N-terminal specificity of PEGylation of human bone morphogenetic protein-2 at acidic pH

Site-specific PEGylation offers the possibility to modify a therapeutic protein without interfering with its biological activity. Previously, a preferential N-terminal PEGylation has been reported for several proteins when the reaction was performed at acidic pH. In the present study it was explored if acidic pH favors N-terminal PEGylation of bone morphogenetic protein-2 (BMP-2). PEGylation by poly(ethylene glycol) aldehyde (PEG-AL) or poly(ethylene glycol) carboxymethyl succinimidyl ester (PEG-NHS) was performed at moderate acidic pH of 4. Comparing with PEG-NHS, PEG-AL converted more BMP-2 mainly to mono- or di-PEGylated derivatives at much less molar excess and shorter duration. Analysis of Tryptic fragments of the PEGylated derivatives indicated a partial N-terminal PEGylation specificity. PEG-AL exhibited higher specificity than PEG-NHS. UV spectrometry proved that PEGylation improved the solubility of BMP-2 in PBS. Surface plasmon resonance showed that PEGylation decreased the binding of BMP-2 proteins to a type II receptor. Remarkably, mono-PEGylated BMP-2 with PEG-AL showed higher cellular bioactivity than unmodified protein. Higher N-terminal PEGylation specificity correlates with higher receptor binding affinity and cellular activity. In summary, PEGylation of BMP-2 by PEG-AL and PEG-NHS at acidic pH exhibits a partial N-terminal specificity which however might be sufficient for an efficient site-specific PEGylation process.     

PEG-modified biopharmaceuticals

PEGylation is a process in which one or more units of chemically activated polyethylene glycol reacts with a biomolecule, usually a protein, peptide, small molecule or oligonucleotide, creating a putative new molecular entity possessing physicochemical and physiological characteristics that are distinct from its predecessor molecules. In recent years, PEGylation has been used not only as a drug delivery technology but used also as a drug modification technology to transform existing biopharmaceuticals clinically more efficacious than before their PEGylation. PEGylation bestows several useful properties upon the native molecule, resulting in improved pharmacokinetic and pharmacodynamic properties, which in turn enable the native molecule to achieve maximum clinical potency. In addition, PEGylation results in sustained clinical response with minimal dose and less frequency of dosing, leading to improved quality of life via increased patient compliance and reduced cost. During the course of development of various pegylated protein therapeutics, several new insights have been gained. This review article focuses on the approaches, strategies and the utilization of modern PEGylation concepts in the design and development of well-characterized pegylated protein therapeutics.     

Conformational Stability of Lyophilized PEGylated Proteins in a Phase-Separating System

PEGylation of proteins is of great interest to the pharmaceutical industry as covalent attachment of poly(ethylene glycol) (PEG) molecules can increase protein sera half‐lives and reduce antigenicity. Not surprisingly, PEGylation significantly alters the surface characteristics of a protein, and consequently, its conformational stability during freezing and drying. Freeze concentration‐induced phase separation between excipients has been previously shown to cause degradation of the secondary structure in lyophilized hemoglobin. In this report we show how PEGylation of two proteins, hemoglobin‐and brain‐derived neurotrophic factor (BDNF), influences partitioning and protein secondary structure as determined by FTIR spectroscopy in a system prone to freezing‐induced phase separation. PEGylation of hemoglobin reduces the loss of structure induced by lyophilization in a PEG/dextran system that phase separates during freezing, perhaps due to altered partitioning. The partition coefficient for native hemoglobin favors the dextran‐rich phase (PEG/dextran partition coefficient = 0.3), while PEGylated hemoglobin favors the PEG phase (partition coefficient = 3.1). In addition, we demonstrate that PEGylation alters hemoglobin's stability during lyophilization in the absence of other excipients. In contrast, because native BDNF already partitions into the PEG‐rich phase, PEGylation of BDNF has a less dramatic effect on both partition coefficients and conformational stability during lyophilization. This is the first report on the effects of PEGylation on protein structural stability during lyophilization and points out the need to consider modification of formulations in response to changing protein surface characteristics.     

