Optimized Methods for Analytical and Functional Comparison of Biosimilar mAb Drugs: A Case Study for Avastin, Mvasi, and Zirabev

Bevacizumab is a humanized therapeutic monoclonal antibody used to reduce angiogenesis, a hallmark of cancer, by binding to VEGF-A. Many pharmaceutical companies have developed biosimilars of Bevacizumab in the last decade. The official reports provided by the FDA and EMA summarize the analytical performance of biosimilars as compared to the originators without giving detailed analytical procedures. In the current study, several key methods were optimized and reported for analytical and functional comparison of bevacizumab originators (Avastin, Altuzan) and approved commercial biosimilars (Zirabev and Mvasi). This case study presents a comparative analysis of a set of biosimilars under optimized analytical conditions for the first time in the literature. The chemical structure of all products was analyzed at intact protein and peptide levels by highresolution mass spectrometry; the major glycoforms and posttranslational modifications, including oxidation, deamidation, N-terminal PyroGlu addition, and C-terminal Lys clipping, were compared. The SPR technique was used to reveal antigen and some receptor binding kinetics of all products, and the ELISA technique was used for C1q binding affinity analysis. Finally, the inhibition performance of the samples was evaluated by an MTS-based proliferation assay in vitro. Major glycoforms were similar, with minor differences among the samples. Posttranslational modifications, except C-terminal Lys, were determined similarly, while unclipped Lys percentage was higher in Zirabev. The binding kinetics for VEGF, FcRn, FcγRIa, and C1q were similar or in the value range of originators. The anti-proliferative effect of Zirabev was slightly higher than the originators and Mvasi. The analysis of biosimilars under the same conditions could provide a new aspect to the literature in terms of the applied analytical techniques. Further studies in this field would be helpful to better understand the inter-comparability of the biosimilars.


Introduction
Monoclonal antibodies (mAbs) have gained significant attention due to their efficient and specific therapeutic potential for many diseases [1][2][3]. Unlike conventional chemical drugs, the development and standard production of an original mAbs or biosimilars are challenging due to their complex structure. The manufacturers should carefully follow specific quality parameters for the production of mAbs with consistent efficacy, safety, and quality [4,5].
From upstream production to shelf, many parameters can affect the structure, function, immunogenicity, or stability of mAbs. For instance, glycosylation or sequence variants are LC-MS/MS system (Waters, ACQUITY UPLC-ESI-Xevo G2-XS QtoF, Milford, MA, USA). Mobile phase A was MS grade water (Merck, Rahway, NJ, USA), mobile phase B was © (Merck, Rahway, NJ, USA), and mobile phase C was 1% formic acid (Merck, Rahway, NJ, USA). The reverse-phase separation was performed on ACQUITY UPLC-BEH300 C4 1.7 µm column (2.1 mm × 50 mm, Waters, Milford, MA, USA) using a 1 min gradient (5-85% B). During the run, the flow rate and column temperatures were 0.4 µL/min and 80 • C. The mass range was set to 500-4000 m/z and analyzed in ESI-positive and sensitivity mode. The instrument was calibrated using NaCsI (Sigma-Aldrich, St. Louis, MO, USA), and Glu-1-fibrinopeptide B (Waters, Milford, MA, USA) was used as a lock-mass reference.
The tryptic peptides were analyzed by the ACQUITY UPLC-ESI-Xevo G2-Xs QToF system (Waters, Milford, MA, USA). Mobile phase A was composed of MS grade water, mobile phase B was ACN, and mobile phase C was 1% FA. The percentage of mobile C was set to 10%, and the percentage of mobile phase B was increased from 1 to 80% over an 85 min total run time. The instrument was calibrated with NaCsI, and Glu-1-fibrinopeptide B (100 fmol/uL) was used as a lock-mass reference. Data-independent acquisition mode (DIA) was performed by sequential MS, and MS/MS scans with 0.5 s cycle time. All ions within the 50-2000 m/z mass range were fragmented together without selecting precursor ions in sensitivity mode.
The raw data was processed by applying the UNIFI (1.9 SR4, Waters, Milford, MA, USA) peptide mapping workflow parameters. The bevacizumab sequence was retrieved from http://www.drugbank.ca/ (1 June 2020) as a reference database. Trypsin was selected as a digesting reagent with one missed cleavage maximum. Carbamidomethyl-C was set as a fixed modification due to the alkylation step in the sample preparation. In contrast, the other modifications (oxidation-M, oxidation-W, deamidation-N, deamidation-Q, succinimide intermediates, pyroglutamic Acid-N TERM, -lysine C-TERM) were set as a variable. The mass tolerance window was set within 10 ppm. The components greater than 10% matched primary ions (b/y ions), <10 ppm mass error and no in-source fragment were allowed for identification. The percentage of modifications was calculated using the following equation: "%peptide = (Response of modified peptide/Total response of the modified and unmodified peptides) × 100" [22,24].

