Analytical characterization of biosimilar antibodies and Fc-fusion proteins

Accepted Manuscript Analytical characterization of biosimilar antibodies and Fc-fusion proteins Alain Beck, Hélène Diemer, Daniel Ayoub, François Debaene, Elsa WagnerRousset, Christine Carapito, Alain Van Dorsselaer, Sarah Sanglier-Cianférani PII: DOI: Reference:

S0165-9936(13)00100-3 http://dx.doi.org/10.1016/j.trac.2013.02.014 TRAC 14072

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Trends in Analytical Chemistry

Please cite this article as: A. Beck, H. Diemer, D. Ayoub, F. Debaene, E. Wagner-Rousset, C. Carapito, A. Van Dorsselaer, S. Sanglier-Cianférani, Analytical characterization of biosimilar antibodies and Fc-fusion proteins, Trends in Analytical Chemistry (2013), doi: http://dx.doi.org/10.1016/j.trac.2013.02.014

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Analytical characterization of biosimilar antibodies and Fc-fusion proteins Alain Beck, Hélène Diemer, Daniel Ayoub, François Debaene, Elsa Wagner-Rousset, Christine Carapito, Alain Van Dorsselaer, Sarah Sanglier-Cianférani Mass spectrometry (MS) is one of the key analytical techniques to detect and to identify primary sequence differences, to assess similarity and to evaluate batch variability of reference monoclonal antibodies (mAbs). The next generation of highresolution mass spectrometers and the early use of improved MS-based methodologies will help bring biosimilar mAbs and Fc-fusion proteins into highly regulated markets. Keywords: Biobetter; Biosimilar; Cetuximab; Critical quality attribute (CQA); Etanercept; Fc-fusion protein; Mass spectrometry (MS); Rituximab; Therapeutic antibody; Trastuzumab Abbreviations: ADCC, Antibody-dependent cell-mediated cytotoxicity; Ala, Alanine; Asn, Asparagine; CDC, Complement-dependent cytotoxicity; CDR, Complementarity-determining region; CE, Capillary electrophoresis; CE-LIF, Capillary electrophoresis with laser-induced fluorescence; CE-SDS, Capillary gel electrophoresis; CEX, Cationic exchange chromatography; CHMP, Committee for Medicinal Products for Human Use; CID, Collision-induced dissociation; cIEF, Capillary isoelectric focusing; CQA, Critical quality attribute; Cys, Cysteine; Da, Dalton; DTT, Dithiothreitol; CZE, Capillary-zone electrophoresis; ECD, Electron-capture dissociation; ELISA, Enzyme-linked immunosorbent assay; EMA, European Medicines Agency; ESI, Electrospray ionization; ETD, Electron-transfer dissociation; EU, European Union; Fab, Fraction antigen-binding; Fc, Fraction crystallizable; Fd, Heavy chain VHCH1 domain; FDA, Food and Drug Administration; FT-ICR, Fourier transform ion cyclotron resonance; Gln, Glutamine; Glu, Glutamic acid; HC, Heavy chain; HDX-MS, Hydrogen deuterium exchange mass spectrometry; HIC, Hydrophobic interaction chromatography; IdeS, Bacterial cysteine protease of Streptococcus pyogenes ; IgG, Immunoglobulin G; IMGT, International Immunogenetics Information System; IM-MS, Ion-mobility mass spectrometry; LC, Light chain; LC-MS, Liquid chromatography-mass spectrometry; LIF, Laser-induced fluorescence; Lys, Lysine ; mAb, Monoclonal antibody; MS, Mass spectrometry; MS/MS, Tandem mass spectrometry; MW, Molecular weight; NGHC, Non-glycosylated heavy chain; PD, Pharmacodynamic; pGlu, Pyroglutamic acid; PK, Pharmacokinetic; PMF, Peptide-mass fingerprinting; PNGase-F, Peptide-N-glycosidase F; PFF, Peptide-fragment fingerprinting; PSAQ, Protein standard for absolute quantification; PTM, Posttranslational modification; QQQ, Triple quadrupole; Q-TOF, Quadrupole time-of-flight; RP-HPLC, Reversed-phase high-performance liquid chromatography; SDS-PAGE, Sodium dodecyl sulfatepolyacrylamide gel electrophoresis; SEC, Size-exclusion chromatography; SRM, Selected reaction monitoring; TCEP, Tris (2-carboxyethyl) phosphine hydrochloride; TOF, Time-of-flight; UPLC, Ultraperformance liquid chromatography; USD, United States Dollar.

Alain Beck, Daniel Ayoub, Elsa Wagner-Rousset Centre d’Immunologie Pierre Fabre (CIPF), 5 Av. Napoléon III, BP 60497, 74164 Saint-Julien-enGenevois, France Hélène Diemer, François Debaene, Christine Carapito, Alain Van Dorsselaer, Sarah SanglierCianférani* Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, IPHC, 25 rue Becquerel 67087, Strasbourg, France and CNRS, UMR7178, 67037 Strasbourg, France

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*Corresponding author. Tel.: + 33 3 68 85 26 79; Fax: + 33 3 68 85 27 81; E-mail: [email protected]

