B American Society for Mass Spectrometry, 2012DOI: 10.1007/s13361-012-0531-7

J. Am. Soc. Mass Spectrom. (2013) 24:202Y212

RESEARCH ARTICLE

Mass Spectrometric Distinction of In-Sourceand In-Solution Pyroglutamate and Succinimidein Proteins: A Case Study on rhG-CSF

Mukesh Kumar, Amarnath Chatterjee, Anand P. Khedkar, Mutyalasetty Kusumanchi,Laxmi AdhikaryMolecular Characterization Laboratory, Biocon Research Ltd., Bangalore, 560099, Karnataka, India

Insource (MS

)

Inso

lutio

n

succinimide

Pyroglutamate

N

O

NHR

O

O

NHO

O+

Abstract. Formation of cyclic intermediates involving water or ammonia loss is acommon occurrence in any reaction involving terminal amines or hydroxyl groupcontaining species. Proteins that have both these functional groups in abundanceare no exception, and presence of amino acids such as asparagine, glutamines,aspartic acids, and glutamic acids aid in formation of such intermediates. In thebiopharma scenario, such intermediates lead to product- or process-relatedimpurities that might be immunogenic. Mass spectroscopy is a powerful techniquethat is used to decipher the presence and physicochemical characteristics of suchimpurities. However, such intermediates can also form in situ during massspectrometric analysis. We present here the detection of in-source and in-solution

formation of succinimide and pyroglutamate in the protein granulocyte colony stimulating factor. We alsopropose an approach for quick differentiation of such in-situ species from the tangible impurities. We believethat this will not only reduce the time spent in unambiguous identification of succinimide- and/orpyroglutamate-related impurity in bio-pharmaceutics but also provide a platform for similar studies on otherimpurities that may form due to stabilized intermediates.Key words: In-source, In-solution, Pyroglutamate, Succinimide, MS, GCSF

Received: 15 June 2012/Revised: 17 October 2012/Accepted: 29 October 2012/Published online: 3 January 2013

Introduction

Proteins have high susceptibility to a variety of enzymaticand non-enzymatic post-translational modification and

degradation during manufacturing, formulation, and storage,which in effect lead to the loss of their structure andbiological functions [1–5]. Hence it becomes imperative thatthe degradation pathways of therapeutic proteins are mon-itored and deciphered well in advance of human use.

The post-translational modifications include glycosylationand different amino acid modifications, such as cyclization,oxidation, deamination, isomerization, etc. The formation ofpyroglutamic acid (pyro-Glu) by enzymatic (glutaminylcyclase) cyclization of an amino terminus glutamine (Gln)residue is one such post-translational modification that isobserved in many proteins, including antibodies [6, 7]. Theformation of pyro-Glu from Gln eliminates N-terminal amine

that leads to the generation of acidic variants, therebyproducing heterogeneity in pharmaceutics [8]. The Gln wasfound to be most preferred N-terminal residue in case ofsecreted recombinant human proteins. The non-enzymaticcyclization of N-terminal glutamine is facilitated by partialdeamination, which then leads to the formation of cyclicpyroglutamyl residue (2-pyrrolidinone-5-carboxylic acid) [9,10]. It is interesting to note here that a very similarphenomenon of deamination of N-terminal glutamine has beenobserved for protonated peptides undergoing collision-induceddissociation [11]. Though the cyclization of N-terminalresidue may not influence efficacy and immunogenicity,it is important for amino acid sequencing by Edmandegradation, identification of protein by shotgun and top-down proteomics as this heavily relies on the knowledgeof N-terminal residue [12].

The deamidation of asparagine (Asn) and isomerizationof aspartic acid (Asp) through succinimide intermediate isperhaps the most common pathway for spontaneous nonen-zymatic degradation of protein bio pharmaceutics. Thisnonenzymatic degradation happens because of the highpropensity of aspartyl and asparaginyl residues to undergo

Electronic supplementary material The online version of this article(doi:10.1007/s13361-012-0531-7) contains supplementary material, whichis available to authorized users.

