
==== Front
J Pharm Anal
J Pharm Anal
Journal of Pharmaceutical Analysis
2095-1779
2214-0883
Xi'an Jiaotong University

S2095-1779(24)00063-7
10.1016/j.jpha.2024.100966
100966
Original Article
Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry
Shah Arnik ab
Batabyal Dipanwita c
Qiu Dayong a
Cui Weidong a
Harrahy John d
Ivanov Alexander R. a.ivanov@northeastern.edu
b⁎
a Amgen Inc., Cambridge, MA, 02141, USA
b Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, 02115, USA
c Amgen Inc., Thousand Oaks, CA, 91320, USA
d Sanofi, Waltham, MA, 02451, USA
⁎ Corresponding author. a.ivanov@northeastern.edu
16 3 2024
8 2024
16 3 2024
14 8 10096622 9 2023
7 3 2024
13 3 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Biotherapeutic's higher order structure (HOS) is a critical determinant of its functional properties and conformational relevance. Here, we evaluated two covalent labeling methods: diethylpyrocarbonate (DEPC)-labeling and fast photooxidation of proteins (FPOP), in conjunction with mass spectrometry (MS), to investigate structural modifications for the new class of immuno-oncological therapy known as bispecific antigen-binding biotherapeutics (BABB). The evaluated techniques unveiled subtle structural changes occurring at the amino acid residue level within the antigen-binding domain under both native and thermal stress conditions, which cannot be detected by conventional biophysical techniques, e.g., near-ultraviolet circular dichroism (NUV-CD). The determined variations in labeling uptake under native and stress conditions, corroborated by binding assays, shed light on the binding effect, and highlighted the potential of covalent-labeling methods to effectively monitor conformational changes that ultimately influence the product quality. Our study provides a foundation for implementing the developed techniques in elucidating the inherent structural characteristics of novel therapeutics and their conformational stability.

Graphical abstract

Image 1

Highlights

• A novel BABB was investigated for impact of HOS on product quality.

• BABB was characterized using covalent labeling and MS-based footprinting.

• Subtle conformational alterations occurring at the amino acid residue level were discerned.

• The developed approach helps assess the BABB conformational stability and product quality.

Keywords

Covalent labeling/footprinting
Liquid chromatography-mass spectrometry
Fast photooxidation of proteins
Diethylpyrocarbonate
Higher order structure characterization
==== Body
pmc1 Introduction

Protein-based therapies continue to offer innovative treatments for various disease conditions [[1], [2], [3], [4]]. Monoclonal antibodies (mAbs) have garnered significant attention, generating billions in annual revenue [1,5,6]. The higher order structure (HOS) of protein-based biotherapeutics plays a crucial role in determining binding specificity toward antigens and developing new protein-based therapies [[7], [8], [9], [10], [11]].

Bispecific antigen-binding biotherapeutics (BABBs) are a novel class of therapies that are based on mAbs. These molecules consist of two domains, one with a specificity towards antigens characteristic to cancer and the other domain capable of eliciting an immune response. Each domain is composed of variable heavy (VH) and variable light (VL) regions connected with random coil linker peptides consisting of Gly-Gly-Gly-Gly-Ser (G4S) amino acid residue sequences. The entire molecule is engineered to be aglycosylated and with the molecular mass of approximately one-third of traditional mAbs. Given the significant differences observed for the BABB molecule, particularly in its conformational attributes, such as VH and VL regions, and the presence of the linker peptide, it is imperative to understand the structural aspects of BABBs, which can impact their quality and efficacy. Earlier studies reported higher efficacy for the BABB molecule, as compared to its mAb counterparts, which has resulted in increased interest in the development of such therapeutic modalities [[12], [13], [14], [15], [16], [17], [18]].

The multidomain HOS of protein biotherapeutics presents a challenge for characterization [[19], [20], [21], [22]]. Currently, available analytical techniques have limitations and may not be suitable for the efficient structural characterization of all proteins. High-resolution methods such as nuclear magnetic resonance (NMR) and X-ray crystallography can provide information down to the atomic level but are often sample amount- and time-intensive and may only be applied to the characterization of some proteins [[23], [24], [25], [26], [27]]. In contrast, commonly used biophysical methods such as circular dichroism (CD), dynamic light scattering (DLS), and differential scanning calorimeter (DSC) can provide rapid, low-resolution structural information about the global protein conformation [[28], [29], [30]]. However, these techniques do not provide information about localized conformational changes, which can significantly impact the molecule's efficacy and safety profile. Hence, a mid-resolution analytical technique is needed to provide information on subtle changes at the amino acid residue level, allowing for a more comprehensive characterization of HOS.

