# Therapeutic monoclonal antibody - Anti-drug antibody affinity constant determination using capillary electrophoresis - tandem mass spectrometry

Tessa Reinert, Pascal Houzé, Nathalie Mignet, Aynur Naghizade, Lola

Alez-Martin, Oscar Hernandez-Alba, Armand Leclerc, Sarah Cianferani,
Rabah Gahoual, Yannis-Nicolas Francois

► To cite this version:

Tessa Reinert, Pascal Houzé, Nathalie Mignet, Aynur Naghizade, Lola Alez-Martin, et al. Therapeutic monoclonal antibody - Anti-drug antibody affinity constant determination using capillary electrophoresis - tandem mass spectrometry. Analytical Chemistry, 2024, 96 (49), pp.19286-19293. 10.1021/acs.analchem.4c02932. [hal-05237144](https://hal.science/hal-05237144v1)

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Therapeutic monoclonal antibody – Anti-drug antibody
affinity constant determination using capillary
electrophoresis – tandem mass spectrometry

<table><tr><td>Journal:</td><td>Analytical Chemistry</td></tr><tr><td>Manuscript ID</td><td>ac-2024-02932j.R1</td></tr><tr><td>Manuscript Type:</td><td>Article</td></tr><tr><td>Date Submitted by the Author:</td><td>n/a</td></tr><tr><td>Complete List of Authors:</td><td>Reinert, Tessa; Universite de Strasbourg, LSMIS; CNRS UMR8258, INSERM U1022, Paris descartes University, Pharmaceutical Science, UTCBS<br>Houzé, Pascal; Université Paris-Descartes, Laboratoire de Biochimie Mignet, Nathalie; CNRS UMR8258, INSERM U1022, Paris descartes University, Pharmaceutical Science, UTCBS<br>Naghizade, Aynur; Universite de Strasbourg, LSMIS<br>Alez-Martin, Lola; Universite de Strasbourg, LSMIS<br>Hernandez-Alba, Oscar; IPHC, Laboratoire de Spectrométrie de Masse Bio-Organique (LSMBO); Infrastructure Nationale de Protéomique ProFI<br>Leclerc, Armand; ENS de Lyon, Centre de Recherche Astrophysique de Lyon (CRAL), CNRS, UMR5574<br>Cianférani, Sarah; IPHC, Laboratoire de Spectrométrie de Masse Bio-Organique (LSMBO); Infrastructure Nationale de Protéomique ProFI<br>Gahoual, Rabah; CNRS UMR8258, INSERM U1022, Paris descartes University, Pharmaceutical Science, UTCBS<br>Francois, Yannis-Nicolas; Universite de Strasbourg, LSMIS</td></tr></table>

SCHOLARONE™
Manuscripts
Therapeutic monoclonal antibody – Anti-drug antibody affinity
constant determination using capillary electrophoresis – tandem
mass spectrometry

Tessa Reinert* a, b, Pascal Houzé b, c, Nathalie Mignet b, Aynur Naghizade a, Lola Alez-Martina,
Oscar Hernandez-Alba d, e, Armand Leclerc f, Sarah Cianferani d, e, Rabah Gahoual b, Yannis-
Nicolas Francois a

**ABSTRACT:** Monoclonal antibodies (mAbs) therapeutics cannot evade the occurrence of adverse effects. Thus, mAbs are commonly triggering immune responses corresponding to the expression of anti-drug antibodies. Anti-drug antibodies can neutralize mAbs, leading to their inhibition and hasten clearance, which dramatically hampers their therapeutic effects. Therefore, studies concerning the affinity between a mAbs and the corresponding anti-drug antibody are demonstrating a growing interest to further understand the outcome of biotherapeutics after administration.

We describe a novel analytical approach for the determination of the dissociation constant ($K_d$) between a mAb and an anti-drug antibody using capillary electrophoresis coupled to mass spectrometry (CE-MS/MS). The CE-MS/MS method employs a competitive assay followed by the quantification of the residual amount of the active mAbs. The methodology allowed for the measurement of $K_d$ using serum samples without the implementation of immobilization to achieve the protein-protein interaction. This characteristic enabled to generate the interaction in conditions reflecting the physiological environment. A mathematical treatment was developed to calculate $K_d$ from MS/MS data, taking into account the stoichiometry of the mAbs/anti-drug antibody complex and the bivalent properties of the two immunoglobulins. Prior to CE-MS/MS analysis, the interaction of the two proteins was studied using mass photometry to determine equilibrium conditions and complexation stoichiometry. CE-MS/MS was used to investigate the interaction between infliximab and a neutralizing anti-infliximab antibody. Results allowed to measure a $K_d$ of 14.4 ± 2.9 nM. MS instrumentation sensitivity and specificity showed to be relevant to achieve accurate and robust $K_d$ measurements for strong interactions in the nano-molar range.

