The potential role of antibodies (Abs) in the treatment of diseases dates back to the end of 1800, when the German physiologist Emil Adolf von Behring discovered that small amounts of diphtheria toxins were able to immunize animals against the pathology [1]. However, Emil Adolf von Behring did not attribute this propriety to Abs and several years were necessary to well understand their role, structure, and potentiality. With the large-scale monoclonal Ab (mAb) production started with the hybridoma technique [2], mAbs have become one of the most popular drug candidates. The first mAb approved by the United States Food and Drug Administration (FDA) was the Muromonab-CD3 in 1986, while the first fully human mAb (adalimumab) was approved in 2002 for the treatment of rheumatoid arthritis [1]. mAbs are not only a therapeutic instrument, but represent one of the most commonly used tools in laboratory research, being at the basis of many techniques and approaches (e.g., Western blot, immunochemistry, flow cytometry, immunoprecipitation, ELISA). It is clear that more and more interests are growing around mAbs and their potential use. In this framework, processes related to mAbs development, production, and distribution need to be controlled and reproducible. Mass spectrometry (MS) represents one of the most powerful tools to analyze high complex molecules as mAbs. With high reproducibility, specificity, sensitivity and high-throughput features, MS has become a very used technique in the mAbs research field. For example, Arboleda-Velasquez JF et al verified the identity of the ApoE3 monoclonal antibody 1343A before its usage [3]. This review focus on principal quality issues in mAbs research and some MS-based methods useful to check the quality and to quantify mAbs batches during their development and commercial production.

Therapeutic antibodies are glycoproteins belonging to the immunoglobulin (Ig) family. Igs consist of 2 heavy and 2 light polypeptide chains (HC and LC, respectively) covalently linked together to form the typical Y shape. Both HC and LC contain variable domains (VH for HC and VL for LC) and constant domains (CH1, CH2 and CH3 for HC and CL for LC) (Fig. 1A). Antigen binding is mediated by specific sites called complementarity determining regions (CDRs) within the variable domains of both HC and LC. The fragment antigen binding (Fab) and fragment crystallizable (Fc) regions are within the papain cleavage sites. Therapeutic mAbs are commonly based on human or mouse IgG (Fig. 1B). Murine mAbs (suffix: -omab) were produced by hybridomas and introduced in the clinical environment at the end of 1980 [4], but diversity between murine and human immunology led first to the development of chimeric mouse-human mAbs with about 65% human composition (suffix: -ximab) and then to humanized mAbs with about 95% human composition (suffix: -zumab). Fully human mAbs (suffix: -umab) were finally obtained with new technologies such as phage-display and genetic manipulation [5].

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Figure 1. (A) Schematic general structure of mAb. For detail see the main text. (B) Schematic structure of a murine antibody (-omab), chimeric antibody (-ximab), humanized antibody (-zumab) and fully human antibody (-umab). Yellow and blue represent murine and human sequences, respectively.

Being one of the most commonly used tools in transversal research laboratories and one of the most promising biopharmaceuticals, mAbs represent an intersection of interests that often converge on some challenging points related to the quality of these products. How reproducible are different batches? How pure is a preparation? How homogeneous is the product? Are there some modifications, such as post-translational modifications (PTMs), different glycosylation patterns? Is it really the concentration? Those are only a few common questions about mAbs development and production. Even if there are many international guidelines for mAbs production, quality and distribution, recently the urgency for high reproducibility of mAbs stood out [6], along with several suggestions to improve quality issues in antibody research [7].

The European Medicines Agency (EMA)’s guideline about the “Production and Quality Control of Monoclonal Antibodies” clearly requests that “the mAbs should be characterized thoroughly” [8]. The suggested characterization may be accomplished by the analysis of critical quality attributes (CQAs) of biopharmaceuticals that have been described by the International Conference on Harmonization Guideline Q8 as a “physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality” [9]. CQAs thus include several characteristics such as identity, purity, composition, structural features and biological activity. These parameters which should be strictly monitored during bioprocessing steps, can be obtained by combining multiple analytical approaches, always more represented by mass spectrometry-based methods, as discussed below. Chemical and structural characterization is often used to assess the CQAs of mAbs, for product development and regulatory acceptance. Even the characterization of multiple batches is of utmost importance to show to the regulatory body that the production process and the manufacturer have been controlled. This is achieved by a comparative analysis of batches, and thus comparing the obtained data. If present, significant differences among batches need to be investigated. It is clear that a comparative analysis is easier, stronger and more reproducible with high-throughput instruments as modern mass spectrometers.