VEGF Binding Assay
The VEGF binding analyses were performed on VEGF 165A-immobilized (Sigma-Aldrich, St. Louis, MO, USA) CM5 chips with a Biacore T200 SPR Instrument (Cytiva, Marlborough, MA, USA) [25]. The chip surface was prepared with standard EDC-NHS coupling chemistry [26]. VEGF165A was diluted to 5 ng/µL in pH 5.5 in 10 mM acetate buffer. Following the activation of surface carboxylate groups by EDC/NHS injection, the target protein, VEGF 165A, was covalently immobilized through the free primary amine groups. The excess number of activated carboxyl groups on the matrix was blocked with a 1 M ethanolamine-HCl (Cytiva, Marlborough, MA, USA) injection. The final immobilization level for the active flow cell reached approximately 500 response units (RU) for all experiments. An ethanolamine-immobilized flow channel was used as the control surface. The samples at three concentrations (15 nM, 5 nM, 1.66 nM). were prepared in 1× HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% v/v polysorbate 20) at pH 7.4, which was also employed as the running buffer. Single-cycle kinetic analyses were conducted at the flow rate of 30 µL/min at 22 • C. Analytes were injected for 120 s in the association phase, followed by a dissociation phase of the 1800 s with the running buffer. Blank measurements were also performed on the active and control flow channels by running buffer injections under identical conditions. The chip surface was regenerated by injecting 10 mM glycine pH 1.5 buffer for 90 s. Results were obtained by subtracting responses from blank flow cells and zero concentration analyte injection (running buffer). The SPR data were presented as the mean value, calculated from at least three measurements per sample. The equilibrium dissociation constants (K D ) were calculated by Biacore Evaluation Software (3.0 Biacore T200, Shrewsbury, MA, USA) using a 1:1 Langmuir binding model [26]. For similarity assessment, statistical equivalence analysis was used. The difference in means between the two products within the equivalence acceptance criterion [±1.5 standard deviation (SD) of the originator data set] in the 90% confidence interval was accepted as similar.

FcRn Binding Assay
FcRn binding analyses were conducted on Anti-His IgG1 antibody (Cytiva, Marlborough, MA, USA) immobilized CM5 chips (Cytiva, Marlborough, MA, USA). Anti-His IgG1 antibody immobilization procedure was applied by an amine coupling kit based on the manufacturer guide (Cytiva, Marlborough, MA, USA). His-Tagged FcRn (Sigma-Aldrich, St. Louis, MO, USA) protein and all other charge variants or whole molecule samples were prepared in 1× HBS-EP pH 6.0 running buffer with three-fold dilutions (15 nM, 5 nM, 1.66 nM). Recombinant His-tagged FcRn molecule was captured on the active flow cell for 120 s with 10 µL/min flow rate at 22 • C. A blocked flow cell was used as a blank reference during all measurements. Samples were injected over both flow cells (active and blank) at a 30 µL/min flow rate for 120 s, followed by the dissociation phase of 900 s with the running buffer. The chip surface was regenerated with 1× HBS-EP buffer (pH 7.4) for 60 s. Blank buffer injections were also performed on both flow channels, later subtracted from the active surface data before the fitting. The SPR data were presented as the mean value, calculated from at least 3 measurements per sample. One-way analysis of variance, ANOVA, revealed the statistically significant differences between the fractionated sample pairs (p < 0.05 was considered significant and p < 0.005 was considered highly significant). The results were evaluated with Biacore Evaluation Software (3.0 Biacore T200, Shrewsbury, MA, USA) using the steady-state [27,28] and two-state binding models [29,30].

FcγRIa Binding Assay
The interactions between Fc(RIa and the samples were analyzed using the Anti-His capture method on antibody immobilized CM5 chips (Cytiva, Marlborough, MA, USA). Anti-His IgG1 antibody immobilization procedure was applied by an amine coupling kit based on the manufacturer guide (Cytiva, Marlborough, MA, USA). Immobilization levels were found at 8000 RU levels on active and blank flow channels. His-Tagged FcγRIa (R&D Systems, Minneapolis, MN, USA) protein and all samples were prepared in 1× HBS-EP pH 7.4 running buffer with three-fold dilutions (90 nM, 30 nM, 10 nM). Recombinant His-tagged FcγRIa (0.2 ug/mL) molecule was captured on the active flow cell for 60 s with a 10 µL/min flow rate at 22 • C. A blocked flow cell was used as a blank reference during all measurements. Samples were injected over both flow cells (active and blank) at a 30 µL/min flow rate for 60 s, followed by the dissociation phase of 900 s with the running buffer. The chip surface was regenerated with 10 mM glycine pH 1.5 buffer for 60 s. Blank buffer injections were also performed on both flow channels, later subtracted from the active surface data before the fitting. The SPR data were presented as the mean value, calculated from at least 5 measurements per sample. One-way analysis of variance, ANOVA, revealed the statistically significant differences between the samples (p < 0.05 was considered significant and p < 0.005 was considered highly significant). The K D results were evaluated with Biacore Evaluation Software (3.0 Biacore T200, Shrewsbury, MA, USA) using the steady-state binding model and the 1:1 Langmuir binding model for kinetics [31].

