Current advances in the non‐chromatographic fractionation and characterization of PEGylated proteins

For more than 30 years, PEGylation has been used to improve the physicochemical properties of several proteins and therapeutic drugs having a major impact in the biopharmaceutical industry. The purification of PEGylated proteins usually involves two basic challenges: (1) the separation of PEG‐proteins from other reaction products; and (2) the sub‐fractionation of PEG‐proteins on the basis of their degree of PEGylation and positional isomerism. Currently, most PEGylated protein purification processes are based on chromatographic techniques, especially size exclusion chromatography (SEC) and ion exchange chromatography (IEX). Nonetheless, other less frequently used strategies based on non‐chromatographic techniques such as ultrafiltration, electrophoresis, capillary electrophoresis, and aqueous two‐phase systems have been developed in order to fractionate and analyze PEGylated derivates. This review presents current advances in some of the most widely used non‐chromatographic strategies for the fractionation and analysis of PEG‐protein conjugates. Copyright © 2010 Society of Chemical Industry     

Synthesis and Chromatographic Separation of Monomethoxypolyethylene Glycol Modified Insulin

Abstract

Insulin was modified with monomethoxypolyethylene glycol (MPEG)‐succinimidyl succinate and succinimidyl ester of carboxymethyl MPEG. Effects of reaction solvents, initial molar ratio of MPEG derivative to insulin and reaction time on PEGylation of insulin were investigated by 2,4,6‐trinitrobenzenesulfonic acid spectrophotometric assay and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Sephadex G75 size exclusion chromatography (SEC), ion exchange chromatography (IEC) and reversed phase‐high performance liquid chromatography (RP‐HPLC) were applied to separate PEGylated insulin. IEC and RP‐HPLC were proved to be efficient tools on separation of different PEGylated insulin species.     

Potential application of aqueous two‐phase systems for the fractionation of RNase A and α‐Lactalbumin from their PEGylated conjugates

BACKGROUND: PEGylation reactions often result in a heterogeneous population of conjugated species and unmodified proteins that presents a protein separations challenge. Aqueous two‐phase systems (ATPS) are an attractive alternative for the potential fractionation of native proteins from their PEGylated conjugates. The present study characterizes the partition behaviors of native RNase A and α‐Lac and their mono and di‐PEGylated conjugates on polyethylene glycol (PEG)—potassium phosphate ATPS. RESULTS: A potential strategy to separate unreacted native protein from its PEGylated species was established based upon the partition behavior of the species. The effect of PEG molecular weight (400–8000 g mol^?1), tie‐line length (15–45% w/w) and volume ratio (V_R; 0.33, 1.00 and 3.00) on native and PEGylated proteins partition behavior was studied. The use of ATPS constructed with high PEG molecular weight (8000 g mol^?1), tie‐line lengths of 25 and 35% w/w, and V_R values of 1.0 and 3.0 allowed the selective fractionation of native RNase A and α‐Lactalbumin, respectively, from their PEGylated conjugates on opposite phases. Such conditions resulted in an RNase A bottom phase recovery of 99%, while 98% and 88% of mono and di‐PEGylated conjugates, respectively were recovered at the top phase. For its part, α‐Lac had a bottom phase recovery of 92% while its mono and di‐PEGylated conjugates were recovered at the top phase with yields of 77% and 76%, respectively. CONCLUSIONS: The results reported here demonstrate the potential application of ATPS for the fractionation of PEGylated conjugates from their unreacted precursors. Copyright © 2010 Society of Chemical Industry     

Impacts of PEGylation on the gene and oligonucleotide delivery system

Poly(ethylene glycol) (PEG) is the most widely used polymer and also the gold standard in the field of drug delivery. Therapeutic oligonucleotides, for example, are modified with PEG at the terminus to increases nuclease resistance and the circulating half‐lives. The surface of nanoparticle such as micelle and liposome has been also modified with PEG. At present, one PEGylated therapeutic oligonucleotide has been approved for the market and several more PEGylated products including oligonucleotide and liposome are being tested in clinical settings. This review summarizes the methods and effects of PEGylation on gene delivery. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40293.     

