Dr Rabah Gahoual
email: rabah.gahoual@etu.unistra.fr

Alain Beck

Emmanuelle Yannis-Nicolas François

Emmanuelle Leize-Wagner

Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE-ESI-MS/MS

Introduction

Amino acids side chains of proteins and peptides may undergo various chemical reactions occurring at physiological temperature and pH. Those modifications, commonly referred as post-translational modifications (PTMs) includes deamidation, oxidation, phosphorylation, N-terminal acetylation, glutamic acid (Glu) cyclization, isomerization, glycosylation or disulphide bonds exchange for instance [START_REF] Walsh | Protein Posttranslational Modifications: The Chemistry of Proteome Diversifications[END_REF]. PTMs have a determining influence on biochemical activity of proteins. Indeed, they are involved in inhibition or activation of proteins mainly by modifying their tertiary and quaternary structure likewise acting as biochemical signals transmitter or molecular clocks [START_REF] Corti | Isoaspartate-dependent molecular switches for integrin-ligand recognition[END_REF][3]. One of the most frequent PTM, occurring on asparagine (Asn) and marginally on glutamine (Gln) residues, is deamidation which also involves isomerization when forming aspartic acid. Deamidation corresponds to the hydrolysis of the side chain amide bond to a free carboxylic group (Figure 1) [START_REF] Geiger | Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation[END_REF]. Deamidation kinetics is increased at elevated temperature and pH (≥ 8) or if the susceptible amino acid is followed by a small, flexible residue such as glycine whose low steric hindrance leaves the amide bond exposed for reaction [START_REF] Chelius | Identification and Characterization of Deamidation Sites in the Conserved Regions of Human Immunoglobulin Gamma Antibodies[END_REF]. In isomerization/racemization, the α-carbon of aspartic acid can have two different configurations inducing two configuration isomers: D-aspartic acid (Asp) and L-isoaspartic acid (isoAsp) [START_REF] Geiger | Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation[END_REF]. Alongside to Asp isomerization resulting from Asn deamidation to both Asp isomers, Asp residues may also spontaneously experience isomerization by the formation of an instable succinimide intermediate. Due to their significant role in biochemical pathways, deamidation and isomerization have been subject to intensive research activities in order to improve their qualitative and quantitative characterization [START_REF] Wold | In Vivo Chemical Modification of Proteins (Post-Translational Modification)[END_REF][START_REF] Shang | Fragmentations of the heptapeptides containing α-or β-aspartate by ESI-MS and low-energy CAD[END_REF].

Mass spectrometry (MS) has gradually taken a consequent role in the characterization of PTMs due to its outstanding selectivity and sensitivity. Especially tandem mass spectrometry (MS/MS) allows in various cases to characterize PTMs by attributing the mass difference of the fragment corresponding to the modified amino acid [START_REF] Yates | Method to Correlate Tandem Mass Spectra of Modified Peptides to Amino Acid Sequences in the Protein Database[END_REF]. However MS often needs to be used in combination with a separation technique like nanoflow liquid chromatography (nanoLC) or capillary electrophoresis (CE) in order to limit competition during the electrospray ionization (ESI) process and maximize signal sensitivity. Ion suppression and ionization competition are common phenomena in ESI that may hamper PTMs detection: protein modification is usually faint while major part of the sample remains intact. Without a separation prior to the MS analysis, ionization of a weakly abundant modified peptide would be suppressed during ionization leading to the inability to detect the considered PTM. Currently, Asn deamidation and Asp isomerization occurring on protein are characterized by MS using a bottom-up proteomic workflow where the sample of interest is undergoing proteolytic digestion [START_REF] Fornelli | Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry[END_REF]. The peptide mixture generated is then separated and characterized using reverse phase (RP) nanoLC-MS/MS instrumental settings. However, using this analytical strategy, the modification must involve a significant change in hydrophobicity in order to enable the separation of a modified peptide from the intact homologous. Recently, our group developed a method using sheathless capillary zone electrophoresis (CZE) hyphenated to high-resolution MS/MS to perform in a single analysis the simultaneous characterization of different aspects of a protein defining its primary structure [START_REF] Gahoual | Full Antibody Primary Structure and Microvariant Characterization in a Single Injection Using Transient Isotachophoresis and Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry[END_REF]. We also demonstrated successfully application of this methodology to perform the biosimilarity assessment of different approved therapeutic mAbs compared to their respective biosimilar candidate [START_REF] Gahoual | Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry[END_REF].

Since a few years, capillary electrophoresis hyphenated to electrospray ionization mass spectrometry (CE-ESI-MS) is meeting a renewed interest [START_REF] Sun | Over 10 000 Peptide Identifications from the HeLa Proteome by Using Single-Shot Capillary Zone Electrophoresis Combined with Tandem Mass Spectrometry[END_REF][START_REF] Han | Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry for Top-Down Characterization of Pyrococcus furiosus Proteins on a Proteome Scale[END_REF][START_REF] Li | Optimizing capillary electrophoresis for top-down proteomics of 30-80 kDa proteins[END_REF]. Conventional sheath-liquid CE-ESI-MS interfacing tend to suffer from reduced sensitivity, therefore opposing to one of the major characteristic of MS, while innovative CE-ESI-MS coupling strategies provide an optimal sensitivity especially because of operating flow rates (<50 nL/min) enabling the production of a nanoESI [START_REF] Bonvin | Evaluation of a sheathless nanospray interface based on a porous tip sprayer for CE-ESI-MS coupling[END_REF][START_REF] Sun | Ultrasensitive and Fast Bottom-up Analysis of Femtogram Amounts of Complex Proteome Digests[END_REF].

In the present work, we demonstrate the interest of using CZE, in complement to the ESI-MS analysis, for the separation of different type of peptide mixtures in order to detect and characterize Asn deamidation as well as Asp isomerization. A comprehensive study was carried out using different sets of synthetic peptides. Synthetic peptides employed are based on actual proteolytic peptides showing the possibility to separate and characterize independently intact peptides from homologous exhibiting Asn deamidation, Asp isomerization even for peptides containing both modifications in the same experiment. CZE selectivity was further studied for the primary structure characterization of the therapeutic mAb trastuzumab (Herceptin®).

Trastuzumab is a humanized immunoglobulin gamma 1 (HzIgG-1) directed against the HER2/neu receptor which is overexpressed in about 20-30% of invasive breast cancer patients [START_REF] Hudis | Trastuzumab -Mechanism of Action and Use in Clinical Practice[END_REF][START_REF] Carter | Humanization of an anti-p185HER2 antibody for human cancer therapy[END_REF]. A tryptic digest of the mAb was analyzed by sheathless CZE-ESI-MS/MS. MS data were used to characterize several Asn deamidation and Asp isomerization sites of interests. The characteristics provided by the CZE separation are particularly interesting for mAbs characterization because these modifications can influence the potency of therapeutic proteins and therefore are categorized as critical quality attributes (CQA) for biotherapeutics [START_REF] Eon-Duval | Quality attributes of recombinant therapeutic proteins: An assessment of impact on safety and efficacy as part of a quality by design development approach[END_REF][START_REF] Harris | Identification of multiple sources of charge heterogeneity in a recombinant antibody[END_REF]. Finally a complex peptide mixture was characterize in a similar manner to investigate the conservation of CZE selectivity for complex samples composed of several dozens of proteins. Universal Proteomic Standard 1 (UPS1) was selected as complex peptide mixture to evaluate the analytical platform.

The UPS1 is a standard protein mixture designed by the Association of Biomolecular Resources Facilities (ABRF) standard proteomic research group, composed of 48 human proteins ranging from 6 to 83 kDa, in equivalent quantities [START_REF] Jian | A Novel Algorithm for Validating Peptide Identification from a Shotgun Proteomics Search Engine[END_REF]. Sample was spiked with several peptides in order to demonstrate strictly the conserved selectivity of the separation even for complex peptide mixture. Thus we demonstrate the selectivity provided by the electrophoretic separation, between a modified peptide and the intact equivalent likewise emphasizing the exquisite compatibility of CZE conditions to ESI-MS.

Materials & methods

Materials

Chemicals were of analytical grade or high purity grade and purchased from Sigma-Aldrich (Saint-Louis, MO) otherwise if not specified. Water used to prepare buffers and sample solutions was obtained using an ELGA purelab UHQ PS water purification system (Bucks, UK). Trastuzumab sample is the EMA approved product and formulation purchased from Roche Diagnostics GmbH (Penzberg, Germany). Universal Proteomics Standard 1 (UPS1) was purchased from Sigma-Aldrich and RapiGest SF surfactant from Waters (Milford, MA, USA).

Peptide synthesis

Peptide HT-22 (cf. Table 1) and corresponding modified forms were obtained from Bachem (Bubendorf, Switzerland). These peptides correspond to tryptic fragment 22 of the heavy chain of trastuzumab (position 276 -290) as well as to possible related degradation products. Purity was analytically controlled in order to be superior to 95% and masses of each peptide were measured by MALDI-TOF MS. Peptide HT-3 (cf.

