Analytical characterization of a novel degradation product in a PEGylated recombinant protein

Analytical Characterization of a Novel Degradation Productin a PEGylated Recombinant Protein

BING ZHANG,1 EDWARD W. TOWERS,1 LESZEK POPPE,2 STEVEN L. COCKRILL1

1Analytical Sciences, Amgen, Inc., Longmont, Colorado 80503

2Molecular Structure, Amgen, Inc., Thousand Oaks, California 91320

Received 2 May 2011; revised 19 May 2011; accepted 20 May 2011

Published online 6 June 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22661

ABSTRACT: We report the identification and characterization of a novel degradation prod-uct associated with PEGylation of a recombinant protein. After several months of storage at2◦C–8◦C, an unexpected increase was observed in the proportion of an impurity that elutedwith the native unPEGylated protein by size exclusion chromatography-–from less than 0.01%at the start of storage to more than 0.25% at 12 months. An investigation into the nature of theimpurity determined the presence of an N-terminal adduction with a mass increase of +58 Daover the native unPEGylated protein species, demonstrating that this impurity was the resultof degradation. The impurity was subjected to thorough analytical characterization using or-thogonal methods to establish its identity, and a mechanistic model proposed for its formation.The data implicate the presence of a monomethoxy polyethylene glycol (mPEG)–acetal aldehydeimpurity in the mPEG–aldehyde raw material, indicating the need for diligent raw materialtesting prior to use. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J PharmSci 100:4607–4616, 2011Keywords: analytical biochemistry; PEGylation; degradation; protein structure; mass spec-trometry; NMR spectroscopy; adduct

INTRODUCTION

Covalent coupling of synthetic polymers to proteinsubstrates provides beneficial properties for both invivo and in vitro applications. For example, chemi-cal modification with polymeric substrates has beenreported to alter surface and solubility properties,reduce immune response, and modulate the phar-macokinetics and/or pharmacodynamics of therapeu-tic proteins.1–11 Polymer derivitization has even beenused as an alternative to glycoengineering for legacyproducts.12

Both synthetic and natural polymers have beenreported for use in generating conjugated pro-teins or drug substrates, including polysaccharides,polyamino acids such as poly-L-lysine, poly(vinylalcohols), polyvinylpyrrolidinones, poly(acrylic acid)derivatives, polyurethanes, and polyphosphazenes.13

Correspondence to: Steven L. Cockrill (Telephone:+3034011894; Fax: +3034014403; E-mail: [email protected])

Present address: Bing Zhang’s is Boehringer Ingelheim,Ridgefield, Connecticut.Journal of Pharmaceutical Sciences, Vol. 100, 4607–4616 (2011)© 2011 Wiley-Liss, Inc. and the American Pharmacists Association

The two most widely used polymer derivativesfor improved drug delivery strategies are dextranand polyethylene glycol (PEG).14 Several PEGylatedtherapeutic proteins have been successfully com-mercialized, including Adagen R© (Enzon, Piscataway,NJ, 1990), Oncaspar R© (Enzon, 1994), PegIntron R©

(Schering–Plough, Kenilworth, NJ, 2000), Pegasys R©

(Hoffmann-La Roche, Basel, Switzerland, 2002),Neulasta R© (Amgen, Thousand Oaks, CA, 2002),Somavert R© (Pharmacia & Upjohn, Kalamazoo, MI,2002), Macugen R© (OSI/Pfizer, Melville, NY, 2004),Micera R© (Roche, 2007), and Cimzia R© (UCB, Brussels,Belgium, 2008).15 More than a dozen more PEGylateddrug candidates are in development.15 Overall, PEGy-lation has proven to be a safe and effective method forimproving the properties of therapeutic proteins.

Many chemistries have been reported for the at-tachment of PEG to the target protein, offeringa broad selection of specificity and target.2,10,16

Most common is the use of nucleophilic attack bythe electrophilic group of suitably derivatized PEGtargeting the ε-amino group of lysine residues orthe protein’s N-terminus. Control of the reactionconditions, particularly pH, may afford control

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over site-specific PEGylation and avoids or min-imizes positional isomers that result in hetero-geneous PEG conjugates. Linkages through freecysteine residues have been targeted through theuse of PEG–maleimide,17 PEG–vinyl sulfone,18 orPEG–orthopyridyl disulfide.19 Alternatively, conjuga-tion may be directed to the protein’s N-terminal amineusing PEG–aldehyde.20

