PEG-proteins: Reaction engineering and separation issues

Poly(ethylene glycol)-conjugated (or PEGylated) proteins are an increasingly important class of therapeutic proteins that offer improved in vivo circulation half lives over their corresponding native forms. Their production involves covalent attachment of one or more poly(ethylene glycol) molecules to a native protein, followed by purification. Because of the extremely high costs involved in producing native therapeutic proteins it is important that subsequent PEGylation processes are as efficient as possible. In this paper, reaction engineering and purification issues for PEGylated proteins are reviewed. Paramount considerations for PEGylation reactions are specificity with respect to the conjugation site and overall yield. Batch PEGylation reaction methods are discussed, along with innovative methods using packed bed or “on-column” approaches to improve specificity and yield. Purification methods are currently dominated by ion exchange and size exclusion chromatography. Other methods in common use for protein separations, including hydrophobic interaction chromatography, affinity chromatography and membrane separations, are rarely used in PEGylated protein purification schemes. A better understanding of the effects of PEGylation on the physicochemical properties of proteins (isoelectric point, surface charge density and distribution, molecular size and relative hydrophobicity) and interactions between PEGylated proteins and surfaces is needed for the future development of optimal purification processes and media.     

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     

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     

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.     

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.     

Purification of monoclonal antibodies from cell culture supernatants by Gradiflow™ electrophoresis technology

Monoclonal antibodies (mAbs) are used extensively for analytical, diagnostic and therapeutic applications. The purification of mAbs from cell culture supernatants typically consists of protein A, G or L affinity chromatography, often in association with other conventional chromatographic techniques such as ion exchange and gel filtration. We report the application of Gradiflow^? preparative electrophoresis technology, for the separation of mouse and mouse/human chimeric mAbs from cell culture supernatants in their native state. The one‐step purification of murine mAb HuLym3 shows that mAbs can be purified from hybridoma cell culture supernatants to high purity, and is thus an alternative to other purification methods based on conventional and affinity chromatography for the production of mAbs for analytical and diagnostic applications. A mouse/human IgG1 chimeric mAb produced by Chinese hamster ovary cells was also purified from cell culture supernatant, and the purity achieved suggests that Gradiflow^? electrophoresis could replace affinity chromatography in the downstream processing of mAbs for therapeutic use. Gradiflow^? electrophoresis technology is scaleable and thus is applicable to industrial‐scale purification of mAbs. Copyright © 2005 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.     

Synthesis of PEGylated chitosan copolymers as efficiently antimicrobial coatings for leather

In this work, poly(ethylene glcycol)‐grafted chitosan (PEG‐g‐CS) was synthesized by conjugating PEG to the chitosan (CS) backbone. Such PEGylated CS copolymer was further characterized by FTIR and ¹H NMR, and the results demonstrated the successful synthesis. After PEGylation, the water solubility of CS was significantly improved due to the hydrophilicity of the PEG polymer. Therefore, this PEGylated CS was prepared as water borne coating for leather surface. The morphology and hydrophilicity of this coating on leather was studied by SEM and water contact angle measurement. Furthermore, the antimicrobial activity of PEGylated CS coating was investigated by measuring its minimum inhibitory concentration and the inhibition zone of coated leather against Gram‐negative Escherichia coli and Gram‐positive Staphylococcus aureus, respectively. Compared to CS coating, such PEG‐g‐CS coating exhibited better antimicrobial property, which indicated the synergetic effect of the antimicrobial property of CS and the antiadhesive property of PEG. Thus, this PEGylated CS copolymer can be used as efficiently antimicrobial coating for leather product. © 2016 The Authors Journal of Applied Polymer Science Published by Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43465.     