Table 1) was synthesized at the Centre d'Immunologie Pierre Fabre (Saint-Julien-en-Genevois, France) using Fmoc chemistry. Purity was shown to be superior to 95 % and masses were measured by MALDI-TOF MS before use. These peptides correspond to tryptic fragment 3 of the heavy chain of humanized antibody. Sequence and theoretical masses of the peptides and their isoforms are compiled in Table 1.

Trastuzumab sample preparation

A volume corresponding to 100 µg of protein were used form marketed formulation (21 mg/mL). Samples were first diluted using milliQ water to a final concentration of 6.7 µg/µL. Samples were then diluted using 0.1% RapiGest surfactant to reach a final concentration of 3.35 µg/µL and heated to 40ºC for 10 min. Dithiothreitol (DTT) was added to the sample to a final concentration of 25 mM. The samples was then heated for a 5 min incubation at 95ºC. After being cooled down to room temperature (RT), iodoacetamide (2-IDA) was added to the sample to a final concentration of 10 mM and the mixture was kept in the dark for 20 min to allow alkylation of cysteines (Cys) thiol residues. A volume of 1 µL of trypsin (0.5 µg/µL) was added to the sample which was left at room temperature for 3 h. Finally another 1µL volume of trypsin was added and digestion was performed overnight at 37ºC. After digestion completion, 1% (v/v) formic acid (FA) was added to the samples, which were left at RT in order to cleave the surfactant. Samples were finally diluted to a final concentration of 2.2 µM of protein using ammonium acetate 50 mM (pH 4.0).

UPS1 sample preparation

UPS1 sample was reconstituted first in 0.1% RapiGest by adding 30 µL of the solution and heated to 40ºC during 10 min. Trypsin was reconstituted by adding a volume of 40 µL of milliQ water as advised by manufacturer. DTT was added to the sample to a final concentration of 25 mM. Sample was then incubated for 5 min at a temperature 95ºC. After being cooled down to RT, 2-IDA was added to the sample to a final concentration of 10 mM and sample was kept in the dark for 20 min. A volume of 1 µL of trypsin was added to the mixture which was left at RT for 3 h and another 1 µL was added afterward, digestion was carried on overnight at 37ºC. After digestion completion, 1% FA (v/v) was added to the sample and left at RT for 2 hours. A volume of 12 µL was sampled and concentrated to an approximate volume of 1 µL using vacuum concentration (miVAC, NY, USA).

Capillary zone electrophoresis

CZE separations were performed using CESI8000 system from Sciex separations (Breas, CA, USA) equipped with a temperature controlled autosampler and a power supply able to deliver up to 30 kV.

Hyphenation to the mass spectrometer was carried out using the sheathless CE-ESI-MS interfacing equipped on the CESI8000 system. Bare fused-silica (BFS) capillaries (total length 92 cm; 30 µm i.d.) with a characteristic porous tip on its final 5 cm were made available by Sciex separations. New capillaries were flushed at 75 psi (5.17 bar) for 10 min with methanol, 10 min with 0.1 M sodium hydroxide, then 10 min with 0.1 M hydrochloric acid and water for 20 min. Finally, the capillary was flushed 10 min at 75 psi with 10% acetic acid (AA), which was the BGE used for the separation. Unless stated, hydrodynamic injections (410 mbar for 100 sec) corresponding to a total volume of 110 nL were used. Separations were performed by applying a voltage of +20 kV.

Sheathless CE-ESI-MS hyphenation

CESI8000 system was hyphenated to a 5600+ TripleTOF mass spectrometer (AB Sciex, Darmstadt, Germany). The 5600+ MS is equipped with a hybrid analyzer composed of a succession of quadrupoles followed by a time-of-flight (TOF) mass analyzer. ESI source parameters were set as follows: ESI voltage -1.75 kV, gas supplies (GS1 and GS2) were deactivated, source heating temperature 150ºC and curtain gas value 2 psi.

Mass spectrometry

For synthetic peptides analysis, MS/MS experiments were performed in Top10 information dependent acquisition (IDA), accumulation time set to 250 msec for MS scans and 80 msec for MS/MS scan leading to a duty cycle of 1.05 sec. Mass/charge ratio (m/z) range was set to 100-2000 in both MS and MS/MS experiments. For trastuzumab and UPS1 characterization, experiments were carried out in Top20 IDA experiment with similar parameters (duty cycle 1.85 sec).

MS/MS data analysis

Results and Discussions

Modified peptides separation under CZE

In order to investigate the selectivity provided under the electrokinetically driven separation, two different set of peptides incorporating the PTMs of interests were synthesized. Characteristics of those peptides are summarized in Table 1. Each peptide set consists of the considered peptide without modification and the similar peptide bearing Asn deamidation, Asp isomerization or both modifications. Note as these peptides are based on actual tryptic peptides C-terminal extremity is composed of either a lysine (Lys) or an arginine (Arg) in order to emphasize the characterization of a tryptic peptides (Table 1). While peptide synthesis allows to obtain independently the same peptide with different Asp conformation. The first set of peptides (HT-22) contains two distinctive modification sites: an Asp residue in position 6 (D 6 ) and an Asn residue in position 12 (N 12 ) which can undergo through isomerization and deamidation/isomerization respectively. Different mixtures of the considered peptides were prepared in order to determine whether CZE enables separation regarding strictly Asn deamidation and Asp isomerization. As emphasized in Figure 2, the separation of a mixture constituted of HT-22 and HT-22 (deaN 12 ) peptides exhibited two consecutive peaks.

Peptide HT-22 exhibits an apparent mobility of 1.91•10 -5 cm 2 V -1 s -1 while the same peptide with an Asn deamidation exhibited an apparent mobility of 1.93•10 -5 cm 2 V -1 s -1 , leading to a difference of 0.20 ± 0.015 min in migration times corresponding to a resolution of 1.31 (Figure 2). To confirm the separation regarding Asn deamidation, an equivalent mixture was prepared using HT-22 (isoD 6 ) and HT-22 (isoD 6 /deaN 12 ).

Similarly, results obtained showed two consecutive peaks with a difference in migration time of 0.20 ± 0.01 min. Considering their peak width, CZE separation gives a resolution of 1.38 meaning that CZE enables a significant separation of similar peptides which sole difference is an Asn residue experiencing deamidation.

Because deamidation leads to a mass shift of 0.984 Da which can be resolved by high resolution MS, both peaks could be unambiguously attributed and collision induced dissociation (CID) MS/MS spectra even allowed to determine the exact amino acid modified (Figure 2 B. C.). Deamidation does not involve a change of the peptide charge in solution at the BGE pH (2.2). However, difference in migration times is explained by a modification of the hydrodynamic radius of the peptide due to the deamidation. Relevance of the separation of the two peptides is supported by several observations: both peptides are detected with a charge state of (+2) which means that in case of co-migration, the modification would not be characterized due to the overlapping of their respective isotopic profiles. Additionally, samples are commonly showing partial modification which can be extremely weak (< 5%). The ability to separate the modified peptide from the intact one before ESI ionization is therefore interesting because it prevents competition during the ionization process and increase the sensitivity of the signal. CZE-ESI-MS experiments were also performed to study the selectivity of the separation regarding Asp isomerization. Separation of a mixture containing peptide HT-22 (deaN 12 ) and HT-22 (isoD 6 /deaN 12 ), as emphasized in Figure 3, showed two consecutive peaks having a migration time difference of 0.40 ± 0.10 min and a resolution of 2.24. The capacity to separate two peptides composed of the same amino acid sequence but with two different conformation of Asp is certainly a huge advantage in that case. MS instrumental settings used in this study do not permit the characterization of Asp isomerization occurring on a peptide from MS or MS/MS data. Indeed isomerization does not involve a difference in the mass of the tryptic peptide meaning that it cannot be directly distinguished from TOF-MS data. Additionally, y/b fragment ions produced in CID are common to both peptides (Figure 3 B. C.). As peptide mass and charge in solution are not impacted by Asp isomerization, separation between HT-22 (deaN 12 ) and HT-22 (isoD 6 /deaN 12 ) is attributed to a change in the hydrodynamic radius of the peptide due to the different conformation. Complete separation before ionization enables to unambiguously distinguish peptides isoforms and prevent competition effect during ESI process. Baseline separation obtained suggests CZE-ESI-MS is particularly appropriate to perform a relative quantification regarding modification levels of the sample. A mixture of peptides HT-22 and HT-22 (isoD 6 ) was also analyzed using the same conditions.

Results obtained revealed similarly two consecutive peaks with a migration time difference of 0.45 ± 0.04 min. Results recorded for both previous separation enable to conclude that CZE selectivity is conditioned by the presence of two different Asp isomers despite the presence of another PTM on the considered peptide.