PEGylation introduces a significant increase inthe molecular weight of a native protein, affordingready separation of the PEGylated and native pro-tein species by size-based methods such as sodiumdodecyl sulfate polyacrylamide gel electrophoresis orsize-exclusion high-performance liquid chromatogra-phy (SE-HPLC). In the present study, the proportionof an impurity that eluted with the native unPEGy-lated protein by size exclusion chromatography wasfound to increase unexpectedly after several monthsof storage at 2◦C–8◦C. The impurity peak increasedfrom less than 0.01% at the start of the storage tomore than 0.25% at 12 months. Several possible rootcauses for the apparent release of the PEG moietywere considered, including proteolytic cleavage of theprotein backbone and hydrolysis of the bond link-ing the PEG to the protein’s N-terminus. These con-siderations directed the analytical strategy, and aninvestigation into the nature of the impurity wasundertaken. The results of this structural elucida-tion demonstrated the presence of an adduct speciespresent on the N-terminus of the protein that in-creased during the storage.

MATERIALS AND METHODS

Materials

Reagents were sourced as follows: sodium phosphate,dithiothreitol (DTT), and urea were from Sigma–Aldrich (St. Louis, Missouri); trifluoroacetic acid(TFA) and tris(2–carboxyethyl)phosphine (TCEP)were from Pierce (Rockford, Illinois); solvents wereprocured from J.T. Baker (Phillipsburg, NJ); and se-quencing grade endoprotease Glu-C was from Roche(Indianapolis, Indiana).

Material Generation

A recombinant protein was subjected to PE-Gylation using established methods based ontargeted N-terminal derivatization chemistry us-ing commercially available monomethoxy PEG(mPEG)–aldehyde,20 and incubated at 2◦C–8◦C for upto 12 months. Control substrates included the unPE-Gylated recombinant protein starting material and aPEGylated protein derivatized using a different lot ofmPEG–aldehyde.

Isolation of Degradation Product by SE-HPLC

The degradation product impurity species was iso-lated from the PEGylated main peak and high molec-ular weight species by SE-HPLC. Separation was per-formed by loading 100:L of protein solution onto aG-3000SWxl gel filtration column (Tosoh Bioscience,King of Prussia, Pennsylvania) equilibrated with50 mM sodium phosphate (pH 6.4) and 5% reagent-grade alcohol flowing at 0.5 mL/min. Protein elutionwas monitored at 280 nm and the degradation prod-uct peak was collected from 20.3 to 21.5 min from atotal of seven injections of the degraded PEGylatedprotein. The collected fractions were combined andconcentrated by ultrafiltration.

Whole Mass Analysis

The enriched degradation product peak from the de-graded PEGylated protein and the unPEGylated con-trol protein were assessed by mass spectrometry (MS)using an MSD-TOF electrospray ionization time-of-flight instrument (Agilent, Santa Clara, California) inpositive-ion mode. Reverse-phase HPLC (RP-HPLC,Agilent 1200; Agilent) was used to desalt the sam-ples with a Jupiter C4 column (Phenomenex, Tor-rence, California) held at 60◦C. Mobile phases A and Bwere 0.1% TFA in HPLC-grade water and 0.09% TFAin 90% acetonitrile, respectively, and separation wasachieved using a short linear gradient of 40%–80%mobile phase B in 8 min at a flow rate of 0.4 mL/min.

Glu-C Peptide Mapping with MS and MS/MS Detection

Samples were digested with endoprotease Glu-C bydissolving 80:g of each sample in 390:L of diges-tion buffer containing 10 mM DTT and 2.5 M urea.Next, each sample was treated with 2:g Glu-C, over-laid with nitrogen, and incubated at room tempera-ture overnight. The reaction was stopped by additionof 18:L of 5% TFA. Unfractionated samples of theunPEGylated and PEGylated protein were analyzedand compared with enriched degradation productpeak previously isolated by SE-HPLC.