Influence of PEGylation with linear and branched PEG chains on the adsorption of glucagon to hydrophobic surfaces

PEGylation has proven useful for prolonging the plasma half lives of proteins, and since approval of the first PEGylated protein drug product by the FDA in 1990, several PEGylated protein drug products have been marketed. However, the influence of PEGylation on the behavior of proteins at interfaces is only poorly understood. The aim of this work was to study the effect of PEGylation on the adsorption of glucagon from aqueous solution to a hydrophobic surface and to compare the effects of PEGylation with a linear and a branched PEG chain, respectively. The 3483 Da peptide glucagon was PEGylated with a 2.2 kDa linear and a branched PEG chain, respectively, and the adsorption behaviors of the three proteins were compared using isothermal titration calorimetry, fixed-angle optical reflectometry and total internal reflection fluorescence. PEGylation decreased the number of glucagon molecules adsorbing per unit surface area and increased the initial adsorption rate of glucagon. Furthermore, the results indicated that the orientation and/or structural changes of glucagon upon adsorption were affected by the PEGylation. Finally, from the isothermal titration calorimetry and the reflectometry data, it was observed that the architecture of the PEG chains had an influence on the observed heat flow upon adsorption as well as on the initial rate of adsorption, respectively.     

The impact of PEGylation on protein immunogenicity

Covalent attachment of PEG (PEGylation) is widely used to improve the pharmaceutical properties of therapeutic proteins. The applicability and safety of this method have been proven by the use of various PEGylated pharmaceutical proteins approved by the Food and Drug Administration (FDA). One of the properties attributed to PEGylation is immunogenicity reduction of the PEGylated protein. In this study, the impact of PEGylation on immunogenicity was tested and compared for two proteins (chicken IgY and horse IgG) in two strains of mice (Balb/c and C57BL/6) for two routes of administration (i.v. and i.m.) and two sizes of PEG (5 kD and 20 kD). The influence of PEG was shown to be inconsistent between the mouse strains and routes of administration, even with the same tested protein. Consequently, immunogenicity reduction by PEGylation cannot be predicted or assumed; it must be tested on an individual case basis.     

蛋白药物聚乙二醇化技术的研究进展

蛋白药物聚乙二醇化技术是指一项利用聚乙二醇衍生物对蛋白药物进行化学修饰的技术。聚乙二醇的代表药物有：腺苷脱氨酶、干扰素α—2b和干扰素α—2a等。它可以改善药物的可溶性和稳定性，减少免疫原性和蛋白水解，显著增加体内循环时间，以及在作用部位提高药物浓度和延长驻留时间从而提高疗效。本文对该技术进行了综述。     

聚乙二醇化蛋白质多肽类药物定点修饰策略

聚乙二醇修饰具有抵抗蛋白酶降解、提高稳定性、延长体内半衰期、降低免疫原性等优点,能够有效地改善蛋白质多肽类药物的临床药效。而聚乙二醇的定点修饰由于能够获得均一性和高活性保留率的产物,并能提高产物的产率,已经引起了广泛关注。本文概述近年来聚乙二醇定点修饰蛋白质多肽类药物方面的研究进展,并对聚乙二醇定点修饰技术的发展趋势进行了展望。     

Long-acting forms of Sonic hedgehog with improved pharmacokinetic and pharmacodynamic properties are efficacious in a nerve injury model

