Direct Identification of Rituximab Main Isoforms and Subunit Analysis

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Direct identification of rituximab main isoforms and subunit analysis by online selective comprehensive twodimensional liquid chromatography – mass spectrometry Dwight R Stoll, David Christopher Harmes, John Danforth, Elsa Wagner-Rousset, Davy Guillarme, Szabolcs Fekete, and Alain Beck Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01578 • Publication Date (Web): 06 Jul 2015 Downloaded from http://pubs.acs.org on July 13, 2015

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Analytical Chemistry

Direct identification of rituximab main isoforms and subunit analysis by online selective comprehensive two-dimensional liquid chromatography – mass spectrometry

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Dwight R. Stoll1*, David C. Harmes1, John Danforth1, Elsa Wagner2, Davy Guillarme3, Szabolcs Fekete3, and Alain Beck2

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1 – Gustavus Adolphus College Department of Chemistry St. Peter, MN, USA 2 –Center of Immunology Pierre Fabre 5 Avenue Napoléon III BP 60497 74160 Saint-Julien-en-Genevois, France 3- School of Pharmaceutical Sciences University of Geneva University of Lausanne Boulevard d’Yvoy 20 1211 Geneva 4, Switzerland

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* address correspondence to – [email protected]

ACS Paragon Plus Environment


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Abstract

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In this proof-of-concept study, rituximab, a reference therapeutic monoclonal antibody (mAb), was characterized through the implementation of online, selective comprehensive twodimensional liquid chromatography (sLCxLC) coupled with mass spectrometry (MS), using a middle-up approach. In this setup, cation exchange chromatography (CEX) and reversed phase liquid chromatography (RPLC) were used as the first and second separation dimensions, respectively. As illustrated in this work, the combination of these two chromatographic modes allows a direct assignment of the identities of CEX peaks using data from the TOF/MS detector, because RPLC is directly compatible with MS detection, whereas CEX is not. In addition, the resolving power of CEX is often considered to be limited and therefore, this 2D approach provides an improvement in peak capacity and resolution when high performance second dimension separations are used, instead of simply using the second dimension separation as a desalting step. This was particularly relevant when separating rituximab fragments of medium size (25 kDa), whereas most of the resolution was provided by CEX in the case of intact rituximab samples. The analysis of a commercial rituximab sample shows that online sLCxLCTOF/MS can be used to rapidly characterize mAb samples, yielding the identification of numerous variants based on the analysis of intact, partially digested and digested/reduced mAb samples.

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Graphical Abstract

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Introduction Monoclonal antibodies (mAbs) belong to a major class of therapeutic proteins being developed by most of the pharmaceutical companies.1 The main advantage of mAb-based therapy is its high specificity. MAbs are currently used in a number of therapeutic areas including oncology, infectious diseases and immune diseases.1 Due to the increasing number of approved therapeutic proteins in the pharmaceutical area and the number of biosimilars (or follow-onbiologics) potentially entering the market2, the need for analytical techniques that facilitate their detailed characterization is increasing substantially.3 MAbs are glycoproteins and belong to the immunoglobulin (Ig) superfamily, which can be divided into five isotypes (IgA, IgD, IgE, IgG, and IgM). Since only IgGs are produced for therapeutic purposes through genetic engineering, the terms recombinant mAb and IgG are often used interchangeably. IgGs are large tetrameric glycoproteins (~150 kDa) that are structurally composed of four polypeptide chains: two heavy chains (HC, ~50 kDa) and two light chains (LC, ~25 kDa) connected through several inter- and intra-chain disulfide bonds at their hinge region.4 Each chain is composed of structural domains according to their size and function, giving the constant, variable, and hypervariable regions. Functionally, mAbs consist of two regions -the antigen-binding fraction (Fab) and the crystallizable fraction (Fc).5 Fc (~50 kDa) is composed of two truncated HCs and is responsible for the effector functions. The Fab domain (~50 kDa) is composed of the LC and the remaining portion of the HC (Fd). This domain is primarily involved in antigen binding.5 MAbs exhibit high molecular complexity; they are quite sensitive to changes during the manufacturing processes and storage that can lead to considerable micro-heterogeneity in each individual chain. There are several common modifications leading to antibody charge variants (or isoforms) on the peptide chains (e.g.,deamidation, C-terminal lysine truncation, N-terminal pyroglutamation, methionine oxidation, or glycosylation variants) and size variants (also referred to as ‘proteoforms’6) on the peptide chains (e.g., aggregation or incomplete formation of disulfide bridges). The combination of these micro-heterogeneity sources in the polypeptide chains significantly increases the overall micro-heterogeneity in an entire IgG.7 In general, the identity, heterogeneity, impurity content, and activity of each new batch of mAb has to be thoroughly characterized before drug product release. This examination is performed using a wide range of analytical tools, including ion-exchange chromatography (IEX), reversedphase liquid chromatography (RPLC), hydrophobic interaction chromatography (HIC), hydrophilic interaction chromatography (HILIC), size exclusion chromatography (SEC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), capillary isoelectric focusing (cIEF), capillary zone electrophoresis (CZE), circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR), fluorescence spectrophotometry (FL), and mass spectrometry (MS). The aim of this multi-method strategy is to demonstrate the similarity between production batches by precisely characterizing the primary, secondary, and tertiary structure of the mAbs.8, 9 The first step of a mAb structural characterization is the mass measurement of the intact molecule (top level).10 This step enables a profile spectrum of the whole antibody giving access to the confirmation of elemental composition, the identification of post translational modifications (PTMs), and the characterization of major and minor glycoforms (Fig 1). Middle level analysis refers to the analysis of an antibody after its cleavage into large subunits.11 Therefore, various enzymes, such as pepsin, papain, IdeS and Lys-C, are often used to obtain mAb fragments and facilitate the investigation of its micro-heterogeneity. Papain is often used to create three fragments at the HC hinge region, one Fc and two identical Fab fragments of about 50 kDa



