Analytical Biochemistry 419 (2011) 17–25

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Analytical Biochemistry

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On-line characterization of monoclonal antibody variants by liquidchromatography–mass spectrometry operating in a two-dimensional format

Melissa Alvarez a, Guillaume Tremintin b, Jennifer Wang a, Marian Eng a, Yung-Hsiang Kao a, Justin Jeong a,Victor T. Ling a, Oleg V. Borisov a,⇑a Protein Analytical Chemistry, Genentech, South San Francisco, CA 94080, USAb Dionex, Sunnyvale, CA 94085, USA

a r t i c l e i n f o

Article history:Received 5 April 2011Received in revised form 30 June 2011Accepted 27 July 2011Available online 2 August 2011

Keywords:Mass spectrometryLiquid chromatography–mass spectrometryMonoclonal antibodiesVariantsIon exchange chromatographySize exclusion chromatographyTwo-dimensional liquid chromatography

0003-2697/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ab.2011.07.033

⇑ Corresponding author.E-mail address: oborisov@gene.com (O.V. Borisov)

1 Abbreviations used: MAb, monoclonal antibody; IECphy; SEC, size exclusion chromatography; LC–MS,spectrometry; RP, reversed phase; 2D, two-dimensioliquid chromatography; ACN, acetonitrile; FA, formicultraviolet; TIC, total ion chromatogram; NANA, N-acetchain; LC, light chain; HMW, high-molecular-weight; L

a b s t r a c t

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of bio-therapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requir-ing a battery of analytical technologies for their characterization. However, incompatibility betweenseparation and subsequent detection is often encountered. Here we demonstrate the utility of a genericon-line liquid chromatography–mass spectrometry (LC-MS) method operated in a two-dimensional for-mat toward the rapid characterization of MAb charge and size variants. Using a single chromatographicsystem capable of running two independent gradients, up to six fractions of interest from an ion exchange(IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto aseries of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysisof poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating frac-tions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorpo-rated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis withminimal sample handling. This technology provides rapid data turnaround time, a much needed featureduring product characterization and development of multiple biotherapeutic proteins.

� 2011 Elsevier Inc. All rights reserved.

Recombinant monoclonal antibodies (MAbs)1 are highly hetero- development, production, storage, and handling [7]. These meth-

geneous due to a number of enzymatic and nonenzymatic processesthat occur during their production and storage [1]. Heterogeneity re-lated to these processes commonly includes differential glycosyla-tion, glycation, aggregation, fragmentation, oxidation, deamidation,N-terminal cyclization, C-terminal processing, and sequence alter-ation [2–4]. These are monitored during production, purification,and formulation development of biotherapeutics to ensure consis-tent product quality [5].

Characterization of MAbs typically requires a battery of meth-ods that are specific for individual structural alterations or modifi-cations [6]. Methods such as ion exchange chromatography (IEC)and size exclusion chromatography (SEC) are used to assess chargeand size heterogeneity, respectively. IEC and SEC are commonlyused to monitor the quality and stability of the product during

ll rights reserved.

., ion exchange chromatogra-

liquid chromatography–massnal; HPLC, high-performanceacid; DTT, dithiothreitol; UV,ylneuraminic acid; HC, heavyMW, low-molecular-weight.

ods are designed to reveal key quality attributes that can affectpotency or pharmacokinetic properties of the product [8].

Mass spectrometry (MS) plays an essential role in the structuralelucidation of MAbs because it offers an additional degree of sepa-ration by mass, greatly facilitating the characterization of variants[9,10]. Although IEC and SEC usually provide optimal separation ofMAb variants, these methods employ salts and other nonvolatilebuffers that are generally incompatible with on-line MS detection.In contrast, reversed phase liquid chromatography (RP-LC) offersonly limited separation of MAbs and their variants due to their size,complexity, and general hydrophobicity. Nevertheless, its excellentcompatibility with on-line MS detection has made RP-LC–MS a keytechnology in the characterization of MAbs [11–14].