Preparation and characterization of PEGylated terlipressin

Terlipressin was chemically modified by reaction with succinimidyl propionate‐ monomethoxy polyethylene glycol (mPEG‐SPA). To determine the PEGylated degree, the position and the optimized condition for PEGylated terlipressin, the reactions were monitored in different pH value buffers at different molar ratios by reversed‐phase high performance liquid chromatography (RP‐HPLC). Tryptic digestion and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) was used. The results showed that the amount of mono‐PEG‐terlipressin was higher at lower pH value and lower content of PEG. Meanwhile, the amount of di‐PEG‐terlipressin was higher at higher pH value and higher content of PEG under the conditions investigated. The position of PEGylated terlipressin was confirmed by tryptic digestion. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Kinetic and transport effects on enzymatic biocatalysis resulting from the PEGylation of cofactors

The utilization of cofactor‐dependent redox enzymes in bioprocess technologies requires low cost cofactor regeneration methods. PEGylated NAD(H) (PEG‐NAD(H)) has been utilized in enzyme membrane reactors as a means to recover the cofactor; however, there is a lack of understanding of the effect of PEGylation on enzymatic activity, especially on the relationship between biocatalysis and transport phenomena. To explore this further, two redox enzymes (formate dehydrogenase (FDH) from Saccharomyces cerevisiae and NAD(H)‐dependent d ‐lactate dehydrogenase (nLDH) from Escherichia coli) have been chosen and the kinetic effects caused by cofactor modifications (with PEG of three different chain lengths) have been investigated. The PEGylation did not impact the cofactor dissociation constants and mass transfer was not the rate‐limiting step in biocatalysis for either enzyme. However, the PEG chain length had different impacts on the formation of enzyme/cofactor and/or enzyme/cofactor/substrate ternary complexes for the enzymes. © 2017 American Institute of Chemical Engineers AIChE J, 63: 12–17, 2018     

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.     

单修饰重组水蛭素的设计,制备及体外生物活性测定

Hirudin, the most potent inhibitor of thrombin found in nature, has a short half-life in serum, which sig-nificantly limits its clinical application as an anticoagulant. Recently, PEGylation has been commonly used as an effective method to prolong its half-life in serum. In contrast to the nonspecific pEGylation under basic conditions that targets lysine residues randomly, PEGylation sites under mildly acidic conditions preferably targets histidine residues, and there is only one histidine residue at 51 in r-hirudin; therefore, succinimidyl carbonyl methoxy poly-ethylene glycol （SC-mPEG, 20000） was attached to r-hirudin at mildly acidic pH to favor the formation of mono-PEGylated r-hirudin. The reaction mixture with high mono-PEGylated ratio was easily separated by a one-step ion-exchange chromatographic （IEC） procedure. Approximately 79.71% of the mono-PEGylated r-hirudin was PEGylated at His51, which showed that the acidic PEGylation operation prevented the PEGylation of active center （Lys47） of r-hirudin in pdnciple. Mono-PEGylated.product with purity higher than. 95% was obtained as the pre-dominant product, and 34% of the anticoagulant activity was retained in vitro. The staining method for sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） was improved to obtain perfect electrophoretic pattern in less than 5min. More accurate molecular weight was deduced due to the use of PEGs as molecular weight standards.     

水蛭素聚乙二醇化及其体外抗凝活力分析

Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical anpplication. The PEGvlation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEG ylated hirudin were investigated, wh.en the PEGylation react, on.wasconducted under 4℃ after 10h, in the borate buffer at pH 8.5 .with the molar ratio 230 ： 1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactivity of PEGylated hirudin was measured in vitro.Compared with unmodified hirudin, 26% of anti-thrombin activity was retained.     

Surface modification of protein nanocontainers and their self-directing character in polymer blends

Tailoring the surfaces of a nanocontainer with polymer brushes that have different affinities to the components of a phase-separating polymer blend should impart self-directing properties to the nanocontainers. Such nanocontainers could then be used to deliver a variety of functional species in tunable amounts and in a site-specific manner to polymer systems. This paper describes the surface modification, subsequent characterization of nanocontainers derived from ferritin, and the effects of surface modification on their self-directing properties in a binary phase-separating homopolymer blend. Wild ferritin was either PEGylated or alkylated by zero-length cross-linking to its surface carboxylate groups that were activated by carbodiimide. Modification was confirmed by ion-exchange chromatography, ζ-potential measurement, and electrospray ionization mass spectrometry. FT-IR spectrometry was used to quantify the extent of PEGylation by ratioing the intensity of the C-O-C asymmetric stretching vibration from the grafted PEG to that of the carbonyl stretching vibration (amide I band) from the protein. Importantly, modified ferritin was soluble in the organic solvent dichloromethane (DCM). Modified ferritin was introduced into a polymer blend of hydrophobic and hydrophilic polymers made up of poly(desaminotyrosyl tyrosine dodecyl ester carbonate) (PDTD) and PEG by solvent casting from solution in the common solvent DCM. Polymer thin films with an average thickness of ∼200 μm were obtained upon evaporation of the solvent. Transmission electron micrographs of microtomed polymer films demonstrated remarkable selectivity of PEGylated ferritin to PEG domains, while alkylated ferritin self-directs to the PDTD matrix.     