Mass spectrometry (MS) is a powerful analytical tool that has found broad applications in characterizing biotherapeutics attributes such as primary structure evaluation and post-translation modifications [[31], [32], [33], [34], [35]]. MS has also been utilized for the characterization of HOS of protein biotherapeutics [[36], [37], [38]]. Application of MS in the characterization of covalently labeled proteins can provide structural information by measuring changes in mass rendered by covalent labels [36,[38], [39], [40], [41], [42], [43], [44]]. Apart from covalent labeling, there are several reversible labeling approaches that also investigate protein structural conformations. One of the most widely used techniques is hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), wherein backbone amide hydrogens are exchanged with deuterium isotopologue atoms, and the resulting mass shifts are monitored using MS [[45], [46], [47], [48]]. Crosslinking MS-based (XL-MS) techniques utilize covalent labels that can provide information about protein structure by probing distance constraints in proteins, protein-protein interactions, or multisubunit and macromolecular assemblies [[49], [50], [51], [52]]. Ion mobility spectrometry (IMS) was recently used in applications for identifying global high-order structure changes induced by different modifications that resulted in detectable differences in collisional cross-sections of the analyzed molecules detected in drift tube-based IMS applications [[53], [54], [55]]. Furthermore, alternative separation techniques, such as capillary electrophoresis, are under investigation for the analysis of protein structures. These techniques can be integrated with diverse labeling methodologies to provide comprehensive insights into the higher-order structures of protein therapeutics [[56], [57], [58], [59]].

Covalent labeling, which involves the introduction of a label to surface-exposed amino acid residues in a natively folded protein, has been widely utilized to study protein structure and protein-protein interactions. Two commonly used irreversible covalent labels are diethylpyrocarbonate (DEPC) and fast photooxidation of proteins (FPOP). The hydroxyl radical reacts non-specifically with the side-chain of surface-exposed amino acid residues and is able to generate oxidized or oxidation-based products for 14 different amino acid residues (methionine (Met), tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), histidine (His), arginine (Arg), leucine (Leu), lysine (Lys), serine (Ser), valine (Val), proline (Pro), aspartic acid (Asp), cysteine (Cys), and isoleucine (Ile)), while DEPC specifically targets 6 amino acid residues (Cys, His, Lys, threonine (Thr), Tyr, and Ser). FPOP may introduce various mass shifts in each oxidation reaction of the protein (e.g., +15.994, +31.989, +47.984, +3.994, −22.032, −43.057, +50.001, −10.032, −33.980, and −31.974 Da) [60], while DEPC labeling results in an increase in molecular mass of +72.021 Da for each added covalent tag. Such changes in molecular masses of reagent/solvent-exposed surface amino acid residues of the protein can be measured using MS through a bottom-up proteomics approach [39,[61], [62], [63], [64], [65], [66], [67]]. Relative quantitation of the labeled sites can provide valuable insights into alterations in the overall protein structure or microheterogeneity that can affect the product quality. In this way, covalent labeling and MS-based analysis can be valuable tools in the structural characterization of biotherapeutic proteins.

In this study, for the first time, we evaluated the performance of FPOP- and DEPC-based structural footprinting of BABBs under native and thermal stress conditions. The results obtained from these techniques confirmed their efficiency in assessing structural alterations in BABBs and showed similar trends in labeling for solvent-exposed amino acid residues by two labeling techniques. Additionally, the changes in labeling sites observed under the two explored conditions (native vs. stressed) provided insights into the impact of structural changes on the product quality of the BABB molecule.

2 Materials and methods

2.1 Reagents and materials

The highest purity reagents were used for analysis. MS-grade solvents 0.1% formic acid (FA) in water and 0.1% FA in acetonitrile were obtained from Fisher (Madison, WI, USA). DEPC and 30% (m/m) hydrogen peroxide, high purity l-His, l-Met, and imidazole were obtained from Sigma-Aldrich (St. Louis, MO, USA). The Promega low-pH digestion kit (Promega Corporation, Madison, WI, USA) was used for the peptide mapping procedure.