The therapeutic effects of the Sonic hedgehog (Shh) have been difficult to evaluate because of its relatively short serum half-life. To address this issue polyethylene glycol modification (PEGylation) was investigated as an approach to improve systemic exposure. Shh was PEGylated by a targeted approach using cysteines that were engineered into the protein by site-directed mutagenesis as the sites of attachment. Sixteen different versions of the protein containing one, two, three, or four sites of attachment were characterized. Two forms were selected for extensive testing in animals, Shh A192C, which provided a single site for PEGylation, and Shh A192C/N91C, which provided two sites. The PEGylated proteins were evaluated for reaction specificity by SDS-PAGE and peptide mapping, in vitro potency, pharmacokinetic and pharmacodynamic properties, and efficacy in a sciatic nerve injury model. Targeted PEGylation was highly selective for the engineered cysteines and had no deleterious effect on Shh function in vitro. Systemic clearance values in rats decreased from 117.4 mL/h/kg for unmodified Shh to 29.4 mL/h/kg for mono-PEGylated Shh A192C that was modified with 20 kDa PEG-maleimide and to 2.5 mL/h/kg for di-PEGylated Shh A192C/N91C modified with 2, 20 kDa PEG vinylsulfone adducts. Serum half-life increased from 1 h for unmodified Shh to 7.0 and 12.6 h for the mono- and di-PEGylated products. These changes in clearance and half-life resulted in higher serum levels of Shh in the PEG-Shh-treated animals. In Ptc-LacZ knock-in mice expressing lacZ under regulation of the Shh receptor Patched, about a 10-fold lower dose of PEG-Shh was needed to induce beta-galactosidase than for the unmodified protein. Therapeutic treatment of mice with PEG-Shh enhanced the regeneration of injured sciatic nerves. These studies demonstrate that targeted PEGylation greatly alters the pharmacokinetic and pharmacodynamic properties of Shh, resulting in a form with improved pharmaceutical properties.     

Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates

Peptide and protein PEGylation is usually undertaken to improve the biopharmaceutical properties of these drugs and, to date, several examples of conjugates with long permanence in the body as well as with localization ability in disease sites have been reported. Although a number of studies on the in vivo behavior and fate of conjugates have been performed, forecasting their pharmacokinetics is a difficult task since the pharmacokinetic profile is determined by a number of parameters which include physiological and anatomical aspects of the recipient and physico-chemical properties of the derivative. The most relevant perturbations of the protein molecule following PEG conjugation are: size enlargement, protein surface and glycosylation function masking, charge modification, and epitope shielding. In particular, size enlargement slows down kidney ultrafiltration and promotes the accumulation into permeable tissues by the passive enhanced permeation and retention mechanism. Charge and glycosylation function masking is revealed predominantly in reduced phagocytosis by the RES and liver cells. Protein shielding reduces proteolysis and immune system recognition, which are important routes of elimination. The specific effect of PEGylation on protein physico-chemical and biological properties is strictly determined by protein and polymer properties as well as by the adopted PEGylation strategy. Relevant parameters to be considered in protein-polymer conjugates are: protein structure, molecular weight and composition, polymer molecular weight and shape, number of linked polymer chains and conjugation chemistry. The examples reported in this review show that general considerations could be useful in developing a target product, although significant deviations from the expected results can not be excluded.     

Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase

Transglutaminase (TGase, E.C. 2.3.2.13) catalyzes acyl transfer reactions between the gamma-carboxamide groups of protein-bound glutamine (Gln) residues, which serve as acyl donors, and primary amines, resulting in the formation of new gamma-amides of glutamic acid and ammonia. By using an amino-derivative of poly(ethylene glycol) (PEG-NH(2)) as substrate for the enzymatic reaction with TGase it is possible to covalently bind the PEG polymer to proteins of pharmaceutical interest. In our laboratory, we have conducted experiments aimed to modify proteins of known structure using TGase and, surprisingly, we were able to obtain site-specific modification or PEGylation of protein-bound Gln residue(s) in the protein substrates. For example, in apomyoglobin (apoMb, myoglobin devoid of heme) only Gln91 was modified and in human growth hormone only Gln40 and Gln141, despite these proteins having many more Gln residues. Moreover, we noticed that these proteins suffered highly selective limited proteolysis phenomena at the same chain regions being attacked by TGase. We have analysed also the results of other published experiments of TGase-mediated modification or PEGylation of several proteins in terms of protein structure and dynamics, among them alpha-lactalbumin and interleukin-2, as well as disordered proteins. A noteworthy correlation was observed between chain regions of high temperature factor (B-factor) determined crystallographically and sites of TGase attack and limited proteolysis, thus emphasizing the role of chain mobility or local unfolding in dictating site-specific enzymatic modification. We propose that enhanced chain flexibility favors limited enzymatic reactions on polypeptide substrates by TGases and proteases, as well as by other enzymes involved in a number of site-specific post-translational modifications of proteins, such as phosphorylation and glycosylation. Therefore, it is possible to predict the site(s) of TGase-mediated modification and PEGylation of a therapeutic protein on the basis of its structure and dynamics and, consequently, the likely effects of modifications on the functional properties of the protein.     

