Infliximab biosimilarity assessment in relation to stability

In order to study the biosimilarity between the infliximab innovator (Remicade®) and the corresponding biosimilar products (Remsima®, Flixabi®) in terms of stability, the products were subjected to different stress conditions. The primary structure of the mAbs was then systematically characterized using LC-MS/MS analysis (see “Materials and methods”) in order to identify PTM hotspots and estimate the level of modification. In addition, the aggregation and/or chain fragmentation of the mAbs was characterized using SEC-MALS-UV/RI analysis.

For the freshly reconstituted products, LC-MS/MS analysis enabled sequence coverage of 94.9% to be systematically achieved (Fig. S1). This shows the robustness of the method and the possibility for near complete characterization of the primary structure, providing the opportunity to identify all the PTM hotspots described for infliximab. Therefore, LC-MS/MS data enabled the unambiguous identification of one deamidation and nine different oxidation hotspots. As emphasized in Fig. 1A, the level of oxidation initially ranged from 0.7% to 5% depending on the residue. Levels of oxidation were found to be similar for the different residues except for heavy chain (HC) M18 and M255 residues in addition to light chain (LC) M55, which exhibited higher modification levels for the biosimilar products. The residue M18 is located in the mAb variable domain, and the oxidation of the amino acid M255 is reported to lead to lower affinity with the FcRn receptor [34]. Therefore, the occurrence of such modifications can alter the biological properties of the mAbs. It is important to characterize the occurrence and the proportion of PTMs in the context of biosimilarity assessment, which in this case showed only slight differences. The residue N57 is located in the complementarity-determining region (CDR) of infliximab HC, and therefore deamidation into aspartic acid would have a major impact on the epitope interaction. Also, this residue has been described as being sensitive to deamidation [12]. The level of N57 deamidation was between 1.5% and 2.4% initially for the different products (Fig. 1B). Note that the PTM levels observed for the reconstituted products are consistent with those in other studies [12, 14, 35, 36]. Similarly, the reconstituted products were characterized using SEC-MALS-UV/RI. SEC-MALS results presented in Fig. 1C show that initially the proportion of aggregates represents less than 1% of the total forms, whereas the proportion of aggregates is slightly higher in the case of the two biosimilar products.

Fig. 1

Proportions of oxidation (A) and N57 deamidation (B) determined from LC-MS/MS data and levels of aggregation (C) measured from SEC-MALS-UV/RI experiments in the case of Remicade® (red), Remsima® (green) and Flixabi® (blue) present after reconstitution

Consequently, forced degradation was performed on the three references of infliximab in order to identify potential degradation of the mAbs and to assess their biosimilarity with regard to their stability. To accelerate the oxidation of residues, the different samples were incubated in the presence of H2O2, whereas mAbs were exposed to relatively high temperature in order to generate deamidation and aggregation. Results of LC-MS/MS experiments demonstrated an increase in oxidation in the case of M55, M18, M255 and M431, as presented in Fig. 2A, whereas no significant modification could be observed for other residues characterized (Fig. S2). Thus, the oxidation levels increased rapidly, with modification levels ranging from 39.8% to 76.0% after incubation for 24 h, depending on the residue, and systematically above 62.5% after 48 h of incubation. Regarding deamidation in the CDR, the residue N57 exhibited modification levels that gradually increased over time, with values ranging from 1.9% to 3.8% after 5 days of exposure to a temperature of 40 °C, and from 21.9% to 24.5% after 90 days of incubation (Fig. 2C). The tridimensional structure of infliximab described in the literature was compared to the LC-MS/MS data in order to characterize the differences between the modified residues and the amino acids which remained intact. As emphasized in Fig. 2B, the amino acid residues prone to modification were located at the periphery of the protein, and therefore significantly exposed to the environment. In addition, this observation suggests that amino acids buried deeper inside the tertiary structure of the protein may be less sensitive to endogenous modification. Regarding the oxidation and deamidation hotspots characterized, the data showed similar modification levels among the different products corresponding to infliximab. Therefore, LC-MS/MS experiments showed the relevant similarity of the different infliximab products with regard to their stability under stress conditions in addition to the similarity of their tertiary structure. Indeed, the modified residues are consistent between the different samples, indicating similar exposure to the environment. In the case of SEC-MALS analysis, the proportion of mAb aggregates remained constantly below 1% irrespective of the duration of the stress (Fig. 2D). In contrast, exposure to a temperature of 40 °C led to the fragmentation of infliximab in free heavy chains and light chains, with a proportion of 5% of fragments after 90 days (Fig. 2E). In this case as well, the innovator and biosimilar products corresponding to infliximab demonstrated similar levels of fragmentation throughout the duration of the temperature stress. Consequently, the fragmentation into free HC and LC was determined to be the major degradation pathway regarding size variants of infliximab.

