Charge Variant Analysis of Monoclonal Antibodies Using Direct

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Charge Variant Analysis of Monoclonal Antibodies using Direct Coupled pH Gradient Cation Exchange Chromatography to High Resolution Native Mass Spectrometry Florian Füssl, Ken Cook, Kai Scheffler, Amy Farrell, Stefan Mittermayr, and Jonathan Bones Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05241 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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Analytical Chemistry

Charge Variant Analysis of Monoclonal Antibodies using Direct Coupled pH Gradient Cation Exchange Chromatography to High Resolution Native Mass Spectrometry

Florian Füssl1, Ken Cook2, Kai Scheffler3, Amy Farrell1, Stefan Mittermayr1 and Jonathan Bones1,4 1

NIBRT – The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount

Merrion, Blackrock, Co. Dublin, A94 X099, Ireland. 2

Thermo Fisher Scientific, Stafford House, 1 Boundary Park, Hemel Hempstead, HP2 7GE, United

Kingdom. 3

Thermo Fisher Scientific, Dornierstrasse 4, 82110 Germering, Germany.

4

School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4, D04

V1W8, Ireland. *To whom correspondence should be addressed: Jonathan Bones, tel: +353 1215 8100, fax: +353 1215 8116.

Keywords: Monoclonal antibodies, charge variant analysis, cation exchange chromatography, native mass spectrometry, high resolution mass spectrometry.

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Abstract Charge variant analysis (CVA) of monoclonal antibodies (mAbs) using cation exchange chromatography is routinely used as a fingerprint of the distribution of posttranslational modifications present on the molecule. Traditional salt or pH based eluents are not suited for direct coupling to mass spectrometry due to non-volatility or high ionic strength. This makes further analysis complicated when an alteration in the charge variant profile or the emergence of an additional peak is encountered. Here, the use of pH gradient elution using volatile, low ionic strength buffers is reported with direct coupling to high resolution Orbitrap™ mass spectrometry. The development of a universal method based on pH elution was explored using a number of mAb drug products. Optimized methods facilitated the separation and identification of charge variants including individual glycoforms of the mAbs investigated using the same buffer system but with tailored gradient slopes. The developed method represents an exciting advance for the characterization of biopharmaceuticals as intact entities through the combination of native charge variant separations with high resolution native mass spectrometry.


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Monoclonal antibody (mAb) drug products, derived from industrial scale bioprocessing of mammalian cells, exist in a variety of different isoforms due to posttranslational modification (PTM) events. These occur both, enzymatically and non-enzymatically following biosynthesis of the nascent protein within the cell and subsequent secretion into the conditioned media.1 Regulatory guidelines require characterization of the primary sequence and PTMs present on mAbs to ensure that the recombinant therapeutic protein meets predefined quality specifications.2 Certain primary sequence variations and PTMs present on mAbs, such as C-terminal lysine truncation, N-terminal pyroglutamate formation, sialylation present on N-glycosylation and deamidation of asparagine or glutamine residues, alter the isoelectric point (pI) of the molecule, resulting in the generation of a series of acidic and basic charge variants of the mAb.3 Charge variant patterns, traditionally monitored using cation exchange chromatography or capillary isoelectric focusing, act as a fingerprint of the manufacturing process and deviations in peak areas or the appearance of new peaks require in depth characterization to ensure that no additional or undesired PTM has been introduced.4-7 Such alterations in the molecular surface charge distribution can affect antigen or receptor binding or may cause conformational changes that may increase the risk of aggregate formation and potential induction of immunogenicity.8-10 Current strategies to identify alterations in the charge variant patterns involve fractionation of the peak of interest, multilevel characterization using peptide mapping, intact mass analysis and N-glycan profiling followed by functional assessment using receptor binding or other bioassays, depending on the location of the modification within the primary sequence.11,12 The ability to hyphenate charge variant analysis (CVA) directly to high resolution mass spectrometry (MS) is highly desired so that potential modifications in peaks of interest can be characterized during the analysis based on a change in the proteins average mass and retention time. Charge variant separations are routinely performed using weak or strong cation exchange stationary phases with carboxylate or sulfonate functionalities, respectively.4 Elution can be performed by displacement using either a gradient of increasing salt concentration or by the generation of a pH 3 ACS Paragon Plus Environment