Prevention of benzyl alcohol-induced aggregation of chymotrypsinogen by PEGylation

Objectives Addition of the antimicrobial preservative benzyl alcohol to reconstitution buffer promotes the formation of undesirable aggregates in multidose protein formulations. Herein we investigated the efficiency of PEGylation (attachment of poly(ethylene glycol)) to prevent benzyl alcohol‐induced aggregation of the model protein α‐chymotrypsinogen A (aCTgn). Methods Various PEG‐aCTgn conjugates were prepared using PEG with a molecular weight of either 700 or 5000 Da by varying the PEG‐to‐protein ratio during synthesis and the formation of insoluble aggregates was studied. The effect of benzyl alcohol on the thermodynamic stability and tertiary structure of aCTgn was also examined. Key findings When the model protein was reconstituted in buffer containing 0.9% benzyl alcohol, copious amounts of buffer‐insoluble aggregates formed within 24 h (>10%). Benzyl alcohol‐induced aggregation was completely prevented when two or five molecules of PEG with a molecular weight of 5000 Da were attached to the protein, whereas two or four molecules of bound 700 Da PEG were completely inefficient in preventing aggregation. Mechanistic investigations excluded prevention of structural perturbations or increased thermodynamic stability by PEGylation from being responsible for the prevention of aggregation. Simple addition of PEG to the buffer was also inefficient and PEG had to be covalently linked to the protein to be efficient. Conclusions The most likely explanation for the protective effect of the 5000 Da PEG is shielding of exposed hydrophobic protein surface area and prevention of protein–protein contacts (molecular spacer effect).     

多肽蛋白质类药物聚乙二醇化修饰研究进展

综述多肽蛋白质类药物聚乙二醇化修饰的优势、方法、鉴定与检测及局限性等.     

Steric Repulsion Forces Contributed by PEGylation of Interleukin-1 Receptor Antagonist Reduce Gelation and Aggregation at the Silicone Oil-Water Interface

Silicone oil, used as a lubricating coating in pharmaceutical containers, has been implicated as a cause of therapeutic protein aggregation. After adsorbing to silicone oil-water interfaces, proteins may form interfacial gels, which can be transported into solution as insoluble aggregates if the interfaces are perturbed. Mechanical interfacial perturbation of both monomeric recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and PEGylated rhIL-1ra (PEG rhIL-1ra) in siliconized syringes resulted in losses of soluble monomeric protein. However, the loss of rhIL-1ra was twice that for PEG rhIL-1ra; even though in solution, PEG rhIL-1ra had a lower ΔG_unf and exhibited a more perturbed tertiary structure at the interface. Net protein-protein interactions in solution for rhIL-1ra were attractive but increased steric repulsion because of PEGylation led to net repulsive interactions for PEG rhIL-1ra. Attractive interactions for rhIL-1ra were associated with increases in intermolecular β-sheet content at the interface, whereas no intermolecular β-sheet structures were observed for adsorbed PEG rhIL-1ra. rhIL-1ra formed interfacial gels that were 5 times stronger than those formed by PEG rhIL-1ra. Thus, the steric repulsion contributed by the PEGylation resulted in decreased interfacial gelation and in the reduction of aggregation, in spite of the destabilizing effects of PEGylation on the protein’s conformational stability.     

Anti-cancer PEG-enzymes: 30 years old, but still a current approach

PEGylation (i.e. the covalent link of PEG strands) is a well known technique used to improve pharmaceutical properties of bioactive proteins and peptides. Even in cancer therapy some proteins, in particular enzymes, can find many applications, because of their antiproliferative action or ability to reduce side effects of chemotherapies, but to do so they need to be properly formulated. Unfortunately, formulation alone can not fulfil all the requirements to yield a safe and successful protein preparation for therapeutic applications. In particular, for many proteins fast clearance from the body and potential immunogenicity are severe limitations, which can not be easily overcome without taking into consideration a purposely designed drug delivery system. Among the approaches in the field of drug delivery, PEGylation has so far been the best choice for protein delivery. Here, we describe some examples of PEGylated enzymes useful in antitumoral therapies and the most recent advances in this field.     

