Enzymatic procedure for site-specific pegylation of proteins

We have developed a novel methodology for site-specific pegylation of proteins by use of transglutaminase (TGase). In this methodology, alkylamine derivatives of poly(ethyleneglycol) (PEG) could be site-specifically incorporated into intact or chimeric proteins without decreasing the bioactivities. The incorporation site of the TGase-catalyzed modification is limited to the substrate Gln residues for TGases. The high homogeneity of the constructed conjugates and the ability to design conjugates with suitable incorporation sites will improve the applicability of PEG-protein conjugates for clinical use.     

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。     

聚乙二醇定点修饰蛋白质药物中巯基的研究进展

聚乙二醇(PEG)修饰是解决蛋白质药物溶解度低、稳定性差、半衰期短、具有免疫原性等问题的有效方法,通常在氨基上进行修饰,但在巯基上进行定点修饰更有利于获得结构明确、组成稳定的修饰产物。综述了聚乙二醇定点修饰蛋白质药物中巯基,包括在游离巯基、二硫键和引入的巯基上进行修饰的研究进展。     

Enzymatically catalyzed HES conjugation using microbial transglutaminase: Proof of feasibility

Polymer–drug and polymer–protein conjugates are promising candidates for the delivery of therapeutic agents. PEGylation, using poly(ethylene glycol) for the conjugation, is now the gold standard in this field, and some PEGylated proteins have successfully reached the market. Hydroxyethyl starch (HES) is a water-soluble, biodegradable derivative of starch that is currently being investigated as a substitute for PEG. So far, only chemical methods have been suggested for HES conjugation; however, these may have detrimental effects on proteins. Here, we report an enzymatic method for HES conjugation using a recombinant microbial transglutaminase (rMTG). The latter catalyzes the acyl transfer between the γ-carboxamide group of a glutaminyl residue (acyl donors) and a variety of primary amines (acyl acceptors), including the amino group of lysine. HES was modified with N-carbobenzyloxy glutaminyl glycine (Z-QG) and hexamethylene diamine (HMDA) to act as acyl donor and acyl acceptor, respectively. Using ¹H NMR, the degree of modification with Z-QG and HMDA was found to be 4.6 and 3.9 mol%, respectively. Using SDS–PAGE, it was possible to show that the modified HES successfully coupled to test compounds, proving that it is accepted as a substrate by rMTG. Finally, the process described in this study is a simple, mild approach to produce fully biodegradable polymer–drug and polymer–protein conjugates. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4420–4428, 2009     

PEGylation of cyanovirin-N, an entry inhibitor of HIV

Cyanovirin-N (CV-N) is a potent inhibitor of human immunodeficiency virus and many other viruses. It has a high potential for use as a systemic compound to control viral load or in the development of microbicides to prevent primary viral infection. Due to its cyanobacterial origin it is likely to show the typical drawbacks associated with pharmaceutical use of foreign proteins such as short plasma half-life, proteolysis and immunogenicity. Several strategies were used to covalently bond poly(ethylene glycol) (PEGylate) to CV-N. Random PEGylation at lysine residues resulted in poor retention of antiviral activity. Many site-directed mutants were made to test site-specific PEGylation. One mutant, where glutamine 62 was replaced with cysteine (CV-N(Q62C)) and PEGylated with maleimide activated PEG, retained significant anti-HIV activity in vitro.     

Characterization of a site-specific PEGylated analog of exendin-4 and determination of the PEGylation site

PEGylation has been a successful strategy for improving the pharmacokinetic and pharmaceutical properties of proteins and peptides. However, PEGylated products also create significant challenges for detailed structural characterization. In this work, a site-specific PEGylation strategy was successfully performed on an exendin-4 analog (Ex4C) through a maleimide method. Tricine–sodium dodecylsulfate polyacrylamide gel electrophoresis (Tricine–SDS-PAGE), analytical reversed phase HPLC (RP-HPLC) and MALDI-TOF were applied to verify the accomplishment of the PEGylation. Peptide mapping was investigated after tryptic digestion, and the PEGylaton site was successfully located on the C-terminal fragment of Ex4C. Amino acid analysis (AAA) of cysteine was then applied to verify the block in the thiol group caused by PEGylation. We believe that the combination of proper enzymatic digestion and amino acid analysis of cysteine provided an easy and convincing way to identify the PEGylation site in this maleimide method.     

