A Novel Online Four-Dimensional SECÃSEC-IMÃMS Methodology for

Article pubs.acs.org/ac

Cite This: Anal. Chem. 2018, 90, 13929−13937

A Novel Online Four-Dimensional SEC×SEC-IM×MS Methodology for Characterization of Monoclonal Antibody Size Variants Anthony Ehkirch,†,¶ Alexandre Goyon,‡,¶ Oscar Hernandez-Alba,† Florent Rouviere,§ Valentina D’Atri,‡ Cyrille Dreyfus,∥ Jean-François Haeuw,∥ Heĺ eǹ e Diemer,† Alain Beck,∥ Sabine Heinisch,§ Davy Guillarme,*,‡ and Sarah Cianferani*,†

Downloaded via UNIV OF WINNIPEG on December 4, 2018 at 06:57:22 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France ‡ School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU, Rue Michel-Servet, 1, 1211 Geneva 4, Switzerland § Université de Lyon, Institut des Sciences Analytiques, CNRS, UMR5280, Université de Lyon, ENS, 69100 Villeurbanne, France ∥ IRPF−Centre d’Immunologie Pierre-Fabre (CIPF), 74160 Saint-Julien-en-Genevois, France S Supporting Information *

ABSTRACT: The determination of size variants is a major critical quality attribute of a therapeutic monoclonal antibody (mAb that may affect the drug product safety, potency, and efficacy. Size variant characterization often relies on size-exclusion chromatography (SEC), which could be hampered by difficult identification of peaks. On the other hand, mass spectrometry (MS)-based techniques performed in nondenaturing conditions have proven to be valuable for mAb-related compound characterization. On the basis of the observation that limited SEC performance was observed in nondenaturing MS compatible ammonium acetate buffer compared with classical phosphate salts, a multidimensional analytical approach was proposed. It combines comprehensive online twodimensional chromatography (SEC×SEC), with ion mobility and mass spectrometry (IM-MS) in nondenaturing conditions for the characterization of a variety of mAbs. We first exemplify the versatility of our approach for simultaneous detection, identification, and quantitation of adalimumab size variants. Benefits of the SEC×SEC-native IM×MS were further highlighted on forced degraded pembrolizumab and bevacizumab samples, for which the 4D setup was mandatory to obtain an extensive and unambiguous identification, and accurate quantitation of unexpected high/low molecular weight species (HMWS and LMWS). In this specific context, monomeric conformers were detected by IM-MS as HMWS or LMWS. Altogether, our results emphasize the power of comprehensive 2D LC×LC setups hyphenated to IM×MS in nondenaturing conditions with unprecedented performance including: (i) maintaining optimal SEC performance (under classical nonvolatile salt conditions), (ii) performing online native MS identification, and (iii) providing IM-MS conformational characterization of all separated size variants.

M

standard of care for many types of cancer. The rapid expansion of the IO space is likely to continue, as 940 IO agents were in clinical developments and 1064 additional ones were in preclinical phase in September 2017.4 The analytical characterization of mAbs requires the investigation of multiple critical quality attributes (CQAs), among which the determination of high-molecular-weight species (HMWS) and low-molecular-weight species (LMWS) is of utmost importance.5 In particular, HMWS can inhibit the product efficacy6 or cause hypersensitivity responses such as anaphylaxis.7 A variety of orthogonal analytical methods are often applied to address aggregation issues, among which size-

onoclonal antibodies (mAbs) and related products are the most rapidly growing class of human drugs. In 2017, more than 80 candidates were approved for therapeutic use.1,2 An additional 19 mAbs are currently being evaluated in latestage studies with primary completion dates in 2018.3 The successful application of mAbs in oncology, autoimmunity, and chronic inflammatory diseases has generated high revenues for the biopharmaceutical industry. As an example, the first fully human mAb (adalimumab/Humira), a TNF inhibitor used as an anti-inflammatory drug to treat rheumatoid arthritis and other autoimmune diseases, induced $16,515 M worldwide sales in 2016.1 Since the approval of the first immuno-oncology (IO) mAb targeted against CTLA-4 (ipilimumab/Yervoy) in 2011, five other IO mAbs targeting PD-1 or PD-L1 have been approved, among which the pembrolizumab (Keytruda) and nivolumab (Opdivo) blockbusters have revolutionized the © 2018 American Chemical Society

Received: July 25, 2018 Accepted: October 29, 2018 Published: October 29, 2018 13929

