Article Cite This: Anal. Chem. 2018, 90, 4669−4676
pubs.acs.org/ac
Charge Variant Analysis of Monoclonal Antibodies Using Direct Coupled pH Gradient Cation Exchange Chromatography to HighResolution Native Mass Spectrometry Florian Füssl,† Ken Cook,‡ Kai Scheffler,§ Amy Farrell,† Stefan Mittermayr,† and Jonathan Bones*,†,∥
Downloaded via DURHAM UNIV on July 24, 2018 at 11:56:08 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
NIBRT−The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin, A94 X099, Ireland ‡ Thermo Fisher Scientific, Stafford House, 1 Boundary Park, Hemel Hempstead, HP2 7GE, United Kingdom § Thermo Fisher Scientific, Dornierstrasse 4, 82110 Germering, Germany ∥ School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4, D04 V1W8, Ireland S Supporting Information *
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 nonvolatility 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.
M
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,
onoclonal 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 nonenzymatically 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, © 2018 American Chemical Society
Received: December 15, 2017 Accepted: March 1, 2018 Published: March 1, 2018 4669
DOI: 10.1021/acs.analchem.7b05241 Anal. Chem. 2018, 90, 4669−4676
Article
Analytical Chemistry
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 C-terminal lysine forms, it 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.
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 (CEX) 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 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 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 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 an 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 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 Lowresolution 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-up strategy. In this respect, normal and aged IgG samples were digested with IdeS protease and the resulting (Fc/2)2 and F(ab′)2 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
■
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). 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 consisted 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 h 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 separations was a MAbPac SCX-10 RS 2.1 mm × 50 mm column, 5 μm particles (Thermo Fisher Scientific, Sunnyvale, CA). 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. 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 4670
DOI: 10.1021/acs.analchem.7b05241 Anal. Chem. 2018, 90, 4669−4676
Article
Analytical Chemistry
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 mm × 2.1 mm, 2.2 μm particles (Thermo Fisher Scientific, Sunnyvale, CA) was used. Separations were performed at 25 °C, and a flow rate of 0.3 mL min−1 with 0.1% formic acid in water as buffer A and 0.1% formic acid in acetonitrile as buffer B using a gradient of 2−40% buffer B over 45 min was used. 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 Window 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 the 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 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.
Table 1. Optimized Gradients for the CVA of Five Monoclonal Antibodies Using the MS Friendly Buffer System mAb
time (min)
% buffer B
curve
trastuzumab
0 0.5 10
40 55 100
5 5
adalimumab
0 0.5 10
40 50 100
5 7
infliximab
0 0.5 10
40 45 55
5 5
bevacizumab
0 0.5 6 10
40 45 50 100
5 7 5
cetuximab
0 0.5 7 10
40 45 65 100
5 7 5
column, buffers, gradients, and all run parameters applied were the same as described for gradient optimization. In total, 100 μg of 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 (HESIII) 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 CVA 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. A volume of 15 μL of magnetic bead solution was 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 DTT to yield a 10 mM concentration, incubated for 40 min at 37 °C and immediately analyzed by LC−MS.
■
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. Premade buffer cocktails, very well suiting this requirement, have been commercialized and are available today.28,29 Online 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 4671
DOI: 10.1021/acs.analchem.7b05241 Anal. Chem. 2018, 90, 4669−4676
Article
Analytical Chemistry
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 mm × 50 mm MabPac SCX-10 RS column; further run parameters can be obtained from the Experimental Section.
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
