Disulfide–bridging PEGylation during refolding for the more efficient production of modified proteins

Proteins that are modified by chemical conjugation require at least two separate purification processes. First the bulk protein is purified, and then after chemical conjugation, a second purification process is required to obtain the modified protein. In an effort to develop new enabling technologies to integrate bioprocessing and protein modification, we describe the use of disulfide‐bridging conjugation to conduct PEGylation during protein refolding. Preliminary experiments using a PEG‐mono‐sulfone reagent with partially unfolded leptin and unfolded RNAse T1 indicated that the cysteine thiols underwent disulfide‐bridging conjugation to give the PEGylated proteins. Interferon‐β1b (IFN‐β1b) was then expressed in E.coli as inclusion bodies and found to undergo disulfide bridging‐conjugation during refolding. The PEG‐IFN‐β1b was isolated by ion‐exchange chromatography and displayed in vitro biological activity. In the absence of the PEGylation reagent, IFN‐β1b refolding was less efficient and yielded protein aggregates. No PEGylation was observed if the cysteines on IFN‐β1b were first modified with iodoacetamide prior to refolding. Our results demonstrate that the simultaneous refolding and disulfide bridging PEGylation of proteins could be a useful strategy in the development of affordable modified protein therapeutics.     

Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals

New radiopharmaceuticals are possible using site-specific conjugation of small tumor binding proteins and poly(ethylene glycol) (PEG) scaffolds to provide modular multivalent, homo- or heterofunctional cancer-targeting molecules having preferred molecular size, valence, and functionality. Residence time in plasma can be optimized by modification of the size, number, and charge of the protein units. However, random PEG conjugation (PEGylation) of these small molecules via amine groups has led to variations of structural conformation and binding affinity. To optimize PEGylation, scFvs have been recombinantly produced in a vector that adds an unpaired cysteine (c) near the scFv carboxy terminus (scFv-c), thus providing a specific site for thiol conjugation. To evaluate the general applicability of this unpaired cysteine for PEGylation of scFv-c, conjugation efficiency was determined for four different scFvs and several PEG molecules having thiol reactive groups. The effect of the PEG molecular format on scFv-c PEG malignant cell binding was also addressed. ScFvs produced as scFv-c and purified by anti E-TAG affinity chromatography were conjugated using PEG molecules with maleimide (Mal) or o-pyridyl disulfide (OPSS). Conjugations were performed at pH 7.0, with 2 molar excess TCEP/scFv and PEG-(Mal) or PEG-OPSS, using 5:1 (PEG/scFv). PEG-Mal conjugation efficiency was also evaluated with 1:5 (PEG/scFv). PEGylation efficiency was determined for each reaction by quantitation of the products on SDS-PAGE. ScFv-c conjugation with unifunctional maleimide PEGs resulted in PEG conjugates incorporating 30-80% of the scFv-c, but usually above 50%. Efficiency of scFv-c conjugation to both functional groups of the bifunctional PEG-(Mal)2 varied between the PEG and scFv-c molecules studied. A maximum of 45% of scFv-c protein was conjugated as PEG- (scFv-c)2 using the smallest PEG-(Mal)2 (2 kDa). No significant increase in scFv-c conjugation was observed by the use of greater than a 5 molar excess of PEG/scFv-c. Under the same conjugation conditions, PEG as OPSS yielded less than 10% PEG-scFv-c. PEG-(scFv)2 conjugates had increased binding in ELISA using malignant cell membranes, when compared with unmodified scFv-c. PEGylated-scFv binding was comparable with unmodified scFv-c. In summary, scFv-c can be PEGylated in a site-specific manner using uni- or bivalent PEG-Mal, either linear or branched. ScFv-c was most efficiently conjugated to smaller PEG-Mal molecules, with the smallest, 2 kDa PEG-Mal, usually PEGylating 60-90% of the scFv-c. ScFv-c conjugation to form PEG-(scFv-c)2 reached greatest efficiency at 45%, and its purified form demonstrated greater binding than the corresponding scFv-c.     