DOI: 10.1021/acs.analchem.8b03333 Anal. Chem. 2018, 90, 13929−13937

Article

Analytical Chemistry exclusion chromatography (SEC),8−12 flow field-flow fractionation (A4F),13 and analytical ultracentrifugation (AUC)14 appear as reference methods for measuring HMWS. In SEC, the use of nonvolatile salts (typically in the 100−500 mM range) has been reported to reduce potential secondary electrostatic interactions between the stationary phase and proteins, to achieve more reliable HMWS determination.15 These salts are even more important for aggregates, since these species are more prone to bind to the stationary phase through nonspecific interactions, resulting in an underestimation of the HMWS.16,17 However, for online coupling to mass spectrometry (MS), the use of volatile salt buffers is mandatory. Online SEC-MS has been reported for both the analysis of reduced mAbs under denaturing conditions (20% of ACN, 0.1% of TFA, 0.1% of formic acid in water)18 and more recently for the analysis of intact mAb under nondenaturing conditions (ammonium acetate volatile salt).19 According to the United States Pharmacopeia (USP) monograph 129 released in 2016,20 species eluting prior to the main peak are considered as HMWS, while the species eluted after the main peak are LMWS. However, in some cases, the partially denatured monomers have been shown to elute before the main peak21 and even the oxidized tryptophan degradant of mAb samples,22 which cannot be considered as HMWS. Furthermore, secondary hydrophobic interactions have been shown to increase the elution/retention times of the most hydrophobic mAbs in SEC,23 allowing sometimes the baseline separation of various mAbs having similar sizes, thanks to mixed-mode interactions.24 Therefore, the interpretation of the SEC profile can be misleading and could require the identification of the various species through comparison with orthogonal techniques, such as AUC14 or A4F13. Multiangle light scattering (MALS) can also be hyphenated to SEC, for determining the MW of the eluted species. It allows the differentiation between partially denatured monomers and aggregates,21 even though its mass precision remains insufficient to discriminate a partially denatured monomer from an oxidized species. Conversely, MS performed in classical denaturing conditions has been shown to be a powerful tool to measure the accurate mass of protein species and could potentially allow the identification of oxidized protein species (shift of +16 Da per oxidation).25 When MS is performed in nondenaturing conditions (also called native MS), several CQAs can be determined through accurate mass measurements of intact mAb assemblies, ranging from mAb/ Ag binding stoichiometries,26 oligomeric forms or even drug-to antibody ratio (DAR), and drug load distributions (DLD) for antibody-drug conjugates (ADCs).27 When native MS is combined with ion mobility (IM), additional conformational information can be provided. IM is thus considered as an orthogonal dimension of separation to MS, enabling discrimination of partially denatured monomers from folded monomers and aggregates.28 However, lower overall SEC performance was clearly highlighted by Goyon et al.23 when using MS compatible mobile phases (volatile ammonium acetate), especially when analyzing basic proteins (pI > 7). This could in turn lessen the feasibility of the online direct SEC-MS coupling. To answer the need for keeping optimal SEC performances, while performing an online MS identification in nondenaturing conditions, we propose here an online 2D-LC setup prior to native IM-MS, involving SEC in both dimensions, with the second SEC dimension used as fast desalting step that could be

adapted for the analysis of a broad variety of mAbs. The benefits of such 2D LC-IM-MS setup were highlighted for forced degraded studies of different mAbs. Adalimumab was chosen for its high pI and presence of solvent accessible basic patches;29 pembrolizumab and ipilimumab were selected because they are among the most hydrophobic therapeutic mAbs;23 and finally, bevacizumab was chosen for its known ability of forming large amount of HMWS under specific conditions.30 Limited SEC performance in ammonium acetate was first presented on bevacizumab and ipilumumab. We next describe the developed online 2D-LC setup coupled to native IM-MS on adalimumab, a well-studied mAb. Finally, benefits of the 2D SEC×SEC-native IM-MS were used to unambiguously identify all species generated from forced degraded pembrolizumab and bevacizumab samples.31

■

EXPERIMENTAL SECTION Reagent and Materials. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.): ammonium acetate (A1542), cesium iodide (21004), phosphoric acid (345245), 2-propanol (I9516), potassium chloride (P9333), potassium phosphate monobasic (P0662), and potassium phosphate dibasic (P3786). All the aqueous solutions were prepared using an ultrapure water system (Sartorius, Göttingen, Germany). Antibodies were obtained as European Union pharmaceutical-grade drug products from their respective manufacturers. For thermally stressed mAb samples, antibodies were incubated at 40 °C during 8 weeks in their respective formulation buffer. Size-Exclusion Chromatography with UV Detection. An Acquity UPLC H-class system (Waters, Manchester, U.K.) included a quaternary solvent manager, a sample manager set at 10 °C, a column oven, and a TUV detector operating at 280 and 250 nm. A volatile mobile phase composed of 100 mM ammonium acetate (pH 6.9) was compared to a generic and nonvolatile mobile phase composed of 50 mM of potassium phosphate buffer and 250 mM potassium chloride pH 6.8 at a flow rate of 0.250 mL/min. Size-Exclusion Chromatography with MALS Detection. Antibodies were analyzed by multiangle light scattering (MALS) after separation by size exclusion. HPLC was performed using an Agilent 1260 Infinity with the bioinert quaternary pump; the 1260 Infinity Diode Array Detector; the 1260 Infinity Standard Autosampler; and the 1290 infinity thermostat. This instrument was coupled to the Eclipse DualTec system, the DAWN HELEOS II Multi-Angle static Light Scattering (MALS) detector and the Optilab T-rEX refractive index (RI) detector (Wyatt Technology, Santa Barbara, CA). This combination of instrumentation enabled the measurement of absolute molecular weight (or molar mass) and size eluting species. The mobile phase was composed of 50 mM of potassium phosphate buffer and 250 mM potassium chloride pH 6.8 at a flow rate of 0.300 mL/min. The SEC column was a Yarra SEC-x300 (150 mm × 4.6 mm, 1.8 μm, 300 Å) from Phenomenex (Torrance, CA, U.S.A.). SEC×SEC-IM×MS Instrumentation. The LC×LCIM×MS system consisted in a combination of H-Class and I-Class liquid chromatography systems hyphenated to a Synapt G2 HDMS Q-TOF mass spectrometer, all from Waters (Manchester, U.K.). In the first dimension, the H-Class system includes a high-pressure quaternary solvent delivery pump, an autosampler with a flow-through needle of 15 μL equipped with an extension loop of 50 μL. In the second dimension, the 13930


Article

Analytical Chemistry

Figure 1. Chromatographic profiles obtained in SEC for bevacizumab (a) and ipilimumab (b) by using ammonium acetate buffer (orange lines) or phosphate buffer in the presence of sodium chloride (green lines). (c) Table summarizing quantitation of HMWS and LMWS from SEC data.