Identification of MAb variants often present as minor peaks in anIEC or SEC separation is critical to the understanding of productquality and stability. This is typically achieved by analysis of off-linecollected fractions by RP-LC–MS methods on intact, reduced,partially digested, and peptide map samples, respectively, withincreasing levels of complexity and information [15]. Off-lineapproaches are generally time-consuming and labor-intensive.Fraction collection is often a limiting step in characterizationbecause it requires significant analyst involvement and sample

18 Characterization of MAb variants by LC–MS / M. Alvarez et al. / Anal. Biochem. 419 (2011) 17–25

manipulation. More important, loss, contamination, and degrada-tion of material are often encountered during sample collection.

Characterization of MAb charge and size variants can also beachieved on-line by coupling IEC or SEC separation modes withRP and interfacing the system to a high-resolution mass spectrom-eter. This strategy, also known as on-line two-dimensional (2D)LC–MS [16–19], is commonly used in proteomics for the analysisof complex samples [20]. With IEC and SEC both highly orthogonalto RP [21], fractions can be transferred directly into the RP gradientfor further analysis. Because RP employs MS-compatible solvents,these fractions can be subsequently detected and characterizedby MS [22]. On-line methods can also be automated, thereby sav-ing time and sample. By using this approach, much of the detri-mental sample perturbations often encountered with off-linecharacterization is alleviated. However, on-line system configura-tions, compared with off-line ones, are often technologically chal-lenging; a method involving the simultaneous operation of twohigh-performance liquid chromatography (HPLC) systems withindependent software controls has been reported [22,23].

Here we describe a novel strategy for characterizing individualMAb variants using an on-line automated system operating in a 2DLC–MS format. Chromatographic separation is performed using asingle commercial HPLC system capable of running two independentternary gradients and equipped with two independent temperature-controlled column compartments with built-in switching valves. Upto six fractions of interest from a single IEC or SEC run can be trappedonto a series of RP trap cartridges for rapid desalting and subsequenton-line detection using an electrospray ionization time-of-flightmass spectrometer. This approach includes an optional on-linereduction step incorporated into the workflow to provide chain-spe-cific information for each of the trapped MAb fractions. Analysis ofpoorly resolved and low-level variants is made possible by precon-centrating fractions on the traps using multiple injections prior toMS detection. The HPLC system is controlled by a single softwareprogram enabling a high-throughput mode of operation with mini-mal sample handling. The utility and applicability of this on-line 2DLC–MS method are demonstrated by using it for the characterizationof MAb charge heterogeneity, the identification of a chemicallyunstable MAb variant, the discovery of a low-abundant sequencevariant using an on-line reduction step, and the elucidation of minorsize variants through preconcentration of low-level fractions.

Materials and methods

Materials

Recombinant MAbs used for the studies were of IgG4 (MAb1) andIgG1 (MAb2 and MAb3) subtypes and were produced in-house(Genentech, South San Francisco, CA, USA). HPLC-grade water, aceto-nitrile (ACN), and formic acid (FA) were purchased from J. T. Baker(Phillipsburg, NJ, USA). Dithiothreitol (DTT), 2-(N-morpholino)-eth-anesulfonic acid (Mes), and N-(2-acetamido)-2-aminoethanesulfon-ic acid (Aces) were purchased from Sigma–Aldrich (St. Louis, MO,USA). Tris-(hydroxymethyl)-aminomethane (Tris) hydrochlorideand base, sodium phosphate monobasic monohydrate (NaH2-

PO4�1H2O) and dibasic anhydrous (Na2HPO4), potassium phosphatemonobasic (KH2PO4) and dibasic anhydrous (K2HPO4), sodium chlo-ride (NaCl), and potassium chloride (KCl) were purchased from Mal-linckrodt Baker (Stroudsburg, PA, USA).

Ion exchange chromatography

A Propac (4 � 250 mm) WCX-10 column (Dionex, Sunnyvale,CA, USA) was used for the separation of MAb1 and MAb2 chargevariants by IEC. IEC separation of MAb1 was carried out at a flow

rate of 1 ml/min and a column temperature set at 15 �C. The mobilephases used were 20 mM Aces buffer (pH 6.1) (A) and 50 mM NaClin A (B). A linear gradient from 0% to 55% B for 50 min and then aramp to 100% B for 5 min was employed, followed by a reequilibra-tion step with 100% A.