Fig. 2

Proportions of (A) oxidation for methionine and tryptophane hotspots after incubation in 0.05% H2O2 for Remicade® (red), Remsima® (green) and Flixabi® (blue). (B) Schematic representation of the structure of infliximab showing the localization of oxidized methionine. Proportions of (C) N57 deamidation, (D) infliximab aggregation and (E) free chain fragmentation after incubation at 40 °C for 90 days

Infliximab stability and biosimilarity assessment for in-use conditions

Following the assessment of the different infliximab products with respect to stress stability, the in-use stability of infliximab was evaluated using different conditions, this time after reconstitution in intravenous (IV) bags. The conditions were selected to reflect different scenarios regarding storage temperature, the type of IV bag or the exposure to light (see “Materials and methods”) over a period of 3 months. In addition to assessing the stability of the different products corresponding to infliximab in real-life conditions, this study aimed to provide evidence regarding the possibility of anticipated preparation in a hospital unit in order to manage a continuously growing number of patients. The results obtained from LC-MS/MS experiments concerning the residues sensitive to oxidation are presented in Fig. 3. At a storage temperature of 4 °C, oxidation appeared globally limited, with levels ranging from 0.5% to 4% depending on the residue. Moreover, the oxidation levels did not show a significant increase upon storage at 4 °C even for an extended time (Fig. 3A). Similarly, when the IV preparations were maintained at 25 °C in the dark, oxidation levels did not exhibit any increase, and the modification levels were comparable to the data obtained at 4 °C (Fig. 3B). In the case of IV bags kept at 25 °C exposed to light, the sensitive methionine previously identified showed a gradual increase in oxidation levels, with values ranging from 0.5% to 12.9% in the case of M255, which showed the highest oxidation levels (Fig. 3C). The oxidation of residue M255 is reported to significantly decrease the affinity between the mAbs and the FcRn receptor which is responsible for the preservation of IgG proteins in the endosome [37]. Therefore, the results obtained show that prolonged exposure to light is likely to impact the serum half-life of infliximab. Interestingly, the methionine and tryptophan residues, which were not modified during the stress degradation, also did not exhibit any modification in the IV bag samples. Therefore, this comparison shows that H2O2 represents a relevant oxidation stress assay capable of indicating residues prone to modification. For the different conditions, the biosimilar products generally exhibited similar behavior regarding oxidation hotspots regardless of the type of IV bag. However, in the case of the infliximab innovator, LC-MS/MS data showed that M55 and M255 demonstrated increased oxidation when the mAbs were reconstituted in IV bag B. For instance, in the case of M255, the level of oxidation was 5% after incubation when the mAbs were reconstituted in IV bag A, while the level was 13% if the product was solubilized in IV bag B (Fig. 3C). IV bag A is composed of polypropylene whereas IV bag B is constituted of low-density polyethylene. Therefore, this result could be linked to an interaction between the additives of the IV bag and the mAbs.

Fig. 3

Oxidation levels measured from LC-MS/MS analysis for the infliximab products reconstituted in IV bag A (continuous line) and IV bag B (dotted line) after (A) conservation at 4 °C, (B) storage at 25 °C protected from light and (C) storage at 25 °C exposed to light. Remicade® (red), Remsima® (green) and Flixabi® (blue)

With regard to the deamidation of the amino acid hotspot N57, the results presented in Fig. 4A show that the modification of the residue was not significantly increased when the sample was stored at 4 °C, with modification levels ranging from 1.5% to 2.7%. On the contrary, the samples subjected to a temperature of 25 °C demonstrated a gradual increase in the modification level from 1.5% to 5%. In the case of the deamidation, light exposure did not influence the kinetics of the modification, indicating that temperature is the major factor in this case. Meanwhile, the study of the higher order structure using SEC-MALS-UV/RI analysis did not demonstrate infliximab fragmentation for the samples maintained at 4 °C, whereas the samples subjected to a temperature of 25 °C over an extended period exhibited fragmentation of the proteins in free chains (Fig. 4B).