1. Introduction 1.1. Biosimilar proteins and glycoproteins in Europe: definitions and status The clinical and commercial success of biologic-derived drugs is transforming the pharmaceutical industry. In 2011, the worldwide sales of the top 30 biopharmaceuticals driven by monoclonal antibodies (mAbs) reached USD 110 billion [1]. As a number of these biodrugs will come off patent soon, many companies are producing copies or “generic” versions that are much more complicated than small-drug generics. Most small pharmaceuticals have molecular weights (MWs) of 150–500 Daltons (Da), and chemical synthesis yields copies with structures identical to the original one. As a consequence, molecular equivalence can be assessed using a panel of analytical methods, and bioequivalence can be documented by bioavailability studies yielding approval of generic versions. Biological drugs [e.g., peptides, non-glycosylated proteins (e.g., insulin and somatotropin) or glycoproteins (e.g., granulocyte colony stimulating factor, epoetins and mAbs)] are much larger, with MWs of 5.6–150 kDa. For these complex macromolecules, different manufacturing processes will produce batches with inherent variability. Even originator companies cannot manufacture their own biologics on an absolutely identical lotto-lot basis, as we illustrate below. From a regulatory standpoint, one of the main requests is to demonstrate consistency in manufacture, with attributes that fall within a set of acceptable specifications. During the R&D process, these criteria have been agreed prior to the production of toxicological and clinical batches. They result from extensive testing and characterization of the experimental biologics by state-of-the-art analytical and pharmacological methods [2]. In the European Union (EU), biosimilars are defined as copy versions of an already authorized biological medicinal product. Demonstration of similarity in physicochemical characteristics, efficacy and safety must be based on a comprehensive comparability exercise [3]. Biosimilars are known as follow-on biologics in the United States of America (USA) and subsequent entry biologics in Canada. Biobetter, biosuperior and next generation biologics are different categories of drugs with differences in primary structure and/or major differences in glycosylation patterns [4]. Because it is not possible to produce exact copies of large proteins and glycoproteins {e.g., Abs [5]}, due to their structural complexity added to the inherent variability of bioproduction, the term biogeneric should be avoided. The EMA has pioneered the regulatory framework for approval of biosimilars since 2005, resulting to date in marketing authorization of 14 recombinant drugs. They encompass three product classes, namely human growth hormone or somatotropin, granulocyte colonystimulating factor or filgastrim, and erythropoietins alfa and zeta [6]. In addition, specific guidelines are also available in Europe for biosimilar insulin, interferon and low-molecularweight heparins (LMWHs) [7]. To go a step forward, the EMA released in 2010 a draft guideline on similar medicinal products containing mAbs, following a workshop organized by the EMA [8]. The guideline discusses relevant animal-model, non-clinical and clinical studies that are recommended to establish the similarity and the safety of a biosimilar compared to an originator mAb approved in the EU. The final version was released by the end of 2012 [9]. IgG1 Fc fusion proteins 2

were included in the scope of the final Committee for Medicinal Products for Human Use (CHMP) guidelines on biosimilar mAbs [10] and will be also discussed below. 1.2. Biosimilar antibodies and Fc-fusion proteins in clinical trials MAbs and related products represent the fastest growing segment of the pharmaceutical industry, resulting in approval of more than 40 drugs [11]. Patent expiry in Europe and the USA of major first-generation mAbs {e.g., rituximab, trastuzumab, infliximab, cetuximab, adalimumab, bevacizumab and etanercept (a Fc-fusion protein) [12]} has led to the development of numerous copies [13]. Head-to-head clinical trials are ongoing to assess their biosimilarity with originator versions [14]. By the end of 2011, the EMA was asked for 35 scientific advice procedures for biosimilar mAbs and Fc-fusion proteins and the first marketing-authorization application for a biosimilar of infliximab was recently filed [10].

2. Primary sequence assessment of mAbs and Fc-fusion proteins A biosimilar should be very similar to the reference product in terms of physicochemical characteristics, functional properties and clinical efficacy. Extensive structural and functional comparison of the biosimilar and the reference product is the foundation of biosimilar development. The primary amino-acid sequence should be the same for the biosimilar and the reference product. Small differences in the micro-heterogeneity pattern of the molecule may be acceptable if appropriately justified with regard to its potential impact on safety, pharmacokinetic (PK) and pharmacodynamic (PD). The analytical package for a biosimilar mAb submission is considerably larger than that of a “stand-alone” mAb. Taking the case of rituximab, we illustrate below the analytical technologies and the structure assessment strategies for characterization of biosimilars. 2.1. The rituximab case study 2.1.1. Rituximab primary sequence reported in database. Rituximab is a chimeric mAb that targets the B-cell surface receptor CD20. Rituximab was the first mAb approved for the treatment of cancer in 1997 (B-cell lymphoma) and then for autoimmune diseases (e.g., rheumatoid arthritis). The 2011 global sales for rituximab were USD 6.6 billion. Rituximab is a chimeric IgG1k produced in Chinese Hamster Ovary (CHO) cells. Rituximab’s sequence is referenced in the International Immunogenetics Information System [15] as a 1328 aminoacid protein. The heavy chain (HC) consists of 451 amino acids (calculated MW 49243 Da for the aglycosylated reduced form) while the light chain (LC) comprises 214 amino-acid residues (calculated MW 23057 Da for the reduced form). HCs and LCs are linked by one disulfide bond and the HCs by two S-S bridges located in a short hinge domain. 12 additional cysteine bridges are intramolecular and delimit six different globular domains: one variable (VL) and one constant for the LC (CL); and, one variable (VH) and three constant for the HCs (CH1, CH2 and CH3). Both LC and HC of rituximab have N-terminal glutamine, which is prone to cyclization, forming N-terminal pyroglutamic acid (-17 Da). Another major modification occurring in recombinant mAbs is the clipping of the C-terminal lysine of HCs leading to a mass decrease of 128 Da. Finally, as mAbs are generally glycosylated, peptide Nglycosidase F (PNGase-F) allows N-glycosylations to be removed, and that further results in the deamidation of Asn residues and a subsequent mass increase of 1 Da for each Asn. Taking into account these modifications, the mass of the PNGase-F deglycosylated rituximab would then be 144 244 Da. Based on these data, a model can be drawn (Fig. 1). 3