In order to confirm and further study the selectivity of the separation, similar experiments were performed using a peptide mixture composed peptide HT-3 (Table 1). Peptide HT-3 is constituted of a higher number of amino acids compared to the previously studied peptide. As emphasize in Table 1, peptide HT-3 contains an Asp residue which can experience isomerization. The analysis of a mixture containing the two isoforms of peptide HT-3 showed again two consecutive peaks having a difference in migration times of 0.16 ± 0.01 min, those separation characteristics providing a resolution superior to 1.34. In order to confirm the nature of each peak present on the electropherogram, several mixture of HT-3 peptides isoforms in different ratios were prepared and analyzed. Results demonstrated that unmodified peptide is detected first followed by the modified isoform similarly to peptide HT-22. The electrophoretic separation enables to distinguish the two peptides while their only difference comes from the conformation of a single Asp residue showing that CZE selectivity regarding Asp conformation is not affected by the peptide composition. As mentioned before, peptide HT-22 can be affected simultaneously by Asn deamidation, Asp isomerization or simultaneously both. CZE-ESI-MS/MS analysis of a mixture containing every form of peptide HT-22 was performed. As presented in Figure 4, each forms of the peptide could be separated in CZE with a resolution ranging from 1.29 up to 1.63 therefore showing four independent peaks. This result is particularly interesting as it was possible to distinguish the four different forms of HT-22 in the same experiment. Separation regarding those minor structural changes of the peptide is explained by the conjugation of two different characteristics provided by CZE separation: the selectivity of the electrokinetically driven separation allowing to distinguish analytes regarding their size, likewise the tremendous efficiency of CZE separation. Indeed peak widths at half height are ranging from 0.12 min down to 0.06 min for the second peak (Figure 4) representing a number of theoretical plates ranging from 218105 up to 890837 plates.

Selectivity in tryptic digest characterization

In order to further study CZE selectivity regarding Asn deamidation and Asp isomerization, a tryptic digest of trastuzumab was characterized using similar CZE-ESI-MS conditions. Especially, we demonstrated the ability to systematically characterize, from a single analysis, the complete amino acid sequence of the protein. Alongside glycosylations could be precisely localized and more than 10 glycans structures could be simultaneously identified using MS/MS spectra [START_REF] Gahoual | Full Antibody Primary Structure and Microvariant Characterization in a Single Injection Using Transient Isotachophoresis and Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry[END_REF]. The amino acid sequence of peptide HT-22 is actually part of trastuzumab sequence. Isomerization of HT-22 (isoD 6 ) is described as influencing significantly the immunogenic activity of trastuzumab by modifying higher order structure of the protein and for this reason is considered as a critical quality attribute [START_REF] Walsh | Post-translational modifications of protein biopharmaceuticals[END_REF]. As it can be observed on Figure 5, the extracted ion electropherograms (EIEs) corresponding to the different forms of peptide HT-22 showed three consecutive peaks similarly to the previous experiments involving synthetic peptides. The first peak was identified as the intact peptide HT-22 while the second peak exhibiting a mass shift of 0.984 Da, was identified as peptide HT-22 (isoD 6 /deaN 12 ) confirmed by fragmentation spectra. The third peak was identified as peptide HT-22 (isoD 6 ). Additionally, migration order is consistent with experiments performed employing synthetic peptides. Results obtained clearly indicate that the selectivity contributing to the separation of peptides with Asn deamidation and/or Asp isomerization is maintained during the analysis of tryptic digests. Respective maximum intensities were used to estimate the relative abundance of each isoform. Results show that a large majority of peptide HT-22 is not modified. MS signal allowed to estimate that 94.7% of HT-22 is not experiencing any modification while 3.3% is experiencing Asp isomerization (deaN 6 /D 12 ) and 2.0% both Asp isomerization and Asn deamidation (N 6 /isoD 12 ). Considering the quantity of digested peptides injected and their abundance in MS, HT-22 modified peptides are representing a total amount of 10.6 fmol which reveals the outstanding ionization efficiency provided by the sheathless CZE-ESI-MS interfacing. The excellent ionization efficiency is attributed by the conjugation of several characteristics. The flow rate generated by the electroosmotic flow is significantly below 50 nL/min, which is favorable with the formation of a nanoESI [START_REF] Busnel | High Capacity Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry: Coupling a Porous Sheathless Interface with Transient-Isotachophoresis[END_REF]. The interesting point emerging from CZE-ESI-MS/MS data is that peptide HT-22 isoform exhibiting deamidation is only HT-22 (D 6 /deaN 12 ). In this case, data obtained allowed to specifically characterize the isoform which is concerned by the modification giving new insights about the conditions of occurrence of this modification.

Several Asp residues, potentially experiencing isomerization present on the considered mAb were also studied. CZE-ESI-MS/MS data allowed to confirm the separation of the modified peptide from the unmodified counterpart while showing that isomerization levels for trastuzumab is quite limited, systematically inferior to 5%. Trastuzumab contains in total four Asn and six Asp residues which modification are influencing significantly the potency of this therapeutic protein in case of deamidation and/or isomerization. CZE-ESI-MS/MS data demonstrated for each hotspots separation of the intact peptide from modified equivalents allowing to characterize and perform the relative quantification regarding modification levels in a single experiment. Results obtained therefore demonstrate conservation of selectivity provided by the electrophoretic separation in the case of tryptic digest characterization. Ionization efficiency provided by the instrumental settings and the ability to separate prior to the ionization process peptides having minor modifications, minimizing therefore ion suppression, demonstrated the possibility using sheathless CZE-ESI-MS coupling to detect and characterize those PTMs even in the case of weakly abundant modification levels.

Selectivity in complex sample characterization

In the last two decades, performance of MS have been tremendously improved in terms of mass accuracy, resolution as well as sensitivity, acquisition rate and dynamic range. Those improvements allow the analysis of more complex samples and the development of shotgun proteomic experiments [START_REF] Bensimon | Mass Spectrometry-Based Proteomics and Network Biology[END_REF]. A shotgun proteomic experiment was designed using CZE-ESI-MS/MS for the separation of a moderate complex peptide digest. This experiment was designed in order to determine the suitability of CZE as an alternative to nanoLC for complex samples analysis and to investigate the selectivity regarding Asn deamidation and Asp isomerization for this type of experiment. The sample considered in this experiment was UPS1 standard. UPS1 is commonly used to evaluate the performances of analytical platforms especially regarding sensitivity as well as the capacity to identify successfully proteins having significant similarities regarding their amino acid sequences [START_REF] Tabb | Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography-Tandem Mass Spectrometry[END_REF][START_REF] Ivanov | Interlaboratory studies and initiatives developing standards for proteomics[END_REF]. Samples were prepared in order to be compatible with CZE-ESI-MS analysis conditions (cf. Materials & Methods). During sample preparation, samples were spiked with each forms of peptide HT-22. Injection conditions were adjusted in order to perform CZE-ESI-MS/MS experiments using a quantity of 50 fmol for each protein representing a total quantity of 129 ng of digested peptides. Despise the complexity of this peptide mixture, the EIE corresponding to the m/z of peptide HT-22 and modified homologous showed that the selectivity of the separation is preserved likewise the efficiency of the separation (Figure 6). Indeed resolutions observed during experiments regarding HT-22 isoforms separation were ranging from 1.24 up to 1.92. Likewise in mAbs tryptic digest characterization, the capacity to separate the different forms of peptide HT-22 before their transfer to the ionization source can prevent competition effect, therefore maintaining an optimal sensitivity and preventing the introduction of biases during relative quantification. CZE-ESI-MS/MS data were additionally used to identify proteins composing the mixture and assess the performances of the sheathless CZE-ESI-MS coupling. Results demonstrated that a single analysis of UPS1 sample led to the identification of all 48 proteins composing the sample through database search algorithm. Strictly relying on database search algorithm, maximum sequence coverage was 73 % for lactotransferin (Mth = 78289 Da) while mean sequence coverage was 35 %. Results obtained show the exceptional adequacy of CZE separation in concomitance with high resolution MS for the characterization of complex samples in shotgun bottom-up proteomic experiments as it was also showed by different groups [START_REF] Sun | Over 10 000 Peptide Identifications from the HeLa Proteome by Using Single-Shot Capillary Zone Electrophoresis Combined with Tandem Mass Spectrometry[END_REF][START_REF] Han | Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry for Top-Down Characterization of Pyrococcus furiosus Proteins on a Proteome Scale[END_REF][START_REF] Li | Optimizing capillary electrophoresis for top-down proteomics of 30-80 kDa proteins[END_REF][START_REF] Bonvin | Evaluation of a sheathless nanospray interface based on a porous tip sprayer for CE-ESI-MS coupling[END_REF][START_REF] Sun | Ultrasensitive and Fast Bottom-up Analysis of Femtogram Amounts of Complex Proteome Digests[END_REF][START_REF] Hudis | Trastuzumab -Mechanism of Action and Use in Clinical Practice[END_REF][START_REF] Carter | Humanization of an anti-p185HER2 antibody for human cancer therapy[END_REF][START_REF] Eon-Duval | Quality attributes of recombinant therapeutic proteins: An assessment of impact on safety and efficacy as part of a quality by design development approach[END_REF][START_REF] Harris | Identification of multiple sources of charge heterogeneity in a recombinant antibody[END_REF][START_REF] Jian | A Novel Algorithm for Validating Peptide Identification from a Shotgun Proteomics Search Engine[END_REF][START_REF] Walsh | Post-translational modifications of protein biopharmaceuticals[END_REF][START_REF] Busnel | High Capacity Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry: Coupling a Porous Sheathless Interface with Transient-Isotachophoresis[END_REF][START_REF] Bensimon | Mass Spectrometry-Based Proteomics and Network Biology[END_REF][START_REF] Tabb | Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography-Tandem Mass Spectrometry[END_REF][START_REF] Ivanov | Interlaboratory studies and initiatives developing standards for proteomics[END_REF][START_REF] Wang | Improving the Comprehensiveness and Sensitivity of Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry for Proteomic Analysis[END_REF]. This characteristic explains the excellent results regarding sequence coverage obtained which allowed to distinguish and identify independently proteins having significant parts of their amino acid sequences in common. Such properties of the sheathless CZE-ESI-MS/MS analysis, in particular the selectivity provided by CZE separation enables the characterization of lowly abundant PTMs even for a complex sample of digested proteins, giving the opportunity in some cases to dispense with time and sample consuming enrichment steps during sample preparation [START_REF] Sarg | Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano-Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Posttranslationally Modified Histones[END_REF]. In this work, CZE selectivity allowed to distinguish especially several peptides having different isomers of aspartic acid, enriching the level of the characterization which could be achieved in a conventional shotgun bottom-up proteomic experiment considering the type of mass analyzer used during the experiments.