C1q ELISA Assay
The direct ELISA method was used to evaluate the binding affinity of samples to the C1q molecule. In this method, an Antibody Pair Buffer kit containing coating buffer, blocking buffer (Assay buffer 5×), washing buffer (25×), chromogen, and stop solution was used (CNB0011, Invitrogen, Waltham, MA, USA). Antibodies (0-5 ug/mL) diluted in coating buffer (contains 50 mM carbonate buffer and 0.1% azide, pH 9.4) to Nunc-Immuno 96-well plate with high binding capacity (168194, Thermo Scientific, Waltham, MA, USA) were placed in triplicate and incubated overnight at +4 • C. After the plate was washed once with 1× wash buffer, blocking was done with 1× assay buffer by agitation at 500 rpm for one hour at room temperature (RT) to block the remaining binding sites. Then, human C1q protein (C1740-Sigma Aldrich, St. Louis, MO, USA) prepared in 1× assay buffer at a 2 ug/mL concentration was added to the plate and incubated for 2 h at RT. After washing the plate five times with 1× wash buffer, it was treated with HRP-conjugated anti C1q polyclonal antibody (157277, Invitrogen, Waltham, MA, USA) diluted 1:1000 at RT for 2 h. After washing with 1× wash buffer five times, the substrate/chromogen mixture was added. The reaction was stopped with a stop solution, and absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

Cell Proliferation Assay
Primary Umbilical Vein Endothelial Cells (HUVEC) (ATCC PCS-100-013, Manassas, VA, USA) were used for cell proliferation studies. Cells were grown at 37 • C, 5% CO 2 in Vascular Cell Basal Medium (ATCC ® PCS-100-030, Manassas, VA, USA) supplemented with Endothelial Cell Growth Kit-VEGF (ATCC ® PCS-100-41, Manassas, VA, USA). In addition, a vascular cell basal medium containing no growth factors such as VEGF, EGF, and FGF was used as the starvation medium. When the cells reached 80% confluency, subculturing was performed. HUVEC cells were counted before the cell proliferation studies, washed by centrifugation at 300× g for 5 min, and resuspended with a complete medium. 5 × 10 3 HUVEC cells/well were inoculated into 96 well plates in 200 µL complete media. It was incubated at 37 • C overnight to allow the cells to adhere. The next day the complete medium was discarded, and the starvation medium that did not contain any growth factors was added. The cells were incubated at 37 • C for 24 h in a starvation medium to allow the proliferative effect of the VEGF growth factor in the complete medium to disappear. After 24 h, monoclonal antibodies were introduced onto the cells at concentrations of 5000 ng/mL, and VEGF was added at a concentration of 20 ng/mL per well. Cells treated with antibodies and VEGFs were incubated at 37 • C for 48 h. Comparison of the potency of originators and biosimilars was represented as relative cell proliferation (%), and similarity acceptance was assessed by statistical equivalence analysis.
CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) (G3582, Promega, Madison, WI, USA) was used to determine the proliferation rates of cells after 24 h. The MTS assay is a commonly used test based on metabolic activity assay. 20 µL of MTS reagent was added per well and incubated for 4 h at 37 • C. In addition, the MTS reagent was placed in an empty well and used as a blank during the measurement. After 4 h of incubation, the absorbance values at 490 nm were measured using an ELISA microplate reader (Bio-Rad, Hercules, CA, USA).