Preparation and characterization of active site protected poly(ethylene glycol)–avidin bioconjugates

Avidin was modified with poly(ethylene glycol) in the presence of a biotin binding site protective agent synthesised by imminobiotin conjugation to branched 20 kDa PEG. Avidin was incubated with imminobiotin–PEG and reacted with high amounts of 5, 10 or 20 kDa PEG to modify the protein amino groups. Circular dichroism demonstrated that the extensive PEGylation does not alter the protein conformational structure. The affinity of avidin–PEG conjugates for biotin and biotinylated antibodies depended on the PEG size or the use of a protective agent. Avidin–PEG 10 and 20 kDa prepared in the presence of imminobiotin–PEG maintained 100% of the native affinity for biotin. The 5 kDa PEG derivative and the ones obtained without biotin site protection maintained 79–85% of the native affinity. The affinity for biotinylated antibodies decreased to 35% when the conjugation was performed without imminobiotin–PEG, while the conjugates obtained with high-molecular-weight PEGs in the presence of protective agent displayed high residual affinity. All conjugates possessed negligible antigenicity and immunogenicity. PEGylation greatly prolonged the avidin permanence in the circulation, reduced its disposition in the liver and kidneys and promoted accumulation into solid tumors. PEGylation was found to prevent the protein cell uptake, either by phagocytosis or pinocytosis.     

Site-specific PEGylation at histidine tags

The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.     

PEGylation of native disulfide bonds in proteins

PEGylation has turned proteins into important new biopharmaceuticals. The fundamental problems with the existing approaches to PEGylation are inefficient conjugation and the formation of heterogeneous mixtures. This is because poly(ethylene glycol) (PEG) is usually conjugated to nucleophilic amine residues. Our PEGylation protocol solves these problems by exploiting the chemical reactivity of both of the sulfur atoms in the disulfide bond of many biologically relevant proteins. An accessible disulfide bond is mildly reduced to liberate the two cysteine sulfur atoms without disturbing the protein's tertiary structure. Site-specific PEGylation is achieved with a bis-thiol alkylating PEG reagent that sequentially undergoes conjugation to form a three-carbon bridge. The two sulfur atoms are re-linked with PEG selectively conjugated to the bridge. PEGylation of a protein can be completed in 24 h and purification of the PEG-protein conjugate in another 3 h. We have successfully applied this approach to PEGylation of cytokines, enzymes, antibody fragments and peptides, without destroying their tertiary structure or abolishing their biological activity.     

Synthesis and characterization of PEGylated toll like receptor 7 ligands

Toll-like receptor 7 (TLR7) is located in the endosomal compartment of immune cells. Signaling through TLR7, mediated by the adaptor protein MyD88, stimulates the innate immune system and shapes adaptive immune responses. Previously, we characterized TLR7 ligands conjugated to protein, lipid, or poly(ethylene glycol) (PEG). Among the TLR7 ligand conjugates, the addition of PEG chains reduced the agonistic potency. PEGs are safe in humans and widely used for improvement of pharmacokinetics in existing biologics and some low molecular weight compounds. PEGylation could be a feasible method to alter the pharmacokinetics and pharmacodynamics of TLR7 ligands. In this study, we systematically studied the influence of PEG chain length on the in vitro and in vivo properties of potent TLR7 ligands. PEGylation increased solubility of the TLR7 ligands and modulated protein binding. Adding a 6-10 length PEG to the TLR7 ligand reduced its potency toward induction of interleukin (IL)-6 by murine macrophages in vitro and IL-6 and tumor necrosis factor (TNF) in vivo. However, PEGylation with 18 or longer chain restored, and even enhanced, the agonistic activity of the drug. In human peripheral blood mononuclear cells, similar effects of PEGylation were observed for secretion of proinflammatory cytokines, IL-6, IL-12, TNF-α, IL-1β, and type 1 interferon, as well as for B cell proliferation. In summary, these studies demonstrate that conjugation of PEG chains to a synthetic TLR ligand can impact its potency for cytokine induction depending on the size of the PEG moiety. Thus, PEGylation may be a feasible approach to regulate the pharmacological properties of TLR7 ligands.     

Electrophoretic analysis of PEGylated hemoglobin-based blood substitutes

Polyethylene glycol (PEG)-conjugated hemoglobins, a novel class of blood substitutes, were investigated by a combination of native and denaturing one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) coupled with the microspectrophotometric characterization of single bands and the functional analysis of electrophoretically separated fractions. For these intrinsically heterogeneous products, the molecular mass, the size distribution, and the degree of PEGylation are strictly correlated to their side effects and, therefore, are crucial pieces of information to evaluate their safety and efficacy. The PEGylation pattern was shown to strongly depend on the quaternary conformation of hemoglobin during the reaction, and the degree of conjugation was shown to correlate with the oxygen binding properties of the individual electrophoretically separated fractions. Moreover, small but not negligible fractions of underivatized tetramers, known to be responsible for serious side effects, were detected even in preparations with a high average degree of PEGylation. Overall, this approach might be exploited to characterize other products of protein PEGylation, an increasingly relevant technology for the optimization of the pharmacokinetic properties of protein-based drugs.     