IEC separation of MAb2 was carried out at a flow rate of 0.8 ml/min and a column temperature set at 35 �C. The mobile phasesused were 10 mM sodium phosphate buffer (pH 7.5) (A) and100 mM NaCl in A (B). The gradient program started at 30% B for2 minutes, then a ramp to 90% B for 29 min and to 100% B for4.5 min, followed by a reequilibration step at initial conditions.

Size exclusion chromatography

Two TSK gel (4.6 � 300 mm) Super SW3000 (4 lm) columns(Tosoh Biosciences, Tokyo, Japan) connected in tandem were usedfor the separation of MAb3 size variants by SEC. Separation wasperformed isocratically at a 0.11-ml/min flow rate and ambientcolumn temperature for 120 min using 200 mM potassium phos-phate with 250 mM KCl (pH 6.2) as the mobile phase.

The ultraviolet (UV) detection for both IEC and SEC separationswas set at 280 nm.

On-line 2D LC–MS configuration

Chromatographic separation was conducted using a single Ulti-mate 3000 HPLC system (Dionex) equipped with dual ternary gra-dient pumps, a single UV detector, a temperature-controlledautosampler, and two temperature-controlled column compart-ments, each with a set of built-in switching valves, with the firstcompartment containing a pair of 2-position 10-port valves andthe second compartment containing a pair of 6-position 7-portvalves. The HPLC system was controlled by Dionex Chromeleonsoftware.

The schematic for characterizing MAb variants on-line using a2D LC–MS configuration is illustrated in Fig. 1A. Pump 1 was usedfor IEC or SEC, whereas pump 2 was used for delivering the RP gra-dient. During peak elution, trapping was performed by divertingthe flow from pump 1 onto one of the six polymeric RP trap car-tridges (Protein Microtrap, 1 � 8 mm) from Michrom BioResources(Auburn, CA, USA) (Fig. 1A, solid lines) and then switching the po-sition valve onto the next available trap cartridge for trapping thesubsequent peak. The process was repeated for other peaks ofinterest. When no peak collection was done, the flow (by default)was diverted to waste. After all peaks of interest were collected,each of the trapped fractions was sequentially desalted with 95%solvent A (0.1% FA in water) for 5 min. For peaks trapped from sep-arations using large amounts of nonvolatile salts, such as SEC, long-er desalting times of up to 10 min were needed. Lastly, trappedfractions were sequentially eluted by a step gradient to 65% solventB (0.1% FA in ACN) and diverting the flow into the mass spectrom-eter for detection. As shown in Fig. 1B, elution of six peaks of inter-est produced a single MS total ion chromatogram (TIC).

Poorly resolved and low-abundant peaks in the IEC or SEC profileswere analyzed by trapping the peaks on the same trap cartridge frommultiple sample injections, ensuring effective preconcentration andimproving MS detection. To obtain MAb chain-specific informationfrom the trapped fractions, an additional on-line reduction stepwas incorporated into the workflow immediately following the ini-tial desalting step. Reduction of trapped fractions was accomplishedby flushing trap cartridges with 100% solvent C containing 15 mMDTT in 25 mM Tris buffer (pH 7.5), followed by a second desaltingstep using 95% solvent A for 5 min prior to elution into the massspectrometer for detection.

A QTOF Premier mass spectrometer (Waters, Milford, MA, USA)coupled on-line to the HPLC system was operated in positive

Fig.1. Schematics of the on-line 2D LC–MS system equipped with a set of RP trap cartridges (T1–T6) available for trapping up to six first-dimension fractions of interest (IECor SEC as demonstrated in this work) (A) with its representative MS TIC (B). TOF, time-of-flight; ES, electrospray.

Characterization of MAb variants by LC–MS / M. Alvarez et al. / Anal. Biochem. 419 (2011) 17–25 19

electrospray ionization mode. The instrument was calibratedexternally to provide a mass accuracy of approximately 20 ppmfor the analysis of intact and reduced MAbs. The capillary voltagewas set at 3500 V, and the desolvation and source temperatureswere set at 200 and 90 �C, respectively. For intact and reducedanalyses of MAb fractions, the cone voltages were set at 55 and35 V, respectively. Instrument control and data analysis were per-formed using a Waters MassLynx software package (version 4.1).Deconvolution of multiply charged ions was performed with theMaxEnt 1 software provided with MassLynx.