The EMA mAbs guidelines also highlight the importance of several structural features including aggregation state, N- and C-termini (pyroglutamic acid at the N-terminus and lysine at the C-terminus of the heavy chain), free sulfhydryl and disulphide bridge structures, glycosylation (the degree of mannosylation, galactosylation, fucosylation, and sialylation), and some specific PTMs such as deamidation, oxidation, isomerization. All these parameters can be monitored by using specific MS approaches, especially thanks to the combination of high resolution and accuracy in measurements. MS-based approaches, discussed below, represent a promising approach for mAbs characterization; modifications such as oxidation (+16 Da), deamidation (+1 Da), and reduction of disulfide bridges (+2 Da) can be easily detected.

Mass spectrometry (MS) approaches include several analytical techniques based on the principle that ionized molecules can be sorted on the basis of their mass/charge ratio. Nowadays MS-based and proteomics techniques are widely used in a plethora of fields having both qualitative and quantitative purposes. With high reproducibility, specificity, sensitivity and high-throughput features, MS is one of the most versatile technologies and has become thoroughly used in the biopharmaceutical research field.

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Figure 2. Schematic representation of the components of a mass spectrometer.

Usually, a mass spectrometer consists of three parts: the ion source, the mass analyzer, and the detector (Fig. 2). The ion source is responsible for the ionization of the molecule (or the part of the molecule) of interest in liquid, gas or dried form. There are several kinds of ionization, such as the electron impact (EI), Fast Atom Bombardment (FAB), Electrospray Ionization (ESI, one of the most popular), Matrix Assisted Laser Desorption Ionization (MALDI). Obtained ions are then sorted and separated by electric and/or magnetic fields according to their mass and charge in the mass analyzer. Even in this case, there are several kinds of mass analyzers and the most commonly used include time-of-flight (TOF), quadrupoles, orbitraps and ion traps. The separated ions hit the detector and are then measured, resulting in a typical mass spectrum showing the mass to charge ratio and the relative abundance of the analyzed ions.

MS analysis can be targeted or untargeted. In the targeted approach one single known protein is qualitatively and quantitatively characterized, even if present in complex matrices. Publication guidelines exist for targeted mass spectrometry measurements of peptides and proteins [10]. The untargeted approach aims to characterize the whole proteome, defined as the set of all the proteins expressed by a cell or a tissue in a defined moment [11]. A proteome can be extremely complex, covering several orders of magnitude in terms of protein concentration for thousands of proteins. To reduce this complexity, often MS instruments are coupled with on-line or off-line fractionating systems (e.g., HPLC, 2D-page, etc.) [12].

Both these two kinds of analysis can be applied in two principal proteomics approaches to analyze proteins and thus mAbs of interest: top-down and bottom-up. The top-down approach allows the global study of intact proteins or mAbs by using native ESI, ion-mobility and subsequent fragmentation in the mass spectrometer [13], in order to have an overall view of the molecule. On the other hand, the bottom-up approach is the most classical used for protein identification and the determination of modification such as PTMs. In the classical approach, the molecule of interest is digested by enzymes like trypsin, chymotrypsin, etc. and obtained peptides are analyzed by ESI-TOF or MALDI-TOF.

These two approaches are complementary and their combination results in a compelling strategy able to produce detailed information on CQAs of mAbs. In fact, MS has become one of the principal tools to deep analyze mAbs throughout their process development and quality control [14].

Top-down approaches allow the analysis of intact proteins, even with high molecular weight [15]. This kind of analysis does not require the enzymatic digestion for example with trypsin, thus making sample preparation easier and more reproducible. Moreover, mAbs are more close to their physiological form, preserving information such as aggregation and/or oligomeric state, complexity, PTMs, stoichiometry, shape and high order structure (HOS). In Table 1 are summarized the principal strategies that can be used for the MS top-down analysis of mAbs.

Overall, top-down approaches have several advantages, as a short, less expensive and less invasive sample preparation, the in-depth structural characterization (e.g., identification of isoforms, PTMs site determination), localization of non-covalent ligands, information on the native state of mAbs. However, there are some disadvantages, the first one is the limited sensitivity and throughput in comparison with classical MS bottom-up approaches. Often for a reliable analysis pure samples are required, thus restricting the possibility of study at the proteomic level. Finally, the instrumentation used is often very expensive (Fig. 3).

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Figure 3. Principal advantages and disadvantages of Top-down MS approach for the study of mAbs.

