997, 49:527-37.
42. Sato H. Enzymatic procedure for site-specific pegylation of proteins. Adv. Drug Deliv. Rev. 2002, 54:487-504.
43. Yamamoto Y, et al. Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity. Nat. Biotechnol. 2003, 21:546-52.
44. Monkarsh SP, et al. Positional isomers of monopegylated interferon α-2a: isolation, characterization, and biological activity. Anal. Biochem. 1997, 247:434-40.
45. Lee H, Park TG. A novel method for identifying PEGylation sites of protein using biotinylated PEG derivatives. J. Pharm. Sci. 2003, 92:97-103.
46. Fee CJ. Size-exclusion reaction chromatography (SERC): A new technique for protein PEGylation. Biotechnol. Bioeng. 2003, 82:200-06.
47. Greenwald RB, et al. Effective drug delivery by PEGylated drug conjugates. Adv. Drug Deliv. Rev. 2003, 55:217-50.
48. Graham ML. Pegaspargase: a review of clinical studies. Adv. Drug Deliv. Rev. 2003, 55:1293-1302.
49. Parkinson C, Scarlett JA, Trainer PJ. Pegvisomant in the treatment of acromegaly. Adv. Drug Deliv. Rev. 2003, 55:1303-14.
50. Bory C, et al. Comparison of red cell transfusion and polyethylene glycol-modified adenosine deaminase therapy in an adenosine deaminase-deficient child: measurement of erythrocyte deoxyadenosine triphosphate as a useful tool. Pediatr. Res. 1990, 28:127-30.
51. Conover CD, et al. The ability of polyethylene glycol conjugated bovine hemoglobin (PEG-Hb) to adequately deliver oxygen in both exchange transfusion and top-loaded rat models. Artif. Cells Blood Substit. Immobil. Biotechnol. 1999, 27:93-107.
52. Torchilin VP, Lukyanov AN. Peptide and protein drug delivery to and into tumors: challenges and solutions. Drug Discov. Today 2003, 8:259-66.
53. Francis GE, et al. Polyethylene glycol modification: relevance of improved methodology to tumour targeting. J. Drug Target 1996, 3:321-40.
54. Kitamura K, et al. Polyethylene glycol modification of the monoclonal antibody A7 enhances its tumor localization. Biochem. Biophys. Res. Commun. 1990, 171:1387-94.
55. Leong SR, et al. Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation. Cytokine 2001, 16:106-119.
56. Chapman AP. PEGylated antibodies and antibody fragments for improved therapy: a review. Adv. Drug Deliv. Rev. 2002, 54:531-45.
57. Francis GE, et al. PEGylation of cytokines and other therapeutic proteins and peptides: the importance of biological optimisation of coupling techniques. Int. J. Hematol. 1998, 68:1-18.
58. O'Riordan CR, et al. PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo. Hum. Gene Ther. 1999, 10:1349-58.
59. Crawford J. Clinical uses of pegylated pharmaceuticals in oncology. Cancer Treat. Rev. 2002, 28:7-11.
60. Teske E, et al. Polyethylene glycol-L-asparaginase versus native L-asparaginase in canine non-Hodgkin's lymphoma. Eur. J. Cancer 1990, 26:891-95.
61. Tan Y, et al. Polyethylene glycol conjugation of recombinant methioninase for cancer therapy. Protein Expr. Purif. 1998, 12:45-52.
62. Kozlowski A, Charles SA, Harris JM. Development of pegylated interferons for the treatment of chronic hepatitis C. BioDrugs. 2001, 15:419-29.
63. Belcheva N, et al. Synthesis and biological activity of polyethylene glycol-mouse nerve growth factor conjugate. Bioconjug. Chem. 1999, 10:932-37.
64. Kim TH, Lee H, Park TG. Pegylated recombinant human epidermal growth factor (rhEGF) for sustained release from biodegradable PLGA microspheres. Biomaterials 2002, 23:2311-17.
65. Hokom MM, et al. Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice. Blood 1995, 86:4486-92.
66. Snider J, et al. Characterization of the heterogeneity of polyethylene glycol-modified superoxide dismutase by chromatographic and electrophoretic techniques. J. Chromatogr. 1992, 599:141-55.
67. Tsutsumi Y, et al. PEGylation of interleukin-6 effectively increases its thrombopoietic potency. Thromb. Haemost. 1997, 77:168-73.
68. Katre NV, Knauf MJ, Laird WJ. Chemical modification of recombinant interleukin 2 by polyethylene glycol increases its potency in the murine Meth A sarcoma model. Proc. Natl. Acad. Sci U. S. A 1987, 84:1487-91.
69. Murakami Y, Hoshi R, Hirata A. Borate buffer dramatically enhances the activity of poly(ethylene)glycol-a-chymotripsin complex catalitically active in anhydrous isooctane. Biotechnol. Lett. 2001, 23:125-29.
70. Hu RG, et al. Bioactivities of ricin retained and its immunoreactivity to anti-ricin polyclonal antibodies alleviated through pegylation. Int. J. Biochem. Cell Biol. 2002, 34:396-402.