2.1.2. Rituximab homogeneity assessment by separation methods using liquid chromatography and electrophoresis. A panel of separation techniques based both on liquid chromatography (LC) and electrophoresis are mandatory for mAb characterization and comparability studies [16]. These orthogonal analytical methods aim at assessing homogeneity and at profiling the main isoforms and micro-variants. As an illustration, several chromatograms and electropherograms of rituximab are displayed in Figs. 2 and 3, respectively. Size-exclusion chromatography (SEC) shows the level of dimers, monomer and fragments such as Fab-Fc variants (Fig. 2A). Cationic-exchange chromatography (CEX) is used to assess the profile of charge variants that may be more acidic or basic relative to the main peak (Fig. 2B). Hydrophobic interaction chromatography (HIC) is used to detect micro-variants (e.g., oxidized methionines or succinimides) (Fig. 2C). Capillary gel electrophoresis (CESDS) under non-reduced conditions is used to assess the tetrameric LHHL (150 kDa) structure of an IgG and the level of fragments [HHL (125 kDa), HH (100 kDa), HL (75 kDa), H (50 kDa) and L (25 kDa)] (Fig. 3A). After reduction, the peaks collapse in two fragments [H (50 kDa) and L, 25 kDa) with small amounts of non-glycosylated HC (NGHC)] (Fig. 3B). Capillary isoelectric focusing (cIEF) is used to determine the isoelectric point (pI) and to assess the profile of charge variants (Fig. 3C). Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is used for glycoprofiling (Fig. 3D); the electropherogram shows the main glycoforms (Mannose 5, G0, G0F, G1F and G2F) with a zoom on those linked to enhanced ADCC (Man5 and G0, Fig 3E) and considered as critical quality attributes (CQAs). 2.1.3. Rituximab primary structure assessment by mass spectrometry 2.1.3.1. Intact mass measurement by ESI-MS. The first step in an IgG primary structure assessment is intact mass measurement by ESI-MS or LC-ESI-MS analysis. This experiment gives access to the confirmation of theoretical molecular mass, including major PTMs (e.g., glycosylation, C-terminal Lys clipping, N-terminal Gln or Glu cyclization and disulfidebridge pairings). As illustrated here for rituximab, intact mass measurement, with or without a prior PNGase-F N-deglycosylation step, allows hypothesizing a primary sequence error in the database. Deconvoluted ESI mass spectra of intact glycosylated or deglycosylated rituximab are presented in Fig. 4A. The molecular mass of the PNGase-F treated rituximab sample leads to a mass of 144,190 Da, which is not in agreement with the MW calculated from the IMGT sequence above. Without prior deglycosylation, different glycoforms were detected, as expected, with masses of 147,077 Da, 147,239 Da, 147,401 Da and 147,563 Da, corresponding to mass increments of +2887 Da (in agreement with G0F/G0F forms), +3049 Da (G1F/G0F), +3211 Da (G1F/G1F or G0F/G2F) and +3373 Da (G1F/G2F) compared to the deglycosylated sample, respectively. This intact rituximab mass measurement is a first hint for suspecting a sequence error in the IMGT database. 2.1.3.2. Middle-up LC-MS analysis. To elucidate mass discrepancies between experimental and calculated rituximab masses, the next step is a middle-up approach, which refers to Ab cleavage into smaller fragments. This can be achieved through the reduction of the disulfide bonds generating free HC and LC and followed by LC-MS analysis. Alternatively, limited proteolysis in non-denaturing conditions in the hinge region of the HC yielding Fab or (Fab’)2 and Fc fragments can also be performed. The reduction of these protease-generated fragments results in three smaller fragments of approximately 25 kDa: the LC, the VHCH1 domain of the HC (Fd) and the reduced Fc domain (Fc/2). Different proteases have been used in middle-up approaches; the most common are papain, pepsin, endoprotease Lys-C and, more recently, IdeS (a bacterial cysteine protease of Streptococcus pyogenes that specifically cleaves IgGs under their hinge domain [17]). Except in the case of Fab glycosylation, LC and Fd fragments 4

are free from glycosylation heterogeneity, while the Fc/2 fragment only bears one glycosylation site. An LC-MS chromatogram of rituximab after IdeS treatment followed by TCEP reduction is presented in Fig. 4B. Peak 1 corresponds to the Fc/2 fragment, in which three masses were measured (25,203 Da, 25,365 Da and 25,528 Da) that could be attributed to G0F, G1F and G2F glycoforms of the Fc/2 fragment, respectively. Peak 2 has a MW of 23,041 Da, in agreement with the theoretical mass of the LC with N-terminus pGlu (23,057-17 = 23,040 Da in theory). Peak 3 corresponds to the Fd fragment with a measured mass of 25,329 Da, which reveals a mass difference of -28 Da compared to the mass calculated from the rituximab IMGT sequence (25,357 Da). These middle-up analyses reveal that the error in rituximab primary IMGT sequence is located on the Fd fragment. 2.1.3.3. Bottom-up LC-MS/MS analysis. Finally, for in-depth characterization of mAbs, the third step is still sequence determination by classical bottom-up approaches that refer to the analysis of protein digest mixtures after enzymatic proteolysis. Following denaturation, reduction and alkylation of Cys residues, Abs and Fc-fusion proteins are commonly digested with trypsin or endoproteinases (e.g., Lys-C, Asp-N, chymotrypsin or Glu-C). The resulting proteolytic mixture is then usually analyzed by nanoLC-MS/MS to gain information on amino-acid sequences and PTM localization (e.g., glycosylations, disulfide bridges and deamidations). The nanoLC-MS/MS analysis of chymotryptic peptides allowed us to identify unambiguously the mistake in the IMGT sequence as an Ala residue on the HC (position 219) instead of a Val residue (Fig. 4C). Recalculation of theoretical masses based on Ala219 results in masses in close agreement with experimental data obtained for intact rituximab mass measurement and middle-up analysis. 2.2. Primary structure mistakes in trastuzumab and etanercept biosimilar candidates As shown in the next sub-sections, the primary structure of biosimilar candidates of trastuzumab and etanercept are not always correct in regard of marketed references [18]. In both cases, MS appeared as the key technique for comparability studies in order to assess the correct primary structure and to localize unambiguously the amino-acid differences. 2.2.1. Trastuzumab. Trastuzumab is a humanized IgG1 Ab approved for treating HER2overexpressing breast cancer patients since 1998. The 2011 global sales reached USD 5.8 billion. In a landmark paper, trastuzumab primary structure was compared to a biosimilar candidate version by Mazeo et al. [19]. LC-MS intact mass measurement revealed that the biosimilar mAb had a mass difference of -64 Da compared to the originator one. The mass difference was located on HCs (~32 Da each). Further nanoLC-MS/MS attributed the difference to a sequence variant of tryptic peptide HT35. HC sequences with two different amino acids on HT35 (Asp359 and Leu361 instead of Glu359 and Met361, respectively) produced a 32 Da lower mass for the biosimilar compared to the reference trastuzumab. The identified sequence variants showed that the biosimilar candidate was derived from a different allotype compared to the originator mAb [20]. 2.2.2. Etanercept. Etanercept is a 150 kDa recombinant dimer protein consisting of two soluble Tumor Necrosis Factor Receptor (sTNFR) molecules fused to the Fc fragment of a human IgG1 [12]. The 2011 global sales for etanercept (USD 7.9 billion) were superior to those of many successful IgG. The patent of etanercept is expected to expire in Europe in 2015. In a recent publication, etanercept and two biosimilar versions approved in China were characterized and compared head to head [21]. One biosimilar showed high similarity in most of the CQAs, including peptide mapping, intact mass, charge variants, purity, glycosylation 5