Reverse-phase ultra performance liquid chro-matography (RP-UPLC) using an Acquity UPLC sys-tem (Waters, Milford, Massachusetts) equipped witha 2.0 × 100 mm, 1.7-:m particle size BEH C18 column(Waters) was used for the separation of approximately4:g protein digest. Mobile phases were as for thewhole mass analysis, and the peptide map was exe-cuted using a linear gradient of 5%–50% mobile phaseB over 38 min at 0.3 mL/min. Detection was affordedby ultraviolet absorbance at 215 nm and MS usingan LTQ Orbitrap XL instrument (Thermo Fisher,Waltham, Massachusetts). Three scan events wereused: a survey scan over the range m/z 200–2000;MS/MS using higher-energy C-trap dissociation(HCD) of the most intense ion; and MS/MS using

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 11, NOVEMBER 2011 DOI 10.1002/jps

CHARACTERIZATION OF A NOVEL PEGYLATION DEGRADATION PRODUCT 4609

Figure 1. SE-HPLC of degraded and control proteins.

collision-induced dissociation (CID) of the most in-tense ion. Alternatively, species of interest were col-lected manually from the UPLC eluent for subsequentanalysis.

High-Resolution Nuclear Magnetic Resonance

Approximately 70:g of lyophilized adducted peptideisolated from repetitive Glu-C peptide mapping of thedegradation product impurity fraction was dissolvedin 200:L volume of 90% H2O/ 10% D2O. The unad-justed pH was 3.3. The sample was flushed with argonand a trace amount of TCEP was added to preventoxidation.

Nuclear magnetic resonance (NMR) experimentswere performed on a Bruker Avance-III 800 MHzspectrometer (Bruker, Billerica, Massachusetts),equipped with a triple-resonance TCI CryoProbe(Bruker). NMR data were acquired using standardBruker pulse-program library (Bruker) and processedwith Bruker Topspin 2.0 software (Bruker). Chemi-cal shifts were referenced to sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS).

RESULTS AND DISCUSSION

Enrichment of Degradation Product by SE-HPLC

Size-exclusion high-performance liquid chromatogra-phy was utilized to separate and enrich the degrada-tion product impurity and main peak fractions fromthe degraded PEGylated protein stored at 2◦C–8◦C.The SE-HPLC profiles of the degraded PEGylatedprotein, a control PEGylated protein, and nativeunPEGylated protein are shown in Figure 1.

The separation demonstrates the presence of anelevated peak in the degraded material at approx-imately 20.5 min, which appears comparable to thenative unPEGylated protein in retention time.

Following isolation and enrichment, the main peakand degradation product impurity fractions fromthe degraded protein were reanalyzed by SE-HPLC,which demonstrated that the fractionation process re-sulted in enrichment to more than 90% purity foreach fraction. The enriched fractions were then sub-jected to further characterization with the main peakfraction serving as a control.

Whole Mass Determination

The apparent loss of the PEG moiety (inferred fromthe SE-HPLC elution of the degraded product peak)could be explained by a variety of root causes, includ-ing proteolytic cleavage of the polypeptide backbonenear the PEGylation site or hydrolysis of the bondbetween PEG and the protein’s N-terminus.

Whole mass determination by liquid chromatogra-phy (LC)–MS analysis of native unPEGylated proteinand the enriched fraction of the degradation productisolated by SE-HPLC was performed to derive a pu-tative identification of the degradation product, usingthe native unPEGylated protein as a comparator. Theexpected average mass of the native unPEGylatedprotein is 18,798.70 Da; therefore, the expected m/zvalue for a 10+ charge state is m/z 1880.88. As shownin Figure 2 (top panel), an ion is present with an ob-served m/z of 1880.84, in good agreement with theexpected value.

Unexpectedly, the whole mass determination ofthe enriched degradation product demonstrated the

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 11, NOVEMBER 2011

4610 ZHANG ET AL.

Figure 2. Comparative whole mass determination of (top) native unPEGylated protein and(bottom) degradation product impurity species isolated from degraded material.

presence of a shift to higher molecular weight ratherthan finding a loss of mass, as would be expected fromproteolytic cleavage. As shown in Figure 2 (bottompanel), similar charge envelopes were observed forthe degradation product and the native unPEGylatedprotein. Examination of the 10+ charge state ion forthe degradation product impurity sample reveals them/z to be 1886.64, which is an m/z difference of 5.8.Considering the charge state is 10+, the actual massdifference between the native unPEGylated proteinand the degradation product is 58 Da.