Monoclonal antibodies (mAbs) and their related formats, such as bispecific antibodies, fusion proteins, or
antibody-drug conjugates, currently represent a significant proportion of biopharmaceutical products. Their
development and successful therapeutic applications have undoubtedly contributed to the advent of this
pharmaceutical field, with 175 approved products, including 19 in 2022, and more than 140 reported in
clinical trials ¹. Rationally, therapeutic proteins such as mAbs are expected to have lower toxicity compared
to cytotoxic molecules. However, mAbs are particularly prone to induce immunogenic reactions ². For
instance, the patient's immune system may express neutralizing anti-drug antibodies (NADAs). NADAs
can target the antigen binding fragments ($F_{ab}$), resulting in inhibition of the therapeutic mAb from binding
its corresponding antigen normally. Neutralization of therapeutic mAbs by NADAs can compromise their
efficacy and hasten their clearance, which in most adverse cases could lead to complete inactivation ³. The
expression of NADAs after injection of mAbs remains challenging in several aspects. Indeed, this type of

<footnote>a Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), Université de Strasbourg, CNRS UMR7140, CMC, Strasbourg, 67081, France.</footnote>

<footnote>b Université Paris Cité, CNRS, Inserm, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), Faculté de sciences pharmaceutiques et biologiques, Paris, 75270, France</footnote>

<footnote>c Laboratoire de Toxicologie Biologique, Hôpital Lariboisière, Assistance Publique – Hôpitaux de Paris (AP-HP), Paris, 75010, France</footnote>

<footnote>d Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, CNRS UMR7178, IPHC, Strasbourg, 67037, France</footnote>

<footnote>e Infrastructure Nationale de Protéomique ProFI - FR2048, Strasbourg, 67087, France</footnote>

<footnote>f Centre de Recherche Astrophysique de Lyon (CRAL), Université Lyon 1, CNRS, UMR5574, ENS de Lyon, Saint-Genis-Laval, 69007, France</footnote>
immune response is not systematic among treated populations, without any clear attributions so far regarding
the causalities of this phenomenon. In addition, their episodic presence complicates the therapeutic
management of patients and urges the implementation of dedicated follow-up.

A variety of approaches have been used to determine antibody affinities, including surface plasmon
resonance (SPR)⁴ and indirect and competitive immunoassays⁵. The latter ones aim to determine the
apparent affinities of a system by assessing the concentration of unbound active antibodies in a solution.
This is done after preincubation of the antibody solution with predetermined molar concentrations of the
corresponding antigen and can be performed in the matrix. Bobrovnik *et al.* and Stevens *et al.* introduced a
novel approach that is based on competitive enzyme-linked immunosorbent assays (ELISA), and facilitated
the determination of apparent antigen-mAb affinity derived from the law of mass action ⁶⁻⁸. The strategy
was then applied to determine mAb affinities with receptors or antigens ⁹,¹⁰.

Although the equilibrium state can be achieved in the matrix system, ELISA technologies are known to be
affected by serum interference (¹¹,¹²). Similarly, the immobilization of mAbs on the surface of ELISA wells
required to perform the analysis can lead to epitope alteration or unfavorable presentation. In addition, the
multiple wash steps can dissociate low affinity interactions, resulting in negative results and interfering with
the affinity measurement ¹³,¹⁴. All these issues can alter the quantification of unbound active mAbs by
ELISA and thus affect the determination of affinity. The use of SPR for affinity measurement is quite
robust¹⁵, however it also requires the immobilization of mAbs on SPR sensor. In addition, the
aforementioned approaches were originally established for bivalent antibodies binding to monovalent
antigens, and therefore need to be adapted for mAb-to-mAb affinity assessment.

The current study overcomes these issues to determine the dissociation constant between two antibodies in
serum. After preincubation of the mAbs solution, the absolute quantification of unbound active antibodies
is performed using capillary electrophoresis hyphenated to tandem mass spectrometry (CE-MS/MS)
analysis ¹⁶, allowing the determination of the experimental dissociation constant *K*<sub>d</sub> between the therapeutic
antibody infliximab (IFX) and a neutralizing anti-drug antibody, referred to as neutralizing antibody-to-
infliximab (NATI). IFX is a chimeric mAb targeting tumor necrosis factor alpha (TNF-α) to inhibit the pro-
inflammatory activity of TNF-α, observed in inflammatory bowel diseases such as Crohn disease. Because
of its use in the treatment of chronic disease, IFX must be injected regularly to maintain an optimal
suppression of symptoms, as long as the symptoms last. However, recent studies highlighted that 38 % of
patients expressed NATIs after 12 months of treatment ¹⁷.

Prior to CE-MS/MS experiments, mass photometry (MP) analysis was performed on samples consisting of
a mixture of stable isotope-labeled infliximab (SIL-IFX) and NATI to investigate the stoichiometry and
kinetics of IFX/NATI complexation. Then, CE-MS/MS analysis allowed accurate quantification of the
fraction of active IFX remaining after equilibrium and non-neutralized by NATI. This concentration was
determined using an internal standard consisting of an isotopically labeled adalimumab (SIL-ADM), also
initially introduced into the model serum. To be compatible with CE-MS/MS analysis, active IFX and SIL-
ADM were extracted from serum samples using an affinity purification process developed in-house based
on magnetic beads functionalized with TNF-α. Subsequently, the purified mAbs were submitted to proteolytic digestion, and the resulting peptide mixture obtained was characterized by CE-MS/MS. Finally, the IFX/NATI dissociation constant $K_d$ was deduced from the absolute quantification of active IFX, and was found in the nanomolar range. Several developments regarding the methodology to calculate the $K_d$ value from CE-MS/MS data were also introduced in this study, especially in order to consider the complexity due to the bivalence of IFX and NATI respectively.

