Published: May 14, 2011 r2011 American Chemical Society 1017 dx.doi.org/10.1021/bc2001374 | Bioconjugate Chem. 2011, 22, 1017–1020 COMMUNICATION pubs.acs.org/bc Recombinant Protein Hydrazides: Application to Site-Specific Protein PEGylation Jennifer Thom, † David Anderson, † Joanne McGregor, †,‡ and Graham Cotton* ,† † Almac Sciences, Elvingston Sciences Centre, Gladsmuir, East Lothian, EH33 1EH, United Kingdom b S Supporting Information R ecombinant protein therapeutics have emerged as an effec- tive treatment for a variety of conditions ranging from cancer to metabolic disorders and autoimmune diseases, but they are commonly limited by their poor pharmacokinetics and immu- nogenicity. Arguably, the most widely used and accepted method for improving the pharmacokinetics of proteins is through PEGylation. 1,2 The covalent attachment of poly(ethylene glycol) (PEG) increases the half-life of the protein in vivo by decreasing renal clearance, due to the large hydrodynamic radius of the polymer, as well as masking potentially immunogenic epitopes and protease cleavage sites. Furthermore, PEGylation of a target protein can substantially improve its solubility and stability. There are currently nine PEGylated protein therapeutics ap- proved for clinical use; 1 however, established methods for PEGylation are generally non-site-selective, resulting in a mixture of protein PEG-positional isomers with significantly decreased biological activity. For example, ViraferonPEG/PegIntron (Schering Plough), approved for the treatment of Hepatitis C, is produced by conjugating recombinant interferon alpha2b (IFNalpha2b) to a single chain 12 kDa succinimidyl carbonate PEG. This results in 95% monoPEGylated protein comprising 14 positional isomers, and as a consequence, PegIntron retains only 28% antiviral activity in in vitro assays compared to non-PEGy- lated IFNalpha2b. 3 There is therefore a pressing need for methods to site- specifically PEGylate proteins. By incorporating a single PEG moiety at a defined position within the protein sequence, the deleterious effects associated with nonselective PEGylation may be minimized. In addition, approaches that yield homogeneous PEGylated proteins are potentially attractive from a regulatory perspective. While a number of protein engineering approaches have been applied to this end, 46 one particularly versatile technology for site-specific protein modification is expressed protein ligation (EPL). The basis of EPL is the production of recombinant C-terminal thioester proteins through thiol- mediated cleavage of the corresponding intein fusion protein. 7,8 The protein thioester functionality enables peptides and labels to be chemoselectively attached to the protein using the well- established native chemical ligation reaction. 9 In addition, C-terminal cleavage of the protein thioester with nucleophiles has also been used to introduce reactive moieties into proteins to enable subsequent modification through alternative chemistries. 1012 While the EPL approach has proven extremely powerful, the steps are performed in the presence of thiol additives, which may compromise its use with certain protein families (for example, disulfide bond containing proteins), and the protein thioesters themselves are often labile. Here, we report the development of a versatile complementary protein ligation approach for site-specific protein PEGylation, which is compatible with folded disulfide bond-containing proteins. This process is based on chemical interception of intein-mediated protein splicing, 13 to create C-terminal hydra- zide recombinant proteins. This facile production of recombi- nant hydrazide proteins then facilitates direct site-specific C-terminal PEGylation (or other modifications) of the target protein using hydrazone bond-forming reactions 14,15 (Figure 1). In principle, any PEG derivative (or alternative label) containing the appropriate functionality, namely, a ketone or aldehyde, can be chemoselectively attached to the C-terminus of the protein in this one-step process. For the approach described here, we have developed novel pyruvoyl PEG derivatives for attachment to the Received: March 18, 2011 Revised: May 11, 2011 ABSTRACT: Here, we describe a novel method for the site- specific C-terminal PEGylation of recombinant proteins. This general approach exploits chemical cleavage of precursor intein- fusion proteins with hydrazine to directly produce recombinant protein hydrazides. This unique functionality within the protein sequence then facilitates site-specific C-terminal modification by hydrazone-forming ligation reactions. This approach was used to generate folded, site-specifically C-terminal PEGylated IFNalpha2b and IFNbeta1b, which retained excellent antiviral activity, demonstrating the utility of this technology in the PEGylation of therapeutic proteins. As this methodology is straightforward to perform, is compatible with disulfide bonds, and is exclusively selective for the protein C-terminus, it shows great potential as general technology for the site-specific engineering and labeling of recombinant proteins.