Conclusions

In this work, we describe the use of sheathless CZE-ESI-MS/MS to improve the characterization of Asn deamidation and Asp isomerization of peptides in bottom-up proteomic experiments. CZE separation selectivity regarding those modifications was extensively investigated using several sets of synthetic model peptides. Results demonstrated that CZE conditions employed allowed to separate an intact peptide from the different homologous experiencing Asn deamidation, Asp isomerization or both modifications independently with a resolution systematically higher than 1.29. Selectivity is attributed to the electrokinetically driven separation mechanism of CZE which enables separation of analytes exhibiting different hydrodynamic radii. Separation of peptide isoforms experiencing Asn deamidation and Asp isomerization provides an optimal sensitivity and prevent the introduction of biases that may hamper estimation of sample modification levels. This characteristics was emphasized in the experiments by the ability to detect weak modification levels inferior to 5% and the possibility to estimate modification levels for several PTMs occurring on the same peptide. Results proved the conservation of CZE selectivity even in the case of characterization of complex samples. Additionally, sheathless CZE-ESI-MS/MS data allowed to successfully identify all proteins composing the UPS1 sample in a single analysis using a total injection of 129 ng of digested proteins with excellent sequence coverage up to 73% allowing to identify respectively proteins having extended amino acid sequence homology.

Results obtained demonstrate the adequacy of CZE-ESI-MS/MS conditions with bottom-up proteomics experiments while providing an outstanding sensitivity for detection and identification of faintly abundant PTMs by favoring ionization efficiency. Implementation of CZE separation prior to the ESI-MS analysis allowed to enrich the level of characterization in certain cases. Electrophoretic separation of modified peptides from their intact counter parts, even for modifications inducing only a conformation of a single amino acid appears as a valuable asset for biological samples characterization. Therefore CZE-ESI-MS/MS analysis can be envisage alongside to conventional shotgun proteomics experiments in order to enrich the level of characterization which can be achieved for a given complex sample by taking advantage of the respective separation selectivity provided by reverse phase liquid chromatography and capillary zone electrophoresis.

Figures

For

  protein identification, MS/MS raw data were processed using ProteinPilot software (AB Sciex, Darmstadt, Germany). Database search were carried out using software native algorithm Paragon configured with the following parameters (a) database: Swissprot database, (b) enzyme: Trypsin/P, (c) maximum missed cleavages: 3, (d) variable modifications (amino acid): acetylation (protein N-term), deamidation (Asn), oxidation (Met), carbamidomethylation (Cys), (e) False Discovery Rate (FDR) < 1%. If required, additional data treatment of CESI-MS/MS experiments was performed manually using PeakView software.

Figure 1 -

 1 Figure 1 -Representation of nonenzymatic deamidation leading to the formation of aspartic acid and isoaspartic acid.

Figure 2 -

 2 Figure 2 -A) Extracted ion electropherograms (EIEs) for the m/z values corresponding to peptide HT-22 and peptide HT-22 (deaN 12 ) with MS isotopic profiles corresponding to each peak. B) MS/MS spectrum corresponding to peptide HT-22. C) MS/MS spectrum corresponding to peptide HT-22 (deaN 12 ).

Figure 6 -

 6 Figure 6 -EIEs of m/z values corresponding to different forms of synthetic peptides HT-22 spiked in Universal Proteomic Standard 1 (UPS1) peptide mixture analyzed by sheathless CZE-ESI-MS/MS.

Table 1 -

 1 Compilation of different forms of synthetic peptides used to study CZE-ESI-MS/MS method selectivity regarding Asn deamidation and Asp isomerization (cf. Materials and methods).

	Tables
	Denomination	sequence	modification(s)	denomination	M r (Da)
	HT-22	FNWYVDGVEVHNAK	none	HT-22		1676.7947
		FNWYVDGVEVHDAK	deaN	HT-22 (deaN	12 )	1677.7788
		FNWYVisoDGVEVHNAK	isoD	HT-22 (isoD	6 )	1676.7947
		FNWYVisoDGVEVHDAK	isoD / deaN	HT-22 (isoD	6 /deaN	12 )	1677.7788
	HT-3	GLEWIGYISYDGTNNYKPSLK	none	HT-3		2417.1904
		GLEWIGYISYisoDGTNNYKPSLK	isoD	HT-3 (isoD)	2417.1904

Acknowledgments

Authors would like to thank Sciex separations Inc. for lending a CESI8000 system, Dr. M. Anselme and Dr. S. Locke for their support. LSMIS would like to thank P. Hammann, L. Kuhn and J. Chicher (Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France) for stimulating discussions regarding bottom-up proteomics experiments. The authors would like also to express their gratitude to Dr. E. Wagner-Rousset, MC. Janin-Bussat, O. Colas and Dr. Christine Klinguer-Hamour (Centre d'immunologie Pierre Fabre, St

Julien en Genevois, France) for helpful discussions around peptide and antibody structural characterization.


Rabah Gahoual

Michaël Biacchi

Johana Chicher

Lauriane Kuhn

Philippe Hammann

Alain Beck

Emmanuelle Leize-Wagner

Yannis N Francois

Yannis N François

Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry

Keywords: monoclonal antibodies, biosimilar, biobetter, transient isotachophoresis, sheathless capillary electrophoresis, mass spectrometry, peptide mapping, complete sequence coverage, glycosylations, posttranslational modifications, glycoforms semiquantitative analysis, trastuzumab, cetuximab

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mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between two samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline.

 INTRODUCTION

Although monoclonal antibodies (mAbs) were introduced as treatments for disease in the late 1980s (muromonab-CD3 was approved in 1986), they currently represent the most rapidly growing category of therapeutic molecule. [START_REF] Beck | 8th Annual European Antibody Congress 2012[END_REF] Some of their properties naturally explain such success. For example, their therapeutic efficiency, reduction of side-effects, favorable pharmacokinetic (PK) and pharmacodynamics (PD) lead to an intensive appeal in research and development (R&D) activities regarding this type of protein for the last decade. [START_REF] Beck | Characterization of Therapeutic Antibodies and Related Products[END_REF] Currently more than 40 mAbs have been approved by regulation agencies, such as the US Food and Drug Administration (FDA) or the European Medicine Agency (EMA), and 30 additional candidates are currently in Phase 3 evaluation. [START_REF] Beck | Strategies and challenges for the next generation of therapeutic antibodies[END_REF][START_REF] Reichert | Antibodies to watch in 2013: Mid-year update[END_REF] Their applications are mainly in the field of oncology, inflammation, immune mediated disorders and neurological diseases, including Alzheimer's treatments. [START_REF] Kotsovilis | Therapeutic Human Monoclonal Antibodies in Inflammatory Diseases[END_REF][START_REF] Waldmann | Immunotherapy: past, present and future[END_REF] Patents protecting the first generation blockbuster mAbs will expire in the next five years, giving the opportunity to many companies to produce "biogeneric versions". These copies are referred as biosimilars or in some cases follow-on biologics. A biosimilar is defined by the EMA as a biological medicinal product that contains a version of the active substance of an already authorized original biological medicinal product. 7 Recombinant mAbs are produced from proprietary cell lines; their structural complexity and important micro-variability induced by a given production process explain the greater difficulty that generic companies have in producing exactly reproduction of biologic molecules compared to small molecules. In the past few years, regulation agencies have worked toward establishment of guidelines that, while taking into account such limitations, determine critical quality attributes (CQAs) that must be in common between a reference mAb and its biosimilar suggesting that their immunogenicity, as well as their PK/PD, should not be significantly different. Those guidelines should help demonstrate similarity between a biosimilar candidate compared to the reference product.