Glu-C Peptide Mapping of Enriched Fractionsfrom Degraded Material

To confirm these results further, and to identify thelocation of the +58 Da adduct, comparative peptidemapping with endoprotease Glu–C was performed onthe enriched main peak (PEGylated) and the enricheddegradation product fractions isolated by SE-HPLCfrom the degraded material (Fig. 1). The peptide mapsshown in Figure 3 clearly demonstrate that the differ-ence between the PEGylated main peak and degrada-tion product peak fractions is related to the peptideeluting at approximately 32 min. The peptide map ofnative unPEGylated protein demonstrated a peptideeluting at the same time, identified as the N-terminalpeptide by MS/MS (data not shown).

Mass spectrometric analysis of the peptide peak inthe degradation product peptide map demonstratedthe presence of two ions-–both doubly charged, withthe first at m/z 1066.5627 and the second at m/z

1095.5829 (Fig. 4). The first ion is consistent with theknown N-terminal peptide of the native unPEGylatedprotein; however, the predominant ion at m/z1095.5829 corresponds to a mass difference of58.0404 Da. This confirms that the +58 Da adductspecies is located within the N-terminal peptide andcoelutes with the N-terminal peptide from the nativeunPEGylated protein.

Estimation of Empirical Formula for Adduct Species

Using high resolution MS, it is possible to derivean estimate of the empirical formula of the +58 Daadduct. The known empirical formula of the na-tive peptide is C96H158N22O28S2, corresponding toa theoretical monoisotopic mass of 2131.1057 Da,using monoisotopic atomic mass values from theNational Institute of Standards and Technology(NIST) database (Table 1).

The observed 2+ ion for the native N-terminalpeptide is m/z = 1066.5627, corresponding to an ob-served mass of 2131.1097 Da. The mass error from the

Table 1. Monoisotopic Atomic Masses from NIST Database21

Element Monoisotopic Atomic Mass

Carbon (C) 12.000000Hydrogen (H) 1.007825Nitrogen (N) 14.003074Oxygen (O) 15.994915Sulfur (S) 31.972071



Figure 3. Glu-C peptide maps of main (PEGylated) and degradation product fractions, iso-lated from degraded material by SE-HPLC.

theoretical monoisotopic value is therefore 0.0040 Daor approximately 1.9 ppm.

As noted earlier, the accurate mass analysis re-vealed the net adduct mass to be 58.0404 Da, anduse of the ±0.0040 Da tolerance from the abovecalculation provides an allowable mass range of58.0364–58.0444 Da for the adduct species, or a masserror of approximately 69 ppm. Using an elemen-tal composition calculator with a target mass of

58.0404 Da, a mass error tolerance of ±69 ppm, andconstrained composition to carbon, hydrogen, ni-trogen, oxygen, sulfur, and phosphorus, two puta-tive empirical formulae for the adduct species weregenerated—CH4N3 (theoretical mass 58.0405 Da,mass error–2.1 ppm) or C3H6O (theoretical mass58.0418 Da, mass error–25.2 ppm). Although the em-pirical data cannot preclude either composition,the most likely structure for the derivative was

Figure 4. High resolution mass spectrum of degradation product peptide at approximately32 min.


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considered to be that of propyl alcohol (C3H7O, 59 Da).The net chemical formula for the formation of the+58 Da adduct species is C3H6O (58 Da) due to theconcomitant elimination of hydrogen from the na-tive protein’s N-terminus, resulting in a net massdifference of +58 Da. This identification is also con-sistent with the putative mechanism of formation asdescribed later.

MS/MS Analysis to Localize Residue of Adduction

The CID and HCD fragmentation patterns of them/z ∼1096 ion are presented in Figures 5a and 6,respectively. The data, particularly the CID fragmen-tation pattern, demonstrate a “y” ion series consistentwith the expected N-terminal peptide sequence as faras “y4”. The “y4” ion contains residues 4–20 and im-portantly, it does not include any modification to themasses of these amino acids because these fragmentions are indistinguishable from those of the nativeN–terminal peptide containing residues 1–20 (datanot shown). This confirms the identity of the pep-tide backbone of the +58 Da containing species, anddemonstrates that the +58 Da modification is locatedon residues 1–3.

The HCD fragmentation pattern of the m/z∼1096.08 ion (Fig. 6) yields diagnostic ions that iden-tify the position of the +58 Da modification. The pres-ence of ions “b2” and “b2–H2O” (large peak to the leftof b2, not labeled for clarity) are consistent with thepresence of the +58 Da adduct somewhere on the firsttwo residues. The “a1” ion at m/z = 162.09 is the keyidentifier, demonstrating that the first residue likelyincludes the +58 Da adduct.