the flow-rate by a factor 7 prior entering MS (i.e., inlet flow of 100 μL/min). SEC×SEC-Native Mass Spectrometry. The Synapt G2 HDMS was operated in sensitive mode and positive polarity with a capillary voltage of 3.0 kV. To avoid disruption of weak noncovalent interactions, the sample cone and pressure in the interface region were set to 180 V and 6 mbar, respectively. Source and desolvation temperature were set to 100 and 450 °C, respectively. Desolvation and cone gas flows were set at 750 and 60 L/Hr, respectively. Acquisitions were performed in the m/z range of 1000−15 000 with a 1.5 s scan time. External calibration was performed using singly charged ions produced by a 2 g/L solution of cesium iodide in 2-propanol/water (50/ 50 v/v). SEC×SEC-Native Ion Mobility Mass Spectrometry. For IM-MS measurements, the sample cone voltage was set to 80 V, and the backing pressure of the source was 6 mbar. The Ar flow rate was 5 mL/min and the trap collision energy was set at 4 V in the traveling-wave-based ion trap. Ions were thermalized with a constant He flow rate of 130 mL/min before IM separation. The height and the velocity of the periodic waveform in the pressurized ion mobility cell were 40 V and 923 m/s, respectively. N2 was used as drift gas (45 mL/min) providing a constant pressure of 2.75 mbar. Transfer collision energy was fixed to 2 V to extract the ions from the IM cell to the TOF analyzer. A SEC-IM×MS calibration based on six different proteins (glutamate dehydrogenase, concanavalin A, pyruvate kinase, alcohol dehydrogenase, avidin, and beta lactoglobulin A) in nondenaturing conditions32,33 was used to perform collision cross section (CCS) calculation, as previously described.34,35 Theoretical CCS Calculation. Theretical CCS value of pembrolizumab (PDB code: 5DK3) was obtained using the Impact trajectory method.36 The PDB file was used as an input file and was run on Impact with a convergence value of 1% enabled to determine an average CCS as a mean of six independent calculations. Solvent heteroatoms were here excluded from the theoretical calculation.

I-class system includes a high-pressure binary solvent delivery pump, a column manager composed of two independent column ovens and two 6-ports high pressure two-position valves acting as interface between the two chromatographic separation dimensions. A single-wavelength UV detector and a diode array detector both equipped with 500 nL flow-cell were used for the first and second dimension, respectively. An external two position-switching valve (Vici Valco Instruments, Houston, U.S.A.) was also placed prior to the MS (see Supporting Information S1). Nondenaturing MS and ion mobility experiments were performed on a TWIMS-MS Synapt G2 HDMS instrument (Waters, Manchester, U.K.). Data acquisition and instrument control were performed with MassLynx V4.1 software (Waters). MS-Data were processed with Masslynx 4.1 and chromatography-data were processed with UNIFI. SEC×SEC Chromatographic Conditions. In the first dimension, the SEC column was a Yarra SEC-x300 (150 mm × 4.6 mm, 1.8 μm, 300 Å) from Phenomenex (Torrance, CA, U.S.A.) or two AdvanceBio SEC (150 mm × 4.6 mm, 2.7 μm, 300 Å) coupled in series from Agilent Techonologies (Wilmington, DE, U.S.A.) for the very hydrophobic pembrolizumab.23 The SEC column employed in the second dimension was an AdvanceBio SEC (50 mm × 4.6 mm, 2.7 μm, 300 Å) also from Agilent Technologies. In the first SEC dimension, the mobile phase was composed of 50 mM of phosphate buffer (K2HPO4 and KH2PO4) and 250 mM potassium chloride pH 6.8. The SEC experiment was conducted in isocratic elution at a flow rate of 0.035 mL/min with Yarra SEC-X300 column and 0.070 mL/min for the two AdvanceBio SEC columns coupled in series. The volume of the sample loops used for interfacing both dimensions was 100 μL filled to 35% for the Yarra SEC-X300 column and 70% for the two AdvanceBio SEC columns. The column was set at room temperature, and wavelength and data acquisition rate were set at 280 and 250 nm and 10 Hz, respectively. The injection volume was 5 μL. For the second chromatographic dimension in SEC, the separation was carried out in isocratic mode with an aqueous mobile phase composed of 100 mM ammonium acetate at a flow-rate of 700 μL/min. Column temperature, wavelength, and data acquisition rate were set at room temperature, 210/ 280 nm, and 40 Hz, respectively. The analysis time of the second dimension run, corresponding to the sampling time of the first dimension separation, was 1.0 min. A fraction of 0.25 min (from 0.50 to 0.75 min) was sent to MS thanks to a switching valve, to limit contamination of the ESI source with nonvolatile salts (phosphate). A T-piece flow splitter divided

■

RESULTS Rationale for the Development of a 2D SEC Approach: SEC Performance in Ammonium Acetate vs Phosphate Buffer. Figure 1 shows the chromatographic behavior of two mAbs, namely, bevacizumab and ipilimumab analyzed in SEC conditions using both ammonium acetate (native MS-compatible buffer) and phosphate in the presence of sodium chloride (ideal SEC-UV buffer). Bevacizumab and ipilimumab are quite difficult to analyze in SEC due to their 13931


Article


Figure 2. Flowchart of the SEC×SEC-native IM×MS for mAb analysis. The optimized SEC×SEC method was hyphenated to IM-MS. In the first dimension, SEC with nonvolatile salts allows a proper separation and quantitation of mAb HMWS/LMWS. In the second dimension, a short SEC column used with a volatile mobile phase was employed as a fast desalting step. Online native IM-MS allows conformational characterization and intact mass measurement of each individual 1D-SEC peaks.