The native state of a protein and thus of mAbs is its properly folded and assembled conformation that is operative and biologically functional. This state is strictly linked to the buffer in which mAbs is solubilized. One of the approaches used to keep mAbs in their native state is the exchange of the native buffer with a volatile ammonium acetate solution prior to performing the MS analysis [16]. The ammonium acetate solution favors solvent and salt evaporation during the ionization process by ESI. In Fig. 4 is reported a comparison between normal and native ESI of a 148 kDa antibody. The normal ESI produces a broad charge state distribution, while native ESI produces a narrow distribution.

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Figure 4. Normal and native ESI obtained for an antibody of approximatively 150 kDa. In normal ESI 0.1% acid in H2O/acetonitrile (50/50) was used as a solvent, while aqueous NH4OAc (pH6-9) was used as a solvent for native ESI.

This approach has been revealed to be useful to investigate noncovalent interactions, higher-order structures as dimers, trimers, etc. and the presence of aggregates. Aggregation is one of the most common types of degradation for mAbs and can occur during production, formulation and storage [17]. Temperature variation, pH changes and inappropriate transportation are among the common causes of aggregation [18, 19] along with partial unfolding and other conformational changes [20]. Aggregation of mAbs is highly undesired considering that it can lead to the decrease of the biological activity and solubility, and the increase of immunogenicity [21]. Kukrer et al proposed a combined chromatographic separation of mAbs oligomers with native ESI-TOF MS, thus separating monomer, dimer and trimer/tetramer fractions [22]. Moreover, the Exactive Plus Orbitrap with extended mass range (EMR) product by Thermo Fisher Scientific can be successfully used to characterize mAbs, mAbs conjugated with drugs, mAbs–antigen complexes and mixtures of mAbs. Recently, several detailed protocols for the analysis of the structural heterogeneity in mAbs using native mass spectrometry have been proposed [23, 24]. It is also possible to study the oxidation state of mAbs, as recently demonstrated by Haberger et al, in order to assess the effect of oxidative stress [25] or by Sokolowska I et al to provide quality control for release and stability testing of an antibody [26].

An intact protein introduced in the mass spectrometer into the gas phase can be indeed analyzed by using the ion-mobility spectrometry-mass spectrometry (IMS-MS), a technique widely used for protein structural analysis [27]. In this case, an ionized sample is conducted in a drift tube or in a traveling wave containing an electric field and a carrier buffer gas. Ions are moving through the matrix, being separated according to their size, shape, and charge. The IMS is coupled with a high-resolution mass spectrometer able to fragment in MS/MS gathering more structural and conformational information. The first use of IMS-MS for antibodies was realized by Bagal et al to distinguish between two isoforms of mAbs that differed in the disulphide bridges position [28]. IMS-MS can be applied to separate co-population of protein conformers with the same mass/charge ratio but that differ in shape and physical size [29]. Recently Ferguson et al applied IMS-MS for a comparative analysis of mAbs. They evaluated the reproducibility of IMS-MS measurements within mAbs, manufacturers, and lots, showing that IMS-MS is able to detect differences between mAbs against the same target protein but different production techniques. Ion mobility MS resulted in a very robust and reproducible approach to assess the CQAs of mAbs [30].

Intact ionized mAbs in the gas phase can be further characterized by MS/MS fragmentation onto hybrid high resolution mass spectrometers, gathering information on the primary structure and specific modifications sites. Several techniques can be used to obtain ions: collision-induced dissociation (CID) [31], electron-capture dissociation (ECD) [32], electron-transfer dissociation (ETD) [33], infrared multiphoton dissociation (IRMPD) [34], surface-induced dissociation (SID) [35] and others. This approach is particularly useful in the determination of the exact site of modifications such as PTMs. In fact, even if the monitoring of the mass changes can be used to study PTMs, the only analysis on intact mAbs cannot reveal the amino acid site involved in the PTM. However, the combination of top-down methods and MS/MS fragmentation can furnish the complete structural information about mAbs and PTMs. For example, ECD in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer has been used to characterize flexible regions of antibodies [36], CID in the ion trap of a LTQ-Orbitrap has been used to determine the exact mass, glycoforms, disulphide bonds location and to characterize variable regions of a population of mAbs [37, 38]. Recently, Tran et al have used the FTICR or HPLC-ESI coupled to an Orbitrap with ETD fragmentation to investigate the glycan structure, sites, relative abundance, and structural conformation of therapeutic mAbs [39].