In combination, the MS/MS data demonstrate twokey points: (1) The m/z ∼1096 ion produces the same“y” ion fragmentation pattern as the native peptide(m/z ∼1067), confirming that the nature of the ad-ducted species is related to the native N-terminalpeptide. This is further supported by the fact thatthe two species coelute. (2) The HCD data locate the+58 Da modification to the first residue, that is, at theN-terminus, due to the presence of the “a1” ion. Whatthe MS/MS analysis cannot elucidate is the chemicalstructure of the +58 Da adduct. Therefore, additionalcharacterization and analytical evidence were gath-ered as described in the following sections.

Evaluation of Synthetic Peptides

The identification of the +58 Da species as having aputative empirical formula of C3H6O suggests thatit may be a propanol adduct at the N-terminus ofthe protein. However, given the structural isomers ofpropanol, it was necessary to identify which form waspresent.

Two peptides were synthesized with the sequencecorresponding to the first 20 residues of the nativeprotein. The first synthetic peptide incorporated

Figure 5. CID fragmentation pattern of m/z ∼1096 ion for(a) degradation product peptide, (b) 1-propanol-adductedsynthetic peptide, and (c) 2-propanol-adducted syntheticpeptide. Note that only singly charged fragment ions arelabeled for clarity.

a 1–propanol modification of the N-terminalresidue, whereas the second peptide incorporated a2–propanol modification of the N-terminal residue.These peptides, along with the Glu-C digest ofthe enriched degradation product fraction from thedegraded protein, were analyzed by LC–MS/MS.



Figure 6. HCD fragmentation pattern of m/z ∼1096 ion.

Extracted ion chromatograms (EICs) of each sample,interrogating for both the native (m/z ∼1067) and theadducted (m/z ∼1096) ions, are presented in Figure 7.Both species were monitored, as they coelute in thepeptide map, and both were present in the syntheticpeptides due to incomplete derivitization.

The EICs show the presence of a single peakat 32.14 min for the degradation product impu-rity enriched material, and similarly at 32.13 minfor the 1-propanol-modified synthetic peptide. Im-

portantly, the 2-propanol-modified synthetic peptideshows the presence of two peaks (A and B at 32.11and 32.32 min, respectively; Fig. 7). Evaluation ofthe mass spectral data showed that for the degra-dation product impurity enriched and 1-propanol-modified synthetic peptides, the single peak at ap-proximately 32.1 min contains both the m/z ∼1067and m/z ∼1096 ions as expected. Conversely, however,the peak at approximately 32.1 min (peak A) in the2-propanol-modified synthetic peptide contains only

Figure 7. EICs for enriched degradation product digest and synthetic peptides.


4614 ZHANG ET AL.

Figure 8. Overlay of 2D TOCSY (red) and 2D NOESY (black) spectra of the isolated material,recorded at 273 K. Sequential through-bonds (TOCSY) and through-space (NOESY) correlationsare indicated with arrows.

Figure 9. Schematic assignment of through-bond andthrough-space interproton correlations.

the native peptide (m/z ∼1067 ion), and the peakat approximately 32.3 min (peak B) contains theadducted peptide (m/z ∼1096 ion). Hence, the 2-propanol-modified peptide may be chromatograph-ically separated from the unmodified native pep-tide. Furthermore, the +58 Da adducted peptide inthe degradation product impurity fraction digest isbiochemically indistinguishable from the 1-propanol-adducted synthetic analog using chromatography andMS. Note the fragmentation properties of all peptideswere essentially identical as expected (Fig. 5).

Table 2. 1H and 13C Chemical Shifts of the 1-Propanol AdductObtained from 13C–1H Heteronuclear Single QuantumCoherence Spectrum at 298K

Position 1H Chemical Shift (ppm) 13C Chemical Shift (ppm)

1 3.125, 3.175 47.52 1.953 30.73 3.716 61.6

Chemical shifts are referenced to 2,2-dimethyl-2-silapentane-5-sulfonate.

Confirmation of 1-propanol Adduct by NMR

The adducted peptide isolated from Glu-C map-ping of the enriched degradation product was struc-turally investigated by two-dimensional (2D) NMRspectroscopy.22 Figure 8 shows the overlay of 2Dtotal correlation spectroscopy (TOCSY) and 2D nu-clear Overhauser effect spectroscopy (NOESY) spec-tra, which represent through-bond and short through-space (<5 Å) interproton correlations, respectively.The threonine, methionine, and 1-propanol spin sys-tems are readily identified in the TOCSY spectrum,as indicated in Figure 8.