The work by EMA and FDA led to the publication of guidelines 7 and to the approval for marketing in the EU of a first biosimilar antibody (infliximab), thus paving the way for the whole product class. [START_REF] Beck | Approval of the first biosimilar antibodies in Europe: A major landmark for the biopharmaceutical industry[END_REF] Biosimilarity assessment includes extensive physicochemical characterization likewise PK and PD study, performed in a comprehensive manner. As different structural heterogeneities emerged from comparison of a biosimilar candidate with the reference molecule, more complementary studies should be performed in order to demonstrate the absence of toxicological and clinical effect. [START_REF] Beck | Analytical characterization of biosimilar antibodies and Fc-fusion proteins[END_REF] To perform a complete structural characterization in such a context, innovative analytical methodologies have a major role to play for the development and approbation of biosimilars, as well as for next generation mAbs. The possibility of obtaining a large variety of structural information over different aspects of the protein (e.g. AA sequence, posttranslational modifications, disulfide bonds, glycosylation), in an accurate and robust manner, is obviously necessary and could be an important asset for safety and product development. One illustration of that trend is the recent intensification of research work aiming to address new issues regarding mAbs characterization. [START_REF] Fornelli | Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry[END_REF][START_REF] Rosati | Qualitative and Semiquantitative Analysis of Composite Mixtures of Antibodies by Native Mass Spectrometry[END_REF][START_REF] Beck | Biosimilar, Biobetter, and Next Generation Antibody Characterization by Mass Spectrometry[END_REF] A crucial point that could significantly improve the biosimilar development pipeline is to obtain suitable structural information in order to quickly eliminate unsatisfying candidates. Mass spectrometry (MS) has progressively taken an important role in the characterization of therapeutic mAbs due to its outstanding selectivity, sensitivity and the possibility to obtain structural information. For example, characterization of therapeutic mAbs by MS has been applied to product and process development [START_REF] Ambrogelly | Characterization of antibody variants during process development: The tale of incomplete processing of N-terminal secretion peptide[END_REF][START_REF]Guideline on development, production, characterization and specifications for monoclonal antibodies and related products[END_REF] and batch consistency assessment. [START_REF] Damen | Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab[END_REF] However, MS still needs to be used in concomitance with orthogonal analytical techniques to be able to focus on the different facets of the protein including mainly liquid chromatography (LC). [START_REF] Rustandi | Use of CE-SDS gel for characterization of monoclonal antibody hinge region clipping due to copper and high pH stress[END_REF][START_REF] Michels | Charge Heterogeneity of Monoclonal Antibodies by Multiplexed Imaged Capillary Isoelectric Focusing Immunoassay with Chemiluminescence Detection[END_REF][START_REF] Wollacott | Analytical characterization of a monoclonal antibody therapeutic reveals a threelight chain species that is efficiently removed using hydrophobic interaction chromatography[END_REF][START_REF] Heudi | Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotopelabeled antibody standard and protein cleavage isotope dilution mass spectrometry[END_REF][START_REF] Beck | Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NSO cells[END_REF][START_REF] Vlasak | Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody[END_REF][START_REF] Fekete | Theory and practice of size exclusion chromatography for the analysis of protein aggregates[END_REF] Recently, we described a method using capillary zone electrophoresis (CZE) coupled to tandem MS with a sheathless interface to perform the simultaneous characterization of several aspects of a protein in one injection, including AA sequence and posttranslational modifications (PTMs). [START_REF] Gahoual | Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry[END_REF] The sheathless CE-ESI-MS interface, referred to as CESI-MS, has been developed based on an original design proposed by Moini et al. [START_REF] Moini | Simplifying CE-MS Operation. 2. Interfacing Low-Flow Separation Techniques to Mass Spectrometry Using a Porous Tip[END_REF] Detailed description of this interface has been given by Haselberg et al. [START_REF] Haselberg | Performance of a sheathless porous tip sprayer for capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins[END_REF] The CESI system has been used to perform successful CE-ESI-MS experiments in proteomics, [START_REF] Busnel | High Capacity Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry: Coupling a Porous Sheathless Interface with Transient-Isotachophoresis[END_REF][START_REF] Sarg | Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano-Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Post-translationally Modified Histones[END_REF] metabolomics, [START_REF] Ramautar | Enhancing the Coverage of the Urinary Metabolome by Sheathless Capillary Electrophoresis-Mass Spectrometry[END_REF] intact proteins, [START_REF] Haselberg | Performance of a sheathless porous tip sprayer for capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins[END_REF] therapeutic small molecules [START_REF] Bonvin | Evaluation of a sheathless nanospray interface based on a porous tip sprayer for CE-ESI-MS coupling[END_REF] and as an infusion platform, [START_REF] Gahoual | Novel sheathless CE-MS interface as an original and powerful infusion platform for nanoESI study: from intact proteins to high molecular mass noncovalent complexes[END_REF] and it demonstrated a drastically increased sensitivity compared to sheath-liquid CE-MS interfaces because of its low operating flow rates below 100 nL/min.

In the present work, we developed a transient isotachophoresis CE-ESI-MS/MS methodology, with the CESI-MS system, to first obtain the characterization of several mAbs. This extensive characterization over different levels of the protein, was performed in a single analysis of each sample. In a second time, each mAb was compared to a candidate biosimilar to establish if the methodology developed could be used to assess the biosimilarity between two samples. The comparison was made using the data from a single injection of each sample. Transient isotachophoresis (t-ITP) was introduced in this work: t-ITP is a preconcentration method widely used with CZE. [START_REF] Larsson | Transient isotachophoresis for sensitivity enhancement in capillary electrophoresis-mass spectrometry for peptide analysis[END_REF] The use of t-ITP enables, in the case of bottom-up proteomics, use of sample concentrations similar to those required for nanoLC-MS/MS analysis. [START_REF] Busnel | High Capacity Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry: Coupling a Porous Sheathless Interface with Transient-Isotachophoresis[END_REF] The mAbs included are trastuzumab, cetuximab and their respective candidate biosimilars.

Trastuzumab is a humanized immunoglobulin gamma 1 (IgG-1) directed against the HER2/neu receptor overexpressed in around 20-30% of invasive breast cancer (HER2 positive) and more recently to treat HER2+ gastric cancer. [START_REF] Li | Structural basis for inhibition of the epidermal growth factor receptor by cetuximab[END_REF][START_REF] Gunturu | Gastric cancer and trastuzumab: first biologic therapy in gastric cancer[END_REF] It was first approved as a therapeutic in the US and EU in 1998 and 2000, respectively. Trastuzumab is composed of two heavy chains (HC) and two light chains (LC) representing a total of 1328 AA. Trastuzumab HC bears an N-glycosylation consensus site on the asparagine (Asn) in position Asn-300 2 (Figure 1). Cetuximab is human/murine chimeric IgG-1 directed against the epidermal growth factor receptor (EGFR) overexpressed in advanced-stage EGFR positive colorectal cancer.

Cetuximab was approved in the US and EU in 2004 and 2005, respectively. It is composed of two HC and two LC as well for a total of 1326 AA and bears two N-glycosylation sites, both present on the HC.

One is located on the Asn-299 and the other is present on the Asn-88 [START_REF] Dubois | Immunopurification and Mass Spectrometric Quantification of the Active Form of a Chimeric Therapeutic Antibody in Human Serum[END_REF] (Figure 1). Note that mAbs' glycosylations are subject to extensive characterization and profiling because it significantly influences antibody -dependent cell -mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). [START_REF] Dalziel | Emerging principles for the therapeutic exploitation of glycosylation[END_REF][START_REF] Krapp | Structural Analysis of Human IgG-Fc Glycoforms Reveals a Correlation Between Glycosylation and Structural Integrity[END_REF][START_REF] Jefferis | Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action[END_REF] A tryptic digest of each sample was prepared using an adapted in-solution digestion protocol. The digests were then separated and characterized by t-ITP CESI-MS/MS. The peptide mapping was first performed using a peptide fragment fingerprinting (PFF) strategy similar to conventional bottom-up proteomics. Glycosylations were structurally characterized using MS/MS spectra and a semiquantitative profiling was established. Other posttranslational modifications (PTMs) of interest were investigated to complete the characterization of mAb samples. Finally trastuzumab and cetuximab CESI-MS/MS characterization data were compared to the data obtained for their respective biosimilar candidates in an equivalent perspective as required by regulation agencies to assess biosimilarity. This comparison was performed in order to determine if the developed t-ITP CESI-MS/MS methodology could be applied to demonstrate the biosimilarity between two mAbs in a robust and hastened manner, thereby improving biosimilars development pipeline.

 RESULTS AND DISCUSSION

Amino acid sequences characterization. AA sequence is a key criteria for demonstrating the biosimilarity of a candidate. Indeed, along with other attributes, regulations require that the AA sequence of a candidate is proven to be the same as the reference product in order to be assimilated as biosimilar.

Each mAb sample was digested by trypsin using an adapted digestion protocol in order to obtain a peptide mixture that could be characterized by CESI-MS/MS enabling demonstration/invalidation of the biosimilarity of two mAbs. Another aspect of the sample preparation is the source of the sample, reference mAbs (trastuzumab and cetuximab) in their commercial formulation were compared to two potential biosimilars (trastuzumab-B and cetuximab-B) in development that were sampled directly from the test bioreactor and purified, meaning that matrixes were significantly different. Sample preparation requires then cancelation of matrix effects, which then renders CESI-MS/MS data completely comparable.