Structure‐Based Site‐Specific PEGylation of Fibroblast Growth Factor 2 Facilitates Rational Selection of Conjugate Sites

Polyethylene glycol modification (PEGylation) can enhance the pharmacokinetic properties of therapeutic proteins by the attachment of polyethylene glycol (PEG) to the surface of a protein to shield the protein surface from proteolytic degradation and limit aggregation. However, current PEGylation strategies often reduce biological activity, potentially as a result of steric hindrance of PEG. Overall, there are no structure‐based guidelines for selection of conjugate sites that retain optimal biological activity with improved pharmacokinetic properties. In this study, site‐specific PEGylation based on the FGF2‐FGFR1‐heparin complex structure is performed. The effects of the conjugate sites on protein function are investigated by measuring the receptor/heparin binding affinities of the modified proteins and performing assays to measure cell‐based bio‐activity and in vivo stability. Comprehensive analysis of these data demonstrates that PEGylation of FGF2 that avoids the binding sites for fibroblast growth factor receptor 1 (FGFR1) and heparin provides optimal pharmacokinetic enhancement with minimal losses to biological activity. Animal experiments demonstrate that PEGylated FGF2 exhibits greater efficacy in protecting against traumatic brain injury‐induced brain damage and neurological functions than the non‐modified FGF2. This rational structure‐based PEGylation strategy for protein modification is expected to have a major impact in the area of protein‐based therapeutics.     

Polymer masked-unmasked protein therapy. 1. Bioresponsive dextrin-trypsin and -melanocyte stimulating hormone conjugates designed for alpha-amylase activation

Polymer-protein conjugation, particularly PEGylation, is well-established as a means of increasing circulation time, reducing antigenicity, and improving the stability of protein therapeutics. However, PEG has limitations including lack of polymer biodegradability, and conjugation can diminish or modify protein activity. The aim of this study was to explore a novel approach for polymer-protein modification called polymer-masking-unmasking-protein therapy (PUMPT), the hypothesis being that conjugation of a biodegradable polymer to a protein would protect it and mask activity in transit, while enabling controlled reinstatement of activity at the target site by triggered degradation of the polymeric component. To test this hypothesis, dextrin (alpha-1,4 polyglucose, a natural polymer degraded by alpha-amylase) was conjugated to trypsin as a model enzyme or to melanocyte stimulating hormone (MSH) as a model receptor-binding ligand. The effect of dextrin molecular weight (7700, and 47200 g/mol) and degree of succinoylation (9-32 mol %) on its ability to mask/unmask trypsin activity was assessed using N-benzoyl-L-arginine-p-nitroanilide (L-BAPNA). Dextrin conjugation reduced enzyme activity by 34-69% depending on the molecular weight and degree of succinoylation of dextrin. However, incubation with alpha-amylase led to reinstatement of activity to a maximum of 92-115%. The highest molecular dextrin (26 mol % succinoylation) gave optimum trypsin masking-unmasking. This intermediate was used to synthesize a dextrin-MSH conjugate (dextrin Mw = 47200 g/mol; MSH content 37 wt %), and its biological activity (+/-alpha-amylase) was assessed by measuring melanin production by murine melanoma (B16F10) cells. Conjugation reduced melanin production to 11%, but addition of alpha-amylase was able to restore activity to 33% of the control value. These were the first studies to confirm the potential of PUMPT for further application to clinically important protein therapeutics. The choice of masking polymer, activation mechanism, and the rate of unmasking can be tailored to therapeutic application.     

Coupling purification and on-column PEGylation of tumor necrosis factor alpha analogue

Trends in preparation of PEGylated protein drugs strive for simple, fast, and cheap processes, resulting in well-defined homogeneous products. We investigated the on-column PEGylation of tumor necrosis factor alpha (TNF-α), where purification and conjugation were performed in one step by using immobilized metal affinity chromatography (IMAC). The same quality of the PEGylated product was obtained by the on-column approach starting from either the crude Escherichia coli protein extract or the purified protein. In comparison with the PEGylation in solution, the on-column approach resulted in more homogeneous PEGylated product. The on-column PEGylation reduces the number of production steps, costs, and preparation time.     