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.     

Assay of transglutaminase activity by electrophoretic removal of the unreacted monodansyl cadaverine

Transglutaminases (TGases) catalyze the transfer of acyl groups between the γ-carboxyamide group of a glutamine residue and a primary amine. Rapid and precise determination of TGase activity is an important issue because improper function of TGases has been suggested to be associated with a variety of diseases. There have been tremendous efforts to develop the TGase assay methods to be more rapid, convenient and accurate. In the conventional assay method, fluorescence-tagged amine molecules such as monodansyl cadaverine (MDC) are coupled with casein by the action of transglutaminase. The removal step of unreacted MDC would require time-consuming work-up processes such as acid-precipitation and centrifugation. These processes would also interrupt the precise measurements of enzymatic activities. In this study, we have developed a new fluorometric assay methods to assay transglutaminase activity based on electrodialysis where the unreacted MDC is removed by electrophoresis. We have found the optimized condition to remove the unreacted MDC while preserving the β-casein protein. We also found the linear relationship between fluorescence intensity associated with β-casein and TGase can maintain in the range of 0–1.6 mU as well as 0–0.4 mU. The results show us as few as 0.1 mU of TGase could be detected by this method.     

Effects of hydrazyl group containing drugs on leucocyte functions: an immunoregulatory model for the hydralazine-induced lupus-like syndrome

Isoniazid (INH) and hydralazine (HYD) are transglutaminase (TGase, E.C.2.3.2.13.) substrates containing catalytically recruitable hydrazyl groups. Since they can be expected to inhibit TGase-mediated cell functions by competing with physiological substrates, their effect upon allogeneically and lectin-induced proliferation of mononucleocytes and upon zymosan-induced chemiluminescence of phagocytes was studied. Both compounds inhibited chemiluminescence in a dose-dependent manner. ID50 of HYD was consistently below 20 microM, while that of INH was above 120 microM. Proliferation of immunocompetent cells was suppressed by HYD with an ID50 of 60 microM, INH was inhibitory only above 5000 microM. Analogs of both compounds not containing hydrazyl groups proved to be inactive. Control experiments indicated that inhibition is not due to toxicity or lipophilicity of the compounds, structural analogs lacking a hydrazyl moiety were inactive. It is suggested that, in vivo, HYD interferes with signal-induced TGase-dependent leucocyte functions essential for immunologic stability, and that the resultant dysregulation with disruption of self tolerance contributes to the HYD promoted lupus-like syndrome.     

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.     

Acyl glucuronides: the good,the bad and the ugly

Acyl glucuronidation is the major metabolic conjugation reaction of most carboxylic acid drugs in mammals. The physiological consequences of this biotransformation have been investigated incompletely but include effects on drug metabolism, protein binding, distribution and clearance that impact upon pharmacological and toxicological outcomes. In marked contrast, the exceptional but widely disparate chemical reactivity of acyl glucuronides has attracted far greater attention. Specifically, the complex transacylation and glycation reactions with proteins have provoked much inconclusive debate over the safety of drugs metabolised to acyl glucuronides. It has been hypothesised that these covalent modifications could initiate idiosyncratic adverse drug reactions. However, despite a large body of in vitro data on the reactions of acyl glucuronides with protein, evidence for adduct formation from acyl glucuronides in vivo is limited and potentially ambiguous. The causal connection of protein adduction to adverse drug reactions remains uncertain. This review has assessed the intrinsic reactivity, metabolic stability and pharmacokinetic properties of acyl glucuronides in the context of physiological, pharmacological and toxicological perspectives. Although numerous experiments have characterised the reactions of acyl glucuronides with proteins, these might be attenuated substantially in vivo by rapid clearance of the conjugates. Consequently, to delineate a relationship between acyl glucuronide formation and toxicological phenomena, detailed pharmacokinetic analysis of systemic exposure to the acyl glucuronide should be undertaken adjacent to determining protein adduct concentrations in vivo. Further investigation is required to ascertain whether acyl glucuronide clearance is sufficient to prevent covalent modification of endogenous proteins and consequentially a potential immunological response. Copyright © 2010 John Wiley & Sons, Ltd.     