Fig. 4

(A) Deamidation of amino acid N57 determined from LC-MS/MS analysis and (B) free chain fragmentation determined from SEC-MALS-UV/RI for the infliximab products reconstituted in IV bag A (continuous line) and IV bag B ( dotted line) for the different conservation conditions. Remicade® (red), Remsima® (green) and Flixabi® (blue)

As a consequence, the experiments performed concomitantly using LC-MS/MS analysis and SEC-MALS-UV/RI allowed us to investigate the stability of the different products corresponding to infliximab with respect to different PTM hotspots and aggregation/fragmentation. The study using stressed conditions allowed us to identify the residues prone to modification, especially for oxidation and deamidation. Thus, the study demonstrated that the occurrence of oxidation is driven by the media solubilizing the mAbs. In addition, data demonstrated that residues accessible to the solvent are rapidly modified, in contrast to the amino acid buried in the structure of the protein, which remains intact. For deamidation and free chain fragmentation, the degradation of the protein was attributed to the effect of temperature. The investigation of the infliximab products reconstituted using in-use conditions demonstrated that the residues from modification during real-life conditions were the same as those modified using stressed conditions. The results showed that no significant modification was observed when samples were stored at 4 °C for a period of 3 months. The innovator product and the corresponding biosimilar demonstrated important similarity in terms of stability; however, it is important to note that the innovator samples exhibited significantly higher oxidation for the residues M55, M18, M431 and M255 when the product was reconstituted in IV bags composed of polyethylene, which has not been described before. Such results clearly emphasize that biosimilarity assessment should not be restricted to a simple structural characterization but should also investigate the stability of biosimilar candidates over the different levels characterizing the structure of the mAbs, if possible in conditions as close as possible to real-world use conditions. Indeed, to our knowledge, this is the first study evaluating the evolution of PTM hotspots during hospital in-use conditions of infliximab. Also, it is the first time that biosimilarity assessment was performed in the context of hospital in-use conditions.

Infliximab stability prediction using ASAP modeling

After the stability study, an accelerated stability assessment program (ASAP) was realized for infliximab in order to evaluate the possibility of employing this type of approach in predicting the long-term stability of therapeutic mAbs. Experimentally, the ASAP model is built by subjecting the studied product to different temperature conditions and various relative humidity levels in the case of a solid formulation. The level of the degradation product (DP) generated depending on the conditions is then used to determine the modified Arrhenius equation parameters as illustrated in Eq. 1:

$$Ln\ (k)= Ln\ (A)-\frac+B\times RH$$

(1)

where k represents the degradation rate, Ln (A) the pre-exponential factor, Ea the activation energy, T the temperature, R the gas constant, B the moisture sensitivity factor and RH the relative humidity.

Thus, the model is established by artificial generation of the considered DP, close to the specification limit in each stress condition [26], which makes it possible to overcome the heterogeneous kinetics of degradation [25]. The specification limit corresponds to the maximum amount of degradation product allowed for the therapeutic product. For small chemical synthetic drugs, the limit of specification is generally defined from 0.05% to 1.0% by the regulatory authorities depending on the posology of the molecule [38]. For biotherapeutic products, the limit is defined on a case-by-case basis for each drug based on the regulatory guidelines or the information described in the scientific literature [39]. The time necessary to reach the specification limit is referred to as the isoconversion time. The model developed can then be used to predict the level of DP that would be observed during long-term stability study in the envisaged storage temperature and relative humidity. Note that in order to be able to perform ASAP modeling, it is essential to characterize the DP beforehand and benefit from an analytical method which allows it to be analyzed without any interference. Infliximab and mAbs in general are extremely complex macromolecules which may undergo several modifications simultaneously [9]. Thus, it is usually difficult to predict the long-term stability for biotherapeutic products because of their inherent complexity and the non-Arrhenius behavior for quality attributes such as aggregation [40]. However, the aggregation pathways seem to be significantly mAb-dependent [41]; for instance, a recent study was able to model aggregation using a thermodynamic equation with other types of mAbs [42].

