Comparative analysis of the structural dynamics of diphtheria toxin and CRM197 carrier proteins used in the development of conjugate vaccines

Mathilde Briday, Nicolas Carvalho, Natalia Oganesyan, Min-Ju Chang, Andrew Lees, Sébastien Brier, Alexandre Chenal

► To cite this version:

Mathilde Briday, Nicolas Carvalho, Natalia Oganesyan, Min-Ju Chang, Andrew Lees, et al. Comparative analysis of the structural dynamics of diphtheria toxin and CRM197 carrier proteins used in the development of conjugate vaccines. International Journal of Pharmaceutics, 2025, 675, pp.125535. 10.1016/j.ijpharm.2025.125535. pasteur-05017717

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# Comparative analysis of the structural dynamics of diphtheria toxin and CRM₁₉₇ carrier proteins used in the development of conjugate vaccines

Mathilde Briday<sup>a</sup>, Nicolas Carvalho<sup>b</sup>, Natalia Oganesyan<sup>c</sup>, Min-Ju Chang<sup>c</sup>, Andrew Lees<sup>c</sup>, Sébastien Brier<sup>a, *,</sup> Alexandre Chenal<sup>b, *</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

### Keywords:

Biopharmaceutical products

Comparative analysis

Structural characterization

CRM₁₉₇

HDX-MS

## ABSTRACT

Carrier proteins are chemically linked to poorly immunogenic antigens to generate conjugate vaccines, significantly improving immunogenicity. CRM₁₉₇, a genetically detoxified diphtheria toxin (DT) mutant carrying the G52E mutation, is a widely used carrier protein as it retains lysine residues for antigen conjugation. In the past, CRM₁₉₇ has been expressed in *Corynebacterium diphtheriae*, but low yields and high costs have prompted the exploration of alternative expression systems. Although high-yield expression and native refolding of CRM₁₉₇ in *E. coli* are challenging due to its reducing cytoplasm, recent advances have enabled the production of soluble and well-folded recombinant CRM₁₉₇ proteins, namely EcoCRM® and EcoCRM®(–Met). In this study, we use Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) to compare the structural dynamics of EcoCRM and EcoCRM(–Met) with DT wild-type. Our HDX-MS data show that the presence or the absence of the N-terminal methionine does not affect the structural dynamics of the two recombinant EcoCRM proteins. Furthermore, our results elucidate the molecular mechanism underlying the lack of toxicity of CRM₁₉₇ compared to DT wild-type: the G52E mutation in the CRM₁₉₇ proteins exclusively alters the stability of the NAD-binding pocket and induces allosteric effects within the receptor-binding domain. Altogether, these insights support the substitution of CRM₁₉₇ produced by *C. diphtheriae* with the recombinant EcoCRM and EcoCRM(–Met) proteins produced in *E. coli*, offering a cost-effective solution for use in conjugate vaccines. Data are available via ProteomeXchange with identifier PXD057388.

## 1. Introduction

The introduction of conjugate vaccines, in which a poorly immunogenic antigen is chemically linked to a carrier protein, has led to significant advances in vaccines (Mrsny et al., 2002; Chenal et al., 2002; Adkins et al., 2012; Chenal and Ladant, 2018). One widely used carrier protein is CRM₁₉₇, a diphtheria toxin mutant genetically detoxified with a single mutation, G52E, substituting a glycine residue for a glutamic acid at position 52, which abolishes toxicity by inhibiting the ADP-ribosyl transferase enzymatic activity of diphtheria toxin. As a genetically detoxified protein, CRM₁₉₇ preserves all lysine residues available for conjugation (Bröker et al., 2011). Historically, CRM₁₉₇ has been expressed as a secreted protein using *Corynebacterium diphtheriae*

(Murphy, 1996) and most licensed conjugate vaccines use this source for their carrier protein (Martos et al., 2012). Yields are relatively low (100–200 mg/L), and large amounts of carrier protein are needed as many conjugate vaccines are highly multivalent. For example, ~100–200 µg of CRM₁₉₇ is required to manufacture a single dose of the multivalent pneumococcal conjugate Prevnar®20 (Shirley, 2022). CRM₁₉₇ has been one of the most expensive components of conjugate vaccines. CRM₁₉₇ has also been expressed as a periplasmic protein in *Pseudomonas fluorescence*. Pseudomonas expressed CRM₁₉₇ is currently used as the carrier protein in two licensed vaccines for *S. pneumoniae*, The Serum Institute of India’s Pneumosil® (Alderson et al., 2021) and Merck’s Vaxneuvance™ (Shirley, 2022).