2.2 Samples

BABB drug substance molecule was formulated at Amgen Inc. (Cambridge, MA, USA). BABB drug substance was thermally stressed between 55 and 60 °C by incubating on a heat block for 24 h.

2.3 Fast photochemical oxidation of BABB

An FPOP system was built to perform hydroxyl radical footprinting of proteins, as described previously by Liu et al. [36]. A 248 nm KrF excimer laser by GAM Laser Inc. (Orlando, FL, USA) adjusted to approximately 22 mJ/pulse output was used to irradiate a total of 50 μL of sample solution containing BABB sample diluted to 9.0 μM with phosphate-buffered saline (PBS) and 18 mM hydrogen peroxide, along with 10 mM His, as a scavenger to control the half-life of hydrogen peroxide. The laser was focused onto a fused silica tubing (i.d.: 150 μm and o.d.: 250 μm; Polymicro Technologies, Pheonix, AZ, USA), giving a 2.5–3.0 mm irradiation window. The sample flow rate was adjusted to 7 μL/min to provide 20% exclusion volume. A pulse generator was used to externally control the laser frequency from B&K Precision Corporation (Yorbal Linda, CA, USA). Following irradiation at room temperature, the sample was collected in an Eppendorf tube containing a quench solution, consisting of 10 μL of 70 mM Met and 1 μM of catalase, and immediately placed on ice after collection. Control samples were prepared under identical conditions without firing the laser. Each sample was prepared in triplicate.

2.4 DEPC labeling of BABB

The BABB sample was diluted to 9.0 μM with formulation buffer. The DEPC was prepared in acetonitrile. DEPC was spiked in protein solution at 4 M excess of protein concentration, and care was taken to ensure the final concentration of acetonitrile was less than 1% (V/V). The final volume of the sample was 100 μL. Following incubation of protein solution with DEPC at 37 °C for 4 min, the reaction was quenched with imidazole at 50-fold molar excess of DEPC concentration. Control samples were prepared without the labeling reagent. Each sample was prepared in triplicate and analyzed.

2.5 Near-ultraviolet circular dichroism (NUV-CD) analysis of BABB samples

The NUV-CD spectra were obtained on a qCD Chirascan spectropolarimeter (Applied Photophysics, Leatherhead, UK) at ambient temperature. The protein samples were analyzed at a 0.2 mg/mL concentration. Using cuvettes with a pathlength of 1 cm, the spectra were corrected for concentration and contributions from the buffer and are reported as mean residue molar ellipticity. Each spectrum is an average of four scans and was smoothed with a seven-point smoothing function using OMNIC 32 software by Thermo Fisher Scientific Inc. (Waltham, MA, USA). Background nitrogen blanks and buffer blanks were measured to eliminate the signals from the nitrogen, cuvette, and buffer. The parameters for the NUV-CD were: 1 cm path length, 240–350 nm wavelength range, 4s exposure time, 1 nm bandwidth, 0.5 nm step, and averaged over four runs using a 900 μL sample volume. Spectral similarity values were quantitatively determined using the BABB sample spectrum as a reference and applying a spectral similarity mathematical algorithm. The similarity between any two spectra was analyzed using the OMNIC™ software QC compare function (Thermo Scientific, Waltham, MA) with a seven-point smooth and 4-nm resolution. The result is reported between 0% and 100%, which shows how well the sample spectrum matches the reference spectrum. A value of 100% indicates the spectra are identical. Our internal studies show 95% is the lower tolerance limit, with 95% coverage and 95% confidence [68]. Triplicate analysis was performed.

2.6 Binding assay for BABB

Biacore™ T200 biosensor system by Biacore Inc. (Piscataway, NJ, USA) was utilized to identify relative binding affinities of BABB samples. Interaction between the BABB samples and its antigen was performed by immobilizing the antigen on a carboxymethylated dextran (CM5) chip using covalent amine coupling chemistry. Each sensor chip contained four flow cells (2.43 mm × 0.53 mm × 0.05 mm dimensions) wherein antigen at 10 μg/mL was bound to two cells of the CM5 chip, and the two other cells were used as reference cells. BABB samples were then injected over each cell of the CM5 chip to measure binding in response units (RU). The surface of the CM5 chip was cleaned with 10 mM glycine using a contact time of 30 s at 30 μL/min, followed by injection of BABB sample at 18 μM for 90 s at 37 °C for 10 μL/min. After the measurements were complete, the chip surface was regenerated using 10 mM glycine with a contact time of 30 s at 30 μL/min. The final response was recorded as the difference between binding and reference cells. Furthermore, relative binding (%) was calculated based on the response observed for the non-stressed BABB molecule. Triplicate analysis was performed to calculate a coefficient of variance.