For trastuzumab, the analysis of the tryptic digest using the CESI-MS/MS method allowed acquisition, in a single analysis, of 100% sequence coverage on both HC and LC while injecting only 32 ng of digested protein. In a previous work, we demonstrated the capacity to obtain the complete sequence coverage in a single analysis using CESI-MS/MS. [START_REF] Gahoual | Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry[END_REF] It is important to note, however, that significant improvement of the methodology enabled us to obtain the complete AA sequence characterization exclusively through the identification of peptides, without miscleavages nor posttranslational modifications, as emphasized in Figure 2. Trastuzumab-B was characterized using the same methodology. As shown in Figure 2, the AA sequence characterization of this biosimilar allowed demonstration of a complete AA sequence similarity to the reference protein except for HC lysine 217 (lys-217). Even by considering tryptic peptides with miscleavages, the AA lys-217 could not be confirmed, suggesting that this particular AA may be substituted in trastuzumab-B compared to the reference mAb. The interpretation of unassigned MS/MS spectra allowed us to demonstrate that the AA composing trastuzumab-B in position 217 was an arginine 8 instead of a lysine, as illustrated by the spectra in Figure 3. Several ions pointed out this particular AA substitution reinforcing the confidence of this interpretation (Figure 3). From a regulatory standpoint, the substitution of one AA does not fit with the highly similar paradigm, and thus trastuzumab-B would not be considered biosimilar by either the EMA or FDA. The same experiment was performed for cetuximab and cetuximab-B head-to-head. Once again, one single injection of cetuximab digest allowed the complete characterization of the AA sequence in this case as well through peptides without modifications nor miscleavages, demonstrating the robustness of the methodology. Cetuximab-B was produced in Chinese hamster ovary cells (CHO) in contrast to the reference product, which was produced in mouse myeloma cells (Sp2/0), but based on the same aminoacid sequence. The comparison of the CESI-MS/MS data, from unique injections of each sample, proved the complete equivalence of the AA sequence between cetuximab and its biosimilar candidate.

The experiment was repeated in three technical replicates and the same results were obtained in a single analysis of each mAb sample, demonstrating the reproducibility in term of characterization of the developed methodology. The CESI-MS/MS interface demonstrates its capacity to provide an impressive sensitivity, as it allows advanced characterization of the AA sequence using only 32 ng of digested mAbs.

Even more, the MS/MS data exhibited the capacity to retrieve systematically more than 70% of the y/b fragment ions. In the case of trastuzumab, more than 90% of the y/b ions were obtained, which resulted into the possibility to unravel the totality of the AA organization over the variable domain. That trend in MS/MS spectra quality, even on a MS using a TOF analyzer enabling ions accumulation only on a reduced time, is explained by the excellent ionization efficiency when coupling CE to MS through sheathless interfacing. Indeed, the low flow rate induced by the electroosmotic flow (< 40 nL/min) and the intrinsic characteristics of the CESI interface contribute to the formation of a nano electrospray (nanoESI). The nanoESI improves analyte ionization and reduces ion suppression effects, therefore impacting positively on MS/MS spectra quality. [START_REF] Gahoual | Novel sheathless CE-MS interface as an original and powerful infusion platform for nanoESI study: from intact proteins to high molecular mass noncovalent complexes[END_REF] The capacity to obtain the totality of the y/b ions, meaning by deduction the AA succession order, for the variable domain could be a huge asset in mAbs development because this part of the protein is directly responsible for antigen epitope recognition, and is therefore crucial for mAbs potency.

Glycosylation structural characterization and semiquantitative profiling. Glycosylation is a PTM that occurs naturally during excretion of antibodies from the expression system to the extracellular medium. It only represents ~ 3% of the total mass of the protein but is subject to extensive studies due to its significant influence on effector functions of mAbs, i.e., ADCC and CDC.

No glycan release was performed during the sample preparation and glycans could be characterized directly on the corresponding digested peptide. In the case of the trastuzumab/trastuzumab-B biosimilarity study, the injection of the reference mAb allowed identification of 16 different glycoforms borne by trastuzumab. Alongside of glycans structural characterization, the semiquantitative glycoforms profiling was performed for trastuzumab and its biosimilar candidate as shown in Figure 4. To estimate glycoforms distribution, maximum intensities of each charge states of the glycopeptides were considered. The glycoprofiling established for the protein demonstrated the possibility to access lowly abundant glycans which is of interest in comparability and biosimilarity studies.

These results suggest the compatibility between the MS dynamic range and the CE loading capacity.

Note also that the glycosylation profiling established is consistent to data available in the literature. [START_REF] Stadlmann | Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides[END_REF] Even subtle glycoforms distribution differences between trastuzumab and the candidate biosimilar could be distinguished from the results. Trastuzumab-B was also shown to exhibit a rather different glycoforms distribution while carrying no glycans containing sialic acid (Figure 4). domain and common to all IgGs, while the second one in the Fd domain. [START_REF] Qian | Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrixassisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion[END_REF] Focusing on the glycopeptide allowed us to distinguish glycans with regard to their glycosylation sites, which cannot be done when using methodologies involving glycans release. In this study as well, glycosylation profiles established demonstrated the identification of a significant number of glycosylation on each sites as shown in Figure 4-B and Figure 5. Significant differences in the glycosylation profile between cetuximab and its biosimilar candidate could be characterized, showing the applicability of the method to biosimilarity assessment (Figure 4-B). Several cases of hypersensitivity to cetuximab were reported in the literature; the cause of that side-effect was related to the galactose-α-1,3-galactose present on the Fd glycosylation site of the protein. [START_REF] Chung | Cetuximab-Induced Anaphylaxis and IgE Specific for Galactose-α-1,3-Galactose[END_REF] For the marketed version of cetuximab produced in SP2/0 cells, 18 different glycoforms were identified, with 88% capped by at least one alpha-1,3-galactose residue, 23% capped by a Nglycolylneuraminic acid (NGNA) residue and traces of oligomannose. Development of a cetuximab biobetter should take into account that side-effect in order to prevent the occurrence of such glycosylation and provide an improved product, e.g. by production in CHO cells. Fd glycosylation profiles established from CESI-MS/MS data demonstrated that cetuximab-B would not be a suitable biosimilar candidate but a possible biobetter. Thus, the glycans identified on the Fd glycosylation site of cetuximab-B were significantly different from those present on the innovator mAb. The profile established for cetuximab-B therefore demonstrated that the abundance of glycoforms containing galactose-α-1,3-galactose could be significantly reduced (Figure 5). Highly sensitive glycosylation semiquantitation is often performed by removing the glycans using specific enzymes. [START_REF] Wagner-Rousset | The way forward, enhanced characterization of therapeutic antibody glycosylation: Comparison of three level mass spectrometry-based strategies[END_REF] Results proved that the methodology used in this study additionally to reduce sample treatment, lowering the potentiality of artefacts, allows researchers to specifically locate the glycosylation sites and study them independently, with a sensitivity challenging already applied methods. The adopted strategy has allowed, concomitant with AA sequence characterization, to demonstrate/disconfirm the biosimilarity regarding the glycosylation profile between reference mAbs and their candidate biosimilar.

PTMs hot-spots comparison study. Other PTMs besides glycosylation must also be considered in biosimilarity assessment. PTMs referred as hot-spots are described in the literature as being induced by changes of the protein structure. Some of them classified as CQAs may influence the immunogenicity, the PK/PD of the protein which explains the analytical developments achieved to perform their characterization [START_REF] Harris | Identification of multiple sources of charge heterogeneity in a recombinant antibody[END_REF][START_REF] Diepold | Simultaneous Assessment of Asp Isomerization and Asn Deamidation in Recombinant Antibodies by LC-MS following Incubation at Elevated Temperatures[END_REF] . It is therefore necessary to prove that none of those PTMs are over represented in a biosimilar candidate, including N-terminal glutamine/glutamic acid cyclization leading to the formation of N-terminal pyroglutamic acid (pE), asparagine deamidation (deaN), methionine oxidation (oxiM) and aspartic acid isomerization (isoD) on various position depending on the considered mAb. 2 CESI-MS/MS data were also used to characterize the PTM hot-spots of the different mAbs samples prepared. PTMs occurrence levels were estimated from the data and finally compared between a biosimilar candidate and the corresponding originator antibody. Data showed that every single PTM hot-spot monitored could be successfully characterized (Table 1).