For the long-term stability study of infliximab, the ASAP approach was performed concomitantly for different degradation processes in order to investigate the possibility of modeling the stability of the mAbs regarding different aspects defining the structure of the protein. As illustrated in Eq. 1, the implementation of the modified Arrhenius equation makes it possible to study the degradation influenced by temperature and/or humidity. The results obtained previously for stability studies showed that oxidation of methionine hotspots were influenced by the level of oxygen and the exposure to light, whereas the storage temperature had no influence on the level of their oxidation when increased from 4 °C to 25 °C (Fig. 3). On the contrary, the stability study showed that for the different infliximab products, the deamidation of N57 and the fragmentation in free chains are impacted by the temperature (Fig. 4). Therefore, the ASAP modeling was envisaged only for these two types of degradation. Thus, a vial of infliximab corresponding to the marketed formulation was reconstituted and consequently split into equal-volume samples, which were subjected to temperature ranging from 30 °C and 45 °C for up to 30 days. During the incubation, the samples were regularly characterized regarding N57 deamidation using LC-MS/MS analysis and concomitantly regarding chain fragmentation of mAbs by the intermediate of SEC-MALS-UV/IR analysis. Note that the incubation temperature was limited, because above 45 °C, rapid precipitation generating non-soluble particles could be observed. The specification limits were fixed at a value of 5.0% in the case of deamidation and 1.0% for free chain fragmentation. The monograph of the pharmacopoeia for infliximab did not mention the limits in terms of acceptable PTM levels; therefore, the specification limits were determined using minimal values considering modification levels previously described in the literature. In addition, 5% modification was considered as a maximum acceptable level of PTMs, in order to maintain 95% of the original form as is commonly acceptable [16, 43].

As emphasized in Fig. 5A, the deamidation of the residue N57 demonstrated a gradual increase from 0.7% initially up to 8.6% for incubation at 45 °C for 15 days. Results also showed an increase in the kinetics of the reaction when the incubation temperature was increased. With regard to the fragmentation of infliximab in free chains, no fragments were initially detected (Fig. 5B). Subsequently, the proportion of free chains increased over time when the mAbs were exposed to increasing temperatures up to 1.6% and incubated at 40 °C for 30 days. In this case as well, the kinetics of the free chain fragmentation increased when the temperature was higher, showing that the degradation process is impacted by the temperature. For each temperature condition, the isoconversion time was calculated or extrapolated from the experimental data. A linear regression was used in the case of N57 deamidation (Fig. S3) and a diffusion regression (Fig. S4) for mAb free chain fragmentation, which is consistent, as the two processes of degradation showed different evolutions (Fig. 5). The isoconversion times and isoconversion ratios obtained for the different conditions are detailed in Table 2. The isoconversion ratio corresponds to the ratio between the latest measurement time and the calculated isoconversion time. It enables us to estimate the extent of the extrapolation required to determine the isoconversion time. Thus, it tends to be closer to 0.0 when an important extrapolation is required to calculate the isoconversion time. The value is above 1 when the specification limit is reached during the experiment and extrapolation is not necessary. Therefore, the isoconversion ratio makes it possible to limit excessive extrapolation in order to build a valid model. Generally, isoconversion ratios lower than 0.1 are considered out of range, and the data should be excluded from the model. For the N57 deamidation hotspot, the lowest isoconversion ratio calculated was 0.62 at 30 °C, whereas for the free chain fragmentation, the lowest isoconversion ratio was 0.41 (Table 2). Thus, the data obtained using the different conditions were compatible with the model for infliximab N57 deamidation and free chain fragmentation.

Fig. 5

(A) Proportion of N57 deamidation estimated from LC-MS/MS analysis and of (B) infliximab chain fragmentation determined from SEC-MALS-UV/RI analysis for the ASAP stress samples. The red discontinuous lines indicates the specification limit in each case

Table 2 Isoconversion time and isoconversion ratio calculated from ASAP stress samples for deamidation of the amino acid N57 and infliximab chain fragmentation for the different temperature conditions