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique widely used for the analysis of peptides, proteins, nucleic acids and sugars along with large organic molecules. MALDI coupled with in source decay (ISD) is an MS method used for the simultaneous sequencing of protein N- and C-terminal through the fragmentation of ions directly in the mass spectrometer ion source. The verification of the presence of modification at N- and C-terminal of mAbs is very important. In some proteins or peptides, comprising antibodies, the glutamine or glutamic residue at the first position (N-terminal) can rearrange forming the cyclic form pyroglutamate. In antibodies this process can occur spontaneously in vitro in light or heavy chains, thus affecting the safety and structural properties of mAbs [40]. At the C-terminal of mAbs it is possible to observe the presence or absence of a lysine residue that may affect the structure and biological function [41]. In both cases, an incomplete reaction at N- and/or C-terminal in different batches of the same mAbs produces heterogeneity leading in a lack of production process control. MALDI-ISD can be applied to rapidly gather sequence information on these modifications on therapeutic mAbs for quality control (Bruker Daltonics, application note). Moreover, MALDI-ISD is used to obtain the N-terminal sequence of mAbs, which is a requirement according to the ICH Q6B Guideline for the characterization of recombinant proteins for clinical testing, showing batches to be comparable and consistent. In other applications, MALDI-ISD can be coupled to MALDI imaging technique, thus providing at the same time identification and localization of a protein of interest. This approach has been used to monitor therapeutic mAbs in brain tumor [42]. Detailed information on glycosylation profile and heterogeneity of glycoforms can be obtained by using top-down MALDI-ISD [39, 43].

Bottom-up proteomics probably represents the most classical approach used in the proteomics field. This approach refers to the characterization of proteins through peptides analysis: proteins of interest are ad hoc digested by proteolysis and then loaded onto mass spectrometers. Samples can be very complex and often a fractionation system (e.g., SDS-page, 2D-page, liquid chromatography) is associated with MS analysis. When bottom-up is performed by using as sample a protein mixture as a total cell lysate or serum, it is called shotgun proteomics, as proposed by the Yates lab for the analogy to shotgun genomic sequencing [44]. With enzymatic digestion, analyzed proteins and so mAbs lose their native state and also all the information on high order structures, aggregates, etc. However, some conformational information can be preserved by fixing them into peptides before the fractionation step and/or the MS analysis. The fixation can be achieved by labelling methods, limited hydrolysis, and protein crosslinking.

Overall, bottom-up approaches have the important advantage of being high throughput with high-resolution separation. Required instrumentation is less expensive than top-down and many applications have been developed. Bottom-up quantitative approaches can be used for quantitative analysis of mAbs, as targeted strategies to monitor and quantify mAbs in complex mixtures. However, there are some issues regarding first the confidence in protein identification, strictly dependent on user’s parameters and particularly challenging for small proteins. Moreover, information about PTMs and structural proprieties are lost, except for specific approaches (e.g., Hydrogen-deuterium exchange (HDX) MS) (Fig. 5).

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Figure 5. Principal advantages and disadvantages of Bottom-up MS approach for the study of mAbs.

Structural characterization of a protein of interest is a pivotal point in understanding its biological e physiological role. Not only the function of a protein is affected by its structural state, but even its aggregation state, solubility and other chemical-physical properties. This is true especially for mAbs, in which also the immunogenicity, clearance and cytotoxicity are linked to their conformation. The in-depth structural characterization of mAbs is thus of utmost importance, even if particularly challenging. mAbs are large and dynamic proteins, present several isoforms and complex high order structures [5]. As discussed above, the combination of native MS and ion mobility (the top-down approach) may furnish measurements of size and shape of mAbs. However, more detailed information regarding the mAbs footprint can be gathered by using the hydrogen/deuterium exchange (HDX) coupled to MS. HDX was studied in proteins in 1954 using gravimetric techniques by Hvidt and Linderstrom-Lang [45]. HDX is a chemical reaction in which covalently linked hydrogen of a biomolecule is replaced by a deuterium atom present in solution, or vice versa. In particular, in proteins HDX concerns three kinds of hydrogens: those in carbon-hydrogen bonds, in side-chain groups, and in amide groups, namely backbone hydrogens. However, the exchange rates of the first two kinds of hydrogens too slow and too fast to detect, respectively. Only the backbone hydrogens are useful to be measured giving information on protein structure and dynamics.