## ■ MATERIALS AND METHODS

**Chemicals.** The experiments were performed with isotopically labeled analogues of infliximab and adalimumab, which are expected to have identical reactivities and affinities as the unlabeled mAbs. Stable isotopes labeled infliximab (SIL-IFX) and adalimumab (SIL-ADM), labeled on arginine (¹³C₆; ¹⁵N₄) and lysine (¹³C₆; ¹⁵N₂) residues, were supplied by Sigma-Aldrich (Saint-Quentin Fallavier, France). A full-length human anti-idiotypic antibody against IFX (clone AbD17841_hIgG1) used as a model NATI was obtained from Bio-Rad Laboratories (Marnes-La-Coquette, France). Streptavidin M-280 Dynabeads™ and biotinylated TNF-α were obtained from Fisher Scientific Invitrogen (Illkirch, France). Sequencing-grade modified trypsin was purchased from Promega Corporation (Madison, WI, USA).

**Preparation of IFX quantification calibration samples.** Standard solutions used to construct IFX calibration curves were prepared from blank serum, obtained from the French Institute of Blood (Paris, France), spiked with increasing IFX concentrations ranging from 1 to 25 µg·mL⁻¹ and ADM at a fixed concentration of 10 µg·mL⁻¹.

**$K_d$ determination samples elaboration.** Model serum samples used for $K_d$ determination were prepared from 100 µL of blank serum spiked with a fixed concentration of ADM and IFX (10 µg·mL⁻¹ each) and increasing concentrations of NATI ranging from 1 to 30 µg·mL⁻¹. Afterward, samples were incubated for 1h at 20°C prior to further treatment.

**CE-MS/MS sample preparation.** The preparation of serum samples was derived from the protocol recently published by our group ¹⁸. Briefly, ferromagnetic beads (Dynabeads™) and biotinylated TNF-α were mixed and incubated for 1h at 20°C. The serum solution was then added to the functionalized beads and incubated for 1h at RT to allow the TNF-α inhibitors IFX and ADM to bind to the beads. After elimination of the serum, acidic dissociation was performed by adding MeOH/H₂O/FA (48.5/48.5/3; v/v/v) solution to elute mAbs from the surface of the beads. The supernatant was dried, and consequently, 70 µL of ammonium bicarbonate buffer (50 mM, pH 8) was used to reconstitute the sample prior to the denaturation-reduction-alkylation-digestion workflow, which is commonly used for the bottom-up strategy of mAbs ¹⁹. Finally, the peptide digest was dried and reconstituted in 5 µL ammonium acetate, 100 mM, pH 4.

**Mass photometry analysis.** MP experiments were carried out using a TWOMP mass photometer (Refeyn Ltd, Oxford, UK) at room temperature. Microscope coverslips (24×50 mm, Fisher Scientific and 24×24 mm, Globe Scientific) were prepared by sequential rinsing with isopropanol and H₂O, followed by drying under ultrapure nitrogen. PBS solutions containing NATI (3 µg·mL⁻¹) and IFX (3 µg·mL⁻¹) were incubated for different times (1-60 min) before being loaded into the six-well reusable silicone gaskets (CultureWellTM,
50-3 mm DIA x 1mm Depth, 3-10 µL, Grace Bio-Labs, Inc., Oregon, USA) assembled on the cover slide. Images were processed using the DiscoverMP software (Refeyn Ltd, Oxford, UK). The relative intensities of the identified species were calculated with the individual number of counts obtained for each population upon performing a Gaussian fitting.

**Capillary electrophoresis-tandem mass spectrometry analysis.** CZE-ESI-MS/MS experiments were performed using a CESI8000® capillary electrophoresis system (Sciex separations, Darmstadt, Germany) coupled to a Sciex 5600 TTOF mass spectrometer (Darmstadt, Germany) with a sheathless interface. The separation capillary, filled with the background electrolyte (BGE, 10 % acetic acid), was made of bare fused silica (total length 100 cm; 30 µm i.d.). For each analysis, a volume of 68 nL was hydrodynamically injected (5 psi, 100 s) and a voltage of +23 kV was applied for the separation. The ESI voltage was -1.5 kV, the source heating temperature was 100 °C, and the curtain gas was set to 2. Experiments were performed in Top20 information-dependent acquisition (IDA, Sciex) with a total duty cycle of 1.9 s during 60 min of analysis.