Pegnology: a review of PEG-ylated systems

Polyethylene glycol conjugation or linking with the system is called PEGylation. Many novel drug systems are used for the delivery of drugs and bioactive substances to particular sites in a controlled or sustained manner, but various side effects or shortcomings restrict their use for the intended purpose. The shortcomings such as RES uptake, drug leakage, immunogenicity, stability, hemolytic toxicity etc. can generally be overcome by PEGylation of novel drug delivery systems such as liposomes, proteins, enzymes, drugs, nanoparticles etc. In this article the whole aspect of PEGylation starting from activation and derivatisation of poly (ethylene glycol) to the linking and designing of systems and their purification and characterization is discussed. The various properties of Pegylated systems are also discussed.     

药物聚乙二醇化的研究进展

聚乙二醇化是化学分子变构中重要的技术之一,是药物研究和开发的里程碑。本文介绍了聚乙二醇修饰药物的优化条件以及优化后药物在体内药动学和药效学等性质的改变,举例说明聚乙二醇化技术在蛋白多肽及纳米脂质体等方面的研究应用,并展望聚乙二醇修饰技术在国内外医药领域的应用前景。     

Chemistry for peptide and protein PEGylation

Poly(ethylene glycol) (PEG) is a highly investigated polymer for the covalent modification of biological macromolecules and surfaces for many pharmaceutical and biotechnical applications. In the modification of biological macromolecules, peptides and proteins are of extreme importance. Reasons for PEGylation (i.e. the covalent attachment of PEG) of peptides and proteins are numerous and include shielding of antigenic and immunogenic epitopes, shielding receptor-mediated uptake by the reticuloendothelial system (RES), and preventing recognition and degradation by proteolytic enzymes. PEG conjugation also increases the apparent size of the polypeptide, thus reducing the renal filtration and altering biodistribution. An important aspect of PEGylation is the incorporation of various PEG functional groups that are used to attach the PEG to the peptide or protein. In this paper, we review PEG chemistry and methods of preparation with a particular focus on new (second-generation) PEG derivatives, reversible conjugation and PEG structures.     

PEG conjugates in clinical development or use as anticancer agents: An overview

During the almost forty years of PEGylation, several antitumour agents, either proteins, peptides or low molecular weight drugs, have been considered for polymer conjugation but only few entered clinical phase studies. The results from the first clinical trials have shared and improved the knowledge on biodistribution, clearance, mechanism of action and stability of a polymer conjugate in vivo. This has helped to design conjugates with improved features. So far, most of the PEG conjugates comprise of a protein, which in the native form has serious shortcomings that limit the full exploitation of its therapeutic action. The main issues can be short in vivo half-life, instability towards degrading enzymes or immunogenicity. PEGylation proved to be effective in shielding sensitive sites at the protein surface, such as antigenic epitopes and enzymatic degradable sequences, as well as in prolonging the drug half-life by decreasing the kidney clearance. In this review PEG conjugates of proteins or low molecular weight drugs, in clinical development or use as anticancer agents, will be taken into consideration. In the case of PEG-protein derivatives the most represented are depleting enzymes, which act by degrading amino acids essential for cancer cells. Interestingly, PEGylated conjugates have been also considered as adjuvant therapy in many standard anticancer protocols, in this regard the case of PEG-G-CSF and PEG-interferons will be presented.     

Obstacles and pitfalls in the PEGylation of therapeutic proteins

In recent years, PEGylation has become widely used as a post-production modification methodology for improving the biomedical efficacy and physicochemical properties of therapeutic proteins. Several marketed drugs that have already been in use for more than a decade have proved the applicability and safety of this technology and, with the successes already achieved, it is expected that PEGylation will be applied to other potential therapeutic proteins. The non-biodegradable nature of PEG, however, may become a limiting factor for the next generation of protein pharmaceuticals, for which use in high concentrations and in the long term is the aim, especially considering the trend for the use of branched and high molecular mass PEGs. This review addresses various obstacles and pitfalls in the production of PEGylated biopharmaceuticals, in particular, the specificity of PEGylation reactions, separation and purification issues, the analysis of inherently polydisperse PEG reagents and PEGylated products, the consistency of products and processes, and the accurate determination of pharmacokinetic properties.