Intact Protein Analysis of Bevacizumab and Its Biosimilars
The structural characterization of the bevacizumab originators, Avastin (AVT) and Altuzan (ALT), and its biosimilars, Zirabev (ZIR) and Mvasi (MVA), was initially performed by intact protein analysis using mass spectrometry. Intact protein analysis is one of the most critical structural elucidation tools for mAbs. The intact mAb analysis reveals the exact molecular weight of the sample. The method also helps to define the modifications that cause a significant molecular weight shift (>100 kDa), such as glycosylation (>1200 Da) and C-terminal lysine clippings (128 Da) [32]. As one of the best-studied modifications, glycosylation is found at the highly conserved Asn303 residue in each heavy chain of Bevacizumab ( Figure 1A). It is already known that the major N-linked glycoform of Bevacizumab is a biantennary fucosylated and agalactosylated structure (G0F:G0F) with fully clipped C-terminal lysine residues (molecular mass:~149.2 kDa) [33]. The most abundant glycoforms of Bevacizumab are G0F:G0F, G0F:G1F, G1F:G1F, respectively.
Avastin and Altuzan are originator products; they are used as reference samples in biosimilarity analysis. All products were analyzed by intact analysis under the same experimental conditions to determine whether there were any glycoform differences between the originator and biosimilar products. The reverse-phase (RP) chromatography performed before mass analysis provides information about the hydrophobicity of the analytes. The retention times of AVT, ALT, ZIR, and MVA on the RP column were the same in all runs and observed as 2.44 min ( Figure 1B) without any shift, which shows that all molecules' hydrophobicity was the same. Similar raw MS spectrums of multiple charged ZIR and MVA were observed compared to the ALT, as represented in Figure 2A,B. The overlay deconvoluted MS spectrums of each sample were shown, and the molecular mass of the main peak in each spectrum was listed in Figure 2C. The intact molecular mass range was determined according to mass values observed in these lots of AVT and ALT and accepted as 149,200.2-149,200.5 Da. The molecular masses of predominant peaks of ZIR and MVA were observed in this reference mass range.
All samples contained one dominant mass peak identified by matching the observed mass and expected theoretical mass with ±50 ppm mass error. G0F: G0F with clipped C-term lysine (149,200 Da) was identified as the predominant glycoform in all originator and biosimilar products. The percentage of G0F:G0F is between 35-50% in AVT and ALT, while it is 38-40% in ZIR and MVA samples. The other glycoforms, including G0F:G1F (149,362 Da), G1F:G1F (149,522 Da) and G0F:G0F, K1 (149,328 Da), which are lower than 20%, were accepted as minor glycoforms and several trace glycoforms (<5%) were identified in all samples in variable quantities. Figure 2D shows the percentage of the major and minor glycoforms found in the indicated samples. It is seen that the amount of the main glycoform varies around 10% even in the originator products, and both ZIR and MVA are in this range. The percentage of G0F:G0F, K1 (one unclipped Lys residue) was higher in Zirabev than in others, as reported in its FDA product quality review [34]. Although C-terminal lysine can lead to the formation of basic charge variants, it is known that it has no impact on in vitro potency, effector function, or pharmacokinetics of mAbs. [35,36]. major and minor glycoforms found in the indicated samples. It is seen that the amount of the main glycoform varies around 10% even in the originator products, and both ZIR and MVA are in this range. The percentage of G0F:G0F, K1 (one unclipped Lys residue) was higher in Zirabev than in others, as reported in its FDA product quality review [34]. Although C-terminal lysine can lead to the formation of basic charge variants, it is known that it has no impact on in vitro potency, effector function, or pharmacokinetics of mAbs. [35,36].

Peptide Mapping Analysis of Bevacizumab and Its Biosimilars
Peptide mapping is one of the crucial analyses to characterize the monoclonal antibodies' primer structural features, including sequence variation, signal sequences, disulfide bonds, and posttranslational modifications (PTMs). Although intact (MS) analysis helps analyze several modifications, assigning chemical modifications with minor mass differences is challenging. Additionally, revealing the posttranslational modification site and rate by a reliable and accurate analysis is vital for developing and