the significant increase in elution times, the peaks were much broader with ammonium acetate, probably due to chemical interactions (above all the ionic ones) subject to slow kinetics. The adsorption of mAbs onto the SEC stationary phase was also more pronounced in the presence of ammonium acetate, leading to a 2-fold and a 4-fold sensitivity reduction for bevacizumab and ipilimumab, respectively. Again, the most basic mAb (ipilimumab) was more affected, suggesting that this adsorption phenomenon was mainly related to ionic interactions. However, thanks to the above-mentioned chemical interactions, some of the peaks observed in SEC (e.g., those eluting just before the main isoforms of bevacizumab and ipilimumab) were better separated in ammonium acetate. The multimodal nature of the elution mechanism in ammonium acetate (separation based on size, ionic character and hydrophobicity) could explain the better selectivity achieved in acetate vs phosphate, for ionic and/or hydrophilic variants. Altogether, our results indicate that SEC quantitation performed in ammonium acetate would lead to incorrect HMWS and LMWS quantitation (0.6% HMWS and 0.5% of LMWS in ammonium acetate versus 7.8% HMWS and 0.8% LMWS in phosphate buffer for bevacizumab, Figure 1c). In conclusion, our results demonstrate interesting separation capabilities, but limited overall performance of SEC with ammonium acetate as buffer (i.e., broader peaks, longer elution times, lower sensitivity, more adsorption, and underestimation of HMW and LMW species) for basic and/or hydrophobic mAbs. Development of a SEC×SEC-Native IM×MS Setup for Adalimumab Characterization. In order to limit the possible drawbacks previously mentioned, which are more pronounced with the use of ammonium acetate in SEC, we adapted a four dimensional approach recently developed in our laboratories.32 In the present work, a multidimensional SEC×SEC-native IM-MS setup was developed (Figure 2). In

high pI (8.3 and 9.2, respectively) with a suspected basic patch for bevacizumab,29 which can make them prone to ionic interactions with the SEC material (ion exchange) at neutral pH. In addition, these two mAbs can be considered as some of the most hydrophobic commercial mAbs, based on the measured apparent retention times in hydrophobic interaction chromatography (HIC), according to the gradient used in Goyon et al.,23 meaning that they could also interact with the SEC column through hydrophobic interactions. A lastgeneration SEC column packed with sub-2 μm particles and 300 Å pore size (Phenomenex Yarra SEC X-300 1.8 μm) was selected for its high kinetic and separation performance in comparison with columns packed with sub-3 μm particles, as described in Goyon et al.37 (see Experimental Section). As shown in Figure 1, the SEC chromatograms were quite different when using ammonium acetate vs phosphate buffer. First, the main peaks of bevacizumab and ipilimumab eluted much later in ammonium acetate, suggesting that the separation with ammonium acetate buffer was not solely based on the size of the proteins. Due to both their high pI and their important hydrophobicity, the elution/retention times of these two mAbs were possibly affected by nonspecific interactions with the SEC stationary phase, resulting in a mixed-mode chromatographic behavior. In fact, although the mobile phase pH was comparable in both buffers (i.e., around 6.8), the ionic strength in ammonium acetate was lower (100 mM) vs phosphate (50 mM + 250 mM KCl). Therefore, the resulting ionic interactions were more pronounced in ammonium acetate, leading to higher elution/retention times and potential protein adsorption, known to be often more pronounced with HMWS.38 The most basic mAb (i.e., ipilimumab, pI of 9.2) eluted three times later in ammonium acetate (tr > 20 min), while the increase in bevacizumab elution time was less important (8 min in ammonium acetate vs 6 min in phosphate/sodium chloride buffer). In addition to 13932


Article


Figure 3. Online SEC×SEC-IM×MS of adalimumab (Humira). (a) Comparison between chromatograms using nonvolatile mobile phase (50 mM phosphate buffer +250 mM NaCl, blue trace) and volatile mobile phase (100 mM ammonium acetate, orange trace) for unstressed adalimumab. (b) Overlaid SEC chromatograms of forced degraded (solid line) and unstressed adalimumab (dashed line) using Yarra SEC-x300 (150 mm × 4.6 mm, 1.8 μm, 300 Å) from Phenomenex (Torrance, CA, U.S.A.). (c) Native mass spectra of each individual SEC separated peak for the stressed sample. (d) Ion mobility arrival time distributions (ATDs) of dimeric, monomeric, Fc-Fab, and Fab species along with their collision cross section (TWCCSN2) (39+ charge state for the dimer, 26+ charge state for the monomer, 22+ charge state for Fc-Fab, and 13+ charge state for Fab).

dimeric aggregate (296 705 ± 23 Da), while peaks III and IV, eluting after the main peak in the LMWS region, were identified as Fc-Fab (100 876 ± 15 Da) and Fab (44 248 ± 10 Da) fragments. The increased amounts of HMWS and LMWS obtained upon stress were quantified by integrating the peaks observed in the SEC-UV chromatograms. For the stressed adalimumab, HMWS and LMWS represented 3.2% and 6.7%, respectively, and 0.8% and 0.4%, respectively, for the unstressed sample. For conformational characterization, IM data were additionally recorded. Arrival time distributions (ATDs) of each individual species were isolated in the IM cell, allowing conformational characterization through collisional cross section (TWCCSN2) of the different size variants (Figure 3d). The order of magnitude of the TWCCSN2 value for the 26+ charge state of monomeric adalimumab (79.5 ± 0.1 nm2) was found to be in good agreement with those published for other mAbs.33,39 Conformational characterization was also obtained for dimeric HMWS (130.8 ± 0.1 nm2) along with LMWS FcFab (64.3 ± 0.1 nm2) and Fab (33.3 ± 0.1 nm2) fragments. Of note, the measured TWCCSN2 for adalimumab dimer was slightly (130.8 ± 0.1 nm2) higher than the one predicted from the mass and considering proteins as spheres40 (108 nm2). More interestingly, the IM-MS calculated TWCCSN2 value was lower than the one calculated considering that the CCS of a dimer should be twice the CCS of the monomer (2 × CCSmonomer = 159 nm2) and that protein shape could be assimilated as a sphere, suggesting a significant conformational compaction upon mAb aggregation, as already observed for pH-stressed trastuzumab dimeric species.33