and bioactivity. In contrast, the intact mass and MS/MS analysis of the second biosimilar candidate showed a number of variations, including a mass difference indicative of a two amino-acid residue variance in the Fc part of the sequence. Etanercept consists of a 2x467 amino-acid protein dimer. Of the 467 amino acids, 235 amino acids form the extracellular portion of the soluble TNFR. The remaining 232 amino acids form the Fc domain of human immunoglobulin G1, including the hinge region, CH2 and CH3 domains. In addition to the conserved N-glycosylation in the Fc’s CH2 region, etanercept has several N-linked and O-linked glycosylations in the sTNFR region that represent onethird of the whole molecular weight. To reduce the complexity caused by PTMs, etanercept was split into two parts using papain digestion (Fc and sTNFR fragment). Then, PNGase-F deglycosylated Fc fragment was analyzed by LC-MS intact mass measurements. Compared with an Fc sample of the reference product, the first biosimilar had the same intact mass (49,930 Da), but the second biosimilar had a -64 Da mass difference for the main peak (49,866 Da) that was demonstrated, as in the case of trastuzumab, to be derived from an allotype different from the originator mAb [20].

3. Micro-heterogeneity variation in therapeutic mAbs Unlike small-molecule generic products, biosimilar products can exhibit a range of structural micro-differences to the original product [22]. Small differences in the micro-heterogeneity pattern of the molecule may be acceptable, if appropriately justified with regard to their potential impact on safety, PK and PD. Therapeutic mAbs vary over time with modifications of the manufacturing process. Biosimilar mAbs and Fc-fusion proteins are developed through head-to-head studies with a specifically identified, previously approved reference product. Hence the first step in developing a biosimilar is to examine carefully multiple samples of that reference product collected over its lifetime. Biologics vary from batch to batch and with any manufacturing change (e.g., additional facilities added to increase capacity) [23]. In the next sub-sections, we review recent papers showing the variability over time of originator trastuzumab, cetuximab, rituximab and etanercept. 3.1. Reported in the literature 3.1.1. Trastuzumab glycosylation variability. As for other IgGs, trastuzumab glycans represent an average of only 2−3% of the total Ab mass. IgGs contain a N-glycosylation site at a conserved Asn in the CH2 domain in both HCs involved in a consensus sequence [24]. The N-glycans are complex biantennary oligosaccharides containing 0–2 non-reducing galactoses (Gal) with or without fucose (Fuc) attached to the reducing end of Nacetylglucosamine (NAcGlc). Fc glycosylation is important for antibody-dependent cellmediated cytotoxicity (ADCC) and for complement-dependent cytotoxicity (CDC) functions through modulating the binding to Fcγ receptors and C1q, respectively. Removal of core fucose can significantly enhance the binding affinity to Fcγ receptors, resulting in increased ADCC [25]. LC-ESI-MS was used for trastuzumab characterization [26]. Based on the deconvoluted masses, peaks observed were found to be very close to the calculated masses and assigned to trastuzumab main glycoforms: G0/G0F (147,920 Da), G0F/G0F (148,059 Da), G0F/G1F (148,216 Da), G0F/G2F or G1F/G1F (148,376 Da), G1F/G2F (148,535 Da) and G2F/G2F (148,680 Da). Four different production batches were compared in terms of global glycosylation profiles and variability at each glycosylation site. Interestingly, results showed that each batch of trastuzumab shared the same types of glycoform but that relative abundance 6

of each glycoform was relatively variable (e.g., the non-fucosylated G0 glycoform, which is involved in ADCC, was in the range 4.9–9.6%). 3.1.2. Cetuximab glycosylation and variability of charge variants. Cetuximab is a chimeric mouse-human IgG1 mAb targeting epidermal growth factor receptor (EGFR) and approved worldwide as a treatment for colorectal cancer (2004) and subsequently for head and neck squamous cell carcinoma. The 2011 global sales reached USD 1.8 billion. As other IgGs, cetuximab has a conserved N-glycosylation site in the CH2 domain, but also a second consensus site within framework 3 of the variable portion of the HC (-Asn88AspThr-). The detailed structures of these oligosaccharides have been investigated based on MALDI-TOF MS and MS/MS. These orthogonal MS-based methods revealed the presence of at least 21 distinct oligosaccharide structures differing in the degree of sialylation with Nglycolylneuraminic acid (NGNA) and in the extent of galactosylation (zero-, mono-, di-, and α-1,3-galactose) [27]. Sundaram et al. also reported important batch variability in terms of glycosylation and C-terminal Lys residue clipping with no measurable impact in functional tests. On the clinical side, a high prevalence of hypersensitivity reactions to cetuximab has been reported in some areas of the USA. Among 76 cetuximab-treated subjects, 25 had a hypersensitivity reaction to the drug. The immune response was shown to be specific for oligosaccharides containing the galactose-α-1,3-galactose glycotype. This motif is present on the Fab portion of the cetuximab HC when the molecule is produced in the murine SP2/0 cell line used for commercial manufacturing, but not in the CHO cells used as control [28]. 3.1.3. Rituximab and etanercept glycosylation and charge variants variability. Schiestl et al. [29] reported data that revealed substantial alterations of the glycosylation profile of different batches of rituximab and etanercept. Significant changes were also shown at the N- and Ctermini. Importantly, rituximab also showed variation in ADCC activity among different batches. Because of the abruptness and the magnitude of the observed alterations, the authors suggested possible changes in the manufacturing processes. As discussed above in the case of rituximab, pGlu is a cyclic amino-acid residue found at both N-termini of LC and HC [30]. Cyclization occurs through the rearrangement of the parent reactive Gln residues. Because of the loss of a primary amine in the Gln to pGlu conversion, rituximab becomes more acidic. Incomplete conversion produces heterogeneity in the Ab that can be observed as multiple peaks using charge-based analytical methods (e.g., CEX or IEF) (Figs. 2B and 3C). This variant is naturally present in IgGs, does not have an impact on safety, PK and PD, and is not considered a CQA. Lys C-terminal clipping is another major source of charge variant in rituximab, etanercept and in other mAbs. This is also a common phenomenon occurring both in natural and recombinant IgGs and not considered a CQA. Conversely, even if glycosylation has no effect on antigen binding but is considered a CQA for rituximab, as glycosylation is required for the Fc-mediated effector functions (e.g., ADCC and CDC), an important part of rituximab mechanism of action. Together, these four examples illustrate the range of micro-variants that were accepted by health authorities for the same brand following changes in the manufacturing process.