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.     

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.     

Site-Specific PEGylation of Therapeutic Proteins

The use of proteins as therapeutics has a long history and is becoming ever more common in modern medicine. While the number of protein-based drugs is growing every year, significant problems still remain with their use. Among these problems are rapid degradation and excretion from patients, thus requiring frequent dosing, which in turn increases the chances for an immunological response as well as increasing the cost of therapy. One of the main strategies to alleviate these problems is to link a polyethylene glycol (PEG) group to the protein of interest. This process, called PEGylation, has grown dramatically in recent years resulting in several approved drugs. Installing a single PEG chain at a defined site in a protein is challenging. Recently, there is has been considerable research into various methods for the site-specific PEGylation of proteins. This review seeks to summarize that work and provide background and context for how site-specific PEGylation is performed. After introducing the topic of site-specific PEGylation, recent developments using chemical methods are described. That is followed by a more extensive discussion of bioorthogonal reactions and enzymatic labeling.     

Preparation of protein microarrays on non‐fouling and hydrated poly(ethylene glycol) hydrogel substrates using photochemical surface modification

BACKGROUND: Conventional protein microarrays prepared on hard, dry substrates, such as glass and silicone, have several limitations, as proteins may easily denature and lose their structure. To overcome such problems, the fabrication of wet protein microarrays on non‐fouling and hydrated PEG‐based hydrogels was investigated. RESULT: Bovine serum albumin (BSA) and glucose oxidase (GOX), chosen as model proteins, were covalently immobilized on PEG hydrogel surfaces via 5‐azidonitrobenzoyloxy N‐hydroxysuccinimide, a photoreactive bifunctional linker. Successful fixation of the bifunctional linker and subsequent immobilization of the proteins on the PEG hydrogel surfaces were confirmed with X‐ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) studies. GOX immobilized on the hydrogel surface maintained approximately 50% of its initial activity after 24 h when left in dry conditions, but maintained only 20% when immobilized on a dry substrate. Photochemical fixation combined with photolithography produced well‐defined protein micropatterns with sizes ranging from 50–500 µm, and molecular recognition‐mediated specific binding between biotin and streptavidin was successfully assayed using microarrays on PEG hydrogels. CONCLUSION: A protein‐repellent PEG hydrogel surface was photochemically modified to covalently immobilize proteins and create protein microarrays. The use of hydrated hydrogels as substrates for protein microarrays could minimize the deactivation of proteins in dry conditions, and the non‐fouling property of PEG hydrogels allows the passivation step of protein microarray preparation to be skipped. Copyright © 2008 Society of Chemical Industry     

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.     

Delivery of PEGylated drugs from mucoadhesive formulations by pH-induced disruption of H-bonded complexes of PEG-drug with poly(acrylic acid)

We formed viscous, mucoadhesive gels by complexing PEGylated proteins with poly(acrylic acid) (PAA) at pH 3. These complexes dissociate at pH 7.4, and under this condition the PEGylated protein is released from the gel. We propose this system as a means for prolonged mucosal delivery of a PEGylated protein to nasal, ophthalmic or vaginal sites. In this paper we have studied the effect of the molecular weight of the PEG used in the PEGylated protein (5 kD, 20 kD and 40 kD PEG) and the effect of added free PEG (18.5 kD) on the in vitro release rate of the PEGylated protein from complexes with PAA. The PEGylated proteins we studied included papain, MW = 23.7 kD and pI 8.75, and soybean trypsin inhibitor (STI), MW = 20.1 kD and pI 4.6.