Correspondence to: Amarnath Chatterjee; e-mail: Amarnath.Chatterjee@biocon.com, Laxmi Adhikary; e-mail: laxmi.adhikary@biocon.com

intramolecular cyclization to form a five-member succini-mide ring [13, 14]. This phenomenon is well studied for itsorigin, formation, and stability of succinimide using modelpeptides as well as proteins [6, 7, 15, 16]. The rate ofsuccinimide formation is influenced by many factors,including nature of the flanking residue, three-dimensionalglobal and local conformation, and environmental factorssuch as pH, temperature, and dielectric strength [17–19].The presence of glycine as the flanking residue to the C-terminal of aspartyl or asparginyl residue imparts minimalsteric interference and maximum flexibility , allowing thereaction site to adopt a conformation that is most favorablefor cyclization [20, 21]. Earlier studies have shown thataccumulation of succinimide is favored at mildly acidiccondition and the succinimide moiety is relatively unstablein alkaline pH [22–24]. However, a recent study onsuccinimide-mediated degradation products using high fi-delity trypsin digestion has shown that succinimide fromaspartyl residue is stable for 48 h at a pH of 8.4. Further, theauthors concluded that protein conformation plays a key rolein deciding the fate of succinimide hydrolysis [25]. Theracemization and hydrolysis of succinimide at either the α-or the β-carbonyl group leads to the generation of mixture ofL-Asp, D-Asp, L-isoAsp, and D-isoAsp, with L-isoAspbeing the most abundant [6]. The physiological significanceof succinimide formation is indicated by the presence of ahighly conserved repair enzyme, protein L-isoaspartyl O-methyltransferase (PIMT), which convert isoAsp into Asp[26, 27]. The mechanism of pyroglutamate and succinimideformation has been explained previously. Scheme 1 showsthe schematic of pyroglutamate and succinimide formationfor ready reference. The techniques that are commonlyemployed to detect succinimide and pyroglutamic acids are:ion exchange chromatography, hydrophobic interaction

chromatography, capillary electrophoresis, and mass spec-trometry [28–30]. Amongst these biophysical techniques,higher sensitivity and rapid data acquisition makes massspectrometry (MS) unique. While conventional MS enablesthe determination of molecular mass, tandem MS (MS/MS)methods facilitate in identifying the molecule and sites ofmodification. Apart from characteristic sequence specificfragment ions, the neutral loss of ammonia and water is acommon phenomenon observed during peptide fragmenta-tion under collision induced dissociation (CID) condition[31]. It is well established that gaseous protonated N-terminal glutamine and glutamic acid readily undergoesdeamination and dehydration respectively to form pyro-Glusimilar to in-solution reaction [32]. The extent of neutral lossof ammonia and water from protonated peptides underelectrospray ionization mass spectrometry, as a function ofin-source voltage, peptide structure, ion charge and collisionenergy have been documented in the literature [23–25]. Suchlosses can reveal or obscure important structural informationand hence need to be monitored carefully.

In this background, the observation of in-source and in-solution formation of pyroglutamic acid and succinimide inone of the Glu-C digested peptide of recombinant humangranulocyte-colony stimulating factor (rhG-CSF) is pre-sented. The rhG-CSF is one of the well characterizedhematopoietic growth factors that selectively stimulatesproliferation, differentiation, and functional activation ofneutrophils. The rhG-CSF is a 175-amino acid singlepolypeptide chain with two intramolecular disulphide bonds[33–35]. Recombinant rhG-CSF, produced using E. coli as ahost, has been successfully used to treat cancer patientssuffering from chemotherapy-induced neutropenia [36]. Weenvisage that the observations presented in this study willhelp in distinguishing the formation of pyroglutamic acid

Scheme 1. Formation of succinimide and pyroglutamic acid from aspartic acid and Glutamine, respectively

M. Kumar et al.: Source or Solution Induced Change in Proteins 203

and/ or succinimide due to in-source or process-induced in-solution reaction from such moieties that are inherentlypresent in the solution because of degradation and/or PTM.Further, this will facilitate the fast screening of the source ofpyroglutamic acid and/or succinimide formation-relatedimpurities in proteins aimed for use as a therapeutic agent.