2.7 Peptide mapping of BABB samples and data analysis

Samples for peptide mapping were prepared following the instructions of the Promega low-pH digestion kit (Promega Corporation). The digests were analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) on a Waters ultra performance liquid chromatography (UPLC) equipped with a binary pump (Waters Corporation, Milford, MA, USA) and column heater set at 65 °C. A BEH C18 column (130 Å, 1.7 μm, 2.1 mm × 150 mm) from Waters Corporation was used for peptide separation with solvent A as 0.1% formic acid in water and solvent B as 0.1% formic acid in acetonitrile. The gradient was established at 0.25 mL/min linearly at a 0.3% increase of solvent B/min. A Biopharma Orbitrap Q-Exactive + by Thermo Scientific (Bremen, Germany) was used to acquire data-dependent MS/MS spectra. Instrument settings are listed in Table S1. All peptide map results were analyzed using an internal Amgen Inc. software MassAnalyzer (equivalent to BioPharma Finder) [[69], [70], [71]]. The MS/MS spectra and the resulting matches were manually examined to confirm the ID assignments for peptides with modified amino acid residues. The chemical labeling efficiency was calculated using relative abundance measurements determined based on the extracted ion chromatogram (EIC) peak areas of the unmodified/native and modified peptides in each sample using Eq. 1. The efficiency of labeling for the FPOP-treated sample was determined by assessing the quantitative difference between the control and FPOP label samples. An illustration of how modifications related to DEPC and FPOP were calculated using peak areas determined for EIC of selected peptide peaks is shown in Fig. S1. To assess the difference between the stressed and native samples, we calculated the ratio difference (%), as shown in Eq. 2.(Eq. 1) Labeling%=EICpeakareaoflabeledpeptideEICpeakareaofunlabeled and labeledpeak×100%

(Eq. 2) Ratiodifference%=Labelingstress%−labelingnative%Labelingnative%×100%

3 Results

3.1 Method development for FPOP and DEPC labeling

The workflow for the structural characterization of the antigen-binding domain of the BABB molecule is illustrated in Fig. 1. In this study, we employed DEPC- and FPOP-based labeling techniques to label the surface-exposed amino acid residues of the antigen-binding domain of the BABB molecule under native and stressed states. Following the labeling, the samples were subjected to analysis using a bottom-up proteomic approach followed by reversed-phase liquid chromatography (RPLC)-MS analysis, allowing identification of labels at amino acid residues. Differences in labeling patterns/trends between the two techniques are observed for various amino acid residues under the native and stressed states of the antigen-binding domain of BABB molecule. These findings provide valuable insights into the effects of conformational changes at the amino acid residue level and its potential impact on product quality.Fig. 1 Workflow for covalent labeling and mass-spectrometry (MS) approaches utilized to characterize structural changes of bispecific antigen-binding biotherapeutic (BABB) molecule. DS: drug substance; DEPC: diethylpyrocarbonate; K: lysine; H: histidine; Y: tyrosine; T: threonine; C: cysteine; S: serine; FPOP: fast photooxidation of proteins; RPLC: reversed-phase liquid chromatography.

Fig. 1

Prior to analyzing the samples, we optimized the conditions for labeling by varying the reagent-to-protein concentration ratios. The overall modifications of carbethoxy for DEPC labeling and oxidation in the case of FPOP on surface-exposed amino acids were evaluated for residues in 10 detected peptides L1−L10 corresponding to the antigen-binding domain of BABB, as seen in Figs. S2 and S3.