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each, while IdeS and pepsin generate F(ab′)2 fragments (~100 kDa).12 These types of digestion are referred to as limited proteolysis (LP) or partial digestion. In addition, the reduction of disulfide bonds is also commonly used to produce two LCs and two HCs.3 IEX is a nondenaturing separation technique that has been widely used historically for the characterization of charge variants of therapeutic proteins. Among the different IEX modes, cation-exchange chromatography (CEX) is the most commonly used for the purification and characterization of mAbs.13–18 CEX is considered the gold standard for the analysis charge variants. However the resolving power of IEX is quite limited compared to RPLC, therefore not only the intact but also the reduced and/or digested forms (limited proteolysis or peptide mapping) of the mAbs are commonly characterized by IEX.13, 14, 18 The potential for direct coupling of RPLC with MS detection is an obvious advantage over other chromatographic approaches (e.g., IEX, SEC). This detection technique has become increasingly popular for the characterization of mAbs thanks to (i) the introduction of two ‘soft’ ionization techniques that enable the transfer of intact proteins into the gas phase without fragmentation (i.e., electrospray ionization (ESI), matrix-assisted laser desorption/ionization [MALDI]), and (ii) the continuous improvements of MS analyzers that provide high mass resolution, high mass accuracy, high mass range, and highly sensitive detection (e.g., TOF-MS, Orbitrap, etc.). Complete proteolytic digestion of a mAb (peptide mapping, bottom level) followed by gradient RPLC-MS/MS analysis (‘bottom-up’ approach) is generally the method of choice for the identification and quantification of chemical modifications of mAbs.19, 20 On the other hand, the limited proteolysis approach followed by RPLC–MS analysis has also seen some application with mAbs and peptide-Fc fusion proteins21–25, as well as direct analysis of intact mAbs.9 Alvarez et. al. first demonstrated the utility of a generic on-line LC–MS method operated in a two-dimensional (2D) format toward the rapid characterization of mAb charge and size variants.26 Using a single chromatographic system capable of running two independent gradients, the contents of up to six fractions of interest from an IEX or SEC separation could be identified by trapping and desalting the fractions onto a series of RP trap cartridges with subsequent on-line analysis by MS. Analysis of poorly resolved and low-level peaks in the IEX or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulphide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified mAbs. Examples of other two-dimensional approaches have involved SEC x Mixed-Mode separation27, as well as off-line fraction collection between dimensions.28 Most recently, a comprehensive two-dimensional (LCxLC) approach was proposed as a novel tool for the peptide mapping of therapeutic mAbs by Vanhoenacker et. al.29 Trastuzumab (Herceptin) tryptic digest was analyzed on a commercially available twodimensional 2D-LC system, and three different combinations of separation modes were used and compared: CEXxRP, RPxRP and HILICxRP. To the best of our knowledge, online selective comprehensive two-dimensional liquid chromatography30–32 – mass spectrometry (sLCxLC-MS) has not been applied to the analysis of intact and partially digested mAbs. The selective approach to 2D separation is superior to both heartcutting (defined as one fraction per first dimension [1D] peak) and fully comprehensive (defined as analyzing everything that elutes from the first dimension using the second dimension [2D] separation) when a detailed 2D separation is desired for peaks eluting from specific regions of the 1D separation. In other words, the selective approach allows us to strategically use the available 2D analysis time for only regions of the 1D separation that are of interest. In this proof-of-concept study, sLCxLC-MS was used for the analysis and direct identification of rituximab main isoforms and variants, using the multi-level approach shown in


