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     

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.     

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.     

Improvement of Drug Therapy by Covalent PEG Conjugation: An Overview From a Research Laboratory

In the last two decades, efforts focused in the field of drug delivery have made it possible to reach important goals, especially with proteins and peptides. The story of the evolution of this matter is vast and it is difficult to summarize its various aspects in a single review. On the other hand, it could be really interesting to look at the inside story of a typical academic research lab that has dedicated almost all its resources to drug delivery. In our case, an initial interest in the issue of protein conformation stability soon paved the way for a new area of study: the modification of proteins with synthetic polymers. We have seen this technique transformed from a crude and inhomogeneous procedure into a well-recognized and successful approach. This great advance has been possible thanks to the development of dedicated chemical coupling methods and to the better understanding of the behavior of polymers in vivo. In particular, among the several polymers investigated, PEG has became the best polymer for protein modification. Exploiting the unique properties of this polymer, we have conducted several investigations in the field of protein PEGylation and then transferred the acquired know-how to the development of conjugates with low molecular weight drug. This last aspect still presents several unmet needs that are awaiting proper solutions.     

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.     

Terminal‐specific PEGylation of polypeptides in a dilute solution

PEGylations of polypeptides with a four‐arm PEG (polyethylene glycol) in a dilute DMSO solution resulted in a successful conjugation with terminal specificity, which supports an intriguing PEGylation mechanism via conformational and kinetic control. © 2010 Crown in the right of Canada. J Appl Polym Sci, 2010     

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.     

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     

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.     

Lipid-based nanoparticles for siRNA delivery in cancer therapy: paradigms and challenges

RNA interference (RNAi) is a specific gene-silencing mechanism that can be mediated by the delivery of chemical synthesized small-interfering RNA (siRNA). RNAi might constitute a novel therapeutic approach for cancer treatment because researchers can easily design siRNA molecules to inhibit, specifically and potently, the expression of any protein involved in tumor initiation and progression. Despite all the potential of siRNA as a novel class of drugs, the limited cellular uptake, low biological stability, and unfavorable pharmacokinetics of siRNAs have limited their application in the clinic. Indeed, blood nucleases easily degrade naked siRNAs, and the kidneys rapidly eliminate these molecules. Furthermore, at the level of target cells, the negative charge and hydrophilicity of siRNAs strongly impair their cellular internalization. Therefore, the translation of siRNA to the clinical setting is highly dependent on the development of an appropriate delivery system, able to ameliorate siRNA pharmacokinetic and biodistribution properties. In this regard, major advances have been achieved with lipid-based nanocarriers sterically stabilized by poly(ethylene glycol) (PEG), such as the stabilized nucleic acid lipid particles (SNALP). However, PEG has not solved all the major problems associated with siRNA delivery. In this Account, the major problems associated with PEGylated lipid-based nanoparticles, and the different strategies to overcome them are discussed. Although PEG has revolutionized the field of nanocarriers, cumulative experience has revealed that upon repeated administration, PEGylated liposomes lose their ability to circulate over long periods in the bloodstream, a phenomenon known as accelerated blood clearance. In addition, PEGylation impairs the internalization of the siRNA into the target cell and its subsequent escape from the endocytic pathway, which reduces biological activity. An interesting approach to overcome such limitations relies on the design of novel exchangeable PEG-derivatized lipids. After systemic administration, these lipids can be released from the nanoparticle surface. Moreover, the design and synthesis of novel cationic lipids that are more fusogenic and the use of internalizing targeting ligands have contributed to the emergence of novel lipid-based nanoparticles with remarkable transfection efficiency.     

Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding

MUC1 mucin expressed in epithelial cancer, such as prostate and breast, is aberrantly glycosylated providing unique targets for imaging and therapy. In order to create a broadly applicable construct to target these unique epitopes on metastatic cancer, we selected an antibody fragment (scFv) that binds both synthetic MUC1 core peptide and epithelial cancer cell-expressed MUC1, and developed a recombinant bivalent molecule (di-scFv). Genetically engineered modifications of the di-scFv were constructed to create five molecular versions, each having a free cysteine (di-scFv-c) at different locations for site-specific conjugation. The effects of the engineered cysteine in the varied sites were studied relative to tumor binding and polyethylene glycol-maleimide (PEG-Mal) conjugation (PEGylation). Escherichia coli production as well as binding to MUC1 core peptide, human tumor cell lines and human tumor biopsies, were comparable. However, the location of the engineered cysteine in these di-scFv-c did influence PEGylation efficiency of this free thiol; higher PEGylation efficiency occurred with this cysteine in the inter-scFv linkage. Di-scFv-c PEG, with the cysteine engineered after the fifth amino acid in the linker, was used as an example to demonstrate comparable antigen-binding to non-PEGylated di-scFv-c. In summary, novel anti-MUC1 di-scFv-c molecules can be efficiently produced, purified and conjugated by site-specific PEGylation without loss of immunoreactivity, thus providing flexible multidentate constructs for cancer-targeted imaging and therapy.     

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     

RNA-TAG Mediated Protein-RNA Conjugation

Combinations of biological macromolecules can provide researchers with precise control and unique methods for regulating, studying, and manipulating cellular processes. For instance, combining the unmatched encodability afforded by nucleic acids with the diverse functionality of proteins has transformed our approach to solving several problems in chemical biology. Despite these benefits, there remains a need for new methods to site-specifically generate conjugates between different classes of biomolecules. Here we present a fully enzymatic strategy for combining nucleic acids and proteins using SNAP-tag and RNA-TAG (transglycosylation at guanosine) technologies via a bifunctional preQ1-benzylguanine small molecule probe. We demonstrate the robust ability of this technology to assemble site-specific SNAP-tag – RNA conjugates with RNAs of varying length and use our conjugation strategy to recruit an endonuclease to an RNA of interest for targeted degradation. We foresee that combining SNAP-tag and RNA-TAG will facilitate researchers to predictably engineer novel macromolecular complexes.     

Rational Design of Self-Assembling Artificial Proteins Utilizing a Micelle-Assisted Protein Labeling Technology (MAPLabTech): Testing the Scope**

Self-assembling artificial proteins (SAPs) have gained enormous interest in recent years due to their applications in different fields. Synthesis of well-defined monodisperse SAPs is accomplished predominantly through genetic methods. However, the last decade has witnessed the use of a few chemical technologies for this purpose. In particular, micelle-assisted protein labeling technology (MAPLabTech) has made huge progress in this area. The first generation MAPLabTech focused on site-specific labeling of the active-site residue of serine proteases to make SAPs. Further, this methodology was exploited for labeling of N-terminal residue of a globular protein to make functional SAPs. In this study, we describe the synthesis of novel SAPs by developing a chemical method for site-specific labeling of a surface-exposed cysteine residue of globular proteins. In addition, we disclose the synthesis of redox-sensitive SAPs and their systematic self-assembly and disassembly studies using size-exclusion chromatography. Altogether these studies further expand the scope of MAPLabTech in different fields such as vaccine design, targeted drug delivery, diagnostic imaging, biomaterials, and tissue engineering.     

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     

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.     

Half‐Life Extension of Biopharmaceuticals using Chemical Methods: Alternatives to PEGylation

Peptides and proteins constitute a vast pool of excellent drug candidates. Evolution has equipped these molecules with superior drug‐like properties such as high specificity and potency. However, native peptides and proteins suffer from an inadequate pharmacokinetic profile, and their outstanding pharmacological potential can only be realized if this issue is addressed during drug development. To overcome this challenge, a variety of half‐life extension techniques relying on covalent chemical modification have been developed. These methods include PEGylation, fusion to unstructured polypeptide‐based PEG mimetics, conjugation of large polysaccharides, native‐like glycosylation, lipidation, fusion to albumin or the Fc domain of IgG, and derivatization with bio‐orthogonal moieties that direct self‐assembly. This review provides an overview of available conjugation chemistries, biophysical properties, and safety data associated with these concepts. Moreover, the effects of these modifications on peptide and protein pharmacokinetics are demonstrated through key examples.     