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4. Emerging protein analytical methods used for comparability studies Recent reports from the EMA or the FDA have pointed out that there is an increasing need for the early use of novel precise techniques for characterizing structure. This includes not only protein primary sequence assessment, higher-order structures and mAb dynamics characterization but also accurate mAb quantification in plasma for comparability studies between originator products and biosimilars. 4.1. Emerging separation methods Additionally to MS, mAb characterization and homogeneity assessment require a plethora of orthogonal separation techniques mostly based both on chromatographic and electrophoretic techniques (for extensive review, see [16,31]). These separation techniques are based on differences of mass, charge and hydrophilic/hydrophobic balance. Chromatographic methods comprise SEC, ion exchange chromatography (IEX), and reversed-phase HPLC (RP-HPLC) mostly used for peptide mass or fragment fingerprinting (PMF or PFF) while electrophoretic methods include capillary electrophoresis (CE-SDS), capillary isoelectric focusing (cIEF) and, the most straightforward, capillary-zone electrophoresis (CZE) that can be coupled to MS detection. Among recent progress in chromatography techniques, the development of ultraperformance LC (UPLC) pumps was of major importance for improved mAb primary sequence characterization by increasing sequence coverage and leading to more precise characterization of impurities. LC-MS or LC-MS/MS works quite well for the analysis of Abs, generally producing high sequence coverage (>95%). Even higher sequence coverages (100%) can be achieved in this case by using additional complementary proteases. However, LC methods are relatively slow (1 h/run to several hours/run), and generally cannot detect small, hydrophilic peptides due to coelution with the column void volume. The loss of these short peptides (especially in the complementarity region) is of utmost importance as complete mAb sequence coverage is needed for verification of the amino-acid sequence, and/or identification of PTMs. Even if the combination of HPLC with MS/MS analysis is still the gold standard for generating PMF or PFF maps, an interesting novel alternative combining sheathless CE (with increased sensitivity compared to liquid-sheath instruments) with MS [32] or MS/MS [33] was recently described. Whitmore and Gennaro used the sheathless CE prototype to develop a fast (25 min/run), simple, reproducible CE-MS method to PMF of a mAb, allowing detection of small, hydrophilic tryptic peptides that were not detected by typical RP-HPLC/MS peptide maps [32]. Similarly, Gahoual et al. described a novel sheathless CE-ESI-MS platform (named CESI) that allowed 100% sequence coverage by PFF for both HCs and LCs of trastuzumab in a single run [33]. 4.2. Top-down MS for intact mAb measurement Mass measurement of the gas-phase fragments of intact proteins is referred to as top-down MS. Given both the large size and the structural complexity of mAbs, their extensive fragmentation and subsequent sequence determination by means of top-down MS is still challenging with only limited success [34]. In theory, top-down MS approaches combining intact mAb measurements and direct fragmentation of protein ions should provide complete protein sequence coverage (including precise determination of PTM type and position, C- or N-terminus truncations and mutations). However, as mAb fragmentation yields a large amount of highly-charged fragment ions, until recently, dedicated high-resolution FT-ICR mass spectrometers with superior resolving power and ECD fragmentation possibilities were 8

required. This approach accesses the sequence confirmation of terminal regions and variable domains [35] for identification and localization of major modifications and characterization of major glycoforms [36]. Limitations of top-down MS on FT-ICR instrumentation mostly relate to increased signal-to-noise ratio with increased MWs, which both affect intact protein MW measurement and MS/MS performance [37] and the tedious maintenance required for FT-ICR magnet. The new generation of high-resolution instruments with Orbitrap or Q-TOF technologies (more common in biopharmaceutical laboratories) available in combination with ETD fragmentation instead offers new perspectives for top-down MS. It has already been demonstrated that ETD fragmentation generally provides larger sequence coverage than CID in top-down MS analysis of intact proteins [38,39]. The ETD-based top-down obtained on the HPL timescale on Q-TOF instruments demonstrated higher sequence coverage than CID on Orbitrap mass spectrometers, reaching sequence coverage of 21% for mouse IgG1 and 15% for human IgG [40]. Even more recently, the same group showed improvement in ETD-based top down on a linear ion trap Orbitrap [41], leading to a sequence coverage of about 33% for a commercial IgG, signifying an almost two-fold increase in IgG sequence coverage relative to prior ETDbased analysis. These results suggest the potential application of the methodology developed to other classes of large proteins and biomolecules. Another way to improve top-down MS analysis is to reduce sample complexity, which can be achieved in case of mAbs by middledown MS approaches that will enable mAbs to be reduced to three 25 kDa fragments, for which appropriate resolutions and fragmentations (ETD) are available with Q-TOF and Orbitrap instruments. 4.3. Native MS, IM-MS and HDX-MS experiments for higher order structure assessment For mAbs and Fc-fusion proteins, classical biophysical techniques (e.g., X-ray crystallography or nuclear magnetic resonance) are difficult to use in the context of comparability studies due to strong limitations (size of mAbs, presence of flexible regions, amount of material required and time to perform analysis). Recently, some MS-based techniques have emerged to complement classical structural methodologies and at least partially circumvent some of their actual limitations. Those technologies include native MS, IM-MS and hydrogen/deuterium exchange MS (HDX-MS) experiments. The goal is to detect not only subtle changes between different batches but also minor populations that could have a different conformation compared to the major one or even to monitor conformational changes within this minor population that may impact the efficacy of therapeutics. 4.3.1. Native MS. Native MS (also called supramolecular or non-covalent MS) has attracted interest and is now ready to use for not only intact routine mAb analysis but also mAb/Ag characterization. This approach also benefits from progress in MS instrumentation with the implementation of high-resolution instruments (the latest Q-TOF and dedicated Orbitraps), opening new doors for even more detailed characterization [42–44]. Native MS will thus play a central role in the next-generation characterization of biopharmaceutical products [e.g., bispecific Abs, Ab mixtures, Ab-drug conjugates (ADC) and obviously biosimilar mAbs]. We, and others, have contributed to the implementation of native MS at the early stages of development and quality control of biopharmaceuticals in the past five years. For analysis of intact mAbs, native MS allows accurate mass measurements, glycoform identification [45], and assessment of higher order structure and oligomeric status (dimer, trimer, tetramer), providing a robust, fast, reliable first-line characterization [46]. For instance, the clarification/simplification of native mass spectra due to the presence of fewer charges states 9