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Fig.1. In our view this approach is complementary to other analytical approaches, including high resolution MS methods coupled with 1D-RPLC (e.g., Orbitrap, FTICR). Indeed, high resolution MS methods are very effective at detecting and identifying many chemical modifications of mAbs (e.g., single or double oxidation)11, however it is inadequate for detection and identification of other more subtle modifications (e.g., deamidation33). The benefit of the sLCxLC-TOF-MS approach involving CEX and RP separations in the first and second dimensions is that highly detailed information about the charge states of different mAb isoforms and fragments (which can easily reveal a deamidation33) can be obtained along with high quality mass information, all in a single analysis, and at lower cost compared to some high-end techniques. Rituximab was chosen for this study because it is considered the ‘gold standard mAb’, and any new methodology should be able to produce results consistent with previous work. It was the first mAb approved by the Food and Drug Administration (FDA) for the treatment of cancer in 1997 (B cell lymphoma) and then for autoimmune diseases (e.g., rheumatoid arthritis) and is one of the top 10 highest selling pharmaceuticals. In addition many biosimilar versions of rituximab are currently being investigated in clinical trials.34

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Figure 1. Antibody LC-MS characterization flowchart (top and middle levels). Rituximab was characterized as whole antibody and subunits following IdeS digestion (F(ab’2)) and Fc/2) and reduction (light chain, Fd and Fc/2)

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Experimental

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Reagents and sample preparation

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All reagents were used as obtained from their respective manufacturers: Acetonitrile (ACN, Chromasolv LC-MS grade; Sigma-Aldrich, St. Louis, MO), trifluoroacetic acid (TFA; part number T6508; Sigma-Aldrich), 2-(N-morpholino)ethanesulfonic acid (MES; part number M3671; SigmaAldrich), and dithiothreitol (DTT, part number 43815; Sigma-Aldrich). Water (hereafter, MQ water) was purified in-house using a MilliQ water purification system (Billerica, MA). The IdeS protease enzyme was from Genovis AB (part number A0-FR1-008; Lund, Sweden). Rituximab (Rituxan) was from Roche (Penzberg, Germany).

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Intact rituximab (hereafter, Rtx) was analyzed following dilution from the 10 mg/mL stock concentration to 0.5 mg/mL with MQ water. As illustrated in Fig. 1, Rtx was partially digested with IdeS protease by adding 5 µL of the 10 mg/mL Rtx stock solution to a polypropylene



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centrifuge tube containing 100 µg of lyophilized IdeS enzyme, followed by 95 µL of digestion buffer (50 mM sodium phosphate, 150 mM sodium chloride, pH 6.6). This solution was incubated at 37 ˚C for 30 min. The resulting sample was split into two 50 µL portions. One portion was diluted with 50 µL of MQ water, and then analyzed by 2D-LC. The second portion was first diluted with 50 µL of MQ water, and then reduced by adding ca. 100 µg of DTT and incubating at 37 °C for 30 min prior to analysis.

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Instrumentation

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A diagram of the instrument used for sLCxLC-MS separations is shown in Fig. 2. All instrument modules were from the 1290 Infinity line from Agilent Technologies (Waldbronn, Germany) unless otherwise indicated. The 1D and 2D pumps were binary pumps (G4220A), the autosampler was model G4226A, the thermostated column compartments were model G1316C, and the diode-array UV detectors (DAD) were model G4212A. A standard flow cell was used in the 1D DAD detector (G4212-60008), and a low dispersion flow cell (800 nL volume, G421260038) was used in the 2D DAD. The two circles labeled ‘PR’ at the outlets of the 1D and 2D flow cells in Fig. 2 are pressure relief valves (G4212-60022) that are normally closed, but will open when (due to tubing blockage or other obstructions) the downstream pressure exceeds 100 bar to avoid breaking the upstream flow cell. All three active valves involved in the 2D separation functionality shown in Fig. 2 (Duo (5067-4214), Deck One (5067-4233, 8-column selector), and Deck Two (5067-4233)) were mounted on external valve drives (G1170A). The salt diversion valve was also mounted on an external valve drive, and was model 5067-4117 (2-position, 6port). The sampling valves Deck One and Deck Two were each fitted with eight nominally identical stainless steel capillaries with nominal volumes of 40 µL (5067-5926). The mass spectrometer was a model 6230 Time-of-Flight MS equipped with an Agilent JetStream electrospray ionization source (Agilent Technologies). The mass analyzer was calibrated using a standard tuning compound mixture (G1969-85000, Agilent). Under these conditions, the observed average mass accuracy for the calibrants was 0.4 ppm, and the mass resolution (at m/z of 3000) was about 10,000.

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The 2D-LC instrument was controlled by OpenLab Chromatography Data System (C.01.07), with the 2D-LC Add-on (G2198AA) installed (Agilent Technologies). The sLCxLC functionality was implemented using specially modified firmware provided by Agilent. The TOF-MS was controlled using MassHunter (B.05.01, Agilent Technologies). Protein mass spectra were deconvoluted using BioConfirm software (B.06.00, Agilent Technologies).