CRM₁₉₇ has been challenging to express at high yields in a low-cost

<footnote><sup>a</sup> Institut Pasteur, Université Paris Cité, BioNMR and HDX-MS Facility, Centre for Technological Resources and Research, Chemistry and Structural Biology Department, UMR CNRS 3528, F-75015 Paris, France</footnote>

<footnote><sup>b</sup> Institut Pasteur, Université Paris Cité, Biochemistry of Macromolecular Interactions Unit, Chemistry and Structural Biology Department, UMR CNRS 3528, F-75015 Paris, France</footnote>

<footnote><sup>c</sup> Fina BioSolutions LLC, 9430 Key West Avenue, Suite 200, Rockville, MD 20850, USA</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: sebastien.brier@pasteur.fr (S. Brier), alexandre.chenal@pasteur.fr (A. Chenal).</footnote>
host like E. coli as the bacterium has a reducing cytoplasm (Goffin et al., 2017). When CRM197, which has two disulfide bonds, is expressed in the cytoplasm, it accumulates in inclusion bodies, necessitating costly refolding. CRM197 is also easily nicked by protease and is shear and pH sensitive. Furthermore, CRM197, like diphtheria toxin, can undergo domain swapping (Bennett and Eisenberg, 1994), resulting in dimer formation. Periplasmic expression of CRM197 in E. coli has been successful, but yields are relatively low, as mentioned above (circa 100–200 mg/L). A recently developed E. coli strain, Δgor, has an oxidative cytoplasm and grows to high cell densities in a fermenter, resulting in production of more than 3 g/L of properly folded, soluble CRM197 in the cytoplasm (Oganesyan and Lees, 2018). However, there is generally insufficient endogenous methionine amino peptidase to remove all the N-terminal methionine residue from over-expressed heterologous proteins. Indeed, partial removal of N-terminal formyl-methionine results in a heterogenous pool of recombinant CRM197 proteins with and without N-terminal methionine (Hickey et al., 2018). A second-generation strain, dubbed “GorMet” E. coli, has been developed and combines the *gor* deletion with an extra copy of E. coli methionine peptidase integrated into the genome at the *gor* locus, that improves the removal of N-terminus methionine from CRM197 (Oganesyan et al., 2021). CRM197 expressed in the GorMet strain has essentially no N-terminal methionine (see results below). To distinguish CRM197 expressed in Δgor and GorMet E. coli, we will refer to them here as EcoCRM® and EcoCRM®(–Met), respectively (Oganesyan et al., 2021; Oganesyan and Lees, 2018).

In this work, we use Hydrogen/Deuterium eXchange Mass Spectrometry (Wales and Engen, 2006; O'Brien et al., 2015, 2017; Oganesyan et al., 2018; Zheng et al., 2019; O'Brien et al., 2018, 2019, 2020; Narang et al., 2020; James et al., 2022) (HDX-MS) to compare the structural dynamics of EcoCRM and EcoCRM(-Met) and to gain further insights into the effects of the G52E mutation. Combined with the recently solved X-ray crystal structure of monomeric EcoCRM (Gallagher et al., 2023), this work demonstrates that the absence or presence of the N-terminal methionine does not affect the structural dynamics of CRM197 in solution. In addition, this study reveals that the G52E substitution leads to structural changes within the catalytic domain, including the NAD-binding pocket, and induces allosteric effects in the DT receptor-binding domain. This new finding allows a better understanding of the molecular mechanism associated with the lack of toxicity (Bröker et al., 2011; Salmas et al., 2016; Sauvé et al., 2018; Malito et al., 2012) and the alteration in CRM197 binding to the heparin-binding EGF-like growth factor cell-surface receptor (Mekada and Uchida, 1985). Finally, this work also provides regulatory support for the interchangeability of CRM197 expressed in C. diphtheriae with EcoCRM and EcoCRM(-Met) as carrier proteins for conjugate vaccines.

## 2. Materials and methods

### 2.1. Proteins

CRM197 was expressed in two E. coli strains: Fina Δgor (Oganesyan and Lees, 2018) and Fina GorMet (Oganesyan et al., 2021). The cloning, expression, and purification of CRM197 in each E. coli strain was similar, as described (Gallagher et al., 2023). EcoCRM® is the Fina Bio trade name for CRM197 expressed in the Fina Δgor strain created by deleting the glutathione reductase gene from the BL21 genome. EcoCRM®(-Met) was produced in the Fina GorMet strain containing an extra copy of the homologous methionine amino peptidase gene inserted into the Fina Δgor strain. Proteins were purified as described in (Gallagher et al., 2023) and stored at a concentration of 4.4 mg/mL (circa 75 µM) in 20 mM HEPES, pH 7.4, 10 % sucrose. Molecular mass of EcoCRM with and without the N-terminal methionine is 58 539 kDa and 58 408 kDa, respectively. DT wild-type was purchased from Sigma-Aldrich (#D0564, unnicked form).