the first dimension, an optimized and generic SEC method was performed with nonvolatile salts, for a proper characterization and quantitation of mAb HMWS/LMWS. An online fast SEC desalting step (less than 1 min using a SEC cartridge) was then performed in the second dimension to achieve the buffer exchange, thereby making the 2D-separation fully compatible with online native MS/IM-MS analysis. As a first example, a temperature-stressed adalimumab sample (see Material and Methods) was compared to an unstressed one. Adalimumab is difficult to analyze by SEC due to its high pI and suspected presence of solvent accessible basic patches.29 Therefore, the choice of the mobile phase is crucial to perform a correct SEC separation, as depicted in Figure 3a. When an optimal phosphate mobile phase is chosen, HMWS and LMWS can be estimated at 0.8% and 0.4% for unstressed adalimumab, respectively. Conversely, with ammonium acetate, no HMWS (0%) could be detected, while the amount of LMWS is largely overestimated (24.5%). On the basis of these observations, we generated a temperature stressed adalimumab sample (see Experimental Section) and benchmarked our SEC×SEC-native IM×MS setup for the comparison of stressed and unstressed adalimumab. The comparison of stressed and unstressed adalimumab chromatograms revealed the presence and the increment of signals related to HMWS and LMWS species in the stressed sample (Figure 3b). Native MS allowed the unambiguous identification of each SEC-separated species (Figure 3c). As expected, the most intense peak (peak II) was attributed to the monomer (148 249 ± 7 Da) with a good mass accuracy (47 ppm). Peak I, eluting in the HMWS zone, was identified as 13933


Article


Figure 4. Online SEC×SEC-IM×MS of pembrolizumab (Keytruda). (a) Comparison between chromatograms using nonvolatile mobile phase (blue trace) and volatile mobile phase (orange trace) for unstressed pembrolizumab, adapted from Goyon et al.23 (b) Overlaid 1D-SEC chromatograms of forced degraded (solid trace) and unstressed pembrolizumab (dashed line). (c) Native mass spectra of each individual SEC separated peak for stressed pembrolizumab. (d) Ion mobility arrival time distributions (ATDs) of 26+ charge state of monomeric species detected for stressed pembrolizumab, along with their collision cross section (TWCCSN2).

peak I (149 092 ± 7 Da) and minor peak II (149 076 ± 4 Da) as monomeric species instead of multimers. Of note, both monomeric forms of peaks I and II have a slightly increased mass (+31 Da and +15 Da, respectively) compared with the main peak III, suggesting that those species may correspond to oxidized monomeric forms as already published for pembrolizumab.22 To validate our hypothesis of increased oxidation in pembrolizumab thermally stressed sample, we performed peptide mapping using enzymatic digestion (see Supporting Information S2) of both stressed and unstressed pembrolizumab samples. An increase of oxidation was observed in the thermally stressed sample (∼30%) vs the unstressed (∼23%) sample (see Supporting Information S3), confirming that oxidation could account for monomers I and II. It is thus clear from these results that the sole use of SECUV would have provided a misleading HMWS quantitation because of incorrect peak identification. Finally, from our SEC×SEC-native IM×MS experiments, it could be concluded that the amount of monomer I increases upon thermal stress (from 7% to 19.8%). At this stage, IM-MS analyses were of utmost importance, considering the different measured TW CCSN2 values of the 26+ charge state. While very similar for monomers II and III (79.5 ± 0.1 nm2), the TWCCSN2 value of monomer I was significantly different (78.9 ± 0.1 nm2). These results provide definitive explanations that monomers II and III exhibit very closely related conformation, while monomer I presents a significantly different conformation. Monomer I, which is the most oxidized and also the most abundant conformer, presents a TWCCSN2 decrease of 0.6 ± 0.1 nm2, which is far more than the CCS difference expected from the mass increase of +32 Da (0.01 nm2), suggesting a strong conformational compaction of monomer I compared to III due to oxidation. Of note IM-MS measured TWCCSsN2

Altogether, this first comparative SEC×SEC-native IM×MS experiment emphasizes the ability of our 4D setup to simultaneously identify HMWS and LMWS under similar native MS/IM-MS conditions, providing a comprehensive characterization of forced degraded mAb samples within a single run. The benefits of the 4D SEC×SEC-native IM×MS are the following: (i) maintaining optimal SEC performance (under classical non volatile salt conditions), (ii) performing online native MS identification, and (iii) providing IM-MS conformational characterization of all-separated size variants. SEC×SEC-Native IM×MS Provides Unambiguous Identification and Quantitation of Unexpected Species in Forced Degraded Samples. To illustrate the benefits of the SEC×SEC-native IM×MS approach compared to standalone SEC-UV or native IM-MS techniques for size variant characterization, we focused on forced degraded studies of two mAbs: pembrolizumab (Keytruda) and bevacizumab (Avastin). Pembrolizumab is challenging to analyze in SEC with ammonium acetate, due to its basic pI (7.5) leading to severe peak broadening, tailing (As = 1.8) and strong adsorption (Figure 4a). Both temperature-stressed and unstressed pembrolizumab were analyzed by SEC×SEC-native IM×MS. As shown in Figure 4b, SEC chromatograms revealed three peaks. The most intense one (peak III) was attributed to the monomer: a mass of 149 061 ± 5 Da (mass accuracy: 67 ppm) was measured along with a homogeneous conformational population of monomers with a TWCCSN2 of 79.5 ± 0.1 nm2 for the 26+ charge state. According to SEC-UV alone, peaks I and II would be classified as HMWS, since they elute before the monomer, thus leading to an estimation of 21.1% HMWS for stressed pembrolizumab. Surprisingly, native MS allowed to identify 13934