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Figure 2. Schematic of instrument setup used for sLCxLC separations. Each of the two ‘sampling decks’ is a 8-column selector valve fitted with stainless steel sample loops that are nominally 40 µL. On both decks the loops connecting ports 1 and 1’ are bypass paths that are used when the deck is not active (i.e., during sampling or re-injection). A detailed diagram showing the timing of all valve switching events relative to the chromatographic timescale is shown in Fig. S1, along with a detailed description of how the instrument works.

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Separation and Detection Conditions

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First dimension cation-exchange separations were carried out using a 250 mm x 2.1 mm i.d. Bio Mab NP5 column (Agilent Technologies, PEEK column hardware, 5 µm non-porous particle). A salt gradient was used for elution with an A solvent containing 10 mM MES buffer at pH 5.5, a B solvent composed of solvent A plus 0.5 M NaCl, and an elution program of 15-25-37-15-15 %B at 0-14-50-50.01-60 min. The column temperature was 40 °C, the flow rate was 0.16 mL/min, and injection volumes were either 10 (digests) or 15 µL (intact Rtx). Second dimension reversed-phase separations were carried out using a 50 mm x 2.1 mm i.d. Advance Bio RP-Mab column (Agilent Technologies, 450 Å pore size, 3.5 µm core-shell particle, C4 stationary phase ligand). An organic solvent gradient was used for elution, with an A solvent made of 0.1% TFA in water, and B solvent using pure ACN, and an elution program of 28-37-50-28-28 %B in 0-2.02.6-2.61-3.0 min. The gradient conditions used in the first and second dimensions were selected on the basis of preliminary studies where the gradient steepness was varied with the goal of using as much of the available separation space as possible. The 2D column temperature was 75 °C, the flow rate was 0.6 mL/min, and the volume of each fraction of 1D effluent injected into



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the 2D column was 40 µL. The effluent from the 2D column was diverted to waste in the interval between 10 and 25 s following each injection into the 2D column to avoid contamination of the MS ionization source with sodium chloride.

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UV detection was based on absorption of light at 280 nm (with a reference wavelength of 360 nm). In the first dimension the acquisition rate was set to 10 Hz, and in the second dimension 80 Hz. TOF-MS data were acquired from from m/z 500 to 10,000 at an acquisition rate of 5 Hz. The drying gas temperature and flow rate were 325 °C and 8 L/min, while the sheath gas temperature and flow rate were 350 °C and 11 L/min. The nebulizer gas pressure was 35 psi. The capillary and nozzle voltages were 5.5 and 1.0 kV, respectively, and the fragmentor voltage was 350 V.

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The timing of 1D sampling and 2D separation events associated with the different mAb samples are shown in Figs. 3 and S1. Generally, 15-s fractions of 1D effluent were captured and reinjected into the 2D column at 3-min intervals. The only differences between the timing events used for different samples were the starting times of sampling and re-injection into the 2D column.

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Results and Discussion

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Intact rituximab

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Chromatograms from the 1D CEX separations of the three samples of rituximab prepared as shown in Fig. 1 are given in Fig. 3, along with blue rectangles indicating the regions of these chromatograms that were targeted for further resolution by the 2D RP separation and MS detection. In panel A, the intact rituximab peak is observed as expected with one major peak flanked by several minor peaks that are attributed to more acidic (lower retention) or more basic (higher retention) microvariants. In this case, a high resolution 2D chromatogram of species eluting in the window between 31.00 and 44.75 min of the 1D separation was assembled by compiling the data from five different sLCxLC analyses, each focused on a 2.75-min. region of the 1D separation. The five regions targeted in this way are labeled R.A – R.E in Fig. 3A. In other words, the 60-min sLCxLC separation is repeated five times, each time targeting a different sub-section (R.A – R.E) in the 1D separation. The high degree of repeatability of the 1D separation (about 0.2% RSD in retention time of the main rituximab isoform) makes this compilation process feasible. The alternatives to doing multiple sLCxLC analyses are to either sample the rituximab elution envelope non-comprehensively, which results in missed information, or to extend the 1D separation to several hours, which results in 1D effluent fraction volumes that are prohibitively large (e.g, several hundred microliters each). In our view the approach employed here involving multiple sLCxLC analyses of the same sample is a good compromise between these extremes that provides good performance and analytical efficiency, while retaining flexibility in the instrument setup that allows targeting of both very wide and very narrow regions of the 1D separation. Within a single sLCxLC analysis, 11 fractions of 1D effluent in a given region (e.g., R.A) were stored in the loops of sampling Decks 1 and 2 shown in Fig. 2, and subsequently injected into the 2D column for further separation. In all of the sLCxLC analyses described here, fractions of 1D effluent were collected at 15-s intervals – an example with this level of detail is shown by the red vertical lines in Fig. 3A. The series of 11 2D chromatograms that results from this process is shown in Fig. 4B for the specific case of the