Transcript
Published: May 14, 2011
r 2011 American Chemical Society 1017 dx.doi.org/10.1021/bc2001374 | Bioconjugate Chem. 2011, 22, 1017–1020
COMMUNICATION
pubs.acs.org/bc
Recombinant Protein Hydrazides: Application to Site-Specific ProteinPEGylationJennifer Thom,† David Anderson,† Joanne McGregor,†,‡ and Graham Cotton*,†
†Almac Sciences, Elvingston Sciences Centre, Gladsmuir, East Lothian, EH33 1EH, United Kingdom
bS Supporting Information
Recombinant protein therapeutics have emerged as an effec-tive treatment for a variety of conditions ranging from cancer
to metabolic disorders and autoimmune diseases, but they arecommonly limited by their poor pharmacokinetics and immu-nogenicity. Arguably, the most widely used and accepted methodfor improving the pharmacokinetics of proteins is throughPEGylation.1,2 The covalent attachment of poly(ethylene glycol)(PEG) increases the half-life of the protein in vivo by decreasingrenal clearance, due to the large hydrodynamic radius of thepolymer, as well as masking potentially immunogenic epitopesand protease cleavage sites. Furthermore, PEGylation of a targetprotein can substantially improve its solubility and stability.There are currently nine PEGylated protein therapeutics ap-proved for clinical use;1 however, established methods forPEGylation are generally non-site-selective, resulting in amixtureof protein PEG-positional isomers with significantly decreasedbiological activity. For example, ViraferonPEG/PegIntron(Schering Plough), approved for the treatment of Hepatitis C,is produced by conjugating recombinant interferon alpha2b(IFNalpha2b) to a single chain 12 kDa succinimidyl carbonatePEG. This results in 95%monoPEGylated protein comprising 14positional isomers, and as a consequence, PegIntron retains only28% antiviral activity in in vitro assays compared to non-PEGy-lated IFNalpha2b.3
There is therefore a pressing need for methods to site-specifically PEGylate proteins. By incorporating a single PEGmoiety at a defined position within the protein sequence, thedeleterious effects associated with nonselective PEGylation maybe minimized. In addition, approaches that yield homogeneousPEGylated proteins are potentially attractive from a regulatoryperspective. While a number of protein engineering approacheshave been applied to this end,4�6 one particularly versatiletechnology for site-specific protein modification is expressed
protein ligation (EPL). The basis of EPL is the production ofrecombinant C-terminal thioester proteins through thiol-mediated cleavage of the corresponding intein fusion protein.7,8
The protein thioester functionality enables peptides and labels tobe chemoselectively attached to the protein using the well-established native chemical ligation reaction.9 In addition,C-terminal cleavage of the protein thioester with nucleophileshas also been used to introduce reactive moieties into proteins toenable subsequent modification through alternativechemistries.10�12 While the EPL approach has proven extremelypowerful, the steps are performed in the presence of thioladditives, which may compromise its use with certain proteinfamilies (for example, disulfide bond containing proteins), andthe protein thioesters themselves are often labile.
Here, we report the development of a versatile complementaryprotein ligation approach for site-specific protein PEGylation,which is compatible with folded disulfide bond-containingproteins. This process is based on chemical interception ofintein-mediated protein splicing,13 to create C-terminal hydra-zide recombinant proteins. This facile production of recombi-nant hydrazide proteins then facilitates direct site-specificC-terminal PEGylation (or other modifications) of the targetprotein using hydrazone bond-forming reactions14,15 (Figure 1).In principle, any PEG derivative (or alternative label) containingthe appropriate functionality, namely, a ketone or aldehyde, canbe chemoselectively attached to the C-terminus of the protein inthis one-step process. For the approach described here, we havedeveloped novel pyruvoyl PEG derivatives for attachment to the
Received: March 18, 2011Revised: May 11, 2011
ABSTRACT: Here, we describe a novel method for the site-specific C-terminal PEGylation of recombinant proteins. Thisgeneral approach exploits chemical cleavage of precursor intein-fusion proteins with hydrazine to directly produce recombinantprotein hydrazides. This unique functionality within the proteinsequence then facilitates site-specific C-terminal modificationby hydrazone-forming ligation reactions. This approach wasused to generate folded, site-specifically C-terminal PEGylated IFNalpha2b and IFNbeta1b, which retained excellent antiviralactivity, demonstrating the utility of this technology in the PEGylation of therapeutic proteins. As this methodology isstraightforward to perform, is compatible with disulfide bonds, and is exclusively selective for the protein C-terminus, it showsgreat potential as general technology for the site-specific engineering and labeling of recombinant proteins.