is of major advantage for exact mass measurements of mAbs [47]. When considering mAb/Ag complexes, native MS is also straightforward, providing additional information including mAb/Ag binding stoichiometries [48], specificities and affinities that can be interesting to use during the comparability exercise between an originator and a biosimilar candidates. 4.3.2. IM-MS. Another evolution of native MS is the combination of IM-MS to native MS. Commercial IM-MS instruments appeared on the market in 2005, bringing new perspectives for structural characterization of intact proteins and protein complexes, especially in biopharmaceuticals [49]. Combining ion mobility, which separates ions according to their size and shape, to MS brings an additional level of conformational characterization of protein complexes, through measurement of collision cross-sections [50]. Ion-mobility separation is fast (ms measurements), sensitive (few nanomoles), and amenable to high-throughput automation. In classical MS conditions (direct infusion or LC-MS experiments), IM-MS can provide routine batch-to-batch characterization of therapeutics, mAbs glycosylation profiling [51], and heterogeneous disulfide bridge pairing monitoring [45] or can even detect protein aggregation. In native conditions, IM-MS can routinely fingerprint higher order structures of mAbs to provide information on conformational changes induced upon Ag binding or even distinguish several isoforms [52]. As an illustration, native MS and IM-MS data of rituximab are displayed in Fig 5. 4.3.3. HDX-MS. HDX-MS can bring insights into subtle conformational effects, flexibility and solution dynamics when classical structural methodologies fail. In a typical HDX-MS experiment, the protein sample is diluted in a deuterated buffer and all amide-exchangeable protons become replaced with deuterium [53] (Fig. 6). As exchange is a function of protein structure and dynamics, a differential HDX-MS experiment is often designed to deduce information on higher order structures, to determine epitope/paratope in mAb/Ag complexes [54] and to monitor conformational changes in therapeutic molecules. HDX-MS has the sensitivity of classical proteomic approaches, can be fully automated [55] with the unique additional advantages of not only revealing conformational changes but also detecting where changes have occurred at the peptide level and, maybe in the near future, at the amino-acid level [56]. Among the issues that have been overcome recently, progress in method automation through implementation of robotics and data interpretation with development of dedicated software has definitely pushed the methodology forwards. For mAbs and related products, HDX-MS can be used to study the conformation and the conformational dynamics of a recombinant IgG, to demonstrate that PTMs present on IgGs can affect conformation, biological function and that the Ab can initiate a potential adverse biological response [57], and to study biopharmaceutical comparability [58]. Even if the commercially available fully automated HDX-MS system relies on the automation of the manual experiment (automation and coordination of pipetting in labeling and quenching steps), the next generation of microfluidic HDX platforms is on the way [59]. A dual-channel microfluidic cell for continuous H/D labeling has been developed to avoid sample dilution and to increase accuracy with short incubation times [60]. Thanks to its ability to monitor subtle changes in conformation and to study conformational dynamics, HDX-MS in conjunction with native MS and IM-MS has the potential to be a pivotal technique for higher-order structure determination in comparability studies of originator and biosimilar candidates. 10

4.4. MS methods for mAb quantitation in plasma PK comparability of originator and biosimilar mAbs is another key feature. Quantitation of mAbs in biofluids is most commonly carried out by enzyme-linked immunosorbent assays (ELISAs) or other specific immuno-binding-based methods (e.g., radioimmunoassay and immunofluorescence) [61]. Immunoassays offer the advantage of being high-throughput, sensitive, robust methods. However, method development consumes resources and time (>6 months) and immunoassays may suffer from non-specific binding, interference from endogenous IgGs, and anti-mAb Abs, so that matrix proteins compromise assays selectivity and accuracy. Proteomic MS-based methods for absolute protein quantitation present an interesting, promising alternative to immunoassays for mAbs quantitation and PK [62]. These methods rely on isotope dilution (ID), used for decades for small molecule quantitation. ID involves using stable-isotope-labeled internal standards (ISs) that are identical to the analytes of interest. For protein quantitation, peptides, generated by protease digestion, are used as surrogates for the proteins of interest. Quantitation is therefore carried out at the peptide level by highly selective, accurate selected reaction monitoring (SRM) experiments. Due to its high multiplexing capability, an SRM assay combined with stable ID is the most suitable strategy for mAb quantitation in biological fluids and total-cell lysates. SRM is primarily performed on triple quadrupole (QQQ) instruments and monitors signals of pairs of precursor ion (peptide)/specific fragment ion called transitions. Transition intensities of peptides and their isotope-labeled counterparts are compared to achieve absolute quantitation [63]. Furlong et al suggested recently a universal surrogate peptide for the quantitation of mAbs and Fc-fusion proteins in preclinical animal studies [64]. Synthetic stable-isotope-labeled peptides are generally used as ISs, but, in order to avoid variability introduced by samplepreparation processes and its effects on quantitation accuracy [65], isotope-labeled proteins can be introduced as early as the sample is collected (PSAQ strategy – protein standard for absolute quantitation) [66]. Finally, as well as demonstrating comparable performances to ELISA techniques, MSbased quantitation assays are faster to develop than immunoassays and circumvent unspecific matrix-binding effects [67]. Their application represents a clear advance for Ab-purity assessment and comparability.