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gradient that results in analyte desorption when the mobile phase pH equals the apparent pI of each variant. The working principle is similar to chromatofocusing with the difference that proteins are not focused along an immobilized pH-gradient but elute as the buffer pH changes over time.13-17 A limitation to both these traditional approaches is the use of non-MS compatible salts and components in the mobile phase which prevents direct coupling of CVA to MS, often requiring the use of a desalting intermediary step such as reversed-phase chromatography.18 An additional limitation of this approach is the denaturation of the protein, resulting in the generation of complex mass spectra containing highly charged mAb ions due to the loss of tertiary structure and exposure of all the charged residues. The potential for direct coupling of CVA with native mass spectrometry, wherein tertiary structure is maintained and the charge states observed by MS are reflective of the surface charge of the mAb in solution is highly desirable.19,20 The ability to separate and analyze large and complicated molecules such as mAbs in the native state is attractive as alterations in the surface charge distribution may provide insight into potential changes in molecular conformation which may affect function, thereby providing the analyst with the tools to potentially link sequence, structural and functional information.21 Limited reports of the direct hyphenation of CVA to MS exist in the literature, salt gradient elution using an increasing concentration of ammonium acetate at pH 7 was used for the CVA-MS analysis of lysozyme and β-interferon allowing for the detection of induced modifications such as deamidation induced following molecular stress.22,23 CVA-MS analysis of IgG2 mAbs was described using a single pH unit gradient change on a weak cation exchanger using ammonium hydroxide eluents containing 20% methanol to promote desolvation.24 Low resolution native spectra of the intact mAbs were obtained on the time of flight (ToF) instrument used, however, the low instrument resolution and a high degree of ammonium adduction present on the ions prevented deconvolution and spectral annotation.24 CVA-MS using ammonium acetate salt gradient elution applying solutions of different pH and salt concentration was also recently reported using a middle down strategy. In this respect normal and aged IgG samples were digested with IdeS protease and the resulting (Fc/2)2 and F(ab’)2 4 ACS Paragon Plus Environment

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fragments were separated on a strong cation exchanger hyphenated to QToF-MS.25 The native structures, including a preserved interaction of the (Fc/2)2 domain as dimer post digestion, were determined with good mass accuracy. Spectral quality however was poor, presumably as a result of insufficient desolvation or high levels of adduct formation in the high ammonium containing eluent. Acidic species were predominantly on the F(ab’)2 fragment and consisted of oxidation and deamidation, which were observed to be significantly increased in the aged sample.25 In 2015 Stoll et al. developed a 2D-LC approach for the intact and middle-up characterization of rituximab charge variants employing CEX chromatography before RPLC-TOF-MS, which in a follow-up study was utilized for the middle-up characterization of several mAbs and their biosimilars by Sorensen et al.26,27 Whereas this method has successfully been proven applicable to the identification of charge variants such as different lysine forms, it though requires an elaborate instrumental setup due to the 2D-LC approach employed. In the current report, we investigate the use of low ionic strength pH gradient elution of mAbs from a strong cation exchanger coupled to high resolution native Orbitrap mass spectrometry for high resolution, high definition characterization of mAb charge variants. pH linearity across the gradient range was first investigated, demonstrating the applicability of the proposed approach for mAbs of different pI using the same LC-MS setup. The utility of this method for the assignment of antibody variants such as different glycosylation and C-terminal lysine forms is exemplarily shown using a number of mAb drug products. The proposed CVA-MS approach represents a simple, yet powerful method for the characterization of complex biopharmaceuticals on the intact level without the need for sample preparation.