PEGylation of deuterohaemin–alanine–histidine–threonine–valine–glutamic acid–lysine and its influence on activity,stability, and aggregation

Deuterohaemin–alanine–histidine–threonine–valine–glutamic acid–lysine (DhHP‐6) is a synthetic heme‐containing peroxidase mimic that exhibits a high peroxidase enzyme activity. Compared to other microperoxidases, DhHP‐6 has a poor stability and tends to aggregate in aqueous solutions. In this study, poly(ethylene glycol) (PEG) was used to improve the properties of DhHP‐6. Factors that affected the PEGylation product yield were investigated. PEGylated DhHP‐6 (mPEG–DhHP‐6) was characterized by reversed‐phase high‐pressure liquid chromatography (RP‐HPLC), matrix‐assisted laser desorption/ionization time of flight mass spectra (MALDI‐TOF‐MS), and ultraviolet–visible (UV–vis) spectroscopy. The results show that the optimal PEGylation reaction conditions were achieved when the PEGylation was conducted in a borate buffer solution at pH 8.0 and 25°C for 4 h with a feeding ratio of 2 equiv of active PEG. After PEGylation, mPEG–DhHP‐6 showed a great improvement in its stability with little activity loss. The UV–vis spectra of DhHP‐6 and mPEG–DhHP‐6 in different pH solutions showed that the aggregation of DhHP‐6 was partly suppressed after PEGylation. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

胰高血糖素样肽-1的聚乙二醇定点修饰

采用分子量为5 kDa的单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(mPEG-SC)修饰胰高血糖素样肽-1(GLP-1),建立了GLP-1的定点修饰反应及分离纯化工艺,考察了修饰反应各因素对单修饰产物得率及体外活性的影响,得到优化修饰条件为：pH 7.5, 50 mmol/L Na2HPO4-NaH2PO4缓冲液,GLP-1浓度1 mg/mL, PEG/GLP-1摩尔比2:1, 4℃下反应1 h. 在此条件下,单修饰PEG-GLP-1得率达50%. 反相高效液相色谱分离纯化所得单修饰产品纯度达98%,体外活性保留为未修饰GLP-1的86%,其在Sprague-Dawley远交群大鼠体内的循环半衰期为60 min,比原肽提高了20倍.     

Development of stealth nanoparticles coated with poly(2-methoxyethyl vinyl ether) as an alternative to poly(ethylene glycol)

Poly(ethylene glycol) (PEG) modification, also known as PEGylation, has been extensively used to improve the stability of nanoparticles for nanomedical applications. However, PEG exhibits antigenicity in some formulations, motivating researchers to explore alternative polymers. Herein, poly(vinyl ether) (PVE) derivatives are highlighted as promising alternatives to PEG because they form intermediate water molecules that suppress non-specific protein adsorption and platelet adhesion to the material surface. We prepared a water-soluble PVE derivative, poly(2-methoxyethyl vinyl ether) (PMOVE), and utilized it as a surface modifier for gold nanoparticles (AuNPs) as model nanoparticles. PMOVE with a thiol terminus was synthesized and confirmed to form an intermediate water molecule using differential scanning calorimetry. Similar to the synthesis of PEGylated AuNPs (PEG-AuNPs), PMOVE-modified AuNPs (PMOVE-AuNPs) were successfully fabricated with an appreciably high density of PMOVE palisades via a thiol-gold coordination reaction. Similar to PEG-AuNPs, PMOVE-AuNPs showed reduced serum protein adsorption and prolonged blood circulation. Additionally, no significant cytotoxicity was observed after incubation of a murine macrophage cell line, RAW264.7, with PMOVE-AuNPs. Our results indicate that the PMOVE modification increases the stealthiness of nanoparticles that is equivalent to that achieved by PEGylation.     

Production of lipid microparticles containing bioactive molecules functionalized with PEG

The functionalization of bioactive principles is a relevant issue as it allows increasing their stability, to raise the hydrophilic properties of hydrophobic materials, to reduce the absorption from macrophages and the proteolytic degradation. For this reason, we have functionalized a model protein (Ribonuclease A) with polyethylene glycol (PEG₅₀₀₀).We have investigated the production of lipid/PEG particles incorporating this active principle by a supercritical fluid technology (particles from gas saturated solution, PGSS) to obtain solid micro- and nanoparticles.Runs were carried out to study the process conditions; thus, the effects of the operative variables, such as temperature, pressure and organic solvent, were optimized to obtain micrometric particles.The particles produced were characterized by static light scattering (SLS) to determine their mean and distribution size. The better operative conditions were employed to produce microparticles incorporating Ribonuclease A (RNAse) as both native and PEGylated form.The BCA test was applied to determine the maximum amount of protein incorporated in the particles and the in vitro release. The retained enzymatic activity of proteins after the PEGylation and after the micronization process was also determined by spectrophotometric evaluation.     