Results and discussion

Development of an on-line system operating in the 2D LC–MS format

This on-line 2D LC–MS method was designed for the rapidcharacterization of charge or size variants of interest by MSwithout the need for additional sample manipulation. During ini-tial method development, a C3 column was tested for separationby RP. However, this resulted in long run times due to the

extended RP gradients needed to resolve components in trappedIEC or SEC fractions with no apparent improvement of the dataquality. Typical RP methods generally fail to resolve intactMAb isoforms, with hydrophobicities not significantly affectedby modifications and small sequence alterations, and requiredevelopment of custom separation conditions for each individualsample. Instead, RP trap cartridges were chosen for the sole pur-pose of rapid desalting of fractions prior to MS analysis, ensuringgeneric conditions of the method. Several RP trap cartridges fromdifferent vendors were evaluated, and Michrom trap cartridgeswere chosen due to their low carryover, good stability, and rea-sonable loading capacity (20 lg). Trap cartridges enabled fastconditioning and reequilibration and also provided minimalbackpressure during column switching events [24]. More impor-tant, the use of RP trap cartridges maximized both the speed andcharacterization power of the method given that data from mul-tiple fractions can be obtained within a short timeframe. Never-theless, an RP column can always be incorporated into theworkflow if additional peak separation prior to MS detection isneeded for a particular application.

Fig.2. IEC profile of MAb1 in-process sample with wide (dotted lines) and narrow (dashed lines) trapping windows for major peaks of interest (A) and correspondingdeconvoluted mass spectra for the main (A, inset), acidic 1 (B), and acidic 2 (C) fractions. Zero mass shift refers to the intact MAb1 antibody with G0F/G0F glycans.

20 Characterization of MAb variants by LC–MS / M. Alvarez et al. / Anal. Biochem. 419 (2011) 17–25

Typical analysis workflow included a scout IEC or SEC analysisduring which trapping times for the peaks of interest were deter-mined. Trapping intervals for these peaks were chosen carefullyto minimize interference from neighboring peaks and to preventtrapping of erroneous peaks due to subtle shifts in elution times,especially when the original sample characterization was per-formed on a different HPLC system. The main peak was always in-cluded in the analysis, and its data were used as a reference tocompensate for minor drift in MS calibration.

Rapid characterization of MAb major charge isoforms

Glycosylation on the conserved asparagine (Asn) residue withinthe Fc (fragment, crystallizable) domain of a typical IgG1 molecule isa characteristic feature of antibodies produced in mammalian celllines. Typical MAb oligosaccharides are of the complex biantennary

type consisting of a fucosylated (F) core with zero (G0), one (G1), ortwo (G2) terminal galactose residues and can be further elongatedby sialic acid (N-acetylneuraminic acid, NANA) residues, a commonsource of charge-related MAb heterogeneity due to the presence ofthe carboxyl group [25]. In general, carbohydrates play a significantrole in various biological functions of proteins, including modula-tion of interaction with receptors, protein folding, stabilization, reg-ulating activity, binding, and pharmacokinetic properties ofproteins they modify. Here we demonstrate the utility of the on-line2D LC–MS method by rapidly identifying major charge variantspresent in the IEC profile of an in-process sample of MAb1, as shownin Fig. 2A.

The masses observed in the main IEC fraction (Fig. 2A, inset)corresponded to intact MAb1 molecule modified with typical bian-tennary glycan structures [26], with the G0F/G0F component as themajor glycoform. According to the theoretical mass shifts in

Table 1Mass shifts corresponding to various combinations of glycans.

G0F G1F G2F G1F-NANA G2F-NANA G2F-2NANA

G0F 0G1F 162 324G2F 324 486 648G1F-NANA 453 615 777 906G2F-NANA 615 777 939 1068 1230G2F-2NANA 906 1068 1230 1359 1521 1812

Note: Zero mass shift refers to the intact MAb1 antibody with G0F/G0F glycans.

Characterization of MAb variants by LC–MS / M. Alvarez et al. / Anal. Biochem. 419 (2011) 17–25 21

Table 1, the minor components in this fraction matched the G1F/G0F and a combination of G1F/G1F and G0F/G2F with mass shiftsof 162 and 324 Da, respectively, relative to the main component.