To determine the optimal conditions for DEPC labeling, we monitored the intensities of the DEPC-labeled sites at varying concentrations. The dose-dependent curve for DEPC labeling shows first-order kinetics, leading to an increase of carbethoxy (+72.021 Da) uptake on labeled amino acid residues as the concentration of DEPC is increased. As shown in Fig. S2, a linear trend was observed for all the tested concentration ratios of DEPC to BABB at 1:1, 2:1, 4:1, and 8:1. It was also observed that an increase in DEPC concentration did not lead to labeling of different amino acid residue sites that were not observed in labeling at lower DEPC concentrations. Moreover, the addition of the carbethoxy group led to an increase in the hydrophobicity of the peptide, leading to the elution of the modified peptide later than the unmodified peptide, when analyzed by reversed-phase chromatography. To ensure that DEPC labeling did not affect the overall HOS, biophysical and binding assays were conducted, as discussed in the subsequent section.

FPOP can induce oxidation on 14 amino acid residues with multiple various reaction products for different amino acid residues [72,73]. This results in a wide range of labeling-induced mass differences for different amino acid residues [60,[74], [75], [76], [77], [78], [79]]. The propensity of each amino acid residue towards oxidation varies, as reported in previous studies [74]. Amino acid residues modified using FPOP oxidation resulted in either earlier or later elution of the modified peptide in comparison to the unmodified peptide in peptide mapping analysis by reversed-phase chromatography coupled with MS [60]. As seen in Fig. S3, a linear trend was observed for most of the amino acid residues monitored at four levels of H2O2 concentrations resulting in the following H2O2:BABB ratios: 500:1, 1000:1, 2000:1, and 4000:1. Exceptions were the two specific sites in L3 and L8 peptides, showing a curved trend for the four H2O2 concentrations, wherein a negative linear trend was observed with up to three concentrations of peroxide. Such trends might be explained by the limited accessibility of a residue in its conformational microenvironment. Based on these data, an FPOP condition was established as 2000:1, which fell within the curve. The rapid, sub-millisecond timescale of the FPOP reaction under native conditions allowed for minimal interference with protein conformation, thereby mitigating concerns about changes in higher-order structure induced by labeling or over-labeling [80,81].

3.2 Biophysical characterization of BABB samples by NUV-CD spectroscopy

In general, the near UV-CD technique provides an average population property and allows one to monitor protein tertiary structure changes. The spectra of native proteins are characterized by distinct features at around 286 nm and 293 nm attributable to Trp, at 270−285 nm attributable to Tyr, and 250−265 nm attributable to Phe, superimposed over the disulfide signal from 250 to 280 nm. In this study, we employed near UV-CD spectroscopy to monitor changes in the overall tertiary structure of BABB under native and thermal stress conditions and to observe any changes in structure resulting from DEPC labeling. As shown in Fig. 2A, a significant difference in overall profiles was observed between native and thermal stress BABB samples, suggesting an overall change in the HOS of the BABB sample after thermal stress. However, we did not observe any significant changes in the overall structure when samples were labeled with DEPC, as traces for labeled and unlabeled samples are highly similar for native and thermally stressed samples, respectively. This data indicates that DEPC label did not induce changes in HOS. Since this technique only provides the weighted average of the modification, site-specific modifications are not known.Fig. 2 Near-ultraviolet circular-dichroism (NUV-CD) and surface plasmon resonance (SPR) analysis of the bispecific antigen-binding biotherapeutic (BABB) molecule. (A) Trace for BABB native molecule (blue) and BABB molecule labeled with diethylpyrocarbonate (DEPC) (dotted-green) shows that labeling does not affect the overall structure while the thermally stressed BABB molecule (red) trace indicates an overall change in structure, whereas no change was observed between thermally stressed (red) and DEPC labeled thermally stressed sample (dotted-orange). (B) Binding data shows that the BABB molecule (green) and BABB labeled with DEPC (blue) did not affect binding, while the thermally stressed BABB molecule (red) shows a decrease in binding, whereas no change in binding was observed for DEPC labeled thermally stressed sample (orange) and non-labeled thermally stressed material (red). DS: drug substance.

Fig. 2

3.3 Binding assay for BABB samples by surface plasmon resonance (SPR)

We utilized the SPR method to investigate the effect of structural change on antigen-binding. The antigen was immobilized on a CM5 plate at a fixed concentration of 10 μg/mL. Native BABB samples were injected at a fixed concentration of 18 μM to assess the response to the binding. Additionally, thermally stressed BABB samples were injected at the same concentration as the native BABB samples, and the resulting responses were recorded and compared to the native BABB samples. As shown in Fig. 2B, a decrease in binding was observed for the thermally stressed sample compared to the native BABB samples. The reduction in binding can be attributed to an overall change in structure, which is consistent with NUV-CD experiments. Furthermore, DEPC-labeled BABB samples, native and thermally stressed, did not show any difference in binding compared to non-labeled BABB samples, indicating that labeling did not affect the overall structure, as seen in Fig. 2B.