The assignment process is depicted in Figure 9,where the through-bond and through-space correla-tions are indicated with red and black arrows, re-spectively. Using both mechanisms of magnetizationtransfer instead of just through-bond correlations,was necessary due to the NH proton of the N-terminalamino acid residue being in fast chemical exchange



Figure 10. mPEG–aldehyde derivatization.

with water. This exchange prevented the observationof this proton in the NMR spectra, even at tempera-tures as low as 273 K.

The entire structural assignment consists of the fol-lowing steps: First, the spin system of a single thre-onine (denoted “T2” in Fig. 8) residue can be readilyidentified in the TOCSY spectrum. From the NH pro-ton of this residue, the through-space correlation tothe N-terminal Ha proton (“M1(Ha)” in Fig. 8), andfrom the M1(Ha) proton, the through-space correla-tion to Pr(11′) protons of the 1-propanol moiety areclearly identified.

In order to confirm that H11′, H22′, and H33′

protons belong to the 1-propanol spin system, werecorded a 1H–13C correlation spectrum. Both 1H and13C chemical shifts as shown in Table 2, and the pro-ton coupling pattern perfectly match those expectedfor a 1-propanol moiety substituted on the secondaryamine nitrogen atom.

Putative Mechanism of Formation

The presence of the 1-propanol adduct on the N-terminal residue and the concomitant loss of thePEGylation is believed to be the degradation productof the species formed when mPEG–acetal aldehyde (araw material impurity) is coupled with the native (un-PEGylated) protein. Acetal cleavage occurs at a slowrate during storage, even under recommended stor-age conditions. Normally, the PEGylation reaction oc-curs as shown in Figure 10; however, the mPEG–ac-etal aldehyde impurity reacts similarly with the pro-tein N-terminus, forming a PEGylated protein witha labile acetal linker (Fig. 11). The resulting by-products of hydrolysis of this linker species includemPEG-aldehyde, ethanol, and the 1-propanol-adducted protein (Fig. 12). This mechanism is con-sistent with the recent reports of novel copolymers

for controlled drug delivery.23 The mPEG–acetal alde-hyde and associated degradation product were corre-lated to a specific batch of raw material.

CONCLUSIONS

We report, here, the identification and characteriza-tion of a novel degradation product observed on aPEGylated protein after storage at 2◦C–8◦C. Thedegradation was indicated by the increased presenceof an impurity species using SE-HPLC, and analyticalcharacterization of these species identified the pres-ence of a +58 Da adduct to the protein’s N–terminalpeptide with a concomitant loss of the PEG moiety.

Mass spectrometric analysis via various modes offragmentation determined that the +58 Da adductwas present on the N-terminal amino acid residue,and accurate mass analysis using an Orbitrap instru-ment (Thermo Fisher) facilitated the determination ofa putative empirical adduct formula of C3H6O, sug-gesting the N-terminal adduct to be propanol.

In comparison with synthetic analogs, chromato-graphic differences were observed between the N-terminal peptide bearing the +58 Da modification anda peptide synthesized with a 2-propanol N-terminalmodification; however, no biochemical distinctioncould be made when compared with a synthetic pep-tide with a 1-propanol N-terminal adduction. Identi-fication of the adduct as 1-propanol was confirmed byNMR analysis of the isolated N-terminal peptide fromthe degradation product.

In consideration, a putative mechanism for 1-propanol adduct formation was proposed, suggestinga raw material impurity of mPEG–acetal aldehyderesulting in the introduction of a labile acetal linkergroup between the PEG and protein N-terminus. Thisstudy highlights the need for stringent screening of

Figure 11. mPEG–acetal aldehyde reacts with protein N-terminus to form a PEGylated pro-tein with a hydrolytically labile acetal linker (boxed region).

Figure 12. By-products of acetal linker hydrolysis reaction.


4616 ZHANG ET AL.

raw materials and appropriate control and character-ization of the intended therapeutic agent. This is ofparticular importance to ensure the quality of substi-tute therapies, as PEGylated innovator molecules exitpatent protection, and pathways for biosimilar licen-sure are authorized by governments and regulatoryagencies.