**Active IFX quantification.** The quantification of active IFX was based on the area of the peak corresponding to the proteospecific peptide IFX\*LC01 (Table 1). In order to provide a robust IFX absolute quantification, ADM was used as an internal standard and measured by the intermediate of the peptide ADM\*HCO1 (Table 1). The MS and MS/MS spectra were studied to ensure unambiguous identification of the two compounds. The IFX peptide IFX\*LC01 is located on the variable part of infliximab, and its specificity was verified by a blast search against a human proteome database. Further details are discussed in the Supporting Information

<table><caption>**Table 1. Ions of surrogate's peptides for SIL-IFX and SIL-ADM**</caption><thead><tr><th>Peptide sequence</th><th>Name</th><th>Analyte</th><th>Role</th><th>Precursor (m/z)</th><th>Product ions (m/z)</th></tr></thead><tbody><tr><td>DILLTQSPAILSVSPGER*<br>(<sup>13</sup>C,<sup>15</sup>N)</td><td>IFX*LC01</td><td>SIL-IFX</td><td>Quantifier</td><td>953.53</td><td>y9-967.54;<br>y8-854.34;<br>y7-741.37;</td></tr><tr><td>EVQLVESGGGLVQPGR*<br>(<sup>13</sup>C,<sup>15</sup>N)</td><td>ADM*HCO1</td><td>SIL-ADM</td><td>Internal<br>standard</td><td>817.94</td><td>y8-909.45;<br>y7-746.39;<br>y6-645.34;</td></tr></tbody></table>

■ RESULTS AND DISCUSSIONS

**1. IFX/NATI complexation mass photometry study.** A multi-step approach was used to characterize the interaction between IFX and NATI. First, mass photometry (MP) analysis was performed to provide insights on the interaction between the different mAbs included in the study. MP is a relatively new technique based on the interferometric scattering microscopy principle (iSCAT) as a means for detecting and measuring the mass of biomolecules and their complexes<sup>20,21</sup>. Briefly, the molecular entities in solution bind the microscope cover glass surface in a non-specific manner. This interaction alters the local reflectivity, generating a contrast signal as a result of the interference between the scattered and reflected lights. The magnitude of the interference (or contrast) can be correlated to the mass of the entity of interest upon calibration with biomolecules of known masses.<sup>22</sup> Therefore, MP is a label-free method that detects biomolecules and their interactions in solution at the single molecule level. The versatility, and sensitivity of this measurement have spurred the use of MP to characterize a wide range of biological samples, such as adeno
associated virus <sup>23</sup>, membrane proteins<sup>24,25</sup>, monoclonal antibodies <sup>26</sup>, ribosomal particles, and ribonucleotides <sup>27,28</sup>.

In our study, MP analysis was performed on samples containing solely IFX or NATI diluted in PBS. As emphasized in Figure 1a., the MP spectrum in the case of IFX alone showed a single peak presenting an average molecular mass of 159 kDa (standard deviation σ: 13kDa). Similarly, the analysis of NATI demonstrated a single peak presenting an average mass of 164 kDa (σ: 20kDa, Figure 1b.). Therefore, the MP experiments showed that both spectra were governed by the monomeric structure of the mAbs, with no experimental evidence supporting the presence of higher molecular weight species (HMWS). Consequently, equimolar mixtures of IFX and NATI in PBS were incubated for 1 hour at 20°C and analyzed by MP. As shown in Figure 1c., the mass of the most intense species was calculated at 145 kDa (σ: 13kDa), corresponding to free IFX, and NATI mAbs. Several signals were detected beyond 200 kDa as a result of the interaction between both mAbs. The mass of the most intense complex signal was 290 kDa (σ: 14kDa), which was attributed to a 1:1 IFX–NATI complex. In addition, the MP spectra exhibited a weak signal at mass 433 kDa (σ: 15kDa), which could be attributed to the formation of complexes presenting 1:2 and/or 2:1 stoichiometries. Finally, the MP data showed the identification of higher molecular weight species with a mass of 577 kDa (σ: 18kDa), potentially corresponding to a 2:2 stoichiometry between both mAbs.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Mass photometry spectra for **a.** SIL-IFX at 3 µg·mL⁻¹, **b.** NATI antibody, **c.** SIL-IFX and NATI mixture at 3 µg·mL⁻¹ and **d.** Incubation of NATI and SIL-ADM both at 3 µg·mL⁻¹</figcaption></figure>

To demonstrate the specificity of NATI to neutralize IFX, a mixture of adalimumab (ADM) and NATI was analyzed under similar conditions as a negative control. ADM is another type of mAb targeting TNF-α. After incubation, MP data revealed a single signal at 170 kDa corresponding to the mixture of ADM and NATI, suggesting that both mAbs do not interact to form HMWS (Figure 1d.), thus corroborating the specificity of NATI to bind IFX F<sub>ab</sub>. Therefore, the MP data revealed a predominant 1:1 stoichiometry in the interaction between IFX and NATI. Drawing from this finding, a critical aspect required further exploration due to the bivalence inherent in both proteins owing to their IgG1 nature. Hence, in the presence of NATI, IFX can exist in an unliganded state, a singly liganded state, or a doubly liganded form, as illustrated in Figure 2. In its singly liganded configuration, IFX can bind to a NATI on one F<sub>ab</sub> and remain active as it can still bind its other arm to TNF-α.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Schematic representation of the different forms of infliximab/NATI interaction for a 1:1 stoichiometry. Non-complexed infliximab or experiencing single Fab complexation remains active.</figcaption></figure>