3.4 Monitoring conformation change using DEPC labeling of BABB

RP-HPLC-MS was used to analyze trypsin-digested peptides to identify amino acid residue-level modifications. Sequence coverage of 95% was achieved, allowing for a comprehensive analysis of all regions. The limit of quantification (LOQ) was determined experimentally, as shown in the Supplementary data. The lowest peak height observed for the lowest modified peptide with DEPC labeling was above the determined LOQ. The detection of the modified peptide at these label concentrations, coupled with comprehensive structural coverage, provides the capability to monitor subtle changes in the BABB molecule under thermal stress treatment.

Complementarity determining regions (CDRs) located on the surface of BABB play a critical role in the binding of BABB to its antigen. Their distribution at various sites determines the overall structure of BABB [[82], [83], [84]]. Monitoring changes in the DEPC labeling of these regions under native and thermal stress conditions can provide insights into their activity. BABB antibodies are designed to have six CDR regions, as depicted in Fig. 3A, and five CDR regions (L1, L6, L7, L9, and L10) were successfully labeled by DEPC and compared to the FPOP results in Fig. 3B. The L2-CDR peptide, which was not labeled by DEPC, was due to the absence of amino acids that are reactive to DEPC. A significant increase in carbethoxylation was observed for non-CDR peptides, including L4, while the L3 and L5 peptides did not show a significant level of modifications. A decrease in the extent of modification was found for the amino acid residues of peptide L8 in thermal stress samples. In order to understand whether the difference observed in labeling was substantial, we calculated the ratio difference using formulae as described in Eq. 2 and showed that out of nine peptides labeled with DEPC, seven peptides showed significant change, which is demonstrated in Fig. 4. Table 1 shows P-values to be significant for the same seven peptides that demonstrated substantial changes.Fig. 3 Diethylpyrocarbonate (DEPC) and fast photooxidation of proteins (FPOP) modified peptides of bispecific antigen-binding biotherapeutic (BABB) molecule. (A) Bar graph showing the difference in DEPC labeling observed from reversed-phase chromatography coupled to mass spectrometry (RP-LC-MS) for native BABB (green) and thermally stressed BABB (blue) bottom panel, and the zoomed-in view for selected peptides shown on the top panel. Peptides marked with ∗ indicate amino acid residues corresponding to complementarity determining regions (CDRs) that were efficiently labeled. (B) Bar-graph showing the difference in FPOP labeling patterns observed in RP LC-MS for native BABB (green) and thermally stressed BABB molecule (blue; bottom panel), and the zoom view of selected peptides is shown on the top panel. Peptides marked with ∗ include labeled amino acid residues corresponding to CDRs. DS: drug substance.

Fig. 3

Fig. 4 Differences in the labeling efficiency and patterns for specific peptide sequences observed in the diethylpyrocarbonate (DEPC) and fast photooxidation of proteins (FPOP) experiments. Ratio of labeling efficiencies was observed for the peptides labeled with DEPC (red) and FPOP (blue). A zoomed-in view for the selected peptides was shown on the top panel. ∗ Indicates amino acid residues corresponding to complementarity determining regions (CDRs).

Fig. 4

Table 1 Statistical analysis to identify the significance of differences in labeling observed for diethylpyrocarbonate (DEPC) and fast photo-oxidation of protein (FPOP). Amino acid residues labeled by DEPC and FPOP labeling along with corresponding P-values.