protein C-terminus, as the resulting R-oxo hydrazone bonds arestabilized through resonance effects.16,17
IFNalpha2b was chosen as our initial target for this C-terminalPEGylation approach. As discussed above, this therapeuticallyimportant protein is currently administered as the PEG isomermixture, and thus there is scope for improvement through site-specific PEGylation. IFNalpha2bwas expressed inE. coli as a solubleN-terminal fusion to the Mycobacterium xenopi GyrA intein, andpurified from the supernatant by immobilization on chitin beadsusing a chitin binding domain (CBD) fused to the C-terminus ofthe intein domain (Figure 2a lane 3 and Supporting Information).The immobilized IFNalpha2b-intein-CBDproteinwas cleavedwithhydrazine to generate the corresponding IFNalpha2b C-terminalhydrazide (Figure 2a,b). The isolated protein (Figure 2c lane 2)contained two disulfide bonds (Supporting Information) and hadpotent antiviral activity (Figure 2d) indicating that folded IFNal-pha2b C-terminal hydrazide was directly produced after expressionand hydrazine cleavage of the corresponding intein fusion protein.
This is in contrast to standard intein cleavage methods that usethiols such as dithiothreitol (DTT) and sodium mercaptoethylsul-fonate (MESNa) that usually reduce disulfide bonds within thetarget protein.
For the PEGylation reactions, a pyruvoyl derivative of 10 kDaPEGwas synthesized and incubated with IFNalpha2b hydrazide asdescribed in the Supporting Information. This typically afforded60�75% yield of the C-terminal PEGylated IFNalpha2b protein(Supporting Information, Figure S1). Importantly, no PEGylationproduct was observed when a C-terminal thioester derivative ofIFNalpha2b was incubated with pyruvoyl PEG under the sameconditions, consistent with site-specific PEGylation via theC-terminal hydrazide group only (data not shown). PEGylatedIFNalpha2b was purified in 2 steps: ion exchange to removeunreacted pyruvoyl-PEG, followed by gel filtration to separatePEGylated IFNalpha2b from any unreacted IFNalpha2b hydra-zide (Supporting Information, Figure S2) to yield pureC-terminal,mono PEGylated IFNalpha2b (Figure 2c, lane 3).
The antiviral activities of the C-terminally PEGylated IFNal-pha2b and IFNalpha2b hydrazide control purified under the
Figure 1. Use of intein technology to generate recombinant C-terminalhydrazide proteins for site-specific PEGylation. (a) The recombinantprotein of interest (IFNalpha2b shown here) is genetically fused to theN-terminus of an engineered GyrA intein and chitin binding domain(CBD), expressed in E. coli and purified by immobilization onto chitinbeads. (b) The first residue of this intein domain is a cysteine. Theproperties of the intein domain are such that it induces an N to S acylshift at this protein-intein junction to form a branched thioesterintermediate. (c) This thioester intermediate is chemically cleaved usinghydrazine to liberate the corresponding C-terminal hydrazide derivativeof the target protein. The intein-CBD remains bound to the chitin beads.(d) The protein C-terminal hydrazide chemoselectively reacts withketone or aldehyde containing moieties. Here the reaction with apyruvoyl functionalized PEG is shown, resulting in site-specific C-term-inal PEGylation of the target protein via the resonance stabilized R-oxohydrazone linkage.
Figure 2. Generation and antiviral activity of site-specifically C-terminalPEGylated IFNalpha2b. (a) SDS PAGE analysis of IFNalpha2b-intein-CBD fusion protein expression in E. coli, purification using chitin beadsand hydrazine cleavage. Protein standard (lane 1), cell lysate (lane 2),IFNalpha2b-intein-CBD purified by immobilization on chitin beads(lane 3), supernatant from hydrazine cleavage (lane 4), supernatantfrom washed beads (lane 5), cleaved and washed chitin beads (lane 6).Arrows indicate IFNalpha2b-intein-CBD fusion protein (i), intein-CBD(ii), and IFNalpha2b C-terminal hydrazide (iii). (b) Electrospray massspectra of purified IFNalpha2b hydrazide, expected mass (without theN-terminal methionine) is 19 340 Da. The higher observed massprobably reflects methionine oxidation, previously reported for IFNal-pha2b and shown not to significantly affect the activity of the protein.24
(c) SDS PAGE analysis of IFNalpha2b hydrazide (lane 2) and purifiedC-terminal PEGylated IFNalpha2b (lane 3). (d) Antiviral activity ofIFNalpha2b derivatives in a cytopathic effect inhibition assay usinghuman A549 lung carcinoma cells and EMC virus. Measured activitiesare in million international units, MIU/mg IFNalpha2b protein ( SDwhere appropriate; IFNalpha2b hydrazide and C-terminal PEGylatedIFNalpha2b (n = 8), IFNalpha2b standard (n = 2) and ViraferonPEG (n= 2). For reported values, see Supporting Information. IFNalpha2bstandard and ViraferonPEG activities are in line with previous reports.3.