Article


highlighted the existence of two main different monomeric forms of pembrolizumab with an estimated mass of 155 kDa. We next compared SEC-MALS and native IM-MS results (Table 1). MALS uses the intensity and the angular dependence of the scattered light to measure absolute molar mass and size of the molecules through the estimation of the hydrodynamic radius (Rh) from the refractive index detector (RI). For the comparison of IM-MS results with MALS, the IM-MS deduced radius (RIM‑MS) of each monomer was derived from the corresponding TWCCSN2 assuming a spherical shape of the mAbs. For the main peak of monomer III, RIM‑MS (5.03 ± 0.10 nm) was in good agreement with Rh (5.15 ± 0.10 nm) calculated from MALS. For peak I in SEC, a smaller Rh (4.12 ± 0.20 nm) was deduced from MALS data, in agreement with a more compact conformation detected by IM-MS. Finally, we illustrated the benefits of our 4D analytical setup by analyzing bevacizumab, which generates a highly complex SEC chromatogram containing six separated species upon thermal stress (Figure 5a). By relying on the sole use of SECUV data, species eluting before the main peak would have been assigned to HMWS (estimated level of 24.5%), while peaks eluting after the main peak would have been attributed to LMWS (estimated level of 9.2%). By applying the SEC×SEC-native IM×MS method, the main peak (peak III) was attributed to monomeric bevacizumab (149 371 ± 7 Da, 68 ppm), peak I was identified as bevacizumab dimer (298 956 ± 13 Da), while peaks II, IV, V, and VI were identified as monomers (Figure 5b), having similar charge state distributions. Notably, noticeable differences in the low m/z range (m/z 1500−4000) for peaks II and V were observed, with the additional identification of

showed a remarkable agreement with CCS values calculated from the cryo-electron microscopy structure (PDB code: 5DK3) (see Table 1). Table 1. Comparison between SEC-MALS/RI and SEC×SEC-IM×MS Experimentsa technique

physical quantity

peak I

peak III

MALS RI

measured MW (kDa) Rh (nm) measured MW (Da) CCSMw (nm2) TW CCSN2 (nm2) RIM‑MS(nm) CCSPA(nm2) RPA(nm)

155 4.12 149 092 ± 7 68.465 78.9 5.01

155 5.15 149 061 ± 5 68.455 79.5 5.03 78.3 4.99

IM-MS

EM 3D (PDB)

a MW and Rh were obtained with MALS and RI detection respectively. CCSMW values were obtained through the equation CCS = 2.435*M2/3 for spherical proteins.40 RIM‑MS were obtained assuming a spherical shape (RIM‑MS = (TWCCSN2/π)0.5). CCSPA was obtained from cryo-electron microscopy structure by using projection approximation as described in experimental section. RPA was obtained assuming a spherical shape (RPA = (CCSPA/π)0.5).

To confirm the existence of the unexpected monomeric conformers and to rule out possible artifacts coming from electrospray ionization, orthogonal techniques like SEC-MALS and A4F were used for pembrolizumab size variant characterization (Supporting Information S4−S7). Both SEC-MALS and AF4-MALS confirmed the presence of monomers while no higher order oligomers were detected. Of note, in line with our SEC×SEC-native IM×MS results, SEC-MALS unambiguously

Figure 5. Online SEC×SEC-IM×MS of bevacizumab (Avastin). (a) Overlaid SEC chromatograms of forced degraded (blue trace) and unstressed bevacizumab (blue dashed trace). (b) Native mass spectra of each individual SEC separated peak for forced degraded sample. (c) Ion mobility arrival time distributions (ATDs) of dimeric and monomeric species detected for forced degraded sample, along with their collision cross section (CCS) (40+ charge state for dimer and 26+ charge state for monomers). 13935


Article


the following: (i) optimal SEC performance in nonvolatile buffer, (ii) simultaneous HMWS and LMWS profiling, (iii) accurate relative quantitation using SEC data, (iv) unambiguous identification of each size variants through accurate intact mass measurement in nondenaturing conditions (native MS), and (v) conformation characterization of each size variant species (native IM). Altogether, the described 4D approach (SEC×SEC-IM×MS) combines the power of chromatographic separation and specificity of MS identification. The all-in-one analytical strategy permits a deep, straightforward and rapid characterization of complex mAb samples. The online coupling to native MS is mandatory to provide unambiguous identification and quantitation of all the detected species, especially for unexpected monomeric species eluting in HMWS but also in LMWS region as illustrated in this work. At this stage, IM-MS analyses were of utmost importance for the online conformational characterization to emphasize different monomeric conformers for both pembrolizumab and bevacizumab at the intact mAb level. The ability to have a straight online coupling of SEC with nonvolatile mobile phase to nondenaturing MS using our 4D technique might have a strong impact on the analytical characterization of mAbs-related compounds not only for mAb development purposes41 and forced degradation studies, but also more generally for comparability42 and biosimilarity assessment.43 We believe that 2D LC×LC-native IM×MS setups will also be of utmost interest for the broad diversity of next generation empowered mAb formats like ADCs44 or multispecific mAbs (bispecific, trispecific, etc.),45 as well as for protein oligomerization state assessment.

compounds at 11 and 48 kDa, respectively. No identification can be proposed for the moment for these two LMWS. Again, native IM-MS analysis allowed highlighting small but significant differences in TW CCS N2 values among the monomeric species (Figure 5c). The very small difference in the TWCCSN2 values for peaks II and IV (79.70 ± 0.1 nm2 versus 79.50 ± 0.1 nm2, respectively) along with no difference for peaks V and VI (79.10 ± 0.1 nm2), suggest very close conformations for monomers II and IV and conformers V and VI. Conversely, major differences were observed between the TW CCSN2 values of monomers III, IV, and V (80.7 ± 0.1 nm2, 79.5 ± 0.1 nm2, and 79.1 ± 0.1 nm2, respectively). To conclude, for bevacizumab, the SEC×SEC-native IM×MS approach was highly informative to obtain an extensive and unambiguous identification and accurate quantitation of all the detected species present in a temperature stressed bevacizumab sample. In this specific context, unexpected monomers were detected not only in the HMWS region but also in the LMWS region, highlighting the benefits of IM-MS for the online conformational characterization of different monomer conformers.