Table 1Peptide labeled	Peptide	Amino acid residue labeled with DEPC	P-values	Amino acid residue labeled with FPOP	P-values	
L1a	26–35	Tyr	0.00540	Met	0.00034	
L1a	26–35	His	0.00950	Not labeled	Not available	
L2a	36–48	Not labeled	Not available	Trp	0.00049	
L3	53–60	Tyr	0.96967	Tyr	0.23298	
L4	70–85	Tyr	0.00271	Met	0.00027	
L5	95–105	Tyr	0.87860	Tyr	0.89267	
L6a	142–151	His	0.00627	Met	0.00228	
L7a	170–180	His	0.00564	Trp	0.00023	
L8	190–198	Tyr	0.00655	Tyr	0.00621	
L9a	200–210	Ser	0.00343	Phe	0.00007	
L10a	230–240	His	0.00540	Met	0.00023	
a Indicates amino acid residues corresponding to complementarity determining regions CDRs. Tyr: tyrosine; Met: methionine; His: histidine; Trp: tryptophan; Ser: serine; Phe: phenylalanine.

Additionally, all the changes observed on CDRs were significant, as seen in Table 1. These determined changes may be indicative of a substantial impact on the binding and activity of the BABB molecule. As previously studied, changes in His and Lys residues are associated with changes in the overall solvent-accessible surface area (SASA), while changes in Ser, Thr, and Tyr residues are more attributed to the micro-environment [62]. As seen in Table 1 from DEPC experiments, out of nine peptides that showed DEPC labeling, four peptides underwent labeling of the His residue, where the observed change in labeling between native and thermally stressed species was significant, as shown in Fig. 4 and Table 1. Such changes may lead to alteration in binding affinities or the overall capability of binding to the target antigens, as seen in Fig. 2.

3.5 Monitoring conformational changes using FPOP on BABB

Fast photochemical oxidation of proteins generates hydroxyl radicals, which in turn induces oxidation on surface solvent-exposed amino acid residues. As per the current studies, oxidation can be induced to 14 amino acids based on their microenvironment and accessibility. Amino acid residues, including Cys, Met, Trp, His, Tyr, and Phe are more reactive with hydroxyl radicals than others. Cys is usually involved in disulfide bond formation, which does not generate oxidation products [[85], [86], [87]]. Met residues exhibit the highest reactivity level and behave as a trap for hydroxyl radicals, typically resulting in the reduced modification rate of the nearby residues. The creation of dose-dependent curves revealed that a 2000 M excess of peroxide was able to generate signals for modified species that were above the LOQ, as shown in the Supplementary data.

Data acquired in the FPOP experiments showed that different amino acid residues were labeled in the same peptides as compared with the DEPC experiments, as demonstrated in Table 1. Particularly, modifications of Met residues dominate the labeling in the FPOP experiments, even when other FPOP-friendly sites of Tyr and His residues are nearby, e.g., in the case of peptide L1. Additionally, FPOP labeling-friendly Trp and Phe amino acid residues are not reactive to DEPC. The CDR peptide L2 does not contain residues that are reactive to DEPC. However, a Trp residue in that region was picked up by FPOP, showing a decrease of oxidation in the thermally stressed state, which potentially indicates conformational changes in this CDR region.

Both, the FPOP and DEPC labeling techniques demonstrated a similar modification trend for CDR and non-CDR peptides of the thermally stressed BABB. An increase in oxidation levels was observed for certain sites, including L1, L4, L6, L7, L9, and L10 peptides. This is consistent with the increase of carbethoxylation for L1, L4, L6, L7, L9, and L10. Likewise, a decrease in the oxidation level was observed for the L2 and L8 peptides. The results for the L8 peptide correlated with the results of the DEPC experiment, wherein a decrease in the oxidation level was observed for the thermally stressed sample. The ratio difference approach was used to calculate changes in oxidation levels observed in FPOP experiments. Out of ten labeled amino acid residues in tryptic digest peptides, eight peptides showed substantial changes, as demonstrated in Fig. 4. This was corroborated by calculated P-values, which showed that the identical eight amino acid residues underwent significant changes as seen in Table 1. Interestingly, the determined P-values from DEPC and FPOP experiments showed that the L3 and L5 peptides exhibited insignificant changes in modification levels under the same confidence criteria. We found that the assessed two chemical labeling approaches complimented and validated each other, and the combined results provided more thorough and informative coverage of the structural features of the BABB molecule and the structural alterations induced by thermal stress.