Analysis of MAb1 acidic peaks revealed additional heterogene-ity, with mass shifts consistent with the presence of glycans withvarying degrees of sialylation. The masses observed in the acidic1 fraction (Fig. 2B) showed the presence of glycans with two andthree sialic acids per MAb molecule, with the dominant glycoformhaving tentative structures of G1F/G2F-2NANA and/or G1F-NANA/G2F-NANA resulting in a nominal mass shift of 1068 Da. The acidic2 fraction (Fig. 2C) contained primarily singly sialylated species,with the nominal mass shift of 453 Da due to G0F/G1F-NANA, of615 Da due to G0F/G2F-NANA and/or G1F/G1F-NANA, and of777 Da due to G1F/G2F-NANA and/or G2F/G1F-NANA glycoforms.The presence of main peak components in this fraction was attrib-uted to the elution of acidic 2 on a rising background of the mainpeak.

Due to the limited dynamic range and resolution of RP-LC–MS,its application is restricted to the characterization of only majorMAb variants when analyzed intact or even reduced to polypeptidechains [27]. Given that acidic 1 and acidic 2 accounts for 2% and13%, respectively, of the total IEC peak area, the identification ofthe individual minor glycoforms found in each of these fractionscan be easily overlooked when the sample is analyzed by typicalRP-LC–MS without prior sample treatment. Assessment of MAbglycoforms can be useful in evaluating cell lines, selecting clones,and optimizing cell culture processes to ensure appropriate andconsistent production [28]. In this work, IEC provided additionalselectivity for the characterization of minor glycoforms by separat-ing charged glycoforms due to the presence of sialic acid prior toRP-LC–MS analysis even on the intact protein level.

Characterization of a partially resolved and chemicallyunstable MAb variant

For a typical intact antibody, the natural isotopic distribution isapproximately 22 Da at the full width at half maximum (FWHM)and is further broadened to approximately 30 Da for a mass spec-trometer with a resolving power of 10,000 [29]. Therefore, modifi-cations with mass shifts falling within this mass range cannot beeffectively resolved for an intact MAb molecule. This obstaclewas encountered during the 2D LC–MS analysis of the basic frac-tion of MAb1 eluting at the tailing edge of the main peak. Becausethe main component was designed to contain pyroglutamic acid(pGlu) residues at both N termini of the heavy chains (HCs), itwas speculated that the basic fraction was due to the uncyclizedglutamine residue on one of the HCs as the result of its incompleteconversion to pGlu during production. The expected mass of thisvariant would be 17 Da heavier compared with the main peakcomponent. In our initial attempt to characterize the basic fraction(Fig. 2A, dotted lines), the MS data revealed a component with amass shift of 4 Da compared with the major component in themain peak (data not shown). Because there was a possibility thatthe presence of the main component in the trapped fraction was

biasing the result, the width of the trapping window was narrowedto obtain a higher purity basic fraction, as indicated by the dashedlines in Fig. 2A. The MS data of this variant showed a 10-Da shift(data not shown). Thus, it was concluded that this basic fractionwas heterogeneous due to a significant contribution from the mainspecies, resulting in mass spectral data that was a weighted aver-age of two values.

Furthermore, when the same basic fraction was collected andenriched off-line, the IEC profile was surprisingly dominated bythe main component species on its reinjection onto the IEC column(data not shown). Further investigations revealed chemical insta-bility of the basic peak variant, exhibiting a rapid conversion tothe main peak isoform, likely during the process of peak collection,enrichment, and/or exchange from the IEC mobile phase into theformulation buffer. Furthermore, this process was determined tohave a strong temperature dependence [30] and was catalyzedby the presence of phosphates [31] in the formulation buffer.Therefore, the fractionation process was optimized by collectingthe fraction on dry ice, followed immediately by lyophilization tominimize the conversion rate, thereby preserving the basic peakvariant. However, this ‘‘preserved’’ fraction was still found to con-tain a large amount of the main peak component (Fig. 3A, solidline), possibly due to its coelution (as discussed above) and/orconversion.