5. Conclusion and future directions Extensive structural and functional comparison of biosimilars and reference products is the foundation of biosimilar development. The comparison exercise must be scientifically tailored using state-of-the-art analytical tools and sensitive tests to detect small product-related differences between the biosimilar and the reference product. Structural differences determine the amount of biological, non-clinical and clinical studies that are needed for development of biosimilar mAbs and Fc-fusion proteins. The primary amino-acid sequence must be the same for the biosimilar and the reference product in Europe and in the USA. As reviewed here, a number of biosimilar candidates of trastuzumab, rituximab and etanercept do not fulfill these criteria. MS evaluation of the degree of similarity between a biosimilar and a reference innovator product should therefore be done at the beginning of a new biosimilar project. As highlighted here, the correct sequence selection results in saving significant time and money. In addition to the correct primary sequence, small differences in the micro-heterogeneity pattern of mAbs may be acceptable if appropriately justified with regard to their potential impact on safety and 11

efficacy. Finally, intact Ab, and middle-up and bottom-up mass measurements in combination with a panel of electrophoretic and chromatographic methods must be used for a fast evaluation of the small differences between the biosimilar and the originator mAb. Among the emerging MS methodologies, HDX-MS, native MS, IM-MS and quantitative MS will play increasing roles in comparability studies of biosimilars and originators. Acknowledgments The authors thank and acknowledge all their colleagues involved in analytical, functional, and MS characterization of mAbs at the CIPF (M.C. Janin-Bussat, O. Colas, M. Excoffier, L. Morel-Chevillet, G. Terral, L. Tonini, C. Huillet, M. Rompais, A. Bourmeaud, M. Trauchessec, T. Champion, C. Klinguer-Hamour) and the LSMBO (C. Atmanene, S. Petiot, J.M. Strub, C. Schaeffer, G. Terral), and MS researchers whose original research work could not be cited because of space limitations. This work was supported in part by the OptimAbs network bioclusters (Lyon Biopole and Alsace Biovalley) and sponsors (DGCIS, Oséo, Feder, Régions Rhône-Alpes and Alsace, Communauté Urbaine de Strasbourg, CNRS, and University of Strasbourg). References [1] http://www.pipelinereview.com/index.php/2011030447751/FREE-Reports/-TOP-30Biologics-2011.html. [2] S.A. Berkowitz, J.R. Engen, J.R. Mazzeo, G.B. Jones, Nat. Rev. Drug Discov. 11 (2012) 527. [3] M. Weise, M.C. Bielsky, K. De Smet, F. Ehmann, N. Ekman, G. Narayanan, H.K. Heim, E. Heinonen, K. Ho, R. Thorpe, C. Vleminckx, M. Wadhwa, C.K. Schneider, Nat. Biotechnol. 29 (2011) 690. [4] A. Beck, mAbs 3 (2011) 107. [5] A. Beck, E. Wagner-Rousset, M.C. Bussat, M. Lokteff, C. Klinguer-Hamour, J.F. Haeuw, L. Goetsch, T. Wurch, A. Van Dorsselaer, N. Corvaia, Curr. Pharm. Biotechnol. 9 (2008) 482. [6] M. McCamish, G. Woollett, mAbs 3 (2011) 209. [7] Z. Kálmán-Szekeres, M. Olajos, K. Ganzler, J. Pharm. Biomed. Anal. 69 (2012) 185. [8] J.M. Reichert, A. Beck, H. Iyer, mAbs 1 (2009) 394. [9] M. Weise, M.-C. Bielsky, K. De Smet, F. Ehmann, N. Ekman, T.J. Giezen, I. Gravanis, H.-K. Heim, E. Heinonen, K. Ho, A. Moreau, G. Narayanan, N.A. Kruse, G. Reichmann, R. Thorpe, L. van Aerts, C. Vleminckx, M. Wadhwa, C.K. Schneider, Blood 120 (2012) 5111. [10] C.K. Schneider, C. Vleminckx, I. Gravanis, F. Ehmann, J.H. Trouvin, M. Weise, S. Thirstrup, Nat. Biotechnol. 30 (2012) 1179. [11] A. Beck, T. Wurch, C. Bailly, N. Corvaia, Nat. Rev. Immunol. 10 (2010) 345. [12] A. Beck, J.M. Reichert, mAbs 3 (2011) 415. [13] A. Mullard, Nat. Rev. Drug Discov. 11 (2012) 426. [14] A. Beck, S. Sanglier-Cianferani, A. Van Dorsselaer, Anal. Chem. 84 (2012) 4637. [15] ImmunoGenetics Database (IMGT), Entry IMGT/mAb-DB ID 161. [16] S. Fekete, A.-L. Gassner, S. Rudaz, J. Schappler, D. Guillarme, Trends Anal. Chem. 42 (2013) 74. [17] G. Chevreux, N. Tilly, N. Bihoreau, Anal. Biochem. 415 (2011) 212. [18] X. Wang, T.K. Das, S.K. Singh, S. Kumar, mAbs 1 (2009) 254. [19] H. Xie, A. Chakraborty, J. Ahn, Y.Q. Yu, D.P. Dakshinamoorthy, M. Gilar, W. Chen, S.J. Skilton, J.R. Mazzeo, mAbs 2 (2010) 379. 12

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Captions Figure 1. Rituximab theoretical structure based on data reported in public databases and expected PTMs. The heavy chain (HC) consists of 451 amino acids while the light chain (LC) comprises 214 amino-acid residues. HCs and LCs are linked by one disulfide bond (S-S) and the HCs by two S-S bridges located in a short hinge domain. 12 additional cysteine bridges are intramolecular and delimit six different globular domains: one variable (VL) and one constant for the LC (CL); and, one variable (VH) and three constant for the HCs (CH1, CH2 and CH3). Both LC and HC of rituximab have N-terminal 1 glutamine (Q ), which is prone to cyclization forming N-terminal pyroglutamic acid (-17 Da, labeled 1 Q /pE). Another major modification occurring in recombinant mAbs is the clipping of the C-terminal lysine. Figure 2. Rituximab characterization by chromatographic techniques. Rituximab chromatograms: (A) size-exclusion chromatography (SEC); (B) cationic exchange chromatography (CEX); and, (C) hydrophobic interaction chromatography (HIC). Figure 3. Rituximab characterization by electrophoretic techniques. Rituximab electropherograms: (A) non-reducing and (B) reducing capillary electrophoresis – sodium dodecyl sulfate (nr/rCE-SDS; intact IgG, HHL, HL, H and L fragment; light chain (LC) and heavy chain (HC); non-glycosylated HC); (C) capillary isoelectric focusing (cIEF; pI and charge variants); (D) glyco-profiling by capillary electrophoresis – laser-induced fluorescence (CE-LIF); and, (E) zoom of non-fucosylated glycoforms (G0 and Man5) important for ADCC. Figure 4. MS-based strategies for rituximab primary-sequence validation. A) Intact mAb mass measurement: MaxEnt deconvoluted ESI-MS spectra of rituximab in classical denaturing conditions (H2O/AcN 50v/50v +1% HCOOH) before (left panel) and after (right panel) PNGase-F deglycosylation. B) Middle-up LC-MS analysis after Ides treatment: LC chromatogram of the different middle-up sized fragments. C) Bottom-up nanoLC-MS/MS analyses: MS/MS spectrum of the 203-222 ICNVNHKPSNTKVDKKAEPK peptide (2 306.216 Da) obtained after chymotrypsin digestion that 219 of the HC was allowed the unambiguous identification of an error in the IMGT sequence. Val identified as an Ala. Figure 5. Native MS and IM-MS analysis of rituximab. ESI-MS mass spectra of rituximab obtained in classical denaturing (A) or in native (B) conditions. In denaturing conditions (A), two species are detected: the most intense one corresponds to the mass of intact rituximab and its main glycoforms; free non-covalent bound light chain is also detected as a minor specie. In native conditions (B), rituximab is mostly detected as a monomer and a minor ion series corresponding to rituximab dimers is detected. IM-MS analysis of rituximab in classical denaturing (C) or native (D) conditions. Native IMMS analysis allows the detection of minor amounts of dimeric rituximab. Figure 6. General workflow for HDX-MS experiments. A) H/D exchange and pepsin digestion; B) LCMS analysis; and, C) Data-interpretation workflow. Black circles represent deuterium atoms.