4 Discussion

To facilitate our understanding of the BABB structure, a simulated three-dimensional (3D) model of the antigen-binding domain of the BABB molecule was developed using the acquired data and previous work [88]. The simulated structure was used to map the labeled residues by both methods, as shown in Fig. 5. Because this domain is designed to have both VH and variable VL regions connected with random coil linker peptides consisting of Gly4Ser motif, the overall structure is more flexible than the corresponding domain in a full mAb structure. As a result, most labeled residues are on the loops and the outer surface of the β-sheet cleft. As shown in Fig. 5, amino acid residues labeled in L1, L3, L4, L6, and L8 peptides in the DEPC and FPOP experiments were part of β-sheets. Amino acid residues in peptide L5 on the loop region, on the other hand, were labeled in the DEPC and FPOP experiments. However, the results for peptides L7, L9, and L10 were different between the two treatment experiments. In the DEPC experiments, the labeled amino acid residues were part of loops, while in the FPOP experiments, the labeled residues were part of β-sheets. Most of the labeling changes at the amino acid residues level for the two examined conditions were significant, as shown in Fig. 4 and Table 1, and could have contributed to the overall structure features, as measured in NUV-CD experiments (Fig. 2A). This change in the primary structure and, specifically, at certain amino acid residues in the CDR region would have expectedly impacted the overall binding to its antigen, as follows from the results of the binding experiments (Fig. 2B). Further studies of the complex of BABB with its antigen would provide the necessary information to better understand the specific mechanism underlying these changes.Fig. 5 Overall results of diethylpyrocarbonate (DEPC) and fast photooxidation of proteins (FPOP) labeling mapped on a modeled structure of the antigen-binding domain of the bispecific antigen-binding biotherapeutic (BABB) molecule. Amino acid residues labeled with DEPC are shown in blue, residues labeled with FPOP in red, and residues labeled with both covalent labels in pink. Tyr: tyrosine; Met: methionine; His: histidine; Trp: tryptophan; Ser: serine; Phe: phenylalanine.

Fig. 5

5 Conclusion

In this study, we successfully utilized DEPC and FPOP to covalently label solvent-exposed CDR and non-CDR amino acid residues to structurally characterize the novel BABB molecule and its thermal stress-induced structural alterations. The use of DEPC and FPOP provided a wide amino acid residue repertoire coverage, enabling the identification of stability and structural changes that impact protein function. Cross-validation of the results of structural alterations was conducted using DEPC and FPOP treatments of BABB, where amino acid residues from the same sequence stretches were labeled. Although the two labeling techniques provide complementary results, DEPC labeling is significantly simpler in its implementation, and it can be utilized in the absence of an expensive KrF excimer laser, while FPOP has the advantage of providing broader coverage of amino acid residues that can be oxidized. NUV-CD was used to confirm overall structural changes in BABB, and the SPR assay measured the impact of these changes on antigen binding. Based on the results obtained, we expect that implementation of covalent labeling can provide valuable insights into the structural features of epitopes and paratopes as well as the stability of novel therapeutics derived from single-chain variable fragment-based therapies. In summary, the utilization of DEPC and FPOP, along with NUV-CD spectroscopy and SPR assays, allowed us to delve into the structural characteristics of the BABB molecule under native and thermal stress conditions. This study not only expanded our understanding of the protein's structure-function relationship but also demonstrated the novelty and significance of covalent labeling as a powerful tool for investigating the higher-order structure of emerging therapeutic modalities. Implementation of these methodologies at different stages of drug development can provide valuable information in selecting stable and optimum therapeutic modalities for various disease indications.

CRediT author statement

Arnik Shah: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - Original draft preparation; Dipanwita Batabyal, Dayong Qiu, and Weidong Cui: Methodology, Data curation; John Harrahy: Supervision, Writing - Reviewing and Editing; Alexander R. Ivanov: Conceptualization, Supervision, Writing - Reviewing and Editing.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Acknowledgments

This research is supported by Amgen Inc., USA and the National Institutes of Health, USA (Grant Nos.: R01CA218500 (ARI) and R35GM136421 (ARI)). We would like to thank Dr. Scott Siera for providing a critical review of this manuscript and Drs. Chetan Goudar, Tiffany Thiel, and Susan Burke for their support of this project. We would also like to thank Drs. Mats Wikstroem and Palanisamy Kanakraj for providing valuable inputs and support with biophysical and binding assay, and Drs. Hao Zhang and Mengru Zhang for helpful discussions on FPOP and protein footprinting.

Peer review under responsibility of Xi'an Jiaotong University.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.jpha.2024.100966.
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