Peptide Mapping Analysis of Bevacizumab and Its Biosimilars
Peptide mapping is one of the crucial analyses to characterize the monoclonal antibodies' primer structural features, including sequence variation, signal sequences, disulfide bonds, and posttranslational modifications (PTMs). Although intact (MS) analysis helps analyze several modifications, assigning chemical modifications with minor mass differences is challenging. Additionally, revealing the posttranslational modification site and rate by a reliable and accurate analysis is vital for developing and standardizing a monoclonal antibody since several studies have been reported that the modifications such as oxidation, deamidation, glycation, or N-terminal pyroglutamic acid can affect the stability, function, or immunogenicity of the antibodies [37,38]. Therefore, we performed the peptide mapping to compare the peptide profiles of the originator and biosimilar products and posttranslational modifications (PTMs) such as deamidation (+1 Da), oxidation (+16 Da), and N-terminal cyclization (−17 Da) were analyzed by data-independent acquisition LC-MS E method to reveal the differences beyond the samples. According to peptide mapping analysis of originator and biosimilar products, it was observed that the tryptic peptide profiles of all samples were similar ( Figure 3A) without any extra unidentified peak. The criteria for identification of peptide sequence are (1) % matched primary ions (b/y ions) are greater than 10%, (2) mass error on peptide mass is less than 10 ppm, and (3) no in-source fragment. Even though the sequence coverage was enhanced by combining two or three different proteolytic enzymes for digestion, over 96% sequence coverage for heavy and light chains was achieved by only trypsin digestion ( Figure 3B). CDRs are the antigen-binding sites, and deamidation or other modifications in this region primarily lead to the loss of target binding and biological activity [38,39]. The sequence and position of CDRs of Bevacizumab are listed in Figure 4B. Three CDRs in heavy chains and three CDRs in light chains were identified (100% sequence coverage) with a maximum 0.25 min retention time shift. No significant modification was determined on the CDRs of any samples. At the same time, minor modifications (<3%) were observed, including Trp oxidation on LC-CDR3 and Asp succinimide on HC-CDR3 Figure 4B,C. In the FDA product quality review report of Zirabev, the risk categories of all assays and attributes are listed in a table [34]. According to the tier-based approach, intact and peptide mapping analyses are suggested as tier 3, the lowest risk ranking. In both intact and peptide mapping analyses performed under the same conditions, there was no significant difference between biosimilars and originators (except for C-terminal Lys clipping). It has been extensively reviewed those chemical modifications such as deamidation or oxidation on mAbs may be caused by process or storage conditions and affect its stability or biological function [39]. In addition to the types, the site of modifications, mostly related to their chemical or physical structure, has great importance in estimating their possible effect [40]. For example, the antigen-binding capacity of an IgG1 antibody is usually not affected by oxidation since the most susceptible residues for oxidation are found in the CH2 domain [41]. The Met oxidation formed in this domain primarily affects the interaction with FcRn or Fcγ receptors [29]. Figure 4A is a schematic illustration of the critical modification sites. Several prone sites in the constant region of IgG1 for specific modification are already known, such as "DTLMISR," which contains Met residue, is the most prone site to oxidation, and a higher amount of deamidation is primarily seen in Asn residues of "GQPENNYYK" [9,37,42,43]. It is also reported that complementarity determining regions (CDRs) can undergo deamidation due to their flexibility or solvent exposure [44].

VEGF Binding Assays of Bevacizumab and Its Biosimilars
Bevacizumab is an IgG1 antibody that binds VEGF-A with their Fab region to neutralize them by preventing their interaction with their receptor, VEGFR, on the cell surface [45] (Figure 5A). Although the biosimilar was "similar" to the originator in terms of structure and function, they are products of different processes, and it is known that the binding kinetics can be affected by the heterogeneities formed during manufacturing [46][47][48]. Enzyme-linked immunosorbent assay (ELISA) [49,50], biolayer interferometry (BLI) [25,51], KinExA [52], and SPR-based methods [53,54] are commonly utilized to investigate antibody-antigen interactions. The SPR technique is robust and reliable for characterizing the binding events in real-time [55]. In this study, SPR revealed the binding affinities of originator and biosimilar drugs.  CDRs are the antigen-binding sites, and deamidation or other modifications in this region primarily lead to the loss of target binding and biological activity [38,39]. The sequence and position of CDRs of Bevacizumab are listed in Figure 4B. Three CDRs in heavy chains and three CDRs in light chains were identified (100% sequence coverage) with a maximum 0.25 min retention time shift. No significant modification was determined on the CDRs of any samples. At the same time, minor modifications (<3%) were observed, including Trp oxidation on LC-CDR3 and Asp succinimide on HC-CDR3 Figure 4B,C. In the FDA product quality review report of Zirabev, the risk categories of all assays and attributes are listed in a table [34]. According to the tier-based approach, intact and peptide mapping analyses are suggested as tier 3, the lowest risk ranking. In both intact and peptide mapping analyses performed under the same conditions, there was no significant difference between biosimilars and originators (except for C-terminal Lys clipping).

VEGF Binding Assays of Bevacizumab and Its Biosimilars
Bevacizumab is an IgG1 antibody that binds VEGF-A with their Fab region to neutralize them by preventing their interaction with their receptor, VEGFR, on the cell surface [45] ( Figure 5A). Although the biosimilar was "similar" to the originator in terms of structure and function, they are products of different processes, and it is known that the binding kinetics can be affected by the heterogeneities formed during manufacturing [46][47][48]. Enzyme-linked immunosorbent assay (ELISA) [49,50], biolayer interferometry (BLI) [25,51], KinExA [52], and SPR-based methods [53,54] are commonly utilized to investigate antibodyantigen interactions. The SPR technique is robust and reliable for characterizing the binding events in real-time [55]. In this study, SPR revealed the binding affinities of originator and biosimilar drugs.