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Experimental section Chemicals and Reagents: Deionized water was provided by an Arium® pro UV ultrapure water system from Sartorius Stedim Biotech (Goettingen, Germany). Acetic acid (ACS reagent grade, ≥ 99.7%), ammonium bicarbonate (BioUltra, ≥ 99.5%), ammonium hydroxide solution (BioUltra, 1 M in H2O) and DL-Dithiothreitol (≥ 98%) were purchased from Sigma Aldrich (Wicklow, Ireland). Water with 0.1% formic acid (Optima, LC-MS grade) and acetonitrile with 0.1% formic acid (Optima, LC-MS grade) were obtained from Fisher Scientific (Dublin, Ireland). All reagents and materials required for KingFisher SMART digestion were obtained from Thermo Fisher Scientific (Sunnyvale, CA, USA). The monoclonal antibodies used were kindly provided by the Hospital Pharmacy Unit of the University Hospital of San Cecilio in Granada, Spain.

Chromatographic gradient optimization: The buffer system used for all charge variant separations was consisting of 25 mM ammonium bicarbonate and 30 mM acetic acid as buffer A (pH 5.3) and 10 mM ammonium hydroxide in 2 mM acetic acid as buffer B (pH 10.18). After mixing, buffers were allowed to rest for 24 hours at room temperature. Optimization of chromatographic separations was performed on an Ultimate™ 3000 HPLC system equipped with a variable wavelength detector and an Ultimate™ 3000 PCM-3000 pH and Conductivity Monitor (Thermo Fisher Scientific, Germering, Germany). The column used for all separation was a MAbPac™ SCX-10 RS 2.1 × 50 mm column, 5 μm particles (Thermo Fisher Scientific, Sunnyvale, CA, USA). A flow rate of 400 μL min-1 was applied, the column compartment was held at 25°C and data acquisition was performed utilizing UV absorption at 280 nm. Injection amount for method optimization was 50 µg of antibody per run. The optimized gradients for the separation of all 5 mAbs are shown in Table 1. Table 1


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CVA-MS method parameters: Chromatographic separations for LC-MS experiments were performed on a Thermo Scientific™ Vanquish™ Flex Quaternary UHPLC system (Thermo Fisher Scientific, Germering, Germany). The chromatographic column, buffers, gradients and all run parameters applied were the same as described for gradient optimization. One hundred µg mAb were injected per LC-MS run. For mass spectrometric data acquisition a Thermo Scientific™ Q Exactive™ Plus quadrupole-Orbitrap mass spectrometer, enabled with the BioPharma option allowing for mass detection up to m/z 8,000, equipped with a Heated Electrospray Ionization-II (HESI-II) probe in a standard Ion Max ion source (Thermo Fisher Scientific, Bremen, Germany) was used. MS tune and method parameters can be obtained from Table S1 of the Supporting Information.

Collection of bevacizumab charge variant peaks Bevacizumab separation for peak collection was performed with identical chromatographic run parameters as described for chromatographic gradient optimization using a Thermo Scientific™ Vanquish Flex Quaternary UHPLC system.

Sample preparation for peptide mapping of bevacizumab charge variant peaks In order to obtain sufficient amounts of protein for peptide mapping, peaks of 4 runs of 100 µg respectively were collected and merged. Samples were buffer exchanged to water using VIVASPIN spin filters from Sartorius (Goettingen, Germany) with a cutoff size of 10,000 Da. Digestion was carried out via a Thermo Scientific™ KingFisher™ Duo Prime Purification System under the control of Bindit™ software (version 4.0) (Thermo Fisher Scientific, Vantaa, Finland). Samples were diluted with SMART Digest buffer in a KingFisher Deepwell 96 well plate. Fifteen µL of magnetic bead solution were mixed with 100 µL of SMART Digest buffer. Magnetic beads were picked up with KingFisher Duo 12-tip combs and were washed for 1 min in SMART Digest buffer diluted with water in a ratio of 1:4 (v/v). Digestion was performed at 70°C for 40 min. Digested samples were reduced by addition of 7 ACS Paragon Plus Environment


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DTT to yield a 10mM concentration, incubated for 40 min at 37 °C and immediately analyzed by LCMS.