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analysis of section R.C in Fig. 3A, which contains the majority of the mass of the main isoform of rituximab. The detector signals from this series of 11 2D separations are then reshaped into a 2D array to produce the 2D chromatogram shown in Fig. 4C. Note that the portion of this image enclosed by the green rectangle corresponds to the data obtained from the second dimension in a single sLCxLC experiment – specifically from the analysis of the region indicated by the green rectangle in Fig. 4A, and the region labelled R.C in Fig. 3A. After five different sLCxLC analyses of the same sample, we obtain the composite 2D chromatogram shown in Fig. 4C. Panels D and E of Fig. 4 show representative deconvoluted mass spectra for one highly abundant isoform (D) and a less abundant isoform (E). Detected 2D chromatographic peaks are highlighted in Fig. 4C using white ellipses to aid visualization. The putative identities of rituximab fragments contributing to these peaks are indicated in Table 1. When possible we refer to previous reports of the same isoforms and fragments observed in this work.34, 9, 35, 26 We see that 13 of the glycoforms of rituximab previously reported were directly detected in these separations.26, 9 While there is some indication of chromatographic separation of some of the glycoforms in the 2 D RP separation, the resolution is rather low because each 2D separation is very short. Of course, optimization of the 2D separation conditions should be guided by the goals of the analysis, and if chromatographically resolving the different glycoforms were a high priority, the sLCxLC approach is sufficiently flexible that much longer 2D analysis times could be used for the separation of each fraction of 1D effluent. Again, in this work, we aimed to demonstrate some of the ways in which sLCxLC-TOF-MS methodology could be used to obtain characteristic information about a mAb, rather than trying to discover previously unknown isoforms and variants of the mAb. It is interesting to note that the majority of the peaks in Fig. 4C have very similar 2D RP retention times, despite the fact that they have very different 1D retentions. We attribute this to the significantly lower resolving power of RPLC for the separation of glycoforms compared to CEX. Herein lies the first example in this work of the virtue of the sLCxLC approach for this application – that is the ability to rapidly sample (i.e., fractionate) a small region of interest in the 1D separation that reveals information about the sample and identity of the components that is difficult to obtain efficiently any other way. On one hand, simple heartcutting approaches either only provide information about individual 1D peaks, or cause the remixing of species that had previously been separated by the first dimension if a large fraction of 1D effluent is collected. On the other hand, comprehensive 2D approaches either force the use of very large sampling times (e.g., >> 1 min) to be compatible with 2D analysis times, which again causes remixing of 1D peaks, and therefore information loss.

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Figure 3. Cation-exchange separations of (A) intact rituximab (top level), (B) rituximab partially digested with IdeS, and (C) rituximab digested with IdeS and reduced with DTT (middle levels); signals are from detection by absorption of UV light at 280 nm. Blue boxes indicate regions of these separations that were targeted using sLCxLC, with each blue division indicating the region targeted in a single sLCxLC analysis. 1 2 The red lines indicate the times at which 40-µL fractions of D effluent were transferred to the D column for further separation (only one set of these lines is shown for clarity). In all sLCxLC experiments indicated by blue boxes, 11 fractions were collected in a continuous way, and transferred to the second dimension per analysis. In the ‘flexible sLCxLC’ experiments indicated by the green boxes, the sampling period was 1 split into two parts, so as to focus the sampling on regions of the D separation of interest in a more efficient way. During these discontinuous sampling periods, 4, 5, or 6 fractions were transferred, depending on the experiment. Labelling of the different focus regions of R.A, RI.C, etc. corresponds to the numbering of peaks used in Table 1.

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Figure 4. Detailed view of data from sLCxLC analysis of intact rituximab; signals in panels A-C are from detection by absorption of UV light at 280 nm. Panel (A) shows a more detailed view of the 1D CEX 2 separation of rituximab shown in Fig. 3A. Panel (B) shows the string of D chromatograms that results 1 1 from the 3-min. analyses of fractions of D effluent collected at 15-s intervals during the D time interval indicated by the green rectangle indicated in panel (A). Note that fractions captured by each deck are reinjected in reverse order relative to the way in which they were collected. The two time periods marked by ‘F’ in panel (B) indicate 3-min. gradient cycles that are used to flush the capillaries connecting the sampling decks to the Duo valve, and elute any compounds that had been accumulated on the 2D column prior to and during the sampling period. Panel (C) shows the 2D chromatogram that results from reformatting of the signals shown in panel (B) into a 2D array. Data from the ‘flush gradients’ are omitted. White ellipses indicate locations of chromatographic peaks in the 2D space as identified by manual inspection of the traces shown in panel (B). Representative deconvoluted protein mass spectra are shown in panels (D) and (E), with labels that correspond to those in Table 1.