Following IFX/NATI stoichiometry determination, a kinetic study of the neutralization was performed using MP analysis. Samples consisting of NATI and IFX in PBS were analyzed after different incubation times. As emphasized in Figure 3.a, the relative proportion of the 4 peaks observed for the MP data (Figure 1c.) showed a rapid formation of the 1:1 complex, correlated with a decrease of the peak corresponding to IFX and NATI monomers. After 5 minutes, the relative distribution of monomers and 1:1 complex remained stable, indicating that equilibrium was reached. The MP data exhibited 1:2 and 2:2 stoichiometries that were systematically present below 4% regardless of the incubation time. Based on these results and assuming that IFX-NATI interaction stoichiometry is identical regardless of the sample composition (PBS or serum), only the 1:1 complex was considered to be formed in the serum sample. Consequently, the proportion of each species at equilibrium was performed by varying the initial concentration of NATI to evaluate the relative proportion of the observed species. MP data demonstrated essentially the formation of a 1:1 IFX/NATI complex, even when NATI was present in large excess compared to IFX (Figure 3.b).

This observation was attributed to the specific nature of the interaction between IFX $F_{ab}$ regions and NATI. However, it was observed that higher proportions of NATI (beyond the 1:7 ratio, Figure 3.b) led to increased relative intensities of the free mAb signal. This observation is the direct consequence of the low MP mass resolution, which impairs the differentiation between the two NATI and IFX mAbs with close molecular masses. The relative intensities of 1:2 and 2:2 stoichiometry complexes remained stable and below 10% (6.0 ± 2.3%, and 6.8 ± 2.9%, respectively) with increasing NATI concentrations.

<figure><img src="image_4.png" /><figcaption>**Figure 3** a. Kinetic study of SIL-IFX/NATI complexations using mass photometry in PBS. SIL-IFX and NATI concentration were fixed at 3 µg·mL⁻¹. b. Evolution of species relative proportion depending on NATI initial concentration for fixed SIL-IFX at 3 µg·mL⁻¹ (n=3, error bars represent the standard deviation). Red dots: free mAbs (signal at 145kDa), green dots: 1:1 stoichiometry (signal at 290kDa), blue dots: 1:2 or 2:1 stoichiometry (signal at 433kDa) and black dots: 2:2 stoichiometry (signal at 577kDa)</figcaption></figure>
**2. CE-MS/MS analysis for IFX/NATI interaction K<sub>d</sub> determination.** A dedicated workflow was developed to experimentally measure the IFX/NATI K<sub>d</sub> constant, as represented in Figure 4. The CE-MS/MS analytical strategy was derived from a competitive binding assay. Model serum samples were spiked with known amounts of IFX and NATI and left to generate IFX-NATI complexation in a state as close as possible to *in vivo* conditions. The fraction of active IFX (i) that did not form complexes with NATI was then specifically extracted and quantified using CE-MS/MS. To ensure accurate and precise quantification of this remaining active IFX, an internal standard consisting of stable-isotope labeled adalimumab (ADM) was added to serum samples. ADM accounted for any bias originating from sample preparation and/or analysis, as it was not affected by NATI neutralization (Figure 1d). The active fractions of IFX and ADM were simultaneously extracted by affinity purification using magnetic beads with immobilized TNF-α<sup>29</sup>. The two extracted mAbs were then digested and analyzed using CE-MS/MS conditions recently developed by our group<sup>18</sup>. Finally, CE-MS/MS absolute quantification of the active fraction of IFX (i) as a function of spiked NATI concentration was used to accurately assess the dissociation constant (K<sub>d</sub>) of the infliximab/NATI interaction (Figure 4).

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Workflow designed for the K<sub>d</sub> determination of infliximab/NATI complex using CE-MS/MS analysis. Model serum samples were prepared using IFX and ADM spiked in similar concentrations (10 µg·L<sup>-1</sup>) in different concentrations of NATI (0-30 µg·L<sup>-1</sup>).</figcaption></figure>

**Quantification of active IFX.** Prior to the determination of the K<sub>d</sub> constant between IFX and NATI, the characteristics of IFX quantification using CE-MS/MS and ADM as internal standards were first evaluated without the presence of NATI. Regarding selectivity, the analysis of serum samples spiked with IFX and ADM only showed that the selected two surrogate peptides (Table 1) exhibited significantly different mobilities, leading to their consistent separation in CZE (Figure S1.a., Supporting Information). In addition, no interferences were observed in the extracted ion electropherograms of the two peptides (Figure S1.b., Supporting Information). Consequently, the linearity between the ratios (r) of the surrogate peptides measured by CE-MS/MS as a function of the concentration of active SIL-IFX was investigated. Good repeatability and linearity were achieved for concentrations ranging from 1-25 µg·mL<sup>-1</sup> (Figure 5). In the case of serum spiked with IFX and ADM at an equivalent concentration of 10 µg·mL<sup>-1</sup> (i<sub>0</sub>), the peptide ratio was defined as r<sub>0</sub> = 1, as emphasized in Figure 5. Therefore, the CE-MS/MS data demonstrated that quantification of active IFX was possible over the concentration range considered. Therefore, the
experiments suggested that by introducing IFX and ADM at equivalent concentrations into a serum sample potentially containing NATI, the evolution of the peptide ratios can be used to determine the concentration of active IFX (i) by the intermediate of this linear relationship (Figure 5).