Peptide Mapping Peptide mapping was performed on a Thermo Scientific™ Vanquish Flex Binary UHPLC system coupled online to a Q Exactive™ Plus quadrupole-Orbitrap mass spectrometer. For chromatographic separation an Acclaim™ VANQUISH C18 column 250 × 2.1 mm, 2.2 µm particles (Thermo Fisher Scientific, Sunnyvale, CA, USA) was used. Separations were performed at 25°C and a flow rate of 0.3 ml.min-1 with water in 0.1% formic acid as buffer A and acetonitrile in 0.1% formic acid as buffer B using a gradient of 2% – 40% buffer B over 45 min. Details regarding MS tune and method settings can be obtained from Table S2 in the Supporting Information.

Data processing: Data acquisition and visualization of LC-UV measurements was performed in Thermo Scientific™ Chromeleon CDS 7.2. LC-MS measurements were performed under Thermo Scientific™ Xcalibur 4.0. CVA-MS raw files were exported to Thermo Scientific™ BioPharma Finder™ 2.0 software, and analyzed using the ReSpect™ algorithm via either manual spectra averaging following deconvolution or the sliding windows deconvolution feature. The time windows for manual peak averaging were set to comprise a maximal portion of the peak area of the peaks of interest while excluding possibly present heterogeneities at the front and back ends. All data files were processed with target mass matching utilizing antibody sequences together with modifications such as disulfide bonds, glycosylation and in case of infliximab also lysine truncation. All annotations based on the so generated theoretical masses can be observed in Tables S7-S10 of the Supporting Information. Details on the parameters used for BioPharma Finder analysis can be found in Table S3 of the Supporting Information. Peptide mapping data files were also analyzed via the BioPharma Finder 2.0


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software. For peptide identification bevacizumab light and heavy chain sequences were used and the following variable modifications were specified: lysine loss, deamidation of Gln and Asn, oxidation of Met, glycation of Lys and isomerization of Asp. Search results were filtered for a confidence score ≥ 0.8 and a recovery ≥ 1. Final results are based on average values of triplicate injections.



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Results and Discussion The principle of CVA using pH gradient elution relies on a change in buffer pH per unit time. As soon as the pH reaches a value equal to a proteins apparent isoelectric point (pI), the protein is no longer retained and elutes from the column. This focusing effect concentrates the protein variants at the pH of their apparent pI. Ideally, buffers with considerable buffering capacity are applied to guarantee a linear pH change matching the chromatographic gradient programed. Pre-made buffer cocktails, very well suiting this requirement, have been commercialized and are available today.28,29 On-line hyphenation of CVA to MS does, thereby, not only require the control of the pH slope but also additionally introduces the need for optimization of ion transfer for MS sensitivity. A delicate balance needs to be obtained between low salt concentrations for MS sensitivity while maintaining control of the pH to facilitate reproducible chromatography. As eluents comprised of volatile salts have much less buffering capacity, reduced control of pH makes column choice more important, as the column itself will buffer against pH changes occurring during gradient elution. Hence, low capacity columns, for example packed with pellicular or solid particles, exclusively allowing surface interaction and thus exhibiting fast mass transfer of large molecules for high resolution separations, can be used to minimize such buffering effects. Our column of choice was a MAbPac™ SCX-10 RS column with 5 μm particle size and small dimensions which very well suited these requirements. The short column length contributes to minimizing buffering capacity which allowed more precise control over the pH gradients applied, as well as faster re-equilibration. In addition, column length has a minimal effect on chromatographic resolution using pH gradient elution due to the contribution of chromatofocusing to the separation mechanism.29 Mobile phase compositions with low concentrations of volatile salts (≤ 60 mM) were investigated, which enabled highly repeatable chromatography (RSD < 2% relative peak area and < 1% retention time, n=3) while maintaining MS sensitivity (Figure S2, Table S5). Buffer stability has been evaluated over 24 hours. Whereas relative peak areas remained fairly constant (RSD < 2.5%, n = 8) (Table S6), slight retention time shifts were


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observed in case of the application of a very shallow gradient. In cases where high retention time stability is desired, buffers should be replaced daily.