ACKNOWLEDGMENTS

The authors would like to thank all those who pro-vided insightful input, critical discussion, and in-volvement in data assembly for this manuscript, in-cluding Brent Kendrick, Drew Kelner, Gary Rogers,Julie Lippincott, Jim Seely, Arturo Hornedo, CarlosEscobar, Alejandro Toro, Victor Melendez Colon,Eileen McCarthy, Mike Treuheit, and Karen Miller.We are especially grateful to Ken McRae, AsherLower, Jeroen Bezemer, and Belle Liu for the proposedmechanism.

REFERENCES

1. Hermeling S, Crommelin DJ, Schellekens H, Jiskoot W. 2004.Structure–immunogenicity relationships of therapeutic pro-teins. Pharm Res 21:897–903.

2. Jevsevar S, Kunstelj M, Porekar VG. 2010. PEGylation of ther-apeutic proteins. Biotechnol J 1:113–128.

3. Jain A, Jain SK. 2008. PEGylation: An approach for drug de-livery. A review. Crit Rev Ther Drug Carrier Syst 25:403–447.

4. Constantinou A, Chen C, Deonarain MP. 2010. Modulating thepharmacokinetics of therapeutic antibodies. Biotechnol Lett32:609–622.

5. Hamidi M, Azadi A, Rafiei P. 2006. Pharmacokinetic conse-quences of pegylation. Drug Deliv 13:399–409.

6. Mahmood I, Green MD. 2005. Pharmacokinetic and pharma-codynamic considerations in the development of therapeuticproteins. Clin Pharmacokinet 44:331–347.

7. Caliceti P, Veronese FM. 2003. Pharmacokinetic and biodistri-bution properties of poly(ethylene glycol)-protein conjugates.Adv Drug Deliv Rev 55:1261–1277.

8. Pedder SC. 2003. Pegylation of interferon alfa: Structuraland pharmacokinetic properties. Semin Liver Dis 23 Suppl1:19–22.

9. Kontermann RE. 2009. Strategies to extend plasma half-livesof recombinant antibodies. BioDrugs 23:93–109.

10. Veronese FM, Mero A. 2008. The impact of PEGylation onbiological therapies. BioDrugs 22:315–329.

11. Pasut G, Veronese FM. 2009. PEGylation for improving theeffectiveness of therapeutic biomolecules. Drugs Today (Barc)45:687–695.

12. Schmidt RJ. 2009. Methoxy polyethylene glycol-epoetin beta:Worth waiting for or a novelty worn off? Expert Opin Pharma-cother 10:1509–1514.

13. Duncan R, Kopecek J. 1984. Soluble synthetic polymers aspotential drug carriers. Adv Polym Sci. 57:51–101.

14. Hermanson GT, Ed. 2008. Modification with synthetic poly-mers. In Bioconjugate Techniques . 2nd ed., Boston: AcademicPress, pp 936–960.

15. Kang JS, Deluca PP, Lee KC. 2009. Emerging PEGylateddrugs. Expert Opin Emerg Drugs 14:363–380.

16. Bailon P, Won CY. 2009. PEG-modified biopharmaceuticals.Expert Opin Drug Deliv 6:1–16.

17. Goodson RJ, Katre NV. 1990. Site-directed pegylation of re-combinant interleukin-2 at its glycosylation site. Biotechnol-ogy (NY) 8:343–346.

18. Morpurgo M, Veronese FM, Kachensky D, Harris JM. 1996.Preparation of characterization of poly(ethylene glycol) vinylsulfone. Bioconjug Chem 7:363–368.

19. Woghiren C, Sharma B, Stein S. 1993. Protected thiol-polyethylene glycol: A new activated polymer for reversibleprotein modification. Bioconjug Chem 4:314–318.

20. Molineux G. 2004. The design and development of peg-filgrastim (PEG-rmetHuG-CSF, Neulasta). Curr Pharm Des10:1235–1244.

21. http://physics.nist.gov/cgi-bin/Compositions/stand alone.pl?ele=&all=all&ascii=html&isotype=all( accessed June 24th,2010)

22. K.Wuthrich. 1986. NMR of proteins and nucleic acids. NewYork: John Wiley and Sons.

23. Kim JK, Garripelli VK, Jeong UH, Park JS, Repka MA, Jo S.2010. Novel pH-sensitive polyacetal-based block copolymersfor controlled drug delivery. Int J Pharm 401:79–86.


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Analytical characterization of a novel degradation product in a PEGylated recombinant protein