FcRn Binding Assays of Bevacizumab and Its Biosimilars
The FcRn is a cell surface receptor preventing IgG degradation in endosomes and extends the IgG molecules' half-life in vivo by binding to Fc parts of IgG molecules in a pH-dependent manner [59]. For this reason, the analysis of FcRn kinetics differs from According to the tier-based approach suggested by FDA, VEGF-A binding is evaluated in the tier 1 category, which means the highest risk in terms of product quality. In this category, the analytical biosimilarity range is usually presented as the "mean ± 1.5 *SD" since there is enough reference product sampled from several different lots [34]. It has been reported that Fab mediated binding assays, including the VEGF, were performed by ELISA for Zirabev and Mvasi. In previous studies, the VEGF-165A binding constant for bevacizumab was reported as 58 pM [56], 321 pM [36], and between 75.4-4456 pM at different SPR chip configurations [53]. In the current study, the binding constants were 19.93 ± 3.05, 14.74 ± 6.03, 14.48 ± 9.61, 13.51 ± 5.58 pM for AVT, ALT, ZIR, and MVA, respectively. All samples were analyzed with three replicates. The graph in Figure 5B represents the distribution of individual K D values in each group. The equivalence test was used in the similarity assessment of Zirabev and Mvasi to originators ( Figure 5C). According to analysis, both biosimilars were found statistically equivalent and not different.
It is reported that the extra lysine residues at C-terminal do not affect the antigen binding of the mAbs to their target [36,57]. On the other hand, it is known that the modifications such as deamidation on the CDR region can lead to poor binding of mAbs to their target [38,58]. Surprisingly, the binding kinetics of all products for VEGF seem unaffected by the several minor modifications (<3%) such as deamidation, succinimide formation, or tryptophan oxidation observed in the CDR3 region of all originators (AVT, ALT) and biosimilars (ZIR, MVA) of Bevacizumab ( Figure 4C).

FcRn Binding Assays of Bevacizumab and Its Biosimilars
The FcRn is a cell surface receptor preventing IgG degradation in endosomes and extends the IgG molecules' half-life in vivo by binding to Fc parts of IgG molecules in a pH-dependent manner [59]. For this reason, the analysis of FcRn kinetics differs from other Fc receptors. As reported in product quality reviews of biosimilars, the AlphaScreen method is used for Mvasi, and SPR is used for Zirabev similarity analysis. In this study, all products were investigated by the SPR against captured FcRn ligands to reveal the binding patterns among different samples.
In an SPR-based FcRn-IgG interaction study, the affinity values were reported between 6.58 ± 0.12-49.6 ± 1.78 nM for recombinant IgG and between 9.99 ± 0.43-71.9 ± 15.7 nM for human IgG1 [60]. The FcRn binding interaction of mAbs by SPR is considered in the tier 2 category, with lower risk on the product quality. The analytical biosimilarity range for this interaction was accepted as mean ± 3 × SD [34]. In the current analysis, the overlaid sensograms obtained from all samples at different pH conditions were represented in Figure 6A. K D values for bevacizumab-FcRn interactions were calculated by averaging 3 separate analyses and determined as 30.17 ± 12.13, 24.68 ± 3.11, 25.85 ± 6.25, and 26.5 ± 5.47 nM for AVT, ALT, ZIR, and MVA, respectively, by steady-state binding analysis ( Figure 6B). In contrast, they are calculated as 1.9 ± 0.52, 1.53 ± 0.89, 1.59 ± 0.93, and 1.87 ± 0.89 nM for AVT, ALT, ZIR, and MVA by the Two-State Kinetic Binding model ( Figure 6C). In both analyses, there were no significant differences between originator AVT, ALT, and their biosimilars ZIR and MVA.
It is known that several posttranslational modifications, such as methionine oxidation, can reduce the mAb's interaction with the FcRn receptor by leading to conformational changes in the structure [29,61,62]. On the other hand, deamidation was shown to increase the affinity of the mAbs to the FcRn [63]. Our analysis shows that the modifications even higher than 3% in the Fc region, including DTLMISR oxidation or PENNY deamidation, may not affect the FcRn binding capability of Bevacizumab.