Synthesis and selected properties of polyurethanes with monodisperse hard segments based on hexane diisocyanate and three types of chain extenders

Poly(ethylene glycol) grafted octadecyl quaternized carboxymethyl chitosan (PEG-g-OQC) copolymers were synthesized to both improve the biocompatibility of OQC and form PEGylated cationic polymeric liposomes (CPLs), which composed of the mixture of OQC, cholesterol, and PEG-g-OQC. Structure of the copolymers was characterized by using Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectroscopy (¹H-NMR), and X-ray diffraction (XRD). The methyl tetrazolium (MTT) assay with L929 cell lines confirmed that PEGylation can decrease the cytotoxicity of OQC. PEGylated CPL nanoparticles (NPs) can be prepared by adding different weight ratio of PEG-g-OQC in the mixture. Paclitaxel was successfully incorporated into PEGylated CPLs with high drug encapsulating efficiency (>90%) and drug loading capacity (>15%). Physical stability experiment showed that paclitaxel-loaded PEGylated CPLs was stable with little change of particle size and size distribution in the condition of freeze-dried by adding mannitol or in high temperature and high pressure. Power or reagent of drug-loaded PEGylated CPLs showed a slow steady release profile for paclitaxel. These results show that PEG-g-OQC and CPLs have potential application as a drug delivery vehicle. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Facile purification and characterization of recombinant human antithrombin III from transgenic goat milk

BACKGROUND: Antithrombin III (AT III) is a serine protease inhibitor that inhibits thrombin and the activated forms of factors X, VII, IX, XI and XII. Transgenic expression of therapeutic proteins in animal systems has gradually matured from laboratory scale to industrial practice, demanding efficient and scalable purification processes. The purification and characterization of recombinant human antithrombin III (rhAT III) from transgenic goat milk are described here. RESULTS: The rhAT III was purified by isoelectric precipitation, heparin affinity chromatography, and size exclusion chromatography, resulting in a 90.6% yield and > 99% purity. The goat β‐casein secretion peptide introduced to the rhAT III was cut off using enterokinase and removed by size exclusion chromatography using a Superdex 75 column. The primary structure, disulfide linkages, glycosylation sites, secondary structure and tertiary structure of the rhAT III were measured and found to be the same as those of the plasma‐derived AT III (phAT III). CONCLUSION: A facile process is introduced for the purification of rhAT III from transgenic goat milk. The rhAT III with high purity was achieved after an initial isoelectric precipitation step in which most of the bulk protein impurities are removed, followed by affinity chromatography and size exclusion chromatography. The rhAT III was demonstrated to have the same structure as phAT III. Copyright © 2011 Society of Chemical Industry     

PEGylation of MnO nanoparticles via catechol–Mn chelation to improving T1‐weighted magnetic resonance imaging application

To enhance biocompatibility and physiological stability of hydrophobic MnO nanoparticles as contrast agent of T₁‐weighted magnetic resonance imaging (MRI), dopamine‐functionalized poly(ethylene glycol) (PEG) was used to coat the surface of about 5 nm MnO nanoparticles. Although hydrophilic coating might decrease longitudinal relaxivity due to inhibiting the intimate contact between manganese of nanoparticle surface and proton in water molecules, higher longitudinal relaxivity was still maintained by manipulating the PEGylation degree of MnO nanoparticles. Moreover, in vivo MRI demonstrated considerable signal enhancement in liver and kidney using PEGylated MnO nanoparticles. Interestedly, the PEGylation induced the formation of about 120 nm clusters with high stability in storing and physiological conditions, indicating passive targeting potential to tumor and prolonged circulation in blood. In addition, the cytotoxicity of PEGylated MnO nanoparticles also proved negligible. Consequently, the convenient PEGylation strategy toward MnO nanoparticles could not only realize a good “trade‐off” between hydrophilic modification and high longitudinal relaxivity but also contribute additional advantages, such as passive targeting to tumor and long blood circulation, to MRI diagnosis of tumor. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42360.     

胰高血糖素样肽-1在乙醇中的聚乙二醇修饰

以乙醇为溶剂,单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(mPEG-SC)为修饰剂,对胰高血糖素样肽-1(GLP-1)进行了修饰反应条件的优化,同时对单修饰产物Mono-PEG-GLP-1进行分离纯化,并考察了其体外稳定性和体内活性. 得到的优化反应条件为：GLP-1浓度1 mg/mL, mPEG-SC与GLP-1摩尔比1:1, 37℃下反应24 h,该产物最高转化率达77%. 采用冷冻离心方式对Mono-PEG-GLP-1进行初步分离和浓缩,然后经反相液相色谱进一步高效纯化,纯度可达97%以上. 体外实验表明,Mono-PEG-GLP-1在血清中具有更好的稳定性及更强的抗胰蛋白酶消化能力. 体内动物实验表明,Mono-PEG-GLP-1具有更好的降血糖作用.     