■

CONCLUSIONS We propose here a multidimensional analytical approach combining comprehensive online two-dimensional chromatography (SEC×SEC) to ion mobility and mass spectrometry (IM-MS) for structural characterization of monoclonal antibodies size variants under nondenaturing conditions. In the first dimension, an optimized and generic SEC method was developed with nonvolatile salts, allowing the characterization and quantitation of HMWS and LMWS for a wide range of mAbs (acidic or basic, hydrophobic, etc.). Then, an online SEC fast desalting step has been introduced in the second dimension for replacing the nonvolatile salts with a native IMMS compatible ammonium acetate buffer. Such an online LC×LC methodology presents the advantage of closely mimicking the SEC methods routinely used in QC laboratories while providing an additional level of native MS characterization, which is mandatory for complex SEC profiles. Our methodology can be applied not only to mAbs difficult to analyze in ammonium acetate, as illustrated here, but also to a broad variety of other mAbs independently of their isotype (Supporting Information S8). Our setup offers simultaneous in-depth and detailed information on mAb HMWS and LMWS that cannot be obtained from the use of SEC-UV or nondenaturing MS alone.33 Indeed, with complex SEC profiles, it is difficult to have a correct interpretation of all SEC peaks only based on chromatographic data. On the other side, native MS as standalone is not adapted to perform relative intraspectrum monomer/dimer ratio estimation, due to the different electrospray ionization efficiencies.33 With the goal to highlight its benefits, the online comprehensive SEC×SEC-IM×MS methodology was used to investigate the SEC profiles of mAbs, difficult to analyze in SEC-UV whether because of their high pI, the presence of solvent accessible basic patches, their hydrophobicity or their propensity to form a large amount of HMWS under specific conditions.29,30 However, because of its ease of implementation, our 4D setup can be applied to any mAb variety, with the advantage of direct interfacing of chromatographic conditions used in QC (in nonvolatile salts) to native IM×MS (Supporting Information S8). The proposed 4D setup affords

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b03333.

■

Additional information as noted in text (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Valentina D’Atri: 0000-0002-2601-7092 Sarah Cianferani: 0000-0003-4013-4129 Author Contributions ¶

(A.E., A.G.) These authors contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank GIS IBiSA and Région Alsace for financial support in purchasing a Synapt G2 HDMS instrument. Agilent Technologies is acknowledged for the loan of AdvanceBio SEC columns. A.E. acknowledges the “Association Nationale de la Recherche et de la Technologie” (ANRT) and Syndivia for funding his Ph.D. fellowship. This work was supported by the CNRS, the Université de Strasbourg, the Université de Lyon, the Agence Nationale de la Recherche (ANR) and the French Proteomic Infrastructure (ProFI; ANR-10-INBS-08-03), and 13936


Article


(32) Ehkirch, A.; D’Atri, V.; Rouviere, F.; Hernandez-Alba, O.; Goyon, A.; Colas, O.; Sarrut, M.; Beck, A.; Guillarme, D.; Heinisch, S.; Cianferani, S. Anal. Chem. 2018, 90 (3), 1578−1586. (33) Ehkirch, A.; Hernandez-Alba, O.; Colas, O.; Beck, A.; Guillarme, D.; Cianferani, S. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2018, 1086, 176−183. (34) Bush, M. F.; Hall, Z.; Giles, K.; Hoyes, J.; Robinson, C. V.; Ruotolo, B. T. Anal. Chem. 2010, 82 (22), 9557−65. (35) Waitt, G. M.; Xu, R.; Wisely, G. B.; Williams, J. D. J. Am. Soc. Mass Spectrom. 2008, 19 (2), 239−45. (36) Marklund, E. G.; Degiacomi, M. T.; Robinson, C. V.; Baldwin, A. J.; Benesch, J. L. Structure 2015, 23 (4), 791−9. (37) Goyon, A.; Beck, A.; Colas, O.; Sandra, K.; Guillarme, D.; Fekete, S. J. Chromatogr A 2017, 1498, 80−89. (38) Watson, E.; Kenney, W. C. J. Chromatogr. 1988, 436 (2), 289− 98. (39) Marcoux, J.; Champion, T.; Colas, O.; Wagner-Rousset, E.; Corvaia, N.; Van Dorsselaer, A.; Beck, A.; Cianferani, S. Protein Sci. 2015, 24 (8), 1210−23. (40) Ruotolo, B. T.; Benesch, J. L.; Sandercock, A. M.; Hyung, S. J.; Robinson, C. V. Nat. Protoc. 2008, 3 (7), 1139−52. (41) Jain, T.; Sun, T.; Durand, S.; Hall, A.; Houston, N. R.; Nett, J. H.; Sharkey, B.; Bobrowicz, B.; Caffry, I.; Yu, Y.; Cao, Y.; Lynaugh, H.; Brown, M.; Baruah, H.; Gray, L. T.; Krauland, E. M.; Xu, Y.; Vasquez, M.; Wittrup, K. D. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (5), 944−949. (42) Ambrogelly, A.; Gozo, S.; Katiyar, A.; Dellatore, S.; Kune, Y.; Bhat, R.; Sun, J.; Li, N.; Wang, D.; Nowak, C.; Neill, A.; Ponniah, G.; King, C.; Mason, B.; Beck, A.; Liu, H. MAbs 2018, 10 (4), 513−538. (43) Beck, A.; Debaene, F.; Diemer, H.; Wagner-Rousset, E.; Colas, O.; Van Dorsselaer, A.; Cianferani, S. J. Mass Spectrom. 2015, 50 (2), 285−297. (44) Beck, A.; Goetsch, L.; Dumontet, C.; Corvaia, N. Nat. Rev. Drug Discovery 2017, 16 (5), 315−337. (45) Brinkmann, U.; Kontermann, R. E. MAbs 2017, 9 (2), 182− 212.

the Swiss National Science Foundation (fellowship 31003A_159494).