Because a pure basic fraction could not be obtained even afteroff-line enrichment, coupling IEC on-line with RP-LC–MS was nec-essary to further isolate the basic fraction from the interferingmain peak component for MS analysis. Due to the improved resolv-ing capability of this approach, accurate determination of themolecular weight of the basic component was achieved and wasfound to be 17 ± 1 Da heavier than the main component (Fig. 3Band C). This shift was consistent, within mass determination inac-curacy as determined by the spectral quality and fidelity of thedeconvolution algorithm, with the expected uncyclized residue atthe N terminus of one of the HCs, thereby confirming ourhypothesis.

Peptide mapping and N-terminal sequencing data subsequentlyconfirmed the presence of the uncyclized isoform in the basic frac-tion of MAb1 (data not shown). Although peptide maps provide themost comprehensive information on the molecule’s sequence andits alterations, characterization requirements can often be satisfiedby providing masses for expected variants on the intact level.

Identification of a low-level sequence variant using on-line reduction

During the analysis of an in-process sample of MAb2 by IEC, anew peak with abundance of less than 1% of the total peak areawas detected in the basic region of the profile when compared witha reference material, as shown in Fig. 4A. Confronted with the issueof a new peak, researchers often need to make a prompt identifica-tion to assess possible consequences of its presence. Due to the fastdata turnaround time and on-line nature of 2D LC–MS, this methodwas employed for the analysis of this new basic peak. Intact massanalysis of the fractioned basic peak (Fig. 4C) revealed a dominantspecies with a mass shift of 70 Da compared with the major com-ponent of the main peak (Fig. 4B). This mass shift, within the esti-mated inaccuracy of the deconvolution process of ±3 Da, did notcorrespond to any typically occurring posttranslational modifica-tions (PTMs) on MAbs. In addition, intact data lacked the informa-tion as to whether this was a modification of a single polypeptidechain or the cumulative result of multiple modifications of severalchains. Because chemical reduction of disulfide linkages greatly re-duces the complexity associated with intact mass analysis of MAbs,improves mass accuracy, and provides polypeptide chain-specificinformation [32], on-line reduction of the trapped IEC fractions ofMAb2 was performed to facilitate the identification of this new

Fig.3. (A) IEC chromatogram overlays of the off-line collected basic fraction (solid line) and the original unfractionated profile (dotted line) given as a reference. (B and C)Deconvoluted mass spectra of the main (B) and basic (C) components measured from the characterization of the basic fraction of MAb1.

22 Characterization of MAb variants by LC–MS / M. Alvarez et al. / Anal. Biochem. 419 (2011) 17–25

basic species. Although the current 2D LC–MS method used a stepgradient to elute trapped fractions into the mass spectrometer,resulting in coelution of the light chain (LC) and HC polypeptides,the presence of the two components simultaneously did not inter-fere with their successful mass deconvolution.

The deconvoluted spectrum for the HC of the basic fraction didnot show any new species compared with the main fraction (datanot shown). However, the LC spectrum of the basic fraction(Fig. 4E) was found to contain an additional species that was69.1 Da heavier than the LC component in the main fraction(Fig. 4D). Because the abundance of this variant in the spectrum ofthe LC (Fig. 4E) was lower compared with the spectrum of the intactMAb2 (Fig. 4C), it was concluded that the modification involved asingle LC. Within the estimated mass determination inaccuracy of±0.5 Da, the on-line database of protein modifications (www.uni-mod.org) suggested a substitution of serine (Ser) to arginine (Arg).Because incorporation of an additional Arg residue would increasethe overall basicity of this isoform of MAb2, this correlated well withthe elution of this variant in the basic region of the IEC profile.Clearly, the increased mass accuracy and site-specific information

obtained using on-line reduction greatly complemented data fromintact analysis and strengthened the likelihood of the correctassignment. Furthermore, subsequent analysis of the in-processsample by tryptic peptide mapping confirmed the presence of aSer-to-Arg substitution in the LC (data not shown).

Elucidation of minor size variants using multiple trappings

As a product quality assay, SEC is routinely used to monitor pro-tein aggregation and the presence of other size variants duringdevelopment and manufacturing processes [33]. Careful examina-tion of an SEC profile of a MAb3 sample revealed the presence of ahigh-molecular-weight (HMW) shoulder peak with a peak area of0.4%, partially separated from the main component, as shown inFig. 5A (solid line).