Using the experimental data from the different conditions, the parameters of the Arrhenius equation were calculated for the deamidation of the amino acid N57 and the fragmentation of the mAbs in free chains, as summarized in Table 3. Different parameters were considered in order to evaluate the adequacy of the various ASAP models. The correlation coefficient R2 between the model and the experimental data was 0.934 for the deamidation and 0.968 in the case of the free chain fragmentation, thus demonstrating validity (Table 3). Indeed, regarding adequacy, the ASAP model is considered to be valid when R2 > 0.9 [44]. The cross-validation coefficients Q2 were also calculated yielding a value of 0.576 for deamidation and 0.898 for free chain fragmentation. Due to their respective characteristics, the value of Q2 is expected to be lower than R2; however, it is the difference between the two values which makes it possible to further demonstrate the adequacy of the model compared to the experimental data. For the two types of degradation considered, the values of both R2 and Q2 proved to be satisfactory. In addition, as presented in Fig. 6, the residual plots corresponding to the DP formation rate ln (k) were satisfactory compared to other studies using the ASAP methodology [29], with residues of ln k systematically lower than ±0.4.

Table 3 ASAP model parameters obtained from infliximab stress samples for the deamidation of N57 hotspot and free chain fragmentation and correlations calculated between the present model and the experimental dataFig. 6

Residual plot obtained from the ASAP model (A) for the deamidation of residue N57 and (B) in the case of infliximab free chain- fragmentation

Previous stability studies of infliximab using forced degradation enabled the identification of different alterations of the mAbs. In addition, they identified the degradation processes which could potentially be modeled using an ASAP approach. Thus, the characterization of the samples subjected to different temperature conditions showed the possibility for concomitant modeling of the deamidation of the residue N57 and the fragmentation of the mAbs in free chains. The ASAP models established for the different degradation types were shown to be in agreement with the experimental data, confirming the validity of the model based on common practice regarding ASAP prediction. In addition, the relevance of the models clearly indicates that N57 deamidation and free chain fragmentation are thermodynamically driven alterations of infliximab. The ASAP models further highlighted the possibility of performing mAb stability prediction using the ASAP approach over several degradation processes which may occur concomitantly on the structure of the mAbs. From that perspective, the methodology developed in this work is particularly interesting because it provides the opportunity to perform comprehensive stability prediction regardless of the structural complexity of the mAbs. To the best of our knowledge, this is the first time that ASAP modeling was performed simultaneously with success for degradation over different levels defining the structure of the mAbs, primary structure for the deamidation and tertiary structure in the case of free chain fragmentation.

Comparison between stability prediction and long-term stability data

In order to evaluate the performance of the model, the stability prediction generated using the described ASAP models was compared to the data obtained during the in-use stability study of infliximab with respect to the deamidation of the residue N57 and the free chain fragmentation of the mAbs (Fig. 4). The elaboration of the ASAP model takes into account the variability of the generated model and eventually the variability of the analytical method in order to predict the minimum and maximum quantity of DP generated over time for a designated condition (Fig. 7). Regarding deamidation of the residue N57, at 4 °C the level of deamidation predicted using the model was systematically below 2% and the prediction described a near absence of increase concerning the modification level. However, the level of deamidation determined during the in-use stability study was systematically over the predicted level, even in the initial conditions directly after reconstitution (Fig. 7A). Nevertheless, the predicted and experimental evolution appears similar. This observation was attributed to the fact that infliximab vials used to perform the ASAP modeling and the in-use stability study came from different batches. Indeed, after reconstitution the two batches exhibited different initial deamidation levels of N57: 0.7 % ± 0.1 for the ASAP batch and 1.5 % ± 0.1 for the batch used for in-use stability samples (Fig. 7A). If a correction of the ASAP prediction is applied based on the initial mismatch concerning the deamidation level, all the experimental points of the in-use stability study are comprised in the prediction showing an outstanding correlation. For the storage conditions at 25 °C and 40 °C, the predictions described a gradual increase of the deamidation level after 90 days up to 3% for a temperature of 25 °C (Fig. 7B) and 4% at 40 °C (Fig. 7C). The predictions generated from the ASAP model demonstrated different slopes depending on the temperature applied. The experimental data are showing a relevant correlation with the prediction for the different case in term of deamidation levels and evolution over the duration of the study. Therefore, the correlation of the results obtained from the ASAP prediction and experimental data demonstrated that the ASAP modeling is able to predict the evolution of the modification level of the residue characterized.