Site-specific pegylation of G-CSF by reversible denaturation

A new strategy has been developed for extending the possibility of poly(ethylene glycol) (PEG) modification to accessible thiol groups of biologically active proteins. In particular, thiol-reactive PEGs have been coupled to the cysteine 17 of granulocyte colony stimulating factor (G-CSF), which is known to be partially buried in a hydrophobic protein pocket. The PEG linking was accomplished by partial protein denaturation with 3 M guanidine.HCl in the absence of any reducing agent in order to preserve the native protein's disulfide bridges. PEG coupling occurred also, but at a lower degree, by using a 3 M solution of urea as the denaturing agent. Following the PEGylation, which was carried out in the unfolded state, the conjugated protein was refolded using dialysis or gel filtration chromatography to eliminate the denaturant. Different thiol-reactive PEGs and polymer molecular weights (5, 10, or 20 kDa) were investigated for G-CSF conjugation under denaturation. The secondary structure of the protein in the G-CSF-PEG conjugates, evaluated using circular dichroism and biological activity assay in cell culture, was maintained with respect to the native protein. Unexpectedly, conjugation enhanced the G-CSF tendency to aggregate, a problem that was overcome by a proper formulation.     

重组人睫状神经营养因子的聚乙二醇化修饰简

目的：研究重组人睫状神经营养因子（rhCNTF）突变体的聚乙二醇（PEG）化修饰,对rhCNTF的PEG化产物进行初步分离纯化及相关生物活性检测。方法：采用分子生物学技术经点突变得到rhCNTF的突变体cNm通过实验设计研究CN10的最佳PEG化条件;采用分子筛层析方式对偶联产物进行初步纯化,最后用ELISA和小鼠体重增长抑制法检测PEG化后的CN。。蛋白的生物活性。结果：能运用mPEG—MAL对CN,。进行定点修饰,PEG化后用Superdex200能够分离CN10;PEG化后的CN10每2d腹腔注射1次,对小鼠体重的增长抑制率可达50％,与rhCNTF每天注射2次的体重增长抑制作用相当。结论：CN10蛋白在PEG化修饰后,其减重效应持续时间明显延长。     

New PEGs for peptide and protein modification, suitable for identification of the PEGylation site

New PEG derivatives were studied for peptide and protein modification, based upon an amino acid arm, Met-Nle or Met-beta Ala, activated as succinimidyl ester. PEG-Met-Nle-OSu or PEG-Met-beta Ala-OSu react with amino groups in protein-yielding conjugates with stable amide bond. From these conjugates PEG may be removed by BrCN treatment, leaving Nle or beta Ala as reporter amino acid, at the site where PEG was bound. The conjugation of PEG and its removal by BrCN treatment was assessed on a partial sequence of glucagone and on lysozyme as model peptide or protein. Furthermore, insulin, a protein with three potential sites of PEGylation, was modified by PEG-Met-Nle, and the PEG isomers were separated by HPLC. After removal of PEG, as reported above, the sites of PEGylation were identified by characterization of the two insulin chains obtained after reduction and carboxymethylation. Mass spectrometry, amino acid analysis and Edman sequence, could reveal the position of the reporter norleucine that corresponds to the position of PEG binding.     

Protein carboxyl amidation increases the potential extent of protein polyethylene glycol conjugation

Chemical coupling of polyethylene glycol (PEG) to therapeutic proteins reduces their immunogenicity and prolongs their circulating half-life. The limitation of this approach is the number and distribution of sites on proteins available for PEGylation (the N terminus and the -amino group of lysines). To increase the extent of PEGylation, we have developed a method to increase the number of PEGylation sites in a model protein, recombinant methionine alpha,gamma-lyase (recombinant methioninase; rMETase), an enzyme cancer therapeutic cloned from Pseudomonas putida. rMETase was first PEGylated with methoxypolyethylene glycol succinimidyl glutarate-5000 with a molar ratio of PEG:rMETase of 15:1. The carboxyl groups of the initially PEGylated protein were then conjugated with diaminobutane, resulting in carboxyl amidation. This reaction was catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, a water-soluble carbodiimide. The steric hindrance provided by the PEG chains already coupled to the protein prevented cross-linking between rMETase molecules during the carboxyl amidation reaction. The carboxyl-amidated PEGylated rMETase was hyper-PEGylated at a molar ratio of PEG to PEG-rMETase of 60:1. Biochemical analysis indicated that 13 PEG chains were coupled to each subunit of rMETase after hyper-PEGylation compared with 6-8 PEG chains attached to the non-carboxyl-amidated PEG-rMETase. Approximately 15-20% of the non-PEGylated rMETase activity was retained in the hyper-PEGylated molecule. Immunogenicity of the hyper-PEG-rMETase was significantly reduced relative to PEG-rMETase and rMETase. Initial results suggest that hyper-PEGylation may become a new strategy for PEGylation of protein biologics.     