Material and MethodsThe granulocyte colony stimulating factor (G-CSF) analyzedunder the present study is recombinant human G-CSF (rhG-CSF). The expression system used was E. coli purified usingstandard manufacturing process step at Biocon (Bangalore,India). All the chemicals used were reagent grade or above.TRIS base, TRIS HCl, trifluoroacetic acid, and calciumchloride were purchased from Sigma (St. Louis, MO, USA).Acetonitrile (HPLC grade) was purchased from J. T. Baker(Phillipsburg, NJ, USA). Sequencing grade endoproteaseGlu-C was purchased from Roche (Indianapolis, IN, USA).The sequencing grade modified trypsin and endoproteaseAsp-N were purchased from Promega (Madison, WI, USA).

Endo-Protease Digestions

Glu-C peptide digestion of rhG-CSF was carried out with anenzyme: protein ratio of 1:10 (wt/wt) at a pH of 8.2 andincubation at a temperature of 37 °C for 10 h. Trypsindigestion was carried out with an enzyme:protein ratio of1:10 (wt/wt) at a pH of 7.5–8.0 and incubation at 37 °C for12 h. Digestion with the enzyme Asp-N was carried out withan enzyme:protein ration (wt/wt) of 1:40 at a pH of 8.0 andovernight incubation at 37 °C. The pH adjustments weredone using 1 M Tris-HCl buffer (pH 8.5). The peptidedigestions in all cases were carried out under non-reducedcondition.

LC-ESI MS/MS Data Acquisition and Analysis

The peptides formed after enzymatic digestion were sepa-rated on reverse phase (RP) YMC-Pack ODS-AQ column(100×2.0 mm i.d.) using an Agilent HPLC system (1100series), using H2O (0.1 % TFA)/90%ACN (0.1%TFA) asmobile phase. A linear gradient typically set from 5 % ACNto 95 % ACN over a period of 67 min with a flow rate of0.6 mL/min and column temperature of 45 °C was used. Allthe separated peptides were subjected to MS and subse-quently to MS/MS on an online ESI MS platform [MSD trap(Agilent, Santa Clara, CA, USA) or HCT-Ultra ETDII iontrap (Bruker Daltonics, Bremen, Germany)]. Mass spec-trometers were operated in positive ionization mode with thefollowing source conditions: capillary voltage (3500),nebulizer (35 psi), dry gas (11 l/min), dry temperature(350 °C), and trap drive (147.1). Further mass spectra werealso acquired with different capillary voltages (4500, 3500,2500, and 1500 V), dry temperatures (300 and 350 °C), andskimmer values (20 and 40). The intact mass analysis of

rhG-CSF was carried out using Synapt G2-HDMS (WatersCorporation, Milford, MA, USA) coupled with AcquityUPLC (Waters Corporation, Milford, MA, USA) and QStarXL (AB SCIEX, Framingham, MA, USA) coupled withcapillary LC (Agilent, Santa Clara, CA, USA).

Results and DiscussionSuccinimide and Pyroglutamate Formation Is NotInherent of rhG-CSF Molecules in Solution

The mass spectrum and the corresponding deconvolutedmass spectrum of the intact native rhG-CSF molecule isshown in Supplementary Figure 1a. Upon deconvolution, ityielded an average molecular mass of 18798.0 Da, whichcorroborates with the expected theoretical mass. In the intactmass analysis we did not observe any rhG-CSF species,which has 18 Da less mass compared with intact rhG-CSFand elutes after main peak under the given reverse phase(RP) HPLC condition, thus showing absence of anydetectable modification such as succinimide. Pyroglutamatecannot be associated with the intact molecule because the N-terminal residue in rhG-CSF is methionine. These observa-tions suggest that neither succinimide nor pyroglutamate isinherently present in the rhG-CSF sample analyzed in thepresent study.