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Table 1. Interpretation of observed rituximab isoform and fragment masses1

380 Mass (Da) Sample

Rtx

Rtx:IdeS

Rtx:IdeS:DTT

381

Experiment

Abundance Measured

Calculated2 Difference 3

Mass147,0794

CEX tR (min)

RP tR (min)

Interpretation5

Ref6

R.B.3

minor

148,012

148,018

-6

933

34.3

1.5

Rtx:G2F/G2F-NANA

9

R.B.4

minor

147,105

147,111

-6

26

34.3

1.5

Rtx:G1/G1 or Rtx:G0/G2

9

R.B.4

minor

148,147

148,147

0

1,068

34.3

1.5

Rtx:G1F/G2F-NANA

9

R.B.4

minor

147,462

147,466

-4

383

35.5

1.5

Rtx:Man5/G2F-NANA

9

R.B.4

minor

147,870

147,872

-2

791

35.5

1.5

Rtx:G2/G2F-NANA

R.C.5

minor

25,200

25,202

-2

37.0

1.5

Fc/2-G0F-(s–s)ox (wo C-terminal lysine)

9 35

R.C.5

minor

25,365

25,365

0

37.0

1.5


35

R.C.5

minor

50,398

ND

37.0

1.5

Fc-G0F-(s–s)ox (wo C-terminal lysine)

/

R.C.5

minor

50,725

ND

37.0

1.5


/

R.C.5

minor

75,602

ND

37.0

1.5

H-L

33

R.C.5

minor

76,086

ND

37.0

1.5

H-L

33

R.C.6

minor

121,914

ND

37.0

1.5

H-H-L

33

R.C.6

minor

122,062

ND

37.0

1.5

H-H-L

33

R.C.6

minor

122,224

ND

37.0

1.5

H-H-L

33

R.C.6

major

147,081

147,079

2

2

37.0

1.5

Rtx:G0F/G0F

9

R.C.6

major

147,242

147,241

1

163

37.0

1.5

Rtx:G0F/G1F

9

R.C.6

major

147,404

147,403

1

325

37.0

1.5

Rtx:G1F/G1F

9

R.C.6

minor

147,568

147,565

3

489

37.0

1.5

Rtx:G1F/G2F

9

R.C.6

minor

147,721

147,727

-6

642

37.0

1.5

Rtx:G2F/G2F

9

R.E.9

minor

147,096

147,095

1

17

42.5

1.5

Rtx:G0/G1F

9

R.E.9

minor

147,257

147,257

0

178

42.5

1.5

Rtx:G1/G1F

9

R.E.9

minor

147,416

147,419

-3

337

42.5

1.5

Rtx:G1/G2F

RI.E.1,2

major

25,200

25,202

-2

8.7

0.6


9 35

RI.E.1,2

major

25,363

25,365

-2

8.7

0.6


35

RI.E.1

minor

25,524

25,527

-3

8.7

0.6


35

RI.E.1,2

minor

50,400

ND

8.7

0.6


/

RI.E.1,2

minor

50,724

ND

8.7

0.6


/

RI.E.1

minor

51,048

ND

8.7

0.6


/

RI.E.3

minor

48,356

ND

8.7

1.45

(Fab') (S-S)ox

/

RI.E.4

major

25,200

25,202

-2

13.75

0.6


35

RI.E.4

major

25,363

25,365

-2

13.75

0.6


35

RI.E.4

major

50,400

ND

13.75

0.6


/

RI.E.4

major

50,724

ND

13.75

0.6


/

RI.A.1

minor

96,713

96,720

-7

21.5

1.6

(Fab')2 (S-S)ox

/

RI.A.2

minor

96,713

96,720

-7

22.75

1.6

(Fab')2 (S-S)ox

/

RI.B.3

minor

48,357

ND

24.5

1.4

(Fab') (S-S)ox

/

RI.B.4

major

96,713

96,720

24.5

1.6

(Fab')2 (S-S)ox

/

RI.B.5

minor

96,714

96,720

24.75

1.7

(Fab')2 (S-S)ox

/

RI.B.6

minor

96,745

ND

24.5

1.8

/

/

RI.C.7

minor

96,725

ND

27.0

1.6

/

/

RI.D.8

minor

96,780

ND

29.75

1.6

/

/

RI.D.9

minor

96,748

ND

29.75

1.7

/

/

RID.A.1, RID.D.1

major

25,200

25,202

8.75

0.6


35

RID.A.1, RID.D.1

major

25,363

25,362

1

8.75

0.6


35

RID.A.1, RID.D.1

minor

25,524

25,526

-2

8.75

0.6


35

RID.A.1, RID.D.1

minor

50,400

ND

8.75

0.6


/

RID.A.1, RID.D.1

minor

50,724

ND

8.75

0.6


/

RID.A.1, RID.D.1

minor

51,048

ND

8.75

0.6


/

RID.A.2, RID.D.2

major

23,035

23,039

-4

8.75

0.7

pLC(s-s)ox

35

RID.A.4, RID.D.4

major

25,325

25,329

-4

8.75

1.3

Fd(s-s)red

35

RID.A.5, RID.D.5

major

48,358

ND

8.75

1.4

(Fab') (S-S)ox

/

RID.B.6, RID.D.6

minor

23,036

23,039

12.0

0.75

pLC(s-s)ox

35

-6

-2

-3

RID.B.7, RID.D.7

minor

25,041

ND

12.0

1.35

Fc/2-G0-(s–s)ox (wo C-terminal lysine)