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Evolution of IFX\*<sub>LC01</sub> / ADM\*<sub>HC01</sub> peptide ratio with the concentration of IFX (0-25 µg·mL⁻¹), ADM concentration was fixed at 10 µg·mL⁻¹ (n = 3). Linear regression y= 0.10 x – 0.02 (r² = 0.982).</figcaption></figure>

**Interaction between IFX and NATI paratopes.** To measure the dissociation constant of the IFX/NATI complex further experimentally, as described in Figure 4, a series of serum samples were prepared incorporating IFX and ADM systematically at 10 µg·mL⁻¹ (i₀) with different concentrations of NATI (a₀). The serum samples were then analyzed using the CE-MS/MS analytical strategy developed, in order to determine the ratio r between the surrogate peptides corresponding to IFX and ADM, respectively (Table 1). As previously determined, the value of r denotes the fraction of active IFX compared to the internal standard. Figure 6 shows the ratio depending on the initial concentration of NATI. CE-MS/MS results demonstrated a progressive decline as the concentration of NATI increased. This phenomenon indicated the neutralizing effect of NATI, which resulted in inhibition of IFX extraction during the purification process. This indicates a hampered biological activity of IFX.

<figure><img src="image_7.png" /><figcaption>**Figure 6.** Ratio between IFX/ ADM CE-MS/MS signals as a function of the initial concentration of neutralizing-antibodies-to-infliximab (NATI, a₀) added to serums. Error bars represent the standard deviation among the triplicates.</figcaption></figure>
The results presented in Figure 6 highlight crucial information about IFX/NATI complexation. At equimolar concentration ($i_0 = a_0 = 10 \ \mu g \cdot mL^{-1}$), it appears that approximately 80% of the IFX antibodies were completely neutralized by the NATI (both paratopes bound) and were therefore not extracted by TNF-$\alpha$. This could suggest that under native conditions, most NATI/infliximab interactions occur in a doubly liganded form (Figure 2).

Due to their IgG nature, both IFX and NATI are bivalent proteins. Thus, IFX may potentially be neutralized on the two $F_{ab}$ arms. Similarly, NATI can be involved in the partial neutralization of two independent IFX molecules (Figure 2). As a consequence, the methodology used to calculate $K_d$ values based on CE-MS/MS data was adapted to account for double bivalence. Therefore, to address this complexity, the two paratopes were considered independently for IFX and NATI, respectively. Table 2 presents the reaction table corresponding to the neutralization of IFX paratopes by NATI paratopes when IFX and NATI are introduced into the serum sample.

<table><caption>**Table 2.** Complexation reaction of IFX and NATI paratopes when introduced in a solution. Term x defines the concentration of IFX/NATI paratopes complexes formed at equilibrium.</caption><thead><tr><th>State</th><th>Paratope (IFX) +</th><th>Paratope (NATI) ↔</th><th>Liganded paratopes</th></tr></thead><tbody><tr><td>Initial</td><td>2<i>i</i><sub>0</sub></td><td>2<i>a</i><sub>0</sub></td><td>0</td></tr><tr><td>Equilibrium</td><td>2<i>i</i><sub>0</sub> - <i>x</i></td><td>2<i>a</i><sub>0</sub> - <i>x</i></td><td><i>x</i></td></tr></tbody></table>

$a_0$ the initial concentration of NATI ($\mu g \cdot mL^{-1}$), $i_0$ the initial concentration of IFX ($\mu g \cdot mL^{-1}$), $x$ the concentration of liganded paratopes ($\mu g \cdot mL^{-1}$)

Based on Table 2, the probability for a single IFX paratope to be neutralized at equilibrium can be expressed using equation (1), as described previously for an antibody-antigen system 8:

$$p = \frac{x}{2i_0} \qquad (1)$$

The inactivation of IFX resulted from the simultaneous neutralization of its two paratopes at equilibrium (Figure 2). The probability of such an event could be expressed by the factor $p^2$. Therefore, the probability for IFX to remain potentially active after equilibrium, due to having only 0 or 1 paratope neutralized, is expressed by the intermediate of equation (2):

$$\frac{i}{i_0} = 1 - \left(\frac{x}{2i_0}\right)^2 \qquad (2)$$

The analysis performed using the CE-MS/MS method enabled us to quantify the fraction of active IFX ($i/i_0$). Indeed, inactive IFX evaded the affinity purification step, resulting in the quantification of active IFX alone. Using this characteristic and the proportional relationship between the fraction of active IFX ($i$) and the IFX/ADM peptide ratio ($r$) determined previously (Figures 5 and 6); the concentration of totally bound IFX/NATI paratopes is obtained (further details are discussed in the Supporting Information):

$$x = 2i_0\sqrt{1 - r} \ (\mu g \cdot mL^{-1}) \qquad (3)$$

Based on the law of mass action, the dissociation constant of IFX/NATI paratope interaction is defined as:
$$K_d = \frac{(2a_0-x)(2t_0-x)}{x} \quad (4)$$

Equation (3) was further transposed into equation (4), resulting in the establishment of the following expression:

$$\frac{a_0 - t_0 \sqrt{1 - r}}{K_d} = \frac{\sqrt{1 - r}}{2 * (1 - \sqrt{1 - r})} \quad (5)$$

It was assumed that the extraction of the active IFX from the serum using TNF-α functionalized beads does not readjust the equilibrium of the IFX/NATI neutralization. Equilibrium readjustment was found to be negligible in a previous study for another antigen/antibody complex after extraction in ELISA plates ³⁰. This property was explained by the nanomolar affinities involved in the interactions between IFX and NATI or TNF-α. Therefore, such important affinities favored the formation of complex formation compared to complex disassembly, considering the concentration of IFX and NATI present in serum samples.