Bioorthogonal chemistry for site-specific labeling and surface immobilization of proteins

Understanding protein structure and function is essential for uncovering the secrets of biology, but it remains extremely challenging because of the high complexity of protein networks and their wiring. The daunting task of elucidating these interconnections requires the concerted application of methods emerging from different disciplines. Chemical biology integrates chemistry, biology, and pharmacology and has provided novel techniques and approaches to the investigation of biological processes. Among these, site-specific protein labeling with functional groups such as fluorophors, spin probes, and affinity tags has greatly facilitated both in vitro and in vivo studies of protein structure and function. Bioorthogonal chemical reactions, which enable chemo- and regioselective attachment of small-molecule probes to proteins, are particularly attractive and relevant for site-specific protein labeling. The introduction of powerful labeling techniques also has inspired the development of novel strategies for surface immobilization of proteins to create protein biochips for in vitro characterization of biochemical activities or interactions between proteins. Because this process requires the efficient immobilization of proteins on surfaces while maintaining structure and activity, tailored methods for protein immobilization based on bioorthogonal chemical reactions are in high demand. In this Account, we summarize recent developments and applications of site-specific protein labeling and surface immobilization of proteins, with a special focus on our contributions to these fields. We begin with the Staudinger ligation, which involves the formation of a stable amide bond after the reaction of a preinstalled azide with a triaryl phosphine reagent. We then examine the Diels-Alder reaction, which requires the protein of interest to be functionalized with a diene, enabling conjugation to a variety of dienophiles under physiological conditions. In the oxime ligation, an oxyamine is condensed with either an aldehyde or a ketone to form an oxime; we successfully pursued the inverse of the standard technique by attaching the oxyamine, rather than the aldehyde, to the protein. The click sulfonamide reaction, which involves the Cu(I)-catalyzed reaction of sulfonylazides with terminal alkynes, is then discussed. Finally, we consider in detail the photochemical thiol-ene reaction, in which a thiol adds to an ene group after free radical initiation. Each of these methods has been successfully developed as a bioorthogonal transformation for oriented protein immobilization on chips and for site-specific protein labeling under physiological conditions. Despite the tremendous progress in developing such transformations over the past decade, however, the demand for new bioorthogonal methods with improved kinetics and selectivities remains high.     

PEGylation and aqueous solution behaviour of pH responsive poly(l-lysine iso-phthalamide)

The effect of PEGylation on the aqueous solution properties of a pH responsive pseudopeptide, poly(l-lysine iso-phthalamide), has been investigated using UV and fluorescence spectroscopy, ¹H NMR spectroscopy and dynamic light scattering. It was demonstrated that the level of PEGylation had a critical effect on the pH response of the parent polymer. When the degree of PEGylation was less than 23.4 wt% the modified polymer exhibited a transition from an expanded structure at high degrees of ionization to a compact hydrophobically stabilised structure at low degrees of ionization. The specific pH at which the conformational transition occurred was dependent on the degree of PEGylation. At levels of PEGylation in excess of 25.6 wt% the polymer no longer displayed this pH dependent conformation and existed in a micellar form (100-200 nm) over the whole range of ionization. Both linear and micellar forms of the pseudopeptide have applications in drug delivery.     

PEG修饰蛋白质等高分子

蛋白质等高分子用于人类疾病的治疗近几年得到飞速发展。尽管有许多产品即将上市,但它们却存在诸如半衰期短、免疫原性及溶解性低、被酶水解等弊端。用PEG(聚乙二醇)修饰这些蛋白质等高分子,就会明显消除这些不利因素,使蛋白质等高分子性能得到改善,应用领域更加广阔。