/

RID.B.7, RID.D.7

minor

50,080

ND

12.0

1.4

RID.B.8, RID.D.8

minor

25,324

25,327

12.0

1.4

Fc-G0-(s–s)ox (wo C-terminal lysine) Fd/2(s-s)red

/ 35

RID.B.8, RID.D.8

minor

50,648

ND

12.0

1.4

Fd(s-s)red

/

RID.B.8, RID.D.8

minor

75,971

ND

12.0

1.4

12.0 12.0

0.7 1.5

H-L /

34 /

(Fab') (S-S)ox

/

13.75

0.7


35 35

-3

RID.B.9, RID.D.9

minor

48,074

ND

RID.B.10, RID.D.10

minor

48,358

ND

RID.C.11

minor

25,200

25,202

-2 1

RID.C.11

minor

25,363

25,362

13.75

0.7


RID.C.11

minor

50,400

ND

13.75

0.7


/

RID.C.11

minor

50,724

ND

13.75

0.7


/


Page 13 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

382 383 384 385 386


1) The ‘experiment’ number refers to the labelling in Fig. 3 (e.g., R.A, RI.B, etc.), and the peak numbers indicated in the 2D chromatograms in Figs. 4, 5, and S2-S4; 2) Masses calculated using MassLynx Software; 3) Calculated – Measured mass; 4) 147,079 Da is the mass of the most abundant isoform of rituximab; 5) Full structural characterization would require peptide mapping by 10, 9, 29 LC-MS/MS ; 6) When available.

387

IdeS-Digested Rituximab

388 389 390 391 392 393 394 395 396 397 398 399

The 1D CEX separation of the IdeS digested sample of rituximab is shown in Fig. 3B. The partial digestion with IdeS is commonly employed in biopharmaceutical applications to increase the amount of information that can be obtained from a CEX or RPLC separation. For this sample, two different types of sLCxLC separation were carried out. A series of four separations, indicated by blue rectangles (RI.A–RI.D), were used to focus on the region encompassing the elution of the F(ab’)2 fragment of ~100 kDa in the first dimension. This is similar conceptually to the sLCxLC separation of intact rituximab discussed above, and in the interest of brevity the presentation and discussion of these results are deferred to the electronic Supporting Information in Fig. S2. A second, more flexible sLCxLC separation was used to target different regions of the 1D separation, as indicated by the green rectangles in Fig. 3B. This is conceptually similar to the separation discussed in the next section below, and we provide the results of this separation in the Supporting Information (see Fig. S3 for more detail) as well.

400 401

IdeS-Digested and DTT-Reduced Rituximab

402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425

The 1D CEX separation of rituximab first digested with IdeS and then reduced with DTT is shown in Fig. 3C. Here again, the digestion + reduction approach may be useful to simplify the chromatograms and help with the interpretation of the data. In this case, we comprehensively analyzed the region from 8.00 to 16.25 min in three segments as indicated by the blue rectangles shown in Fig. 3C. The results of these separations are shown in the Supporting Information (see Fig. S4). More interesting in this case is the use of sLCxLC to flexibly target two regions of the 1D CEX separation that are not immediately adjacent to each other in time, as shown by the green rectangles in Fig. 3C. Figure 5A shows the targeted region of the 1D CEX chromatogram in more detail, and Figs. 5B and 5C show the resulting 2D chromatograms obtained for the two targeted regions after collection and subsequent 2D separation of four and six fractions of 1D effluent, respectively. In both cases, we see that the 2D RP separation is very powerful for quickly resolving mAb fragments that co-eluted in the 1D CEX separation. To visualize this more clearly, we show in Fig. 5D one of the six 2D chromatograms that underlies the 2D chromatogram in Fig. 5C. Here, we see that at least five different constituents of the 1D peak at about 12.5 min are visible as distinct peaks in the second dimension. Representative deconvoluted protein spectra are shown in Figs. 5E-5G, corresponding to peaks 1, 4, and 5 in Fig. 5B. The major fragments contributing to the spectra in Fig. 5E are Fc fragments with different glycosylations, as indicated in Table 1. The fragment contributing to peak 4 is interpreted as the Fd’ fragment, while the fragment in peak 5 is interpreted as a light chain with a pyroglutamate residue (pLC). As was the case with the intact rituximab, we see again here that there are some fragments that have the same nominal mass, but elute with very different 1D retention times (e.g., see in Table 1 Fc/2 fragments RID.D.1 and RID.C.11). A deeper understanding of these observations would require more study that is beyond the scope of this work, however we believe that they likely result from fragments that are isomeric. Chemically



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426 427 428 429

Page 14 of 18

reasonable examples are isomeric amino acids, e.g. Asp/IsoAsp and Leu/IsoLeu, and different glycosylation patterns that result in the same nominal mass (e.g., G1F/G1F and G0F/G2F). Indeed, Vlasak et al. have already shown that chromatographic separation of light chain variants differing only in the amino acid Asp or IsoAsp by CEX is straightforward33.