Consequently, equation (5) was used to plot $a_0/K_d$ as a function of the initial concentration of NATI $a_0$, as shown in Figure 7, using data obtained from CE-MS/MS experiments. The dissociation constant $K_d$ between IFX and NATI, corresponding to the inverse of the slope, was found to be 14.4 ± 2.9 nM. The error was calculated as the 95 % confidence interval of the slope. The linear regression and experimental data demonstrated a good correlation, indicating that the experimentally determined $K_d$ constant using CE-MS/MS data was stable for the concentration range of NATI considered. The results showed that the $K_d$ values determined experimentally were in a similar range compared to other isotypes of anti-IFX antibodies described in the literature ³¹. As expected, dissociation constants measured in the nanomolar range demonstrated the important affinity regulating IFX/NATI interaction. Therefore, the CE-MS/MS analytical strategy demonstrated the possibility to determine extremely low dissociation constants. In addition, the experiments were performed in a native environment composed of human serum, which allowed the experiments to be positioned closer to in vivo conditions. ELISA assays and surface plasmon resonance (SPR), conventionally used to characterize protein-protein dissociation constants, rely on the immobilization of mAbs on different types of surfaces ⁴,³². However, these methodologies may hinder the interaction if the mAbs are not properly oriented, exposing their variable domain, which could lead to a systematic bias in the $K_d$ determination.

It is important to note, regarding the CE-MS/MS analytical strategy, that the calculated $K_d$ reflects the global affinity of all IFX paratopes existing in mixed valences of the $F_{ab}$ region. The intrinsic affinity of a local IFX(paratope)/NATI(paratope) interaction may differ depending on whether the two proteins are singly liganded or doubly liganded (Figure 2) ³³. Therefore, the intrinsic affinity of a single IFX paratope may vary depending on whether the second IFX $F_{ab}$ is already involved in an interaction with a NATI paratope.

To conclude, the analytical methodology developed using affinity purification followed by CE-MS/MS analysis, allowed the dissociation constant between IFX and NATI paratopes to be measured in a reliable manner while minimizing artifacts and preserving the native state of the two proteins. The method conditions were designed in order to be compatible with the subsequent CE-MS/MS analysis. Thus, for the first time, this analytical technique could be applied to a peptide-centric approach for the measurement of
protein-protein dissociation constants. Results showed the capacity of the developed strategy to measure the concentration of active IFX in a biological sample, in addition to distinguishing faint changes concerning the levels of active mAbs. This property allowed the consistent calculation of the dissociation constant corresponding to the neutralization of IFX by NATI. The methodology provided valuable insights into the IFX/NATI interaction and demonstrated the possibility of maintaining the natural diffusion of both antibodies within the biological matrix, without any labeling or surface functionalization during the complexation. The incorporation of the ADM internal standard was particularly important, as it allowed us to account for losses and/or biases potentially originating from sample preparation, thereby improving the reproducibility and robustness of the method. Also, it allowed the estimation of the amount of neutralized IFX, and thus the value of the dissociation constant, without any structural information regarding NATI antibody, such as the amino acid sequence or the molecular mass of the protein. Thus, the CE-MS/MS analytical approach was not restricted to the analysis of a single isotype of NATI but can be used to characterize any isotype responsible for the neutralization of IFX. Consequently, it is possible to use the analytical strategy based on CE-MS/MS for the detection of the immune response in samples originating from patients, regardless of the isotype expressed by their immune system.

<figure><img src="image_8.png" /><figcaption>**Figure 7.** Plot of a₀/Kₐ calculated using equation (5) depending on the initial concentration of NATI (a₀) ranging from 0 to 30 µg·mL⁻¹. Linear regression y= 0.46 x – 2.5 (r² = 0.958). Error bars represent the standard deviation among triplicates.</figcaption></figure>

## ■ CONCLUSION

An integrated methodology was developed to characterize the interaction between therapeutic mAbs IFX and a neutralizing antibody-to-infliximab NATI, commonly expressed due to immunogenic reactions in patients. MP analysis was performed on IFX/NATI mixtures in order to determine the stoichiometry of the complex formed consequently to neutralization. In addition, the MP results allowed us to demonstrate the specificity and investigate the kinetics of NATI neutralization. Consequently, a novel analytical strategy based on a label-free and immobilization-free method was designed and implemented to experimentally measure the dissociation constant between IFX and NATI, directly performed in biological matrices. The approach utilized affinity purification prior to CE-MS/MS analysis using an internal standard in order to eliminate any potential bias due to sample treatment analysis. CE-MS/MS was performed to provide an accurate determination of active IFX quantities in the presence of NATI. A robust mathematical model, compatible with CE-MS/MS data and taking into account the bivalent characteristics of both IFX and NATI,
allowed to measure experimentally the apparent dissociation constant of IFX/NATI complexation. As a result, the dissociation constant was calculated from the intermediate of concentrations of active IFX in model serums in the presence of NATI. To our knowledge, CE-MS/MS has never been used to determine the dissociation constant between therapeutic mAbs and their corresponding anti-drug antibodies. The application of the CE-MS/MS analytical strategy allowed to measure an IFX/NATI dissociation constant of $14.4 \pm 2.9$ nM showing relevant affinities for this type of high-binding interaction.