FcγRIa Binding Assay of Bevacizumab and Its Biosimilars
FcγRs are responsible for the effector functions in the immune system through their interactions with the Fc part of IgG [64,65]. FcγRIa has the highest affinity to IgG1 among other FcγRs (FcγRIIa, FcγRIIb, and FcγRIIIa). It is expressed on monocytes, macrophages, neutrophils, and dendritic cells and activates the release of pro-inflammatory molecules and phagocytosis of antibody-coated complexes [65]. In contrast to FcγRIIa and FcRIIIa, FcγRIa could bind monomeric IgGs without forming antigen: IgG complexes [66]. Bevacizumab is reported as it has no effector activity through their interactions with FcγRs [33,67]. The FcγRs interactions is evaluated to show the candidate biosimilar's safety and similarity to the original drug [33]. In the official documentation of bevacizumab biosimilars (Zirabev and Mvasi), it is reported that the interactions between IgG:Fc and FcγRs were evaluated with SPR [67 -69]. Previous studies presented that His capture method ensures an oriented configuration on the chip surfaces for IgG:FcγRI binding analysis [70,71]. With this purpose, the binding analysis of FcγRIa is performed with SPR Biacore T200 via utilizing His tagged FcγRIa ectodomain protein on Anti-His immobilized CM5 chip.
According to steady-state K D results ( Figure 7A), FcγRIa affinities were found comparable between originators (AVT: 95.02 ± 8.14 nM, ALT: 104.74 ± 14.35 nM) and the biosimilars (ZIR: 94.02 ± 9.76 nM, MVA: 84.95 ± 11.47 nM). These findings are also compatible with the literature, which states the K D value as 100 nM for the IgG1 molecule in the steady-state model. Also, the K D values were found as 2.75 ± 1.16 nM, 2.32 ± 0.22 nM, 2.27 ± 0.08 nM, and 2.29 ± 0.13 nM for AVT, ALT, ZIR, and MVA, respectively, using 1:1 Langmuir binding model ( Figure 7B). A study using the same method, anti-His capture, represented a K D value of 52 nM from kinetics for IgG1 and FcγRIa binding [71]. Another study reported that a bevacizumab biosimilar's FcγRIa binding affinity is 90-98% similar to the originator [33].

C1q ELISA Assay of Bevacizumab and Its Biosimilars
The complement system called the complement cascade belongs to the innate immune system and consists of many small proteins. The complement system has many essential functions, such as eliminating pathogens or damaged cells and increasing inflammation. Especially with the cooperation of antibodies, it can perform these tasks properly [72]. IgM and IgG immunoglobulins work effectively with the complement

C1q ELISA Assay of Bevacizumab and Its Biosimilars
The complement system called the complement cascade belongs to the innate immune system and consists of many small proteins. The complement system has many essential functions, such as eliminating pathogens or damaged cells and increasing inflammation. Especially with the cooperation of antibodies, it can perform these tasks properly [72]. IgM and IgG immunoglobulins work effectively with the complement system, developing a membrane attack complex on the target cell and influentially destroying the target [73]. Thanks to this effector function of antibodies, it was aimed to create therapeutic monoclonal antibodies, and CDC activity was started to be used effectively in tumor treatment [74].
One of the effector functions of the IgG antibodies is Complement-dependent cytotoxicity (CDC). They initiate a complement cascade system by binding to C1q with the CH2 domain in their Fc parts [75]. The schematic illustration of C1q binding to an IgG is shown in Figure 8A. In the literature, there are several studies, including C1q binding analysis, which is performed by SPR or ELISA [76][77][78]. According to the product quality assessment reports of both Zirabev and Mvasi, this analysis is performed by ELISA. In the current study, the binding efficiencies of AVT, ALT, ZIR, and MVA were analyzed using direct ELISA under the same conditions. is shown in Figure 8A. In the literature, there are several studies, including C1q binding analysis, which is performed by SPR or ELISA [76][77][78]. According to the product quality assessment reports of both Zirabev and Mvasi, this analysis is performed by ELISA. In the current study, the binding efficiencies of AVT, ALT, ZIR, and MVA were analyzed using direct ELISA under the same conditions. It can be seen that the C1q binding is increased in correlation with the increased antibody concentration ( Figure 8B). Four different C1q concentrations (0, 0.6, 1.25, 2.5, 5 ug/mL) were used, and the C1q binding showed linear increasing. Although Bevacizumab can bind C1q, it is known that it does not demonstrate complement-mediated cytotoxicity [16], and we also confirmed this well-known information by CDC assay (data not shown). On the other hand, the binding efficiency of AVT, ALT, ZIR, and MVA was found to be similar and not differ from each other significantly. It is also known that galactosylation may affect the C1q binding [33], but the minor differences in the glycoforms observed had no impact on C1q binding activity in our analysis.