430

431 432 433 434 435 436 437 438 439 440

Figure 5. Flexible sLCxLC separation of rituximab digested with IdeS and reduced with DTT for the best 1 compromise of information production and analytical efficiency. Panel (A) shows the same D separation shown in Fig. 3C, but magnified to emphasize the region containing the major mAb fragment peaks. Panels (B) and (C) show the 2D chromatograms resulting from targeting the 1D regions from 8.75 to 9.50, 2 and 11.50 to 11.75 min, respectively, in a single sLCxLC analysis. Panel (D) shows one of the D 2 chromatograms that underlies the 2D chromatogram in panel (C) – this chromatogram shows the high D resolving power that enables the chromatographic separation of five different fragment species that all co1 elute from the D CEX column. Panels E-G show representative deconvoluted protein mass spectra for different fragments eluting in this region.

441 442

Conclusions

443 444 445 446 447 448 449 450

As illustrated through the case study of rituximab, the potential for direct identification of mAb isoforms and subunit analysis using online selective comprehensive two-dimensional liquid chromatography - mass spectrometry (sLCxLC-MS) was demonstrated. In this study, a selective two-dimensional approach has been selected rather than a simple heartcutting or a fully comprehensive 2D separation strategy, since sLCxLC provides a path to easily maximize the degree of information that can be obtained from both chromatographic dimensions in a given analysis time, while minimizing information loss due to undersampling. As shown in this study, there are two main advantages in using CEX and RPLC as the first and second dimensions of


Page 15 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


451 452 453 454 455 456 457 458 459 460 461 462 463 464 465

the sLCxLC setup, respectively. First, it is well known that CEX is one of the best strategies for the separation of charge variants in biopharmaceutical analysis, but this approach suffers from incompatibility with MS due to the high concentrations of salts used in the eluent. Thanks to the implementation of an RPLC step in between CEX and MS (desalting step), it becomes possible to directly acquire MS information from a CEX experiment, which is particularly useful to assign all the fragment variants. In the present work, the observed CEX peaks of intact rituximab as well as digested and reduced/digested rituximab peaks were putatively assigned and reported in a data table for a deep characterization of rituximab sample. Second, because the resolving power of CEX may be limited (poor kinetic performance), there is an obvious interest in adding a second dimension (RPLC) to improve peak capacity and resolution. When analyzing intact rituximab, the resolving power of the RPLC step was found to be relatively limited because of the high separation speed used in each 2D separation, but it was much more relevant when analyzing rituximab fragments of limited size (25 kDa). In a forthcoming study, this analytical platform (selective CEX x RPLC-TOF-MS) will be employed to compare FDA and European Medicine Agency (EMA) approved mAbs and biosimilar candidates.36, 37

466 467 468 469 470 471 472 473 474 475 476

In our view the sLCxLC-TOF-MS approach is complementary to other existing high-end methods for mAb characterization (e.g., 1D-LC-Orbitrap-MS) in that it provides a cost-effective, time-efficient way to obtain protein charge state and high quality mass information in a single analysis. While the chromatographic front-end to the MS is more complex and more expensive in the 2D-LC case compared to the 1D-LC case, modern 2D-LC systems are continually becoming more reliable and easy to use, and more widely commercially available. On the other hand, the improved resolving power of the 2D separation (separation by both charge and size/hydrophobicity) relaxes the demands on the MS capabilities of the system such that highly useful mass information can be obtained using a mass analyzer with intermediate mass resolution (i.e., TOF technology) that is considerably less expensive compared to higher resolution mass analyzers.

477 478

Supporting Information Available

479 480

Additional chromatograms and further discussion of those results are available as supporting information. This information is available free of charge via the Internet at http://pubs.acs.org/.

481 482

Acknowledgements

483 484 485 486 487 488 489 490 491 492

We acknowledge substantial technical assistance in configuration of the sLCxLC instrument, development of prototype control firmware, and data visualization from Klaus Witt, Konstantin Choikhet, Herbert Anderer, Uwe Freisler, and Stephan Buckenmaier. DS wishes to dedicate this paper to the memory of Herbert Anderer, who was a champion of 2D-LC, a brilliant scientist, and gentle soul. All of the instrumentation used in this work was provided as a loan from Agilent Technologies. The chromatographic columns were generously provided by Agilent Technologies. The IdeS enzyme was generously provided by Genovis AB. DS acknowledges financial support from a Camille and Henry Dreyfus Teacher-Scholar Award, and from a grant from the Agilent University Relations program. DG wishes to thank the Swiss National Science Foundation for support through a fellowship to SF (31003A_159494).



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Page 18 of 18

Direct Identification of Rituximab Main Isoforms and Subunit Analysis

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