A classical HDX-MS experiment of therapeutic mAbs involves several steps, as reported in Fig. 6. Briefly, the mAb to be analyzed is diluted with deuterated buffer in order to start the exchange of backbone amide hydrogens. The exchange is then quenched at different times decreasing the pH to ∼2.5 and the temperature to 0°C by adding an ice-cold acidic buffer. The analyzed mAb is denatured and disulphide bridges are reduced. The quenched mAb is indeed injected into a chromatographic column with immobilized pepsin. Obtained peptides are trapped, eluted and separated by HPLC/UPLC. Finally, peptides are analyzed by a mass spectrometer.

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Figure 6. Schematic representation of a classical HDX-MS experimental workflow for mAbs.

Houde et al assessed changes in high order structures (HOS) of mAbs as a consequence of their deglycosylation [46]. HDX-MS is a precious tool to investigate the HOS of mAbs, which is strictly dependent on environmental conditions (e.g., pH, temperature, ionic strength) potentially resulting in denaturation or aggregation of mAbs [47]. For example, this approach has been used to study the effect of freeze/thaw process and exposure to high temperature on Bevacizumab (Avastin) aggregates derived from changes in the structural dynamics. Moreover, mAbs aggregates such as dimers that occurred during normal production and storage of mAbs have been investigated by Iacob et al [48]. Even the effect of other processes on HOS of mAbs has been evaluated: chemical and post-translational modifications [49, 50], the presence of specific pharmaceutical excipients as sucrose and arginine [51] and targeted mutations [52]. The assessment of an analytical technique able to monitor in a reproducible manner all the above potential sources of heterogeneity in mAbs quality control processes is very important. HDX-MS can be also useful to investigate and locate the epitopes involved in antibody-antigen interactions. The knowledge of these epitopes is crucial for therapeutic mAbs design and patenting [53]. Several groups have used this approach for this purpose [54-56].

Glycosylation is one of the most common PTMs in mAbs and the different glycan moiety represents one of the principal sources of heterogeneity in mAbs batches [57, 58]. Moreover, glycosylation affects folding and thus the functionality of mAbs, thus it is a crucial issue during quality control in the development or production processes [59]. Hydrophilic interaction liquid chromatography (HILIC) has been commonly used for glycosylation profiling, for example coupled to a QTOF mass spectrometer for a detailed characterization of Rituxan®, a marketed mAbs [60]. As already reported, ion mobility can be applied to characterize glycosylation profile of mAbs: with a hybrid electrospray quadrupole ion-mobility time-of-flight mass spectrometry (ESI-Q-IM-TOF) platform, the global glycan profiles and structure at each glycosylation sites can be obtained, as reported for different batches of transtuzumab [61]. In this framework, the improvement of platforms able to in-depth characterize the glycosylation pattern of mAbs represents a key point of quality control processes, facilitating the analysis of products from different batches. A particular approach to characterize antibody peptides and glycan moiety is based on the use of specific enzymes from Genovis Enzyme Technologies [14] : FabRICATOR® IdeS Protease and IgGZERO™ (EndoS Glycanase). In mAbs with glycosylated Fab regions FaBRICATOR® enzyme cleaves and produce glycans associated with two separate peptide fragments that can be observed by MS analysis. This improves glycan analysis, especially for the effector functions of Fc fucosylation and galactosylation. The enzymatic hydrolysis of antibodies glycans has usually been performed by using enzymes such as PNGase F and other endo and exoglycosidases. IgGZero™ (EndoS), isolated from Streptococcus pyogenes as also FaBRICATOR®, has shown strong hydrolytic activity specific for IgG bound glycans [62].

Quantitative measurement of proteins of interest is an essential analytical task in all laboratories. Considering the increasing interest in the therapeutic mAbs field, the need for most efficient quantitative methods to monitor mAbs improving quality control, pharmacokinetics and toxicokinetics studies are urgent. Ligand binding assay (LBA) that is based on immunocapture and detection, has probably been the most commonly used method for protein quantification for years. However, LBA presents some limitations [63]. Liquid chromatography (LC) MS is a very powerful method for selective (at bottom-up level), accurate, sensitive, and rapid analysis of small molecule as mAbs.

Contrary to the global approach, targeted MS analysis measures selected peptides because a protein may be quantified by measuring proteotypic peptides, which are specific peptides typical of that protein and released following proteolytic digestion. Data are then acquired only for proteotypic selected peptides thus increasing precision, sensitivity, and throughput [64].