In-Source Formation of Pyroglutamic Acidand Succinimide

The peptides generated by endoprotease Glu-C digestion of therhG-CSF sample, termed Glu-C peptides from here onwards,were separated under the RP-HPLC condition described in theSection 2, on an LC-ESI MS platform. The MS2 data wereacquired for each peptide by utilizing the auto MSn technique,and the sequence of all the standard peptides (Table 1) wereconfirmed by CID MS2 (data not shown). Amongst the Glu-Cpeptides of rhG-CSF, the possibility of formation of pyrogluta-mic acid exists only for the peptide 2 (Q1VRKI5QGDG-A10ALQE14) in Table 1. The glutamine being N-terminalresidue in this peptide can form pyroglutamic acid through theloss of ammonia (−17Da). The peptide 2 also has an aspartic acidresidue (Asp8) flanked by glycine residues making the favorablecondition for it to undergo isomerization through succinimideintermediate by the loss of water (−18 Da) and form isoAsp.

The afore-described is confirmed experimentally and isshown in Figure 1, where the mass spectrum of the peptide 2(Figure 1a) shows a species that corresponds to −18 Da (P2-18; m/z0747.7 Da doubly charged ion) besides the doublycharged parent ion (P2 m/z0756.7). On the other hand,peptide 8 (Figure 1b) shows the presence of only the doublycharged parent ion (P8, m/z0834.8 Da). This observationwas true for the other asp-containing peptides, namelypeptide 6 and peptide 7 (Table 1) as well (data not shown).Further, a −17 Da (P2-17; m/z0748.2) doubly charged ion wasalso observed with P2 and P2-18 at 19.8 min (Figure 1a).

204 M. Kumar et al.: Source or Solution Induced Change in Proteins

Extracting the ions P2 (756.7±0.5 Da), P2-18 (747.7±0.5 Da),and P2-17 (748.7±0.5 Da) shows that both P2-18 and P2-17 notonly elute with the peptide P2 at 19.8 min but also elute at twodifferent retention times namely 21.2 and 22. 4 min. This isshown in Figure 2a and b, where the extracted ion chromato-grams (EIC) corresponding to P2 and P2-17 are shown. TheEIC for P2-18 is shown in Supplementary Figure 5c. Further anexpanded EIC overlay of the peaks eluting at 19.8 min isshown in Supplementary Figure 6 in order to unambiguouslyshow the elution of P2-18 and P2-17 at the same RT as P2. Thisindicates that the ions P2-18 and P2-17 might have originatedbecause of at least two different sources.

To further investigate the origin of the peptides P2-18 andP2-17, the MS2 data was analyzed. The MS2 profile of thedoubly charged ion of both the peptides, P2-18 and P2-17 atm/z747.7 and 748.2, respectively, were identical. Figure 3a and bshow the expansion of theMS2 profiles of P2 and P2-18 peptide

at the b3 ion (theoretical b and y ions are given on top of thefigure). Interestingly both show the presence of the parent b3

ion at m/z 384.2 as well as the presence of the b3-17 (m/z367.2 Da) and b3-18 (m/z 366.2 Da) ions. The 18Da loss can beattributed to water loss and 17 Da loss can be assigned to sidechain loss of arginine (Arg) in P2 peptide. This suggests thatboth −17 and −18 Da modification cannot be associated withN-terminus since then the b3 ion should have been absent in theMS2 spectrum of P2-18. Figure 3e show the expansion of theMS2 profile for the peptide P2 at the ion b8. Both b8-18 and b8-17 are also observed as explained earlier for b3 ion (Figure 3a).The expansion of the MS2 profile for the peptide P2-18 at b8 isshown in Figure 3f, where the absence of b8 ion (QVRKIQGD)atm/z 925.2 suggests that the 18 Da modifications is at Asp8. Asimilar observation for the peptide P2-17 suggests that the17 Da modifications is at N-terminus (SupplementaryFigure 2b). All other b ions after b8 show a similar pattern