DT wild-type, EcoCRM and EcoCRM(-Met) were buffer exchanged

against 20 mM HEPES buffer, 20 mM NaCl, pH 7.4, using 7 K MWCO Zeba™ Spin desalting columns (ThermoFisher Scientific). The protein concentration was determined by absorbance at 280 nm using a molar extinction coefficient of 51 730 M⁻¹·cm⁻¹. This value was calculated with ProtParam Expasy computation tool. Aliquots of 20 µL (22.8 µM) were stored at – 20 °C until use. The quality of the protein was assessed by intact mass analysis after buffer exchange (Fig. S1).

### 2.2. Quality controls of CRM₁₉₇ samples

#### 2.2.1. SDS-PAGE analysis

Four µg of CRM197 samples were prepared in 1X LDS Sample Buffer (GenScript, #M00676-10) in the absence and in the presence of 0.05 mM β-mercaptoethanol and incubated at 75 °C for 5 min. The samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis using SurePAGE 4 %-20 % BisTris (GenScript, #M00654) with Tris-MOPS-SDS running buffer (GenScript, #M00138). Broad Multi Color Pre-Stained Protein Standard (GenScript, #M00624) was used to estimate the size of the protein. Protein bands were visualized by using eStain L1 Protein Staining Device (GenScript).

#### 2.2.2. Size Exclusion Chromatography

A Waters 2695 Alliance e2695 Separations Module (Waters Corporation) was used to analyze the samples. Sepax Zenix SEC-HPLC Column 3 µm pore size, 300 x 7.8 mm (Sepax, L-2908) was equilibrated with PBS 1X pH 7.4 with 0.02 % (w/v) sodium azide buffer. Twenty µg of each CRM197 sample were injected onto the column and the run was performed at a flow rate of 1 mL/min for 16 min.

#### 2.2.3. Intact mass measurement by LC-ESI-MS

CRM197 and DT wild-type samples were prepared at 0.2 µM in 0.15 % formic acid, pH 2.5 (Solvent A). Fifty µL (10 pmols) were loaded onto an ACQUITY UPLC BEH C4 Trap column (2.1 µm x 5 mm, Waters Corporation), and desalted for 2 min at 100 µL/min solvent A. Proteins were eluted into the mass spectrometer with a quick linear gradient of acetonitrile from 5 to 90 % in 2 min, at 60 µL/min. Mass spectra were acquired in resolution and positive ion-mode (m/z 400–2000) on a Synapt™ G2-Si HDMS™ mass spectrometer (Waters Corporation). Mass spectra were analyzed in MassLynx 4.2 (Waters Corporation).

### 2.3. Mass photometry analysis

Mass photometry (MP) measurements were performed as described by Wu et al. (2021) (Wu and Piszczek, 2021). Each mass photogram was recorded on glass coverslips for 60 sec using the TwoMP© instrument and Acquire MP software (Refeyn). Briefly, protein solutions at 0.5 µM were equilibrated for 30 min at room temperature in aqueous buffer containing 20 mM HEPES and 150 mM NaCl at pH 7.4. For each measurement, a 19 µL drop of buffer at the desired pH was applied to the glass coverslips. After focusing the microscope, 1 µL of protein was added to the top of the drop. The drop was homogenized by three cycles of 10 µL back and forth pipetting within the drop. After mixing, photograms were taken immediately. Data analysis (Gaussian fitting) was performed using the software Dynamic MP and PhotoMol (Niebling et al., 2022) (https://spc.embl-hamburg.de/app/photoMol) with a bandwidth of 6 kDa. Histograms of the detected masses and Gaussian fits were then plotted using GraphPad Prism (Dotmatics). Mass calibration was performed using BSA monomeric, dimeric and trimeric measurements, yielding an R² value of 0.997.