same conditions were measured in a cytopathic effect inhibitionassay with human A549 lung carcinoma cells and encephalo-myocarditis (EMC) virus (Figure 2d). The site-specificallyC-terminal PEGylated IFNalpha2b retained 74% of the activityof the standard IFNalpha2b protein, more than double that of thecurrent heterogeneous PEGylated IFNalpha2b therapeutic Vir-aferonPEG, clearly demonstrating the advantage of thistechnology.
To further exemplify this site-specific C-terminal PEGylationtechnology, IFNbeta1b was deemed to be an attractive target.This protein is also an important therapeutic, but currently thereare no PEGylated versions approved. Betaseron (Betaferon inthe EU, Bayer Shering Pharma) and more recently Extavia(Novartis) are recombinant IFNbeta1b proteins used in thetreatment of multiple sclerosis, and are under clinical evaluationfor the treatment of other diseases including hepatitis and certaincancers.18,19 IFNbeta1b is rapidly cleared from blood necessitat-ing frequent administration regimes that can result in injectionsite necrosis and decreased patient compliance. Neutralizingantibodies are also problematic, with up to 35% of patientsdeveloping these in one reported study.20 In addition, IFNbeta1bis a very unstable protein, prone to aggregation.21 Work toaddress these issues includes random PEGylation of side-chainprimary amines, which results in a mixture of 5 major positionalisomers, and PEGylation of (predominantly) the N-terminalamine, resulting in products with 22�31% and 19�66% activityof the unmodified IFNbeta1b, respectively.22 Attempts at sitespecific PEGylation using an engineered free cysteine residue ateither position 79 (the natural glycosylation site) or the N- orC-termini were unsuccessful due to insufficient site specificattachment.22 Another strategy used bicin (bis-N-2-hydro-xyethylglycinamide) linkers to randomly attach 2�3 PEG mol-ecules. Under physiological conditions, rapid hydrolysis of thebicin releases the PEG to leave the active IFNbeta1b. However,the activity of these releasable PEGylated forms was onlybetween 7% and 27% that of the unmodified IFNbeta1b.23
We expressed soluble IFNbeta1b-intein-CBD fusion protein inE. coli and purified the target protein through immobilization ontochitin beads. Intein cleavage was induced by overnight treatmentwith hydrazine and the resulting IFNbeta1b-hydrazide was pur-ified by gel filtration (Figure 3a,b; Supporting Information). Aswith IFNalpha2b, IFNbeta1b C-terminal hydrazide was generatedwith the natural disulfide bond intact indicating that the proteinwas correctly folded (Figure 3a and Supporting Information). Togenerate site-specifically C-terminal PEGylated IFNbeta1b, IFN-beta1b C-terminal hydrazide was mixed with the pyruvoyl PEG10 kDa derivative and left overnight at room temperature. Thistypically gave ∼55% yield of the C-terminal PEGylated protein(Supporting Information, Figure S3a). No PEGylation productwas observedwhen IFNbeta1bC-terminal acidwas incubatedwithpyruvoyl PEG under similar conditions, consistent with site-specific PEGylation via the C-terminal hydrazide group only(Supporting Information, Figure S3b).