Figure 1. Mass spectra of the doubly charged ion corresponding to the mass of the peptide: (a) P2; the loss of 18 Da and17 Da mass from the P2 are shown as P2-18 and P2-17, respectively; (b) P8; the absence of the species P2-18 and P2-17 isdepicted by crosses. Both spectra show the presence of sodium adduct which is shown as (M + H + Na)2+

Table 1. List of Expected and Observed Peptides of Endoprotease Glu-C Digested Protein, rhG-CSF. The Mass Given in the Table Represents MonoisotopicMass. The Sequences of All the Peptides were Confirmed by CID MS/MS

Peptide no Peptide range Sequence Expected mass (M + H)+ Observed mass (M + H)+

1 [1–20] MTPLGPASSLPQSFLLKCLE 2132.1 2132.42 [21–34] QVRKIQGDGAALQE 1512.4 1512.43 [35–46] KLCATYKLCHPEE 1532.7 1532.84 [48–94] LVLLGHSLGIPWAPLSSCPSQALQL

AGCLSQLHSGLFLYQGLLQALE4943.8 4943.5

5 [95–99] GISPE 502.2 502.26 [100–105] LGPTLD 615.4 615.47 106–110 TLQLD 589.3 589.38 111–124 VADFATTIWQQMEE 1668.7 1668.79 125–163 LGMAPALQPTQGAMPAFASAFQRRA

GGVLVASHLQSFLE4028.6 4028.6

10 [164–175] VSYRVLRHLAQP 1438.8 1438.8

M. Kumar et al.: Source or Solution Induced Change in Proteins 205

(data not shown). Similarly, y ions before Asp8 did notshow any modification as shown by the presence of theion y6 in Figure 4b. The presence of y10 ion at m/z1001.2 and a corresponding intense y10-18 in the MS2

spectrum of P2-18 (Figure 4f) confirms that the 18 Damodifications are due to succinimide formation fromaspartic acid (Asp8). A similar observation in the MS2

profile of P2-17 confirms that the 17 Da modificationsare due to the formation of pyroglutamic acid from N-terminal glutamine (data not shown). Since both pyro-glutamic acid (P2-17) and succinimide (P2-18) are morehydrophobic compared with the parent peptide, theyshould ideally elute at a different retention time thanthe corresponding parent peptide (P2) under the givenRP-HPLC condition. However, since both P2-18 and P2-17 are seen eluting at the same RT as P2 (i.e., 19.8 min(Supplementary Figure 5), it can be said that themodifications are taking place ‘in-source’ under the massspectrometric condition.

Effect of Mass Spectrometric Parameters:Affirmation of In-Source Loss

To further confirm the observation of the in-source formation ofpyroglutamic acid and succinimide intermediates, we assessedthe effect of few mass spectrometer parameters, specificallycapillary voltage; dry temperature; skimmer voltage; and trapdrive value. Amongst these, the capillary voltage showed asignificant effect on the in-source loss of ammonia and water.The change in capillary voltage and corresponding change in P2-17 and P2-18 represented as relative intensity % with respect toP2, is shown in Table 2, and the corresponding mass spectrum isshown in Supplementary Figure 3e–h. The relative areapercentage of in-solution pyroglutamate (P2-17) and succinimide(P2-18) remains unchanged upon change in different massspectrometric condition, including capillary voltage (data notshown). These observations are in-line with several earlierstudies that have been conducted on model peptides to see theeffect of different mass spectrometric conditions such as cone

Figure 2. Extracted ion chromatogram (EIC) for peptide: (a) P2, (b) P2-18, and P2-17. Mass spectra of the doubly charged ionfor peptide: (c) P2-18 eluting at an RT of 21.2 min and (d) P2-17 eluting at an RT of 22.4 min. The sodium adducts in (c) and (d)are shown as (M + H + Na)2+ or (M + 2Na)2+, as the case may be

206 M. Kumar et al.: Source or Solution Induced Change in Proteins

voltage, collision energy, etc. on neutral loss [11, 32]. Further, itwas observed that at different time points of rhG-CSF incubationwith Glu-C the intensity of the P2-18 and P2-17 peptides elutingat 19.8 min did not change (Supplementary Figure 3a–d). Thusthis endorses that the peptides P2-17 and P2-18 at 19.8 min areformed in-source.