Purification of biomolecules combining ATPS and membrane chromatography

Upstream processes for production of therapeutic proteins have been innovated and fermentation processes have been adopted for the use of recombinant microorganisms with high expression, but the downstream process is still the bottleneck in the biotechnological manufacturing process. A combined process consisting of aqueous two phase extraction (ATPE) and membrane chromatography is suggested to debottleneck downstream processing. ATPE has a large capacity, but the yield of the target product is from 74% to 97%. For this reason the product of ATPE waste stream is captured by membrane chromatography. In this work the binding capacity for the protein on protein A, ion exchange and hydrophobic exchange membrane chromatography was investigated experimentally with different concentration of polyethylene glycol (PEG), salt and protein. Protein A membrane was loaded with solutions resembling waste streams of ATPE for purifying IgG. For ion exchange and hydrophobic interaction membrane chromatography, the membrane was loaded with bovine serum albumin (BSA). PEG shows no significant effect on stability and capacity of membrane process. Even for small amount of BSA/IgG and high salt concentrations membrane adsorption is applicable. In this work it is demonstrated experimentally that a total product recovery of 99.9% for the purification of monoclonal antibody is possible.     

Development of a Spectrophotometric Biphasic Assay for the Estimation of mPEG-maleimide in Thiol PEGylation Reaction Mixtures

Methoxy(polyethylene glycol)-maleimide (mPEG-mal) is a PEG derivative used for thiol PEGylation of protein molecules and finds application in drug delivery studies. The maleimide group undergoes degradation in aqueous media, resulting in the difficult quantitative analysis of mPEG-mal. Routinely employed methods for separation and estimation of mPEG-mal include tedious chromatographic methods like ion exchange, high-performance liquid chromatography with refractive index detector and techniques like mass spectrometry and proton nuclear magnetic resonance. We present a direct and reproducible spectrophotometric method to quantify free and protein bound mPEG-mal in thiol PEGylation reaction mixtures. This method is based on the partitioning of a PEG bound chromophore between an aqueous ammonium isoferrothiocyanate phase to a chloroform phase in the presence of mPEG-mal. Several important parameters influencing the partitioning and stability of the chromophore, volume ratios of liquid phases, ethylenediaminetetraacetic acid concentration in the reaction mixture, mixing time, and chlorinated solvents used for partitioning have been studied.     

微生物酶分离纯化研究进展

在对常规的微生物酶分离纯化方法如沉淀法、疏水层析、凝胶过滤、离子交换层析及亲和层析等的特点、原理及应用进行介绍的基础上,概述了膜处理技术、免疫纯化技术、双水相体系萃取等新颖微生物酶分离纯化技术。指出常规方法和新颖方法的结合为微生物酶带来了高效的分离纯化效果,开发先进、灵活的蛋白质化学技术分离纯化天然酶、重组酶、人工模拟酶及杂合酶势在必行。     

Antitumor efficacy of doxorubicin encapsulated within PEGylated poly(amidoamine) dendrimers

We report here a general approach to using poly(amidoamine) (PAMAM) dendrimers modified with polyethylene glycol (PEG) as a platform to encapsulate an anticancer drug doxorubicin (DOX) for in vitro cancer therapy applications. In this approach, PEGylated PAMAM dendrimers were synthesized by conjugating monomethoxypolyethylene glycol with carboxylic acid end group (mPEG‐COOH) onto the surface of generation 5 amine‐terminated PAMAM dendrimer (G5.NH₂), followed by acetylation of the remaining dendrimer terminal amines. By varying the molar ratios of mPEG‐COOH/G5.NH₂, G5.NHAc‐mPEG_n (n = 5, 10, 20, and 40, respectively) with different PEGylation degrees were obtained. We show that the PEGylated dendrimers are able to encapsulate DOX with approximately similar loading capacity regardless of the PEGylation degree. The formed dendrimer/DOX complexes are water soluble and stable. In vitro release studies show that DOX complexed with the PEGylated dendrimers can be released in a sustained manner. Further cell viability assay in conjunction with cell morphology observation demonstrates that the G5.NHAc‐mPEG_n/DOX complexes display effective antitumor activity, and the DOX molecules encapsulated within complexes can be internalized into the cell nucleus, similar to the free DOX drug. Findings from this study suggest that PEGylated dendrimers may be used as a general drug carrier to encapsulate various hydrophobic drugs for different therapeutic applications. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40358.     

mPEG–NHS carbonates: Effect of alkyl spacers on the reactivity: Kinetic and mechanistic insights