.

15

Extensive biosimilar/reference mAb comparison is required in biosimilar development In EU and USA, biosimilar/reference mAb primary amino-acid sequences must be the same MS, chromatography and electrophoresis are central in biosimilar characterization MS approaches allow detection of small differences in biosimilar/originator mAbs HDX/native/quantitative MS will become pivotal techniques for comparability studies

16

Figure 1

Rituximab major isoform Q1/pE

(chIgG1k)

Q1/pE

(-17 Da)

Q1/pE

(-17 Da) C22

Q1/pE

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(-17 Da)

C96 C148

C23

xV

219A

C204

C88

C224 C230

C133

C193

C214 C265

Hi

Hi C233

C H 2

16 S-S bridges (-32 Da) C325

G0

C371

C H 3

G0F

G1F

G2F

(+1299 Da) (+1445 Da) (+1607 Da) (+1769 Da)

C H 3

N301 Glycosylations

C429

K451 clipping (2 x -128 Da) IMGT aa based calculated raw formula

Modifications included

Calculated MW

Measured MW

Mass difference

Error in HC sequence

C6418H9874N1690O2010S44

4 N-terminal pE, 2 cleaved C-terminal K, PNGase-F deglycosylation, 16 S-S bridges

144 244 Da

144 190 ± 2 Da

+ 54 Da

V219 is an A

Figure 2

0.28

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0.26

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0,080 0.24 0,070

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3,00

4,00

5,00

6,00 Minutes

7,00

8,00

9,00

10,00

11,00

12,00

Figure 3

(A)

LHHL

L

(C)

Basic variants

HL

HH

(B)

L

HHL

Main peak pI = 9.32

(D)

Acidic variants

(E)

H

Figure 4 G1F/G0F 147239

A) Intact mAb mass measurement by (LC-)ESI-MS 147077

144190

G1F/G1F G2F/G0F 147401

PNGase-F

G0F/G0F G2F/G1F 147563

Mass 146700

147200

147700

Mass 143700

148200

144000

144300

144600

B) Mass measurement of middle-up sized fragments by LC-ESI-MS TCEP

IdeS

C) Bottom-up peptide analysis by digestion + nanoLC-MS/MS Enzymatic digestions nanoLC-MS/MS Start 203

–

End 222

M 2306.216

Sequence Y.ICNVNHKPSNTKVDKKAEPK.S

Figure 5

%

A) MS in denaturing conditions 44+

B) Native MS

2011-08-28 ritux 5uM ACNH4 150mM pH7.2 NAP2 120v 6mbar autoQpro

-28 ritux 2uM NAP2 denat 120v 3mbar

O10487FDE 49 (3.300) Sm (Mn, 2x10.00); Cm (1:61) 100

G1F/G0F

2011-08-28 ritux 2uM NAP2 denat 120v 3mbar

28+

TOF MS ES+ 549

G1F/G0F

100

G1F/G1F G0F/G0F G2F/G0F

A: B: C: D: E:

147094.75±2.39 147253.41±2.50 147406.88±5.06 147566.22±6.11 294769.69±25.99

G0F/G0F G1F/G1F G2F/G0F

%

G2F/G1F

G2F/G1F %

44+ 0 3315

3320

3325

3330

3335

0

28+ m/z

3340

3345

5580

3350

3355

5600

3360

3365

5620

3370

3375

5640

3380

3385

5660

3390

3395

5680

3400

3405

5700

3410

3415

5720

3420

5740

3425

3430

3435

5760

3440

5780

3445

3450

5800

3455

3460

5820

3465

3470

5840

3475

5860

5880

5900

5920

5940

5960

5980

6000

6020

6040

6060

6080

1000

1200

1000

1400

1600

1500

1800

2000

6120

6140

6160

6180

m/z 6200

Dimers

Free LC 800

6100

2000

2200

2400

2600

2500

2800

3000

3000

3200

3400

3600

3500

3800

4000

4000

4200

4400

4600

4500

4800

5000

5000

5200

5400

5600

58000 1000

m/z

5500 m/z 1000

m/z 1500

2000

2000

2500

3000

3000

3500

4000

4000

4500

5000

5000

5500

6000

6000

6500

7000

7000

7500

8000

8000

8500

D) Native IM-MS

C) IM-MS in denaturing conditions O14333FDE.raw : 1

O14314FDE.raw : 1

m/z

m/z 4500

8000 4000 6000

3500 3000

4000 2500

5

10

15

20

25

30

35

ms

5

10

15

20

25

30

35

ms

9000

9500

9000 m/z

Figure 6

...... .... . .. ..

A) Sample preparation : D2O labeling and pepsin digestion

D2O pH 7-8 RT

Backbone amide exchange

Quenching pH 2.5 0°C Various t

. . ... ... .

Pepsin digestion pH 2.5 0°C

Mixture of labeled and unlabeled peptides

B) LC-MS analysis LC at 0°C

(

Ion Mobility

)

60 min

MS or MS/MS

30 min 5 min 1 min

Visualization of deuterium incorporation for each peptide

10 sec 0 sec

C) Data interpretation Deuterium incorporation graphs

Comparability plots for differential data interpretation

If known, interpretation in light of structure