Process Induced In-Solution Formationof Pyroglutamic Acid and Succinimide

The observation of the P2-18 and P2-17 at different retentiontimes of 21.2 min (Figure 2b and c) and 22.4 min (Figure 2b and

d), respectively, compared with the parent peptide P2 (elutes at19.8 min; Figures 1a and 2a) prompted further investigations ontheir respective sources. This observation, along with the fact thatno inherent −18 or −17 Da losses were observed for the intactrhG-CSF molecule, clearly depicts the presence of these speciesin-solution, probably as a result of the enzymatic digestionprocess. Figure 3c shows the expansion of the MS2 spectrum ofthe peptide P2-18, eluting at 21.2 min, for the b3 ion (m/z384.2 Da). The observation that b3, b3-18 at m/z 366.2 Da(Figure 3c) and b8-18 at m/z 907.2 (Figure 3g) are present whilethe b8 ion at m/z 925.2 is absent (Figure 3g) confirms that themodification is at Asp8. Further the presence of y6 ion at m/z

Figure 3. MS2 mass spectra expanded at the b3 (a)–(d) and b8 (e)–(h) ion for the peptide: (a) and (e) P2, (b) and (f) P2-18 elutingwith P2, (c) and (g) peptide P2-18 eluting at an RT of 21.2 min, and (d) and (h) peptide P2-17 eluting at an RT of 22.4 min. In thepanels, b3-17 and b3-18, respectively, depict the loss of 17 and 18 Da mass from the b3 ion. Absence of an ion is shown by across. A few representative theoretical ‘b’ and ‘y’ ions corresponding to the MS2 spectrum of P2 is given on top of the panel (a)

M. Kumar et al.: Source or Solution Induced Change in Proteins 207

Table 2. Change in Capillary Voltage and Corresponding Change in the Peptide P2-17 (In-Source) and P2-18 (In-Source) Represented as Relative IntensityPercentages (%) with Respect to the Peptide P2

S.no Capillary voltage (V) Relative abundance of P2 Relative abundance of P2-17 (in-source) Relative abundance of P2-18 (in-source)

1 1500 89.63 8.3 2.072 2500 83.36 12.8 3.843 3500 79.36 16.1 4.544 4500 67.67 25.9 6.43

Figure 4. MS2 mass spectra expanded at the y6 (a)–(d) and y10 (e)–(h) ion for the peptide: (a) and (e) P2, (b) and (f) P2-18 elutingwith P2, (c) and (g) peptide P2-18 eluting at an RT of 21.2 min, and (d) and (h) peptide P2-17 eluting at an RT of 22.4 min.Absence of an ion is shown by a cross. A few representative theoretical ‘b’ and ‘y’ ions corresponding to the MS2 spectrum ofP2 is given on top of the panel (a)

208 M. Kumar et al.: Source or Solution Induced Change in Proteins

588.3 (Figure 4c) suggests that the modification is after Gly6.Furthermore, the presence of the ion y10-18 at m/z 983.3 Da(Figure 4g) confirms that 18 Da modifications is at Asp8. Thisconfirms the formation of succinimide intermediate as a result ofin-solution conditions. Figure 3d shows the expansion of theMS2

spectrum of the peptide P2-17, eluting at 22.4 min. Absence of b3ion atm/z 384.2 and presence of b3-17 atm/z 367.1, suggests thatthe modification is at N-terminus. This is corroborated by theabsence of b8 ion at m/z 925.2 and presence of b8-17 ion at m/z908.3 (Figure 3d). The presence of the y6 and y10 ion atm/z 588.3and 1001.2 respectively (Figure 4d and h) and all other y ionswithout modification (data not shown) confirms the in-solutionformation of pyroglutamate from N-terminal glutamine.