■

REFERENCES

(1) Carter, P. J.; Lazar, G. A. Nat. Rev. Drug Discovery 2017, 17 (3), 197−223. (2) Strohl, W. R. Protein Cell 2018, 9 (1), 86−120. (3) Kaplon, H.; Reichert, J. M. MAbs 2018, 10 (2), 183−203. (4) Tang, H.; Wang, S.; Xiao, G.; Schiller, J.; Papadimitrakopoulou, V.; Minna, J.; Wistuba, II; Xie, Y. Ann. Oncol 2017, 28 (4), 733−740. (5) Lowe, D.; Dudgeon, K.; Rouet, R.; Schofield, P.; Jermutus, L.; Christ, D. Adv. Protein Chem. Struct. Biol. 2011, 84, 41−61. (6) Franco, R.; Daniela, G.; Fabrizio, M.; Ilaria, G.; Detlev, H. Cytotechnology 1999, 29 (1), 11−25. (7) Rosenberg, A. S. AAPS J. 2006, 8 (3), E501−7. (8) Liu, J.; Eris, T.; Li, C.; Cao, S.; Kuhns, S. BioDrugs 2016, 30 (4), 321−38. (9) Magnenat, L.; Palmese, A.; Fremaux, C.; D’Amici, F.; Terlizzese, M.; Rossi, M.; Chevalet, L. MAbs 2017, 9 (1), 127−139. (10) Seo, N.; Polozova, A.; Zhang, M.; Yates, Z.; Cao, S.; Li, H.; Kuhns, S.; Maher, G.; McBride, H. J.; Liu, J. MAbs 2018, 10 (4), 678−691. (11) Lee, J.; Kang, H. A.; Bae, J. S.; Kim, K. D.; Lee, K. H.; Lim, K. J.; Choo, M. J.; Chang, S. J. MAbs 2018, 10 (4), 547−571. (12) Lee, K. H.; Lee, J.; Bae, J. S.; Kim, Y. J.; Kang, H. A.; Kim, S. H.; Lee, S. J.; Lim, K. J.; Lee, J. W.; Jung, S. K.; Chang, S. J. MAbs 2018, 10 (3), 380−396. (13) Litzen, A.; Walter, J. K.; Krischollek, H.; Wahlund, K. G. Anal. Biochem. 1993, 212 (2), 469−80. (14) Carpenter, J. F.; Bain, D. L.; Johnson, G. R. Analytical Ultracentrifugation 2016, 389−395. (15) Goyon, A.; Beck, A.; Veuthey, J. L.; Guillarme, D.; Fekete, S. J. Pharm. Biomed. Anal. 2017, 144, 242−251. (16) Philo, J. S. Curr. Pharm. Biotechnol. 2009, 10 (4), 359−72. (17) Ejima, D.; Yumioka, R.; Arakawa, T.; Tsumoto, K. J. Chromatogr A 2005, 1094 (1−2), 49−55. (18) Liu, H.; Gaza-Bulseco, G.; Chumsae, C. J. Am. Soc. Mass Spectrom. 2009, 20 (12), 2258−64. (19) Haberger, M.; Leiss, M.; Heidenreich, A. K.; Pester, O.; Hafenmair, G.; Hook, M.; Bonnington, L.; Wegele, H.; Haindl, M.; Reusch, D.; Bulau, P. MAbs 2016, 8 (2), 331−9. (20) U.S. Pharmacopeia (USP). USP 39 NF34; USP: Rockville, MD, 2016; pp 204−206. (21) Philo, J. S. AAPS J. 2006, 8 (3), E564−71. (22) Wong, C.; Strachan-Mills, C.; Burman, S. J. Chromatogr A 2012, 1270, 153−61. (23) Goyon, A.; D’Atri, V.; Colas, O.; Fekete, S.; Beck, A.; Guillarme, D. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2017, 1065−1066, 35−43. (24) Yang, X.; Zhang, Y.; Wang, F.; Wang, L. J.; Richardson, D.; Shameem, M.; Ambrogelly, A. Anal. Biochem. 2015, 484, 173−9. (25) Beck, A.; Wagner-Rousset, E.; Ayoub, D.; Van Dorsselaer, A.; Sanglier-Cianferani, S. Anal. Chem. 2013, 85 (2), 715−36. (26) Atmanene, C.; Wagner-Rousset, E.; Malissard, M.; Chol, B.; Robert, A.; Corvaia, N.; Van Dorsselaer, A.; Beck, A.; SanglierCianferani, S. Anal. Chem. 2009, 81 (15), 6364−6373. (27) Beck, A.; Terral, G.; Debaene, F.; Wagner-Rousset, E.; Marcoux, J.; Janin-Bussat, M. C.; Colas, O.; Van Dorsselaer, A.; Cianferani, S. Expert Rev. Proteomics 2016, 13 (2), 157−183. (28) Terral, G.; Beck, A.; Cianferani, S. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2016, 1032, 79−90. (29) Goyon, A.; Excoffier, M.; Janin-Bussat, M. C.; Bobaly, B.; Fekete, S.; Guillarme, D.; Beck, A. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2017, 1065−1066, 119−128. (30) Kahook, M. Y.; Liu, L.; Ruzycki, P.; Mandava, N.; Carpenter, J. F.; Petrash, J. M.; Ammar, D. A. Retina 2010, 30 (6), 887−92. (31) Nowak, C.; Cheung, J. K.; Deallatore, S. M.; Katiyar, A.; Bhat, R.; Sun, J.; Ponniah, G.; Neill, A.; Mason, B.; Beck, A.; Liu, H. MAbs 2017, 9 (8), 1217−1230. 13937


A Novel Online Four-Dimensional SECÃSEC-IMÃMS Methodology for

Recommend Documents

A Novel Online Four-Dimensional SECÃSEC-IMÃMS Methodology for