Different mAbs may contain a considerably varying extent of charge variants spanning diverse pI values. Hence, it is crucial for a generic method to deliver equally good performance across all pH ranges of interest. It is difficult to control the pH with any volatile buffers between pH 7 to 8 as there are none which buffer over this pH range. A sudden jump in a poorly controlled pH gradient will likely result in all the proteins with apparent pI’s in that region eluting simultaneously. For this reason fine-tuned, shallow or curved gradients were applied to partially overcome this effect. With the application of individually optimized gradients, it was possible to establish pH linearity, particularly in those pH regions where it was required for each mAb. As is shown in Figure 1, obtaining a high performance charge variant separation of cetuximab is challenging as the acidic variants elute within the pH range of 7.5 to 8. Applying a shallow convex curved gradient compensates for this providing pH linearity which resulted in a separation of at least eight cetuximab charge variants as well resolved, symmetric peaks. A more detailed history of cetuximab gradient optimization can be obtained from Figure S1a-c of the Supporting Information.

Figure 1

Optimized gradients were developed for five different mAbs, which was supported by pH live acquisition provided by the PCM-3000 (Figure 2a - e). Apparent pI values of the mAbs tested, as were previously determined via imaged capillary isoelectric focusing by Goyon et al., are shown in the supplementary information in Table S4.30 Trastuzumab has a pI close to 9, hence avoiding the critical region between pH 7 and 8 and allowing the use of a linear gradient (Figure 2a). Similar to trastuzumab, adalimumab has a pI of 8.9 and so elutes within a comparable pH region. In case of adalimumab, utilization of a curved gradient resulted in superior chromatographic selectivity for 11 ACS Paragon Plus Environment


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acidic variants whereas the later eluting basic species of comparatively low signal intensity are focused by the increasing steepness of the gradient (Figure 2b). Infliximab and bevacizumab apparent pI values differ by 0.7 pH units. Both antibodies, however, seem to elute at a similar buffer composition (Figure 2c & d). This can likely be contributed to the weak buffering capacity of the buffers especially across the pH range close to both antibody pI values.

Figure 2

The compilation of the optimized separations for all five mAbs tested, shown in Figure 2, demonstrates that this novel method is applicable to mAbs with various pI valuess using the same column and buffer system. It also suggests ease of gradient optimization for new candidates as similar gradients are likely suitable to mAbs with a similar pI and a comparable charge variant complexity. Furthermore, the resolution of the charge variant peaks using the volatile buffers in this study in several cases compares favorably with previous reports using salt- and pH gradients.7,15,24,29,31 A comparison of a chromatographic separation of cetuximab using the volatile buffer system to a separation that can be achieved using the CX-1 pH-gradient buffers can be obtained from Figure S1c & d. Hyphenation with high resolution Orbitrap mass spectrometry was then investigated for each of the developed gradients using the volatile pH buffer system. There are several critical parameters in the acquisition of mass spectra of large proteins under native conditions that relate to source settings since the electrospray process is performed with purely aqueous mobile phase: the probe heater and transfer capillary temperatures, in-source fragmentation (in-source CID) for declustering and desolvation as well as the S-Lens setting for transporting the large protein species through the front end of the ion optics. In addition the resolution setting plays an important role as signals from large proteins decay rather quickly in the Orbitrap mass analyser.32 Thus typically the lowest possible resolution setting is recommended for detection with highest sensitivity since a low resolution setting translates directly into short 12 ACS Paragon Plus Environment

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detection times required to capture the protein signal with highest sensitivity. However when chromatography is performed in the presence of salts, proteins species are prone to form adducts resulting in additional peaks in the spectra. At lowest mass resolution species with and without adducts are not resolved resulting in broader and asymmetrical peaks with decreased mass accuracy. Thus we have performed experiments applying the lowest resolution setting of 17,500 but also at 35,000. Figure 3 shows the benefit of the higher resolution setting in the case of the major variant of adalimumab. The signal intensity in the chromatogram acquired at a resolution setting of 35,000 is decreased by a factor of two but with the benefit of partially resolving the peaks with and without adducts resulting in separated distinct peaks after deconvolution. The ability to resolve adducts is in these particular cases of importance as it enables isoform annotation with much improved mass accuracies. Deconvolution and closer investigation of the adducted forms revealed constant mass difference of around 40 – 42 Da from the main forms, indicating the occurrence of CO2 adduction. That proteins are prone for adduction in the presence of ammonium bicarbonate in solution has previously been demonstrated.