Asp-N and Trypsin Digestions: AncillaryConfirmation of Succinimide and PyroglutamicAcid

Since the succinimide intermediate eventually leads to theformation of isoAsp, monitoring the isoAspmoieties are favoredover the succinimide itself because of their stability and ease ofdetection. The isoAsp typically represents 65 %–85 % ofsuccinimide hydrolysis product; however, this ratio may varydepending on protein conformation. Though the elution order ofisoAsp containing peptides depends on chromatographic con-ditions like the type of ion pairing agent and packing material[37, 38], in the present study the isoAsp-containing peptide isobserved to elute earlier than the corresponding Asp peptide,

most likely because of the use of trifluoroacetic acid (TFA) as anion pairing agent [39, 40]. Amongst the manymethods availablefor isoAsp detection, digestion by endoprotease Asp-N, whichspecifically cleaves at the N-terminus of Asp but not of isoAsp,makes an excellent tool.

In the present study, a careful analysis of the Glu-C peptidesof rhG-CSF revealed that a moiety that has the samemass as P2

(eluting at 19.8 min) elutes at 19.3 min. We termed this moietyas P2*. This observation is corroborated in Figure 5a, whichshows the EIC of doubly charged ion of P2 (m/z0756.7). TheCID MS2 spectrum of both the peptide P2 and P2* wereidentical. Considering the observations presented in theprevious paragraph and the fact that a succinimide like moiety(P2-18) is seen for P2, one could assume that P2* corresponds topresence of isoAsp in the peptide P2. This assumption is provenby the EIC of doubly charged ion of P2 after Asp-N digestion(Figure 5b), which shows the absence of P2 at 19.8 min and thepresence of uncleaved P2* at 19.3 min.

A closer look at the peptides formed by proteolytic digestsof the rhG-CSF sequence in silico shows that the trypticpeptide K4I5QGDGA10ALQE14K15 (P

T; partial cleavage) hasa close sequence similarity to the Glu-C peptide P2

(Q1VRKI5QGDGA10ALQE14). The Tryptic peptide PT isexpected not to have the pyroglutamic moiety (PT-17) since itlacks the N-terminus Gln; however, the presence of thesuccinimide moiety (PT-18) is anticipated. This is confirmedin Figure 5c, where the mass spectrum of the peptide PT

shows a species that corresponds to −18 Da (PT-18; m/z0620.5 Da) doubly charged ion (M + 2H)+2 besides the doublycharged parent ion (PT; m/z0629.3 Da). The species PT-17 is

Figure 5. Extracted ion chromatogram (EIC) for (a) Glu-C peptide P2 and P2* and (b) Glu-C digestion followed by Asp-N of P2*.Absence of P2 is depicted by a cross. (c) Mass spectra of the doubly charged ion corresponding to the mass of the partiallycleaved tryptic peptide PT; the loss of 18 Da from the P2 is shown as P2-18. The presence of sodium adduct is shown as (M +Na)+

M. Kumar et al.: Source or Solution Induced Change in Proteins 209

not observed, proving the absence of pyroglutamic acid kindof moiety as expected. These observations on the Asp-N andtrypsin-digested rhG-CSF peptides prove, albeit indirectly,

that the P2-18 moiety corresponds to P2 with succinimideformation, and the P2-17 moiety corresponds to P2 withpyroglutamic acid formation.

Figure 6. Mass spectra at different time points are shown for the doubly charged peptide P2-17 (a)–(c) and P2-18 (d)–(f)eluting at 22.4 and 21.2 min respectively; (a) and (d) correspond to 4-hour digest. Similarly (b) and (e), and (c) and (f) arefor the 8 and 12 h digestion timepoints, respectively. The presence of sodium adducts is shown as (M + H + Na)2+

Table 3. Glu-C Digest Time Points: Change in the Relative Area Percentages for the Peptides P2-17 (In-Source; In-Solution), P2-18 (In-Solution), and P2*

with Respect to the Peptide P2 in the EIC Spectrum are Tabulated

Time (hours) Relative area % of pyroglutamate (P2-17) and succinimide (P2-18)