Cell Proliferation Assay of Bevacizumab and Its Biosimilars
In vitro proliferation assays using Primary Human Umbilical Vein Endothelial Cells (HUVECs) are highly effective methods for determining the biological activities of the anti-VEGF monoclonal antibodies. HUVECs originate from endothelial cells by isolating them from the umbilical cord vein [79]. It is widely used for the experimental studies of many physiological conditions such as angiogenesis, fibrinolysis, macromolecule transfer, and blood coagulation [80,81]. Due to HUVEC cells being endothelial cells, they can interact with the VEGF via their VEGF receptors, and intracellular signals are transmitted to ensure cell proliferation [82]. In the presence of anti-VEGF mAb, this interaction inhibits cell proliferation, and tumor angiogenesis can be disrupted ( Figure 9A).
In this study, HUVEC proliferation analysis was performed using MTS, a metabolic activity-based method [83]. The MTS assay (5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazole)-3-(4-sulfophenyl) is a commonly used colorimetric method for cell proliferation analysis [84]. The principle of the method is to examine the activation of some mitochondrial enzymes indirectly due to the rapid metabolic activity of rapidly proliferating cells [85]. The increased mitochondrial enzyme activity causes color change by converting the externally added tetrazolium salts into formazan. The absorbance value of this color change is measured with a spectrophotometer, obtaining information about cell proliferation.
The mechanism of action of Bevacizumab is based on the inhibition of proliferation by binding to VEGF. Therefore, biological activity can be assessed by analyzing the effect of Bevacizumab on cellular proliferation in cell culture. In this study, AVT, ALT, MVA, and ZIR samples were evaluated in HUVEC cells for their anti-proliferation activity by using the MTS proliferation assay. Comparison of the potency of originators and biosimilars was represented as relative cell proliferation (%), and similarity acceptance was assessed by statistical equivalence analysis. All samples showed an inhibitory effect on proliferation, but surprisingly, ZIR was the most effective and reduced the proliferation to~40% ( Figure 9B), although there is no significant difference in VEGF binding among all samples. According to equivalence analysis, MVA was found to be statistically equivalent and not different, while ZIR is nonequivalent and different in cell proliferation ( Figure 9C). This is probably the result of the inadequate lot sample tested in the current study; the greater the number of lots, the better the reliability of the test.
According to the FDA, the proliferation assay is evaluated in the tier 1 category and is one of the most critical assays to show the drugs' potency, efficacy, and safety. There is no detailed technical information about the HUVEC proliferation assay in the FDA and EMA report of Zirabev. On the other hand, the only information about the biological assay of Mvasi is that the ATP-specific luminescent reagent was used to detect proliferation. This study has provided complete data to compare these two biosimilars and the originators (AVT, ALT).

Conclusions
The current study aimed to provide inter-comparability of the approved biosimilars by analyzing them under the same conditions. The structural and biological assays were performed for detailed characterization of Avastin, Altuzan, Zirabev, and Mvasi. In the FDA and EMA reports of Zirabev and Mvasi, the analysis methods used and the biosimilarity results were briefly indicated. Nevertheless, the analysis of all products under identical circumstances was provided to get more detailed technical information and compare the biosimilars properly.

Conclusions
The current study aimed to provide inter-comparability of the approved biosimilars by analyzing them under the same conditions. The structural and biological assays were performed for detailed characterization of Avastin, Altuzan, Zirabev, and Mvasi. In the FDA and EMA reports of Zirabev and Mvasi, the analysis methods used and the biosimilarity results were briefly indicated. Nevertheless, the analysis of all products under identical circumstances was provided to get more detailed technical information and compare the biosimilars properly.
The study has similarly identified the glycoforms and their molecular masses except for the unclipped C-terminal Lys in Zirabev, confirmed by peptide mapping analysis. The position and percentages of the posttranslational modifications were determined in a similar range as the originators. There were no modifications to their CDR regions, affecting the antigen-binding efficiency. The VEGF-A binding of the biosimilar products, evaluated by two different binding models, was within an acceptable range.
On the other hand, it is known that several modifications to the Fc region may affect the binding efficiency of mAbs to Fc receptors. Both FcRn and FcγRIa binding ability of Zirabev and Mvasi were found such as the originator despite M-oxidation and Ndeamidation on the Fc region. It can be concluded that the FcRn binding of Bevacizumab may need a higher level of these kinds of modifications. It is known that Bevacizumab has no CDC activity. However, the C1q binding of the biosimilars was also determined such as the originator, even at different concentrations of C1q. The action mechanism of Bevacizumab at the molecular level is based on halting cell proliferation by binding VEGF-A. In vitro proliferation assays are the best-known ways to examine the biological activity of Bevacizumab. One of the most important findings of this study is that MTS assay data revealed that Zirabev had a slightly more significant inhibitory effect on cell proliferation than Avastin, Altuzan, and Mvasi, which was due to the small number of samples evaluated.
The reports provided by the FDA and EMA summarize the analytical comparison of biosimilars to the originator without giving detailed analytical procedures. Thus, the analysis of two different approved biosimilars under the same conditions could provide a new aspect to the literature in terms of the applied analytical techniques. Further studies in this field would be helpful for the inter-comparability of the biosimilars [1].