In tandem MS/MS instruments each proteotypic peptide ion can be selected and then specifically fragmented thus producing a characteristic pattern of b- and y-ions, corresponding to fragments containing the N- and C-termini of the proteotypic peptide, respectively. The combination of proteotypic selected peptide ion, namely the precursor, and resulting fragment ions, namely the products, includes specific transitions for the monitored peptide [64]. The detection of signals created in the selection and fragmentation processes and the transitions is called selected reaction monitoring (SRM). When SRM is applied to multiple product ions, it is referred to as multiple reaction monitoring (MRM). Depending on the instrument utilized, several transitions can be monitored at the same time, within one injected sample. Ideally, each tandem MS instrument can be used for SRM/MRM. However, triple quadrupole (QqQ) and quadrupole-ion trap (Q-Trap) mass spectrometers are most commonly used [65]. In a QqQ, the first quadrupole (Q1) selects the proteotypic peptide ion of choice, which is the precursor, the second quadrupole (Q2) fragments it, and the third quadrupole (Q3) selects a specific fragment ion for the detector (Fig. 7). The presence of interfering ions within ions of interest is reduced thanks to the double mass selection. The integration of the MRM signal area on the obtained mass spectrum allows the quantification of the protein of interest.

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Figure 7. Schematic representation of SRM/MRM process in a triple quadrupole (QqQ).

Other quantitative MS approaches are based on the use of specific labeling agents, such as the isotope-coded affinity tags (ICAT), the tandem mass tags (TMT) and the isobaric tags for relative and absolute quantitation (iTRAQ). All the labeling-based approaches allow obtaining a relative quantification.

Targeted MS analysis is particularly useful to quantify specific modifications, as PTMs. In mAbs quality control is of utmost importance to verify the presence of PTMs occurring during manufacturing, storage, or administration processes. For example, a system based on LC-UV-MS was used to identify and quantify aspartate residue isomerization, a common PTM of mAbs that may impact the target binding [66].

In 2008, Dubois et al developed a sensitive, specific, and accurate absolute quantification SRM method for the human-murine chimeric mAb Erbitux, used against colorectal cancer [67]. The absolute quantification of therapeutic mAbs has been also performed on tissue matrix developing an SRM method using a high-throughput on-the-fly orthogonal array optimization (OAO) strategy [68]. Moreover, mAbs present in serum have been quantified by using a multiplexed strategy based on the use of Protein Standard for Absolute Quantification (PSAQ™) for SRM [69].

Targeted MS assays can be used for quantitative analysis of glycan moiety of mAbs. mAbs contain conserved glycosylation sites and can have different glycosylation patterns, even if some are conserved. N-glycosylation of mAbs affects their stability, immunogenicity and cytotoxicity [14]. In this framework, the FDA released the draft guidance on biosimilar products highlighting the importance of the analysis of glycosylated form. Glycan structures of therapeutic mAbs have been quantitatively analyzed using different techniques such as ESI-MS [70], and MALDI-TOF MS [71]. Therapeutic mAbs and their glycoforms can be quantified in complex matrices as serum [72]. However, the detection of glycoforms is challenging due to their low-abundance and not always efficient ionization [73]. The enrichment of glycans is a good step to introduce in order to improve the sensitivity for glycans analysis.

The quantitative targeted approach can be also used for quality control, for example to evaluate the lot-to-lot microheterogeneity for trastuzumab and bevacizumab mAbs [74].

Recently, a particular MS-based approach to monitor at the same time an array of PQAs important for mAbs has been developed. The proposed multi-attribute method (MAM) optimized analytical solution representing a powerful method in quality control of mAbs.

mAbs represent one of the most promising tools for the treatment of several pathologies, including cancer. In particular, the use of mAbs for cancer therapy has been defined as “one of the great success stories of the past decade” [75]. Growing interests are developing around this field, always requiring better experimental strategies and methodologies to characterize mAbs at each level, from production to the final destination. In this framework, modern MS-based approaches can provide high reproducibility, sensitivity, specificity, precision, accuracy and multiplexing thus representing a powerful tool to go deeper in the therapeutic mAbs research. In fact, in recent years, MS has become a crucial method for the quantitative and qualitative characterization of biotherapeutics in several matrices (e.g., formulated products, body fluids, tissues). With MS-based top-down and/or bottom-up approaches it is possible to address conformational and structural challenges related to mAbs, thus rapidly characterizing parameters such as critical quality attributes (CQAs). Moreover, rapid and reliable targeted MS/MS methods allow the absolute quantification of mAbs even in very complex matrices.

However, a whole vision of such complex macromolecules cannot be achieved by using a single method. Rather, a combination of discussed strategies coupled with classical biochemical/molecular approaches can gather all needed information on mAbs.