Fig. 7

ASAP model prediction concerning the deamidation of the amino acid N57 (A) at 4 °C, (B) 25 °C and (C) 40 °C. ASAP model prediction regarding free chain fragmentation at (D) 4 °C, (E) 25 °C and (F) 40 °C. The mean predicted value is represented by the blue line, the maximum and minimum predicted values are shown in green and red lines, respectively. Experimental data are represented as black dots

Concerning free chain fragmentation, the prediction generated for a temperature of 4 °C indicated a calculated level < 0.02% after 6 months (Fig. 7D) which, considering the performance of the SEC-MALS-UV/RI method, corresponds to an absence of fragmentation. Experimentally, when stored at a temperature of 4 °C, free chain fragmentation of infliximab was not observed, which is in agreement with the prediction of the model. For the conservation conditions at 25 °C, the prediction achieved from the model initially exhibited an absence of fragmentation followed by a constant increase over the duration of the experiment to a maximum value of 0.1% after 6 months. From the comparison with the experimental data, the fragmentation appears to be slightly overestimated by the model prediction (Fig. 7E). The difference in terms of the proportion of free chains was attributed to the extremely low levels generated during the first 60 days of the study, ranging from 0% to 0.015%, which can be difficult to accurately estimate from SEC-MALS-UV/RI chromatograms. When the proportion of free chains was further increased, the experimental data correlated with the model prediction. In addition, the larger discrepancy between the prediction and the experimental data represented 0.01%, which remained negligible and placed the prediction on the safer side compared to the level of free chains effectively characterized (Fig. 7E). Williams et al. reported a similar conclusion in a study using ASAP for small chemicals drugs [27]. As illustrated in Fig. 7F, for a temperature of 40 °C, the model prediction showed a steeper increase in the proportion of free chains above 0.25% after a period of 6 months. The experimental data showed a consistent correlation with the prediction provided by the model regarding the proportion of free chains and the evolution over time. Note that the experimental results from the in-use condition samples exhibited minor differences compared to the prediction of the model, which in this case as well represented a maximal difference of 0.01%, which is lower than the standard deviation provided by the analytical method. Indeed, in addition, the experimental data showed significant variability between the sample triplicates (Fig. 7F). As a consequence, the correlation of the model developed from the ASAP approach with the data obtained from the analysis for the samples stored using in-use conditions was found to be relevant for the long-term fragmentation of the infliximab in free chains.

Finally, the ASAP approach was successfully used to model two different types of alteration occurring on the infliximab mAbs. Thus, the required experiments were performed over a period of 30 days and allowed for the concomitant modeling of the deamidation of the residue N57 and the fragmentation of infliximab into free chains, for a period longer than 6 months (Fig. 7). For the two different alteration processes, the comparison between the prediction generated from the ASAP model and the experimental data obtained from long-term storage of infliximab reconstituted in IV bags demonstrated a relevant correlation. Also, the evolution of mAb degradation over time was relevant with the modeling. Therefore, the results achieved showed the possibility of using an ASAP model in order to predict the stability of mAbs under conventional storage conditions. In addition, the long-term stability prediction generated from the ASAP model could be eventually considered in order to optimize the composition of the formulation regarding mAb stability and help to determine the expiration time of the product. The implementation of the ASAP approach for the long-term stability study of infliximab highlighted the crucial requirements. Indeed, before the development of the model, it is essential to perform a complete characterization of the different degradation products generated by the therapeutic protein investigated. This makes it possible to identify the major degradation products as a preliminary approach, in addition to identifying the degradation processes which can be modeled by a modified Arrhenius equation due to the impact of temperature and/or humidity. In a similar manner, the implementation of the ASAP approach requires an analytical method able to unambiguously separate the investigated DP and accurately estimate the level. In particular, results achieved for the ASAP modeling concerning infliximab demonstrated that the accuracy, sensitivity and variability of the analytical method is crucial for providing a valid model and may influence the correlation with the experimental data. ASAP models could be established concomitantly for two different types of structural alterations of infliximab. Therefore, the workflow implemented in this study showed the methodology required to perform ASAP modeling of therapeutic mAbs. It may be possible to extend the ASAP modeling of mAbs to a wider range of modifications known to influence the characteristics of the protein such as aspartic acid isomerization and N-terminal glutamic acid cyclization in order to provide a comprehensive stability model regarding these complex macromolecules. As such, capillary zone electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) represents a relevant technique for characterization of a large number of PTMs simultaneously [45].