PEGylation, successful approach to drug delivery

PEGylation defines the modification of a protein, peptide or non-peptide molecule by the linking of one or more polyethylene glycol (PEG) chains. This polymer is non-toxic, non-immunogenic, non-antigenic, highly soluble in water and FDA approved. The PEG-drug conjugates have several advantages: a prolonged residence in body, a decreased degradation by metabolic enzymes and a reduction or elimination of protein immunogenicity. Thanks to these favorable properties, PEGylation now plays an important role in drug delivery, enhancing the potentials of peptides and proteins as therapeutic agents.     

Quantification and significance of protein oxidation in biological samples

Protein oxidation is defined here as the covalent modification of a protein induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidative modification of proteins can be induced experimentally by a wide array of prooxidant agents and occurs in vivo during aging and in certain disease conditions. Oxidative changes to proteins can lead to diverse functional consequences, such as inhibition of enzymatic and binding activities, increased susceptibility to aggregation and proteolysis, increased or decreased uptake by cells, and altered immunogenicity. There are numerous types of protein oxidative modification and these can be measured with a variety of methods. Protein oxidation serves as a useful marker for assessing oxidative stress in vivo. There are both advantages and disadvantages to using proteins for this purpose compared to lipids and DNA. Finally, it is important to monitor the degree of oxidative modification of therapeutic proteins manufactured for commercial use. This review will examine various aspects of protein oxidation, with emphasis on using proteins as markers of oxidative stress in biological samples.     

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.     

Advances in PEGylation of important biotech molecules: delivery aspects

BACKGROUND: Although various injected peptide and protein therapeutics have been developed successfully over the past 25 years, several pharmacokinetic and immunological challenges are still encountered that can limit the efficacy of both novel and established biotech molecules. OBJECTIVE AND METHOD: PEGylation is a popular technique to address such properties. PEGylated drugs exhibit prolonged half-life, higher stability, water solubility, lower immunogenicity and antigenicity, as well as potential for specific cell targeting. Although PEGylated drug conjugates have been on the market for many years, the technology has steadily developed in respect of site-specific chemistry, chain length, molecular weights and purity of conjugate. These developments have occurred in parallel to improvements in physicochemical methods of characterization. CONCLUSION: This review will discuss recent achievements in PEGylation processes with an emphasis on novel PEG-drugs constructs, the unrealized potential of PEGylation for non-injected routes of delivery, and also on PEGylated versions of polymeric nanoparticles, including dendrimers and liposomes.     

清蛋白作为药物载体的PEG化修饰研究进展

清蛋白（白蛋白）是一种理想的药物载体,但由于其在体内半衰期短以及易被酶降解等缺点限制了其应用,然而根据其具有多个修饰位点的结构特点,可通过PEG修饰延长循环时间,阻碍酶的作用等。目前,PEG修饰清蛋白仍处于研究阶段,已有较多关于PEG修饰清蛋白的研究,例如PEG修饰所起的作用、对清蛋白及其制剂的影响,以及修饰位点的选择等。本文对清蛋白的PEG化修饰的相关研究进行综述。     

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.     

Site‐Specific Labeling of Proteins and Peptides with Trans‐cyclooctene Containing Handles Capable of Tetrazine Ligation

There is a growing library of functionalized non‐natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non‐natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide‐alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper's toxicity.¹ Here, we report the synthesis of trans‐cyclooctene geranyl diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site‐specially modified with trans‐cyclooctene geranyl diphosphate were subsequently targeted for further modification via tetrazine ligation. As tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.     

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。     

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.