From our optimization studies, it was estimated that at least 1 lgof MAb must be loaded onto a trap cartridge to produce interpretableMS data for intact analysis by 2D LC–MS (data not shown). However,trapping of this low-abundant HMW peak from a single SEC runusing an optimal injection load (without loss of chromatographic

Fig.4. (A) IEC profiles of MAb2 in-process sample (solid line) and the reference material (dotted line) with trapping windows indicated by vertical dashed lines. (B and C)Deconvoluted mass spectra for the main (B) and basic (C) fractions of intact MAb2. (D and E) Deconvoluted mass spectra of the LC obtained after on-line reduction for themain (D) and basic (E) fractions of this sample.

Characterization of MAb variants by LC–MS / M. Alvarez et al. / Anal. Biochem. 419 (2011) 17–25 23

resolution) was not sufficient to provide quality MS spectra for iden-tification. To overcome this problem, preconcentration of the HMWpeak on the same trap cartridge from duplicate injections wasperformed.

Analysis of the main fraction showed masses that correspondedto the intact molecule modified with the typical glycans for theMAb3 antibody (Fig. 5B). In the same fraction, low-molecular-weight (LMW) components were also detected with a mass of23,233 Da, consistent with the expected mass of the free LC of23,234 Da as the dominating LMW species, and some amounts of119- and 305-Da adducts attributed to cysteinylation (Cys) andglutathionylation (GSH) products of the LC, respectively. The pres-ence of several size components otherwise expected to be sepa-rated by SEC, in the same trapped fraction, was postulated to beindicative of their noncovalent association under native conditions,and their dissociation was the result of the denaturing effect of theorganic solvents employed in the RP separation.

Heterodimeric structures of MAbs, consisting of LC and HCpairs, are stabilized by covalent (disulfide bridges) and noncova-lent interactions. Zhang and Czupryn [34] evaluated levels of freesulfhydryls in MAbs that ranged from 0.02 mol/mol in the nativeconformation to 0.1 mol/mol upon denaturation. This implies thatsolutions of antibodies contain fractions where a lack of disulfidebridges is compensated by the noncovalent association of the

domains to produce an intact molecule. The noncovalent intramo-lecular interactions have been demonstrated to be strong enoughto preserve the intact structure of immunoglobulins that did notdissociate under native conditions but was readily revealed underdenaturing conditions [34].

In this work, the free LC observed in the main fraction was as-sumed to be noncovalently associated with a complementary frag-ment consisting of two HCs and one LC (HHL). Association of thefree LC with the intact MAb molecule was considered to be lesslikely. Provided that such a molecular heterogeneity under themain peak exists, intact MAb species with an extra LC would be ex-pected to be enriched in the front portion of the main peak. Never-theless, 2D LC–MS was used to characterize the main SEC peak byfractionating across its elution profile. Examination of the intensityratio of the free LC to the intact MAb revealed a nearly constant ra-tio in all six fractions throughout the elution of the main peak (datanot shown). This result served as indirect evidence for the presenceof noncovalently associated LC and HHL components to give a mol-ecule with mass similar to that of the intact MAb, supporting theabove assumption. It should be noted that the presence of smallamounts of HHL species was not detected under the main peak be-cause its charge state envelope cannot be successfully resolvedfrom the more abundant peaks of the intact MAb. Because chargestate peaks of a typical LC are positioned in the lower m/z range

Fig.5. Overlays of high-resolution SEC chromatograms of MAb3 under native (solid line) and denaturing (dashed line) conditions (A) with deconvoluted spectra of the main(B) and HMW shoulder (C) fractions from native SEC trapped from two consecutive injections of MAb3. The inset in panel A shows the deconvoluted spectrum of the LMWpeak from denaturing SEC. Peaks labeled with an asterisk (�) are due to phosphate adducts. TOF, time-of-flight; ES, electrospray.

24 Characterization of MAb variants by LC–MS / M. Alvarez et al. / Anal. Biochem. 419 (2011) 17–25

of the spectrum, the presence of the free LC can be readily detected.A more detailed examination of noncovalent associations in MAbswill be the subject of a future study.