The use of electrophoretic separation is particularly pertinent to the characterization of PTMs. Indeed, PTMs induced significant change of electrophoretic mobilities between the modified digested peptides and its unmodified homologs. Therefore, those peptides could be separated by the electrokinetically driven separation of CE, from 0.5 min in the case of deaN to several minutes for pE. The use of CE as a separating technique is clearly of great interest. As emphasized in Figure 6, it was possible to separate the same peptide differing solely by the conformation of its aspartic acid. The separation in this particular modification allows additional information regarding the characterization to be obtained. Indeed, the isomerization of aspartic acid does not involve a mass change of the peptide and cannot be identified by MS using a time-of-flight (TOF) analyzer. Moreover, the ability to separate, upfront to MS analysis, a peptide that experienced chemical modifications from the same peptide without modification allows sensitivity to be maximized. In the case of co-migration, those peptides would compete against each other during the ionization process. Signal is improved by their separation and occurrence levels accuracy theoretically optimal. Specificity of CE separation enables the separation of digested peptides experiencing aspartic acid isomerization; that characteristic is particularly valuable as the characterization of such modification requires particular methods giving access solely to this information, [START_REF] Vlasak | Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody[END_REF] in contrast, here the study of aspartic acid isomerization could be incorporated within the framework of the overall primary structure characterization. The excellent MS/MS spectra quality obtained was particularly useful for PTM hot-spots characterization as it was possible to point out precisely the AA affected by the modification. PTM hot-spots semiquantitative measure was performed for each sample and occurrence levels were compared between the reference mAb and its respective candidate biosimilar as emphasized in Table 1 and2. In this part, relative occurrence levels were estimated from the maximum intensities of the ions corresponding to the modified peptides compared to the abundance of the corresponding unmodified peptide. In the context of the trastuzumab/trastuzumab-B biosimilarity study, results showed that endogenous deamidation due to proteolytic digestion conditions could be significantly controlled as several deamidation sites exhibited modifications levels below 7% and even 4% for deaN 55 (Table 1). Results demonstrated the capacity of this methodology to distinguish significant variation of levels. results showed that isoD-170 (LC) abundance was superior to 13.4% in trastuzumab while its candidate biosimilar exhibited only 7.1% on this modification (Table 1). A similar trend could also be unraveled with the presented results for isoD-167 (LC). These high levels of modifications were correlated to the prolonged time the sample of reference trastuzumab had been stored. Regarding deaN, results of trastuzumab/trastuzumab-B showed dissimilarities for deaN-387 (HC) only, indeed trastuzumab-B exhibited less than 1% of this modification. OxiM results obtained from this study proved similar level of oxidation on the designated sites (Table1). Relative occurrence levels of PTM hot-spots were also studied and compared in the case of cetuximab/cetuximab-B. Results obtained were compiled in Table 2. As in the first study, results obtained allowed successful characterization of each of the PTMs hot-spots present on cetuximab. Results of the semiquantitative analysis regarding PTMs hot-spots occurrence were compared. Notably, both mAbs demonstrated specific characteristics supporting their similarity: N-terminal glutamine (Gln) cyclization was shown to be complete because the intact peptide could not be detected in samples of either mAbs.

This trend could also be confirmed on the HC for isoD 403 and deaN 158 (Table 2); indeed those modifications exhibited absolutely no occurrence in cetuximab or its candidate biosimilar, illustrating the similarity of those proteins on such specific aspect. Cetuximab, as opposed to cetuximab-B, demonstrated slightly superior deamidation levels in the case of deaN 172 and deaN 386 for the HC and deaN [START_REF] Wagner-Rousset | The way forward, enhanced characterization of therapeutic antibody glycosylation: Comparison of three level mass spectrometry-based strategies[END_REF] on the LC (Table 2), which were correlated with a longer conservation period for the reference mAb sample.

However, these results illustrate the sensitivity of the method, which allows only minor variations in PTMs occurrence levels to be distinguished. Regarding the biosimilarity assessment of PTM hot-spots, results did not demonstrated overexpressed modifications for the biosimilar candidate compared to their respective innovator mAbs showing they could comply with the guidelines on this particular aspect. The characterization strategy developed in this study involved the use of CESI-MS/MS analysis. Data obtained from only one injection of each sample tryptic digest allowed characterization of each mAbs simultaneously over several levels defining their primary structure: AA sequence, glycosylation structure/relative abundance and specific PTMs hot-spots. The purpose was to study the biosimilarity between two approved mAbs (trastuzumab and cetuximab) and two respective biosimilar candidates.

position sequence PTM Cetuximab distribution Cetuximab-B distribution unmodif modif unmodif modif 1 -5 QVQLK Q 1 /
Biosimilarity study results unraveled that in both cases, biosimilar candidates would hardly be considered as such according to current regulations. In the case of trastuzumab-B, one AA difference compared to the original mAb led to this conclusion, while in the case of cetuximab-B, major glyco-profile variations were highlighted. Among the biosimilarity assessments performed successfully for each mAbs, data collected during the CESI-MS/MS analysis pointed out specifically which attribute of the protein did not comply with the reference mAb giving precious information from an R&D perspective. For example, the difference of one AA for trastuzumab-B strongly supports the need of re-engineering of the vector to produce the right allotype. [START_REF] Jefferis | Human immunoglobulin allotypes: Possible implications for immunogenicity[END_REF] However, in the case of cetuximab-B, each aspect of the primary structure of the mAb demonstrated the biosimilarity except for the glycosylation. Yet recent advances in mAbs glycoengineering permits optimization of the mAbs glycoform expression, thereby suppressing undesired glycosylations. [START_REF] Beck | GlycoFi's technology to control the glycosylation of recombinant therapeutic proteins[END_REF][START_REF] Beck | Marketing approval of mogamulizumab A triumph for glycoengineering[END_REF] In summary, the CESI-MS/MS strategy developed in the context of this study appeared to be fully compatible with use as an orthogonal technique concomitantly to the now commonly used analytical techniques for characterization of mAbs, biosimilar, biobetters and, more generally, biotherapeutic proteins.

 CONCLUSIONS

We report here an innovative strategy involving CE coupled to high resolution tandem MS to characterize mAbs simultaneously over different angles of their primary structure, in order to perform biosimilarity assessments between approved mAbs (trastuzumab and cetuximab) and candidate biosimilars. Prior to analysis, samples were digested by trypsin using an adapted in-solution digestion protocol allowing improvement in digestion yield and a standardized final sample content compatible with CE separation. The analysis of each sample allowed establishment of the similarity/dissimilarities concerning AA sequence between several approved mAbs and their respective biosimilar candidates.

The sensitivity and MS/MS spectra quality obtained, mainly supported by the instrumental setting used in this work, enabled AA sequence characterization on an unprecedented level as 100% sequence coverage could be obtained in a single analysis through identification of only tryptic peptides without miscleavages or PTMs. Furthermore more than 70% of the y/b ions could be systematically retrieved, providing detailed structural data over this aspect of the protein. Along with this characterization, structural and semiquantitative analysis of the glycoforms could be performed, which demonstrated the capacity of the method to locate precisely different glycosylations sites. The glycoforms profiling obtained demonstrated the capacity of the established methodology to detect quite low abundance glycans, enabling the detection of potentially undesired glycoforms. Likewise the profiling accuracy was also highlighted as relative abundances obtained could be correlated to other methods. CESI-MS/MS data enabled subtle differences in glycoforms distribution between reference mAbs and their candidate biosimilars to be distinguished, proving that the dynamic range achievable with CE-ESI-MS coupling is fully compatible with this type of analytical problem. Finally, specific PTMs hot-spots were characterized using the very same CESI-MS/MS data used to characterize mAbs samples previously; results demonstrated the capacity of the method to characterize precisely the targeted sites.

The sensitivity provided by the CESI-MS system allowed, in most cases, the precise identification of the AA affected by the PTMs even in the circumstances of weakly abundant modifications. CE separation specificity proved valuable because peptides with chemical modification, as weak as aspartic acid isomerization, could be separated, enabling their concomitant characterization. The results illustrated the capacity of the method to observe differences in modification levels between the reference mAb samples and their respective candidate biosimilars studied. Data obtained from CESI-MS/MS analysis established dissimilarities that existed amongst the candidate biosimilars compared to the approved mAbs, demonstrating the ability of the developed methodology to address such analytical issues in a structured and comprehensive way while easing sample treatment and reducing the number of experiment required.

Furthermore, results permitted identification of precisely which aspects of the candidate antibody did not comply with their assignment as biosimilars providing important structural information that could be used to strategically plan subsequent R&D work. This study illustrates without ambiguity the possibility of using CE coupled to MS as an orthogonal technique, along with techniques routinely applied, to address complex analytical issues such as mAbs biosimilarity assessment, and points out more generally the capacity of this type of coupling to characterize biotherapeutic proteins.

available by Sciex separation (Brea, CA, USA). Prototype of bare fused-silica capillaries (total length 100 cm; 30 μm i.d.) with a characteristic porous tip on its final 3 cm supplied by Sciex separation, a second capillary (total length 80 cm; 50 μm i.d.) filled during experiments with BGE allows electric contact. New capillaries were flushed at 75 psi (5.17 bar) for 10 min with methanol, 10 min with 0.1 M sodium hydroxide, then 10 min with 0.1 M hydrochloric acid and water for 20 min. Finally, the capillary was flushed 10 min at 75 psi with 10% acetic acid, which is the BGE used for the separation. Hydrodynamic injection (410 mbar for 1 min) corresponding to a total volume of 90 nL of sample injected was used.

Separations were performed using a voltage of +20 kV.

Mass spectrometry. For antibody characterization, the CESI system was hyphenized to a 5600

TripleTOF mass spectrometer (ABSciex, Darmstadt, Germany). The 5600 MS is equipped with a hybrid analyzer composed of quadrupoles followed by a time-of-flight (TOF) analyzer. ESI source parameters were set as follows: ESI voltage -1.75kV, gas supplies (GS1 and GS2) were deactivated, source heating

Figure 1 .

 1 Figure 1. Schematic representation of trastuzumab and cetuximab. Blue parts of the protein represent the constant domain of the mAbs. Yellow parts of the scheme represent the variable domain of the mAbs and red parts represent the complementary determining region (CDR).

Figure 2 .