P2-17 (in-source) P2-17 (in-solution) P2-18 (in-solution) P2*

4 15.9 9.0 2.3 ND8 15.0 9.1 1.1 0.412 14.9 9.5 0.7 0.716 15.7 9.7 0.3 1.120 14.2 9.9 ND 3.524 14.6 10.2 ND 5.2

*Relative area % of isoAsp

210 M. Kumar et al.: Source or Solution Induced Change in Proteins

In-Solution Stability of Succinimideand Pyroglutamic Acid

Evaluating the stability of process induced in-solution pyroglu-tamate and succinimide is important in order to have an unbiasedquantification of eventual degradation products that may beformed due to these. This is especially so for the succinimide,which is an intermediate for the isoAsp degradation product. Inorder to assess the stability, we acquired the mass spectrometricdata for various time points of the Glu-C digestion between 4 to24 h. Figure 6a–c show the mass spectrum of in-solutionpyroglutamate at 4, 12, and 20 h, respectively. There was nosignificant change observed in the amount of process induced in-solution pyroglutamate when rhG-CSF was incubated with Glu-C for up to 24 h. In contrast, the amount of in-solutionsuccinimide was significantly reduced from 4 to 8 h and was notdetectable after 12 h of Glu-C digestion as shown in Figure 6d–f.At the same time, the isoAsp peak at 19.3 min (see previoussection) increased to 5.2 % over a period of 24 h as shown inTable 3. The same is captured qualitatively in SupplementaryFigure 4, which shows the EIC corresponding to P2 and P2* atvarious time-points of Glu-C digestion. These observationsreiterate the importance of optimizing the digestion conditionsduring the development of a method to characterize isoAsp-related impurities in a biopharma drug. If the optimization isprudently done, one can distinguish between process-inducedand product-related impurities by a simple time-dependantexperimentation on the digestion process.

ConclusionIdentifying and characterizing PTM’s and degradation prod-ucts are always critical for well-characterized biologics thatform the key ingredient of biopharmaceutical drugs. Asdescribed earlier, succinimide mediated deamidations form awell-known family of degradation products. Further proteinswith N-terminal glutamine are susceptible to pyroglutamateformation either as a result of PTM or degradation. Hence, thegeneral observation of loss of 17 and 18 Da from any peptideduring the MS analysis should be analyzed carefully. The lossof both 17 and 18 Da was shown to occur from N-terminalglutamine under the mass spectrometric condition [11]. In thepresent study, however, we have seen that the loss of 17 Da isfrom N-terminal glutamine resulting in the formation ofpyroglutamate, and 18 Da loss is from an aspartic acid residuepresent in the same peptide, which leads to the formation ofsuccinimide. The observation that these losses are affected bychange in capillary voltage is in agreement with the proposedin-source modification. Further, it is essential to optimize thetrap drive value and the voltage for such analysis to getunambiguous results and sequence information. We also reporthere the entrapment of succinimide from aspartic acid atalkaline pH. The stability of this succinimide may be attributedto the digestion process, which was carried out under non-

denatured and non-reduced condition. Our observation is wellin agreement with a recent report wherein the succinimide fromaspartic acid at alkaline pH was found to be stable and thedigestion was carried out under similar condition [25].Decrease in the in-solution succinimide concentration accom-panied by an increase in the corresponding isoAsp moietyfurther corroborates this observation. In-source and in-solutionformation of pyroglutamate and succinimide can be differen-tiated and confirmed by closely analyzing the MS andMS/MS.Further, a quick way to differentiate between the in-situ andinherently present pyroglutamate- or succinimide-mediatedimpurities will be to analyze the protein sample at two differentsolution conditions as well as acquiring the ESI-MS data atclosely spaced but different mass spectrometric conditions. Thein-situ species in such a scenario will show an intensity changerandomly, whereas those that are inherent to the moleculeshould behave linearly. The confounding data in the MS andMS/MS should be critically evaluated with the MS conditionsapplied. Our results restate the importance of optimization ofdigestion and MS condition before data acquisition forunambiguous analysis of inherently present impurities inbiopharmaceutical drug products.

AcknowledgmentAuthors would like to thank Biocon Limited for theinfrastructure support.

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