32

The fact that the mass difference observed does not fully correspond to the

expected theoretical mass of CO2, namely 44 Da (Figure S3), can be explained by the merely partial resolution of the adducted species, consequently leading to slight shifts towards the lower m/z region. Another important feature of native mass spectrometry is the reduced number of charge states and the increase in spatial spectral resolution. This allows superior distinction between isoforms of adjacent charged states which could overlap in a denatured charge envelope.

Figure 3

An important feature of therapeutic proteins is glycosylation as it has an impact on protein activity,33 stability33,34 and life time.35,36 mAbs share a highly-conserved site for N-glycosylation at Asn297 in their heavy chains,36 however, the number and relative abundance of different glycans can vary 13 ACS Paragon Plus Environment


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considerably from one to the other. The glycosylation pattern serves a crucial quality criterion for therapeutic mAbs and is monitored rigorously. During intact mass analysis of mAbs, annotation of glycoforms can be compromised due to the co-elution and presence of near isobaric proteoforms of the molecule. Under conditions of higher peak purity such as those attained herein, where charge variants elute separately, CVA-MS enables superior annotation of glycoforms for each charge variant peak. Figure 4 shows the glycoform assignment of the five most abundant glycoforms of the main peak of trastuzumab, all with a mass deviation of less than 10 ppm following spectral deconvolution.

Figure 4

Infliximab exhibits three major charge variants which have been identified to be lysine truncated forms, caused by incomplete carboxypeptidase (CpB) activity.37 Lysine variants show a mass difference of 128 Da which can be challenging to observe using intact mass analysis without efficient separation. The isoform with an additional lysine present at the c-terminus may overlap with the glycoform carrying one additional galactose which may compromise deconvolution and annotation of both species. The presence of a c-terminal lysine on a mAb heavy chain increases the net number of positive charges and consequently increases retention on the cation exchange column which makes it well resolvable in CVA. The traditional route to identify the three major lysine peaks requires either CVA before and after CpB digestion or preparative IEC for fractionation followed by desalting using reversed phase LC-MS to obtain the intact mass data from each of the three fractions. It is demonstrated herein that the required data for a confident identification of lysine variants can be obtained from only one analytical CVA-MS experiment as shown in Figure 5. In addition to isoform identification also quantitative information can be gained. In this regard, the Sliding Windows Deconvolution option of the Biopharma Finder 2.0 facilitates the calculation of individual peak areas by tracking MS intensities over time. This information can be in consequence


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be used to perform relative quantification of protein isoforms as it is shown in the diagram in Figure 5 for the 3 most abundant glycoforms of the 3 infliximab lysine variants.

Figure 5

Bevacizumab exhibits a simple glycosylation pattern and low charge heterogeneity. The charge variant separation however shows a minor portion of acidic variants, whereas the most abundant one is appearing directly in front of the main peak. One very common modification leading to acidic variants is deamidation of asparagine or glutamine residues, which has been shown to naturally occur during antibody downstream processing, formulation and storage.1,38,39 The mass shift induced by deamidation, which is only ~1 Da, makes the modified and non-modified variant undistinguishable by mass spectrometric means, at least if both are entering the MS system at the same time. The fact however, that deamidation results in the generation of one additional acidic residue on the protein, changes its apparent pI in solution, which in consequence reduces the retention on the cation exchange resin.3,38,39 Figure 6 shows the CVA-MS analysis of bevacizumab and a comparison of the masses of the three most abundant glycoforms of both, the main peak and the most abundant acidic species. All three of them show an increase in average mass resembling the mass shift caused by a single deamidation event. The presence of a singly deamidated form is also suggested by means of retention time as deamidation has previously been shown to be a chromatographically distinguishable acidic modification on mAbs3 as well as by a peak collection and peptide mapping conducted in the course of this study (Figure S4). These results suggest that this novel method is not only capable of distinguishing different glycoforms and lysine variants but also low abundant, near isobaric species such as deamidation products.