 2 Figure 2. Sequence coverage obtained by CESI-MS/MS for trastuzumab (left-hand side) and trastuzumab-B (right hand side) showing only one amino acid (Lys-217) unidentified in the case of the candidate biosimilar. Experimental conditions: bare fused silica capillary (95cm; 30µm i.d.); sample mAbs tryptic digest 2.22 µM; injection volume 90nL.

Figure 3 .

 3 Figure 3. Raw MS/MS, extracted from trastuzumab-B CESI-MS/MS analysis, presenting fragmentation of ions m/z 517.3095 (1+) and m/z 314.6937 (2+) characterizing the amino acid substitution between trastuzumab and its candidate biosimilar. VDKK217VEPK for trastuzumab (G1m17 allotype) and VDKR217VEPK in the case of trastuzumab-B (G1m3 allotype).

Figure 4 .

 4 Figure 4. Glycoforms semiquantitative analysis results obtained through the CESI-MS/MS data for the Fc peptide of trastuzumab/trastuzumab-B and cetuximab/cetuximab-B. Glycoform profiling could be compared between original mAbs and their candidate biosimilar.

Figure 5 .

 5 Figure 5. Glycoform semiquantitative analysis results regarding Fd domain glycosylation of cetuximab and cetuximab-B.

Figure 6 .

 6 Figure 6. Extracted Ion Electropherogram (EIE) corresponding to m/z (839.408, 2+) for digested peptide HT22 (position 278-291) experiencing aspartic acid isomerization.

temperature 150°C and curtain gas value 5 .

 5 Experiments were performed in Top15 information dependent acquisition (IDA), accumulation time was 250 msec for MS scans and 100 msec for MS/MS scans leading to a total duty cycle of 1.75 sec. Mass/charge (m/z) range was set to 100-2000 in MS and 50-2000 in MS/MS. Using those parameters, the mean resolution provided by the instrument is 40000 in MS (m/z 485.251) and 25000 in MS/MS (m/z 345.235). MS/MS data analysis. Data obtained from the CESI-MS/MS experiments were analyzed using Peakview software (ABSciex, Darmstadt, Germany). Tryptic peptides (without miscleavages or PTMs except cys carbamidomethylation) were determined theoretically from the mAbs' amino acid sequences available through literature. 23, 47 Additional peptides were identified using Mascot search engine provided by Matrix science; tryptic cleavage rules were applied. Carbamidomethylation of cysteine (+57.02 Da), N-deamidation of aspartic/isoaspartic acid (+0.985 Da) or succinimide intermediate (-17.03 Da), methionine oxidation (+15.99 Da) and N-terminal glutamic acid cyclization (-17.02 Da) were selected as 

Figure 1 .

 1 Figure 1. Schematic representation of trastuzumab and cetuximab. Blue parts of the protein represent the constant domain of the mAbs. Yellow parts of the scheme represent the variable domain of the mAbs and red parts represent the complementary determining region (CDR).

Figure 2 .

 2 Figure 2. Sequence coverage obtained by CESI-MS/MS for trastuzumab (left-hand side) and trastuzumab-B (right hand side) showing only one amino acid (Lys-217) unidentified in the case of the candidate biosimilar. Experimental conditions: bare fused silica capillary (95cm; 30µm i.d.); sample mAbs tryptic digest 2.22 µM; injection volume 90nL.

Figure 3 .

 3 Figure 3. Raw MS/MS, extracted from trastuzumab-B CESI-MS/MS analysis, presenting fragmentation of ions m/z 517.3095 (1+) and m/z 314.6937 (2+) characterizing the amino acid substitution between trastuzumab and its candidate biosimilar. VDKK217VEPK for trastuzumab (G1m17 allotype) and VDKR217VEPK in the case of trastuzumab-B (G1m3 allotype).

Figure 4 .

 4 Figure 4. Glycoforms semiquantitative analysis results obtained through the CESI-MS/MS data for the Fc peptide of trastuzumab/trastuzumab-B and cetuximab/cetuximab-B. Glycoform profiling could be compared between original mAbs and their candidate biosimilar.

Figure 5 .

 5 Figure 5. Glycoform semiquantitative analysis results regarding Fd domain glycosylation of cetuximab and cetuximab-B.

Figure 6 .

 6 Figure 6. Extracted Ion Electropherogram (EIE) corresponding to m/z (839.408, 2+) for digested peptide HT22 (position 278-291) experiencing aspartic acid isomerization.

Table 1 .

 1 Table summarizing the study regarding PTM hotspots occurrence levels for trastuzumab/trastuzumab-B obtained by CESI-MS/MS for each tryptic digest, modification levels (distribution % intact / % modification) were determined through signal intensity for each targeted PTM.

				Trastuzumab	Trastuzumab-B
	position	sequence	PTM	distribution	distribution
				unmodif modif unmodif	modif
	1 -19	EVQLVESGGGLVQPGGSLR	E 1 / pE 1	98.2	1.8	97.3	2.7
	51 -59	IYPTNGYTR	N 55 / deaN 55	89.4	10.6	92.3	7.7
	99 -124	WGGDGFYAMDYWGQGTLVTVSSASTK D 102 / isoD 102	84.9	15.1	86.0	14.0
	252 -258	DTLMISR	M 255 / oxiM 255	95.3	4.7	94.5	5.5
	259 -277	TPEVTCVVVDVSHEDPEVK	D 272 / isoD 272	94.4	5.6	94.5	5.5
	278 -291	FNWYVDGVEVHNAK	D 283 / isoD 283	92.0	8.0	96.5	3.5
	374 -395	GFYPSDIAVEWESNGQPENNYK	N 387 / deaN 387	85.8	14.3	100.0	0.0
	420 -442	WQQGNVFSCSVMHEALHNHYTQK	M 431 / oxiM 431	97.8	2.2	95.2	4.8
	396 -412	TTPPVLDSDGSFFLYSK	D 404 / isoD 404	93.0	7.0	93.9	6.1
	25 -42	ASQDVNTAVAWYQQKPGK	N 30 / deaN 30	96.1	3.9	96.0	4.0
	150 -169	VDNALQSGNSQESVTEQDSK	D 167 / isoD 167	64.0	36.0	91.6	8.4
	170 -183	DSTYSLSSTLTLSK	D 170 / isoD 170	86.6	13.4	92,9	7.1

Table 2 .

 2 Table summarizing the study regarding PTM hotspots occurrence levels for cetuximab/ cetuximab-B. Using the CESI-MS/MS data for each tryptic digest, modification levels (distribution % intact / % modification) were determined through signal intensity for each targeted PTM.

	pQ 1	100.0	0.0	100.0	0.0

Table 1 .

 1 Table summarizing the study regarding PTM hotspots occurrence levels for trastuzumab/trastuzumab-B obtained by CESI-MS/MS for each tryptic digest, modification levels (distribution % intact / % modification) were determined through signal intensity for each targeted PTM.

Table 2 .

 2 Table summarizing the study regarding PTM hotspots occurrence levels for cetuximab/ cetuximab-B. Using the CESI-MS/MS data for each tryptic digest, modification levels (distribution % intact / % modification) were determined through signal intensity for each targeted PTM.

 MATERIALS & METHODS

Materials. Chemicals used were of analytical grade or high purity grade and purchased from Sigma-Aldrich (Saint Louis, MO, USA). Water used to prepare buffers and sample solutions was obtained using an ELGA purelab UHQ PS water purification system (Bucks, UK). Trastuzumab and cetuximab samples are the EMA approved products and formulation purchased from Roche (Penzberg, GE) and Merck KGaA (Darmstadt, GE) respectively. The biosimilar versions were produced at the Centre d'Immunologie Pierre Fabre (Saint Julien en Genevois, FR) for analytical methods development. Marketed mAbs were stored at 4°C for one year before characterization while biosimilar versions was analyzed soon after their reception (approximately 2 months), otherwise all samples were stored at 4°C. RapiGest SF surfactant was purchased from Waters (Milford, MA, USA).

Sample preparation.

A volume corresponding to 100 µg of protein were used. Samples were first diluted using milliQ water to a final concentration of 6.7 µg/µL. Samples were then diluted using 0.1% RapiGest surfactant to reach a final concentration of 3.35 µg/µL and heated to 40°C during 10 min. Dithiothreitol (DTT) was added to the sample to a final concentration of 25 mM. Samples were then heated for a 5 min incubation at 95°C. After being cooled to room temperature (RT), iodoacetamide (IDA) was added to the sample to a final concentration of 10 mM and samples were kept in the dark for 20 min to allow alkylation of cysteines (Cys). A volume of 1 µL of trypsin (0.5µg/µL) was added to the sample which was left at room temperature for 3h and another volume of 1µL was added after this time. Digestion was performed overnight at 37°C. After digestion completion, 1% (v/v) formic acid (FA) was added to the samples, which were left at RT in order to cleave the surfactant. Samples were finally diluted to a final concentration of 2.2 µM of protein using ammonium acetate 50 mM (pH 4.0).

Capillary electrophoresis.

The CE separations were performed with a PA 800 plus capillary electrophoresis system from Beckman Coulter equipped with a temperature controlled autosampler and a power supply able to deliver up to 30 kV. Hyphenation was carried out using a CESI prototype made