The deconvoluted spectrum of the HMW shoulder fraction(Fig. 5C) showed masses that corresponded to an antibody frag-ment modified with the same glycans but approximately 10 kDaless than the intact molecule, matching the mass of the MAb3 frag-ment with a loss of 93 amino acids from the N-terminal end of oneof the LCs. This fraction was found to contain the same free LC spe-cies observed in the main fraction but with greater relativeamounts of cysteinylated and glutathionylated products. More-over, the HMW fraction was significantly enriched with the LCcomponents, with an intensity ratio of the free LC to the MAb frag-ment species approximately 100 times greater compared with therelative amount of the free LC to the intact MAb in the main peakfraction. Assuming similar ionization efficiencies between the frag-ment and the intact MAb, and considering the published data onlevels of free sulfhydryls [34], it was estimated that the species un-der the HMW peak contained at least one free LC per molecule ofthe MAb fragment, presumably existing in a noncovalent assembly.Thus, the resulting noncovalent complex would be at least 13 kDa

larger than the intact antibody and, therefore, eluting in the HMWregion of the SEC profile. It should be noted that resolution of thissize variant from the main peak component can be partially due tothe use of two columns for SEC; however, perturbation of the na-tive conformation of the MAb due to the presence of a clip can re-sult in a significantly larger change in hydrodynamic volume thanwould be expected from the mass shift alone. For example, clip-mediated aggregation has been demonstrated for an IgG2 antibody[35–37], with clipped polypeptide fragments reported to bestrongly noncovalently bound to the intact antibody, giving riseto the occurrence of HMW peaks in SEC profiles.

To examine the nature of the HMW species, MAb3 was furtheranalyzed under denaturing conditions by diluting the sample in20% ACN in water prior to SEC analysis. As expected, the HMWshoulder peak disappeared and an LMW peak increased comparedwith the analysis of the sample under native conditions (Fig. 5A,dashed line). This LMW peak was subsequently characterized bythe 2D LC–MS method and was found to be due to the free LC com-ponents (Fig. 5A, inset). This result strongly suggested the nonco-valent assembly of the free LC species with the HMW fragmentunder native conditions in the original sample.

Characterization of MAb variants by LC–MS / M. Alvarez et al. / Anal. Biochem. 419 (2011) 17–25 25

Conclusions

The fast characterization of MAbs and their related variants wasachieved by using an on-line method operating in a 2D LC–MS for-mat. This generic method was developed to be easily adapted tosatisfy characterization requirements for a range of MAbs. Themethod was relatively high-throughput, conserving not only pre-cious sample but also analysis time, and can be used as the first-line technology in providing intact and chain-specific informationfor rapid characterization of size and charge variants of biothera-peutics. Interfacing the chromatographic system with a high-reso-lution mass spectrometer made it possible to identify componentsin resolved and coeluting fractions.

It should be noted that mass information for intact and reducedMAbs is often not sufficient for the reliable identification of certainvariants and can hardly be recommended as a substitute for pep-tide mapping. Intact and reduced data, however, are readily avail-able and possess a confirmation value if the nature of a certainvariant can be deduced from prior characterization work or generalexperience. In addition, they may help in designing and optimizingsubsequent characterization strategies by indicating chain-specificlocation, and the type of modification that should be examined. Forexample, a potential presence of a Ser-to-Arg basic variant of MAb2suggested that the possible presence of a new trypsin cleavage siteshould be taken into account during evaluation of the peptide map,thereby significantly simplifying the task.

With the entire system operating on-line, there was minimalsample handling involved, thereby reducing contamination, losses,and degradation. The incorporation of an on-line reduction stepwith DTT allowed the characterization of proteins in finer detailand with improved mass accuracy measurements. Analysis oflow-abundant, poorly resolved fractions was made possible byadjusting the trapping time interval and by performing multiplesample injections aimed to further enrich the fractions on the sametrap before characterization by MS. The successful identification ofMAb variants due to differences in their glycosylation, N-terminalcyclization, primary sequence, and size was achieved in the exam-ples described in this article. Simplification of system program-ming and even automation of peak trapping algorithms arecurrently being implemented in the workflow.

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