Figure 6



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Conclusions A method for on-line hyphenation of charge variant analysis to high resolution native Orbitrap mass spectrometry using low ionic strength eluents on a strong cation exchange phase has been described. This new approach offers a simple and universal strategy for mAb characterization on the intact level without any need for sample preparation. Chromatographic resolution in the CVA analysis for several investigated mAbs compared favorably to previous approaches employing nonMS friendly conditions and facilitated the separation of multiple charge variant species even within a narrow pI range. MS data generated from even minor variants was sufficient in quality to achieve component identification with high mass accuracy allowing for the identification of critical quality attributes including accurate intact mass, lysine truncation, glycosylation and deamidation from a single LC-MS injection. The method enables the observation of the glycoform distribution for each separated charge variant allowing for potential interplay between modifications to be investigated. Conveniently, the method bypasses the elaborate sample preparation required by alternative techniques to obtain the same level of information, which saves time and prevents the appearance of unintentionally induced modifications or analytical bias. The developed CVA-MS method offers potential for improvement, for example by further enhancing sensitivity or by avoiding adduction, but represents a significant advance for all fields of intact protein mass spectrometry including biopharmaceutical characterization, proteomics and structural biology.

Acknowledgement The authors gratefully acknowledge funding from Science Foundation Ireland under Grant Number 13/CDA/2196 and collaborators in Thermo Fisher Scientific for instrument access and support.



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Figure Captions Figure 1: Cetuximab gradient optimization. Shown are the UV chromatogram (blue), the actual monitored pH profile (black) and the programed gradient (red). Separation was performed on a 2.1 x 50 mm MabPac SCX-10 column, further run parameters can be obtained from the experimental section.

Figure 2: UV chromatograms of trastuzumab (A), adalimumab (B), infliximab (C), bevacizumab (D) and cetuximab (E). Separations are based on the injection of 100 µg protein respectively. The red traces indicate the individual gradients described in Table 1. Further run parameters can be obtained from the experimental section.

Figure 3: Adalimumab acquired at 2 different resolution settings, namely 17,500 (blue) and 35,000 (magenta). The base peak chromatograms, the mass spectra of the main peak as well as magnifications of charge state 26 are shown.

Figure 4: Glycoform assignment of the trastuzumab main peak, mass analysis was performed with a resolution setting of 35,000: (A) Base peak chromatogram, (B) raw spectrum of the main peak, (C) deconvoluted spectrum with glycoform annotations and mass deviations.

Figure 5: Infliximab glycoform and lysine variant assignment, mass analysis was performed with a resolution setting of 35,000: (A) Base peak chromatogram of infliximab and schematic of the principle of Sliding Windows Deconvolution which was applied, (B) combined deconvoluted spectrum, (C) glycoform assignment and relative quantification.


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Figure 6: Comparison of the main- and an acidic species in the bevacizumab base peak chromatogram. Mass analysis was performed with a resolution setting of 35,000. The mass differences for the three most abundant glycans are indicating the presence of a singly deamidated species and are shown in tabular form.

Table 1: Optimized gradients for the CVA of five monoclonal antibodies using the MS friendly buffer system.

mAb

Time (min) 0 0.5 Trastuzumab 10 0 0.5 Adalimumab 10 0 0.5 Infliximab 10 0 0.5 Bevacizumab 6 10 0 0.5 Cetuximab 7 10

% buffer B 40 55 100 40 50 100 40 45 55 40 45 50 100 40 45 65 100

Curve 5 5 5 7 5 5 5 7 5 5 7 5



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Figure 1


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Figure 2



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Figure 3


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Figure 4

Figure 5



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Figure 6


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For TOC only


Charge Variant Analysis of Monoclonal Antibodies Using Direct

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