The site-specifically C-terminal PEGylated IFNbeta1b waspurified by ion exchange and gel filtration as described in theSupporting Information to generate pure C-terminal PEGylatedIFNbeta1b (Figure 3c lane 3). The antiviral activity was mea-sured in a cytopathic effect inhibition assay with human A549lung carcinoma cells and EMC virus (Figure 3d). The activity ofIFNbeta1b hydrazide was lower than that reported for Betaseron,potentially due to the inherent instability of the protein and thelack of stabilizing ingredients in our formulation. Site-specifically
C-terminal PEGylated IFNbeta1b showed greater activity thanthe hydrazide, probably reflecting the increased protein stabilitybrought by the PEGylation, and this activity was in line with thatreported for the current unPEGylated IFNbeta1b therapeutic,Betaseron. This, to the best of our knowledge, represents themost potent PEGylated IFNbeta1b derivative generated to date.
In summary, we have demonstrated the feasibility of generat-ing active, site-specifically C-terminal PEGylated therapeuticproteins through cleavage of the corresponding soluble inteinfusion proteins with hydrazine, followed by reaction of theresulting protein hydrazide with pyruvoyl functionalized PEG.Importantly, this approach is compatible with proteins contain-ing disulfide bonds, enabling folded proteins to be directlyproduced as the C-terminal hydrazide derivatives in high yield,with the integrity of any disulfide bonds and tertiary structuremaintained during the PEGylation reaction. While the potentialof this technology has been highlighted through the generation of
Figure 3. Generation and antiviral activity of site-specifically C-terminalPEGylated IFNbeta1b. (a) SDS PAGE analysis of IFNbeta1b-intein-CBD fusion protein expression in E. coli and the subsequent purificationand hydrazine cleavage. Samples were reduced withDTT after heating inloading buffer unless stated otherwise: protein standard (lanes 1 and 4),cell lysate (lane 2), IFNbeta1b-intein-CBD purified by immobilizationon chitin beads (lane 3), hydrazine cleavage supernatant without DTT(lane 5), hydrazine cleavage supernatant (lane 6), supernatant fromwashed beads (lane 7), cleaved and washed chitin beads (lane 8). Arrowsindicate IFNbeta1b-intein-CBD fusion protein (i), intein-CBD (ii), andIFNbeta1b C-terminal hydrazide (iii). There is clearly a shift in themigration of cleaved IFNbeta1b hydrazide upon DTT treatment,consistent with the native disulfide bond being intact in the liberatedprotein. (b) Electrospray mass spectra of purified IFNbeta1b hydrazide,expected mass 19 950 Da. The higher observed mass probably againreflects methionine oxidation. (c) SDS PAGE analysis of purifiedIFNbeta1b hydrazide (lane 1) and C-terminal PEGylated IFNbeta1b(lane 3); protein standard (lane 2). (d) Antiviral activity of IFNbeta1bderivatives in a cytopathic effect inhibition assay using human A549 lungcarcinoma cells and EMC virus. Activities are in million internationalunits, MIU/mg IFNbeta protein ( SD; n = 8. The reported antiviralactivity of Betaseron, the current IFNbeta1b therapeutic, is also shown(Betaseron prescribing information; http://berlex.bayerhealthcare.com/html/products/pi/Betaseron_PI.pdf). WHO calibrated IFNbetaprotein was used as a standard in the assays.
highly active C-terminal PEGylated derivatives of IFNalpha2band IFNbeta1b, we have now used this technology to C-termin-ally PEGylate proteins from a variety of classes, including anti-body fragments, with PEGs of different molecular weights. Inaddition to PEGylation, this technology has broader applicationsin protein engineering and has been successfully used to attach anarray of probes and labels, including fluorophores and syntheticpeptides, onto a variety of recombinant proteins in a site-specificfashion (unpublished data). Given the ease of the process, theyields of the associated steps, and the compatibility with disulfidebond-containing proteins, this technology offers an attractiveapproach for the site-specific PEGylation and modification oftherapeutically important proteins and is an attractive addition tothe repertoire ofmethodologies available for protein engineering,opening up new opportunities in this field.
’ASSOCIATED CONTENT
bS Supporting Information. Experimental procedures andsupplementary data. This material is available free of charge viathe Internet at http://pubs.acs.org.
’AUTHOR INFORMATION
Corresponding Author*Graham Cotton, Almac Sciences (Scotland) Ltd, ElvingstonScience Centre, By Gladsmuir, East Lothian, EH33 1EH UnitedKingdom. Tel. þ44 (0)28 3839 5879, Fax. þ44 (0)18 75408151, E-mail [email protected].
We thank Moredun Research Institute (Edinburgh, UK) forMALDI mass-spectrometry analysis, Crystelle Bermand (AlmacSciences, UK) for technical advice on the synthesis of PEGderivatives, and the Scottish Executive for financial support.
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