The CE-MS/MS analytical strategy demonstrated the ability to study IFX/NATI affinity under conditions that are particularly close to *in vivo* conditions. The results showed that the measurement of the quantity of active IFX using an independent internal standard provided the ability to measure the dissociation constant without any structural information regarding NATI. Therefore, the CE-MS/MS analytical strategy could be applied to measure the dissociation constant of other NATI isotypes using the same methodology and method conditions. Also, CE-MS/MS enabled to target neutralizing antibodies in a specific manner compared to commercial ELISA assays that may detect anti-IFX antibodies regardless of their neutralizing capacities. As a consequence, CE-MS/MS analysis could be used for the analysis of patient serums in order to identify unambiguously the occurrence of an immune response in the form of NATI expression. In addition, further developments of the CE-MS/MS analytical strategy could be implemented to provide simultaneous absolute quantification of IFX and NATI, for instance to monitor the occurrence of immune responses and estimate the level of immune response against the administered therapeutic mAbs.

## AUTHOR INFORMATION

**Corresponding Author**

*E-mail: tessa.reinert@univ-lyon1.fr

## SUPPORTING INFORMATION

Additional experimental details of the MS data monitoring of the two selected surrogate peptides.
Further details on the calculation for affinity assessment.

## ACKNOWLEDGMENTS

The authors would like to thank every person that contributed to the achievement of this article. This research work was supported by the Agence National de la Recherche as part of the project **MethAmAbs** (project no. ANR-19-CE29-0009) the CNRS, and the French Proteomic Infrastructure (ProFI; ANR-10-INBS-08-03). OHA acknowledges the “Agence Nationale de la Recherche” for funding his JCJC project «**ConformAbs**» (ANR-21-CE29-0009-01).

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<figure><img src="image_9.png" /><figcaption>Figure 1. Mass photometry spectra for a. SIL-IFX at 3 µg·mL⁻¹, b. NATI antibody, c. SIL-IFX and NATI mixture at 3 µg·mL⁻¹ and d. Incubation of NATI and SIL-ADM both at 3 µg·mL⁻¹</figcaption></figure>

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<figure><img src="image_10.png" /><figcaption>Figure 2. Schematic representation of the different forms of infliximab/NATI interaction for a 1:1 stoichiometry. Non-complexed infliximab or experiencing single Fab complexation remains active.</figcaption></figure>

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<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>Figure 3 a. Kinetic study of SIL-IFX/NATI complexations using mass photometry in PBS. SIL-IFX and NATI concentration were fixed at 3 µg·mL⁻¹. b. Evolution of species relative proportion depending on NATI initial concentration for fixed SIL-IFX at 3 µg·mL⁻¹ (n=3, error bars represent the standard deviation). Red dots: free mAbs (signal at 145kDa), green dots: 1:1 stoichiometry (signal at 290kDa), blue dots: 1:2 or 2:1 stoichiometry (signal at 433kDa) and black dots: 2:2 stoichiometry (signal at 577kDa)</figcaption></figure>

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<figure><img src="image_13.png" /><figcaption>Figure 4. Workflow designed for the Kd determination of infliximab/NATI complex using CE-MS/MS analysis. Model serum samples were prepared using IFX and ADM spiked in similar concentrations (10 µg.L-1) in different concentrations of NATI (0-30 µg.L-1).</figcaption></figure>

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<figure><img src="image_14.png" /><figcaption>Figure 5. Evolution of IFX\*LC01 / ADM\*HC01 peptide ratio with the concentration of IFX (0-25 µg·mL⁻¹), ADM concentration was fixed at 10 µg·mL⁻¹ (n = 3). Linear regression y = 0.10 x - 0.02 (r² = 0.982).</figcaption></figure>

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<figure><img src="image_15.png" /><figcaption>Figure 6. Ratio between IFX/ ADM CE-MS/MS signals as a function of the initial concentration of neutralizing-antibodies-to-infliximab (NATI, a0) added to serums. Error bars represent the standard deviation among the triplicates.</figcaption></figure>

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<figure><img src="image_16.png" /><figcaption>Figure 7. Plot of a0/Kd calculated using equation (5) depending on the initial concentration of NATI (a0) ranging from 0 to 30 µg·mL-1. Linear regression y= 0.46 x - 2.5 (r² = 0.958). Error bars represent the standard deviation among triplicates.</figcaption></figure>

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<figure><img src="image_17.png" /></figure>

Graphical abstract

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