C-terminal site-specific PEGylation of a truncated thrombomodulin mutant with retention of full bioactivity

Addition of poly(ethylene glycol) to bioactive proteins (PEGylation) improves their plasma half-life, enhances stability against proteolytic cleavage, and may also decrease protein immunogenicity. Characteristically, PEGylation usually involves a reaction to available lysine amino groups, some of which may be within or near a bioactive site. Thus, most protocols are nonspecific and result in a loss of protein activity. We report herein a strategy for site-specific PEGylation of a thrombomodulin (TM) derivative at the C terminus. A truncated TM mutant consisting of epidermal growth factor (EGF)-like domains 4-6 was expressed in Escherichia coli with a C-terminal azido-methionine. The TM mutant was site-specifically conjugated to a methyl-PEG-triarylphosphine compound via the Staudinger reaction. Enzymatic activity of the TM construct before and after PEGylation was unchanged, which confirms the utility of this site-specific PEGylation scheme.     

Action mechanism of PEGylated magainin 2 analogue peptide

PEGylation is frequently used to improve the efficacy of protein and peptide drugs. Recently, we investigated its effects on the action mechanism of the cyclic beta-sheet antimicrobial peptide tachyplesin I isolated from Tachypleus tridentatus [Y. Imura, M. Nishida, Y. Ogawa, Y. Takakura, K. Matsuzaki, Action Mechanism of Tachyplesin I and Effects of PEGylation, Biochim. Biophys. Acta 1768 (2007) 1160-1169]. PEGylation did not change the basic mechanism behind the membrane-permeabilizing effect of the peptide on liposomes, however, it decreased the antimicrobial activity and cytotoxicity. To obtain further information on the effects of PEGylation on the activities of antimicrobial peptides, we designed another structurally different PEGylated antimicrobial peptide (PEG-F5W, E19Q-magainin 2-amide) based on the alpha-helical peptide magainin 2 isolated from the African clawed frog Xenopus laevis. The PEGylated peptide induced the leakage of calcein from egg yolk L-alpha-phosphatidylglycerol/egg yolk L-alpha-phosphatidylcholine large unilamellar vesicles, however, the activity was weaker than that of the control peptides. The PEGylated peptide induced lipid flip-flop coupled to the leakage and was translocated into the inner leaflet of the bilayer, indicating that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. The cytotoxicity of the non-PEGylated peptides was nullified by PEGylation. At the same time, the antimicrobial activity was weakened only by 4 fold. The effects of PEGylation on the activity of magainin were compared with those for tachyplesin.     

Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds

The utility of single-chain Fv proteins as therapeutic agents would be substantially broadened if the circulating lives of these minimal antigen-binding polypeptides were both prolonged and adjustable. Poly(ethylene glycol) (PEG) bioconjugate derivatives of the model single-chain Fv, CC49/218 sFv, were constructed using six different linker chemistries that selectively conjugate either primary amines or carboxylic acid groups. Activated PEG polymers with molecular weights of 2000, 5000, 10 000, 12 000, and 20 000 were included in the sFv bioconjugate evaluation. Additionally, the influence of PEG conjugate geometry in branched PEG strands (U-PEG) and the effect of multimeric PEG-sFv bioconjugates on circulating life and affinity were examined. Although random and extensive PEG polymer conjugations have been achievable in highly active derivatives of the prototypical PEG-enzymes, PEGylation of CC49/218 sFv required stringent adjustment of reaction conditions in order to preserve antigen-binding affinity as measured in either mucin-specific or whole cell immunoassays. Purified bioconjugates with PEG:sFv ratios of 1:1 through 2:1 were identified as promising candidates which exhibit sFv affinity (K(d)) values within 2-fold of the unmodified sFv protein. Interestingly, PEG conjugation to carboxylic acid moieties, using a PEG-hydrazide chemistry, achieved significant activity retention in bioconjugates at a higher PEG:sFv ratio (5:1) than with any of the amine-reactive activated PEG polymers. Prolonged circulating life in mice was demonstrated for each of the PEG conjugates. An increase in PEG polymer length was found to be more effective for serum half-life extension than a corresponding increase in total PEG mass. For example, CC49/218 sFv conjugated to either one strand of PEG-20000, or four strands of PEG-5000, displayed about 20- or 14-fold increased serum half-life, respectively, relative to the unmodified sFv. The demonstrated suitability of established random conjugation chemistries for PEGylation of sFv proteins, in conjunction with innovative site-specific conjugation methods, indicates that production of a panoply of sFv proteins with both engineered affinity and tailored circulating life may now be achievable.     