Nowadays, the chemical conjugation to mPEG, also known as PEGylation, is a well-recognized technology used to improve the pharmaceutical properties of the therapeutic proteins. Over the last 20 years, more than 10 PEGylated macromolecules reached the market with tremendous success, whereas various other bioconjugates are under advanced clinical trials. mPEG–N-hydroxysuccinimidyl carbonate is an important reagent of widespread application for the PEGylation of biomacromolecules. One of the most important challenges in this technology is the development of more selective PEGylation reagents aimed to provide more consistent polymer–protein conjugates. One approach followed to improve the selectivity of PEGylation reagents is the design of less reactive derivatives, for example, by incorporation of alkyl spacers between the polymer chain and the terminal reactive group. In this work, we prepared a family of mPEG–N-hydroxysuccinimidyl carbonates bearing spacers of up to 6 carbon atoms. The kinetics of hydrolysis of the carbonates was studied under different experimental conditions, as a straight measure of the influence of the length of the spacer on the reactivity. By DFT calculations, we propose a detailed mechanism for the hydrolysis reaction. The influence of the length of alkyl spacer on the reactivity of the carbonates and related esters is studied and discussed in detail. Finally, to further evaluate the reactivity, selected N-hydroxysuccinimidyl carbonates were studied in the conjugation reaction of bovine lactoferrin. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47028.     

PEGylated chitosan microspheres as mucoadhesive drug‐delivery carriers for puerarin

PEGylated chitosans with different degrees of grafting were synthesized, and the application potential of microspheres based on PEGylated chitosan as mucoadhesive drug‐delivery carriers for puerarin was investigated. Compared with chitosan microspheres, PEGylated chitosan microspheres (PCMs) exhibited better physical stability and higher swelling capacity, and the amount of water uptake increased as the content of poly(ethylene glycol) methyl ether in the microspheres increased. PCMs showed obviously improved mucoadhesive behavior on a mucosa‐like surface. Puerarin was incorporated into the microspheres, and the release experiments in vitro showed that the PEGylation of chitosan accelerated puerarin release from the particles and decreased the retention of the drug. The abilities of all of the tested microspheres to open tight junctions and improve the permeability of puerarin were demonstrated with a Caco‐2 cell monolayer as an in vitro model. The amount of puerarin permeating across the Caco‐2 cell monolayer was significantly increased by the incorporation of puerarin into the PCMs. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42623.     

Alternative antibody Fab' fragment PEGylation strategies: combination of strong reducing agents, disruption of the interchain disulphide bond and disulphide engineering

Antigen-binding fragments (Fab') of antibodies can be site specifically PEGylated at thiols using cysteine reactive PEG-maleimide conjugates. For therapeutic Fab'-PEG, conjugation with 40 kDa of PEG at a single hinge cysteine has been found to confer appropriate pharmacokinetic properties to enable infrequent dosing. Previous methods have activated the hinge cysteine using mildly reducing conditions in order to retain an intact interchain disulphide. We demonstrate that the final Fab-PEG product does not need to retain the interchain disulphide and also therefore that strongly reducing conditions can be used. This alternative approach results in PEGylation efficiencies of 88 and 94% for human and murine Fab, respectively. It also enables accurate and efficient site-specific multi-PEGylation. The use of the non-thiol reductant tris(2-carboxyethyl) phosphine combined with protein engineering enables us to demonstrate the mono-, di- and tri-PEGylation of Fab fragments with a range of PEG size. We present evidence that PEGylated and unPEGylated Fab' molecules that lack an interchain disulphide bond retain very high levels of chemical and thermal stability and normal performance in PK and efficacy models.     

Covalent immobilization of an antimicrobial peptide on poly(ethylene) film

An antimicrobial film was produced by covalently attaching synthetic peptide E14LKK to poly(ethylene) film. E14LKK is a 14 residue, magainin‐class peptide with broad‐spectrum antimicrobial activity. The poly(ethylene) surface was first oxidized with chromic/sulphuric acid, then PEGylated by using carbodiimide chemistry to attach ω‐amino‐α‐carboxyl‐poly(ethylene glycol) (PEG). The peptide was covalently coupled to the free terminus of the PEG, again using carbodiimide coupling. Surface contact angles for distilled water decreased from 101° initially to 61° following oxidation and 45° following PEGylation. Film surface chemistry showed the expected changes during the modifications: dye adsorption assays indicated changes in the number of acidic and basic groups and X‐ray photoemission spectroscopy showed increasing oxygen and nitrogen levels. Antimicrobial activity was demonstrated in broth cultures against E.coli: growth was reduced by atleast 3 log cycles compared to controls. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008