Action mechanism of PEGylated magainin 2 analogue peptide

PEGylation is frequently used to improve the efficacy of protein and peptide drugs. Recently, we investigated its effects on the action mechanism of the cyclic β-sheet antimicrobial peptide tachyplesin I isolated from Tachypleus tridentatus [Y. Imura, M. Nishida, Y. Ogawa, Y. Takakura, K. Matsuzaki, Action Mechanism of Tachyplesin I and Effects of PEGylation, Biochim. Biophys. Acta 1768 (2007) 1160-1169]. PEGylation did not change the basic mechanism behind the membrane-permeabilizing effect of the peptide on liposomes, however, it decreased the antimicrobial activity and cytotoxicity. To obtain further information on the effects of PEGylation on the activities of antimicrobial peptides, we designed another structurally different PEGylated antimicrobial peptide (PEG-F5W, E19Q-magainin 2-amide) based on the α-helical peptide magainin 2 isolated from the African clawed frog Xenopus laevis. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles, however, the activity was weaker than that of the control peptides. The PEGylated peptide induced lipid flip-flop coupled to the leakage and was translocated into the inner leaflet of the bilayer, indicating that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. The cytotoxicity of the non-PEGylated peptides was nullified by PEGylation. At the same time, the antimicrobial activity was weakened only by 4 fold. The effects of PEGylation on the activity of magainin were compared with those for tachyplesin.     

Mono-PEGylation of ribonuclease A: High PEGylation efficiency by thiolation with small molecular weight reagent

PEGylation can improve the therapeutic potential of ribonuclease A (RNase A), a cancer chemotherapeutic agent. However, the common PEGylation that targets at the ?-amino groups of proteins can lead to imprecise control of the stoichiometry of the protein-PEG conjugate (i.e., mono-, di- and multi-PEGylated protein). To prepare a PEGylated therapeutic protein, it is desirable that the protein is mono-PEGylated for industrial production, convenient purification and analytical characterization. Here, N-hydroxysuccinimide esters of S-acetylthioacetic acid (SATA) and 2-iminothiolane (IT) were used to introduce thiol groups on RNase A, followed by maleimide chemistry based PEGylation of the thiolated RNase A. Interestingly, the yield of mono-PEGylated RNase A was higher than 60%, and di- or multi-PEGylated RNase A were absent in the PEGylated product. Presumably, the limited number and low solvent accessibility of the introduced thiol group favored mono-PEGylation of RNase A. As compared to the unmodified RNase A, the mono-PEGylated RNase A showed slightly decreased enzymatic activity, increased anti-proliferative ability and unchanged structural properties. Our study is expected to control the PEGylation process and optimize the industrial pharmaceutical production of PEGylated proteins.     

PEGylation of proteins in organic solution: a case study for interferon beta-1b

Conventional protein PEGylation is carried out in aqueous solution. However, some hydrophobic proteins seem to be stable in organic solution. In this study, a novel approach of PEGylating IFN-β-1b in an organic solution of 2-butanol (2-BuOH) was investigated. Compared with protein PEGylation in aqueous solution, the overall modification yields increased more than 37%, while the yield of mono-PEGylated products could be increased by 36%. Furthermore, the PEGylated IFN-β-1b, which was obtained in organic solution, demonstrated 18% more antiviral potency than those derived from aqueous solution. The PEGylation step could be directly connected to the previous protein separation step for process integration. Dynamic light scattering (DLS) and atomic force microscope (AFM) analysis revealed that IFN-β-1b formed aggregates both in water and in 2-BuOH solutions. However, the aggregates were much smaller and more homogeneous in 2-BuOH than those in aqueous solution, thereby providing larger solvent accessible protein surfaces, which resulted in a more productive PEGylation process. In addition, the results of circular dichroism (CD), fluorescence spectra, and peptide mapping suggested that the increased bioactivity came from the difference in PEGylation site distribution due to solution environment that induced conformational discrepancy. The results of this study show that PEGylation of IFN-β-1b in organic solution is a facile and efficient process, which might find applications for other hydrophobic proteins.