### 2.4. HDX-MS analysis

A summary of the HDX data is provided in Table S1 (Masson et al., 2019).
### 2.4.1. Sample preparation

The concentration of each protein was adjusted to 5 µM in 20 mM HEPES buffer, pH 7.4, supplemented with either 150 mM NaCl (EcoCRM® versus EcoCRM®(-Met)) or 20 mM NaCl (EcoCRM® versus DT wild-type). After 30 min equilibration time at 23 °C, the labeling was initiated by adding 90 µL of deuterated buffer (20 mM HEPES buffer prepared in 99.9 % D₂O, pD 7.4, supplemented with either 150 or 20 mM NaCl) to 10 µL of equilibrated protein solution. The final D₂O/H₂O ratio (90/10) ensures the uni-directionality of the exchange reaction. Continuous labeling was performed at 23 °C for t = 10 sec, 1 min, 10 min, 30 min and 120 min. Aliquots of 20 µL (10 pmols) were removed and quenched upon mixing with 40 µL of ice-cold quench buffer (0.5 % formic acid, 4 M urea, 112.5 mM TCEP-HCl) to reduce the pH to ~ 2.5 (final D₂O/H₂O ratio 30/70). Quenched samples were immediately snap-frozen in liquid nitrogen and stored at -80 °C until use.

Undeuterated protein samples were prepared following the same experimental procedure and used to generate the peptide maps. Fully deuterated (FD) controls were obtained by mixing 10 µL of protein (5 µM) with 90 µL of deuterated buffer supplemented with 8 M urea-d4 (final D₂O/H₂O ratio = 90/10). After 17 h incubation at 23 °C, FD samples were quenched as described above using an ice-cold solution of 0.5 % formic acid, 0.405 M urea, 112.5 mM TCEP-HCl and snap-frozen in liquid nitrogen. All samples were prepared in triplicate for each time point and condition (independent technical replicates, with the exception of the fully deuterated controls).

### 2.4.2. HDX-MS data acquisition

MS analyses were performed with the aid of an HDX manager (Waters Corporation) maintained at 0 °C to limit back-exchange. After 1 min incubation on ice (disulfide bond reduction), quenched samples were injected into the HDX manager (50 µL, 8.3 pmols), and digested for 2 min at 100 µL/min and 20 °C using an in-house packed column (2.0 x 20 mm, 66 µL bead volume) of immobilized pepsin beads (ThermoFisher Scientific). Peptides were trapped, concentrated and desalted at 100 µL/min (0.15 % formic acid, pH 2.5) using a VanGuard™ BEH C18 pre-column (1.7 µm, 2.1 x 5 mm; Waters) and then separated by an 8 min linear gradient of 5–30 % acetonitrile, followed by a 2 min increase from 30 % to 40 % at 40 µL/min on a ACQUITY UPLC™ BEH C18 analytical column (1.7 µm, 1 x 100 mm). After each run, the pepsin column was manually cleaned with two consecutive washes of 1 % formic acid, 5 % acetonitrile, 1.5 M guanidinium chloride. Blank injections were performed after each sample to confirm the absence of carry-over.

The LC flow was directed to a Synapt™ G2-Si HDMS™ mass spectrometer (Waters Corporation) equipped with a standard electrospray ionization source (ESI). Mass accuracy was ensured by continuously infusing a Glu-1-Fibrinogen solution (200 fmol/µL in 50 % acetonitrile, 0.1 % formic acid) through the reference probe of the ESI source at a flow rate of 4 µL/min. Mass spectra were acquired in positive-ion and resolution mode over the *m/z* range of 50–1950. Peptides were identified in undeuterated samples using a combination of data-independent acquisition scheme (MS<sup>E</sup>) and exact mass measurement (< 5.0 ppm mass error) using the same chromatographic conditions as for the deuterated samples. To maximize sequence coverage, three distinct collision energy ramps were used in the trap region of the instrument: Low: 10–30 V, Medium: 15–35 V, and High: 20–45 V.

### 2.4.3. Data processing

Peptide maps were generated by database searching in ProteinLynx Global Server 3.0 (Waters Corporation) using the following processing and workflow parameters: low and elevated intensity thresholds set to 250.0 and 100.0 counts; intensity threshold sets to 750.0 counts; automatic peptide and fragment tolerance; non-specific primary digest reagent; false discovery rate sets to 4 %. Each fragmentation spectrum was manually inspected for assignment confirmation. Peptide maps were refined in DynamX 3.0 (Waters Corporation) using the following import

PLGS results filters: minimum intensity = 10 000; minimum products per amino acid value = 0.4; minimum sum of intensity for products = 5 000; minimum score = 7.0; maximum MH + error (ppm) = 5.0. A total of 96 unique peptides covering 89.0 % of the protein sequence were used to compare EcoCRM® with EcoCRM®(-Met). The sequence coverage increases to 95.5 % (101 peptides) for the comparison of EcoCRM® and DT wild-type (Fig. S2).

The centroid masses of each peptide were extracted in DynamX 3.0. HDX-MS results are reported as relative deuterium exchange level expressed in fractional exchange values. Fractional exchange data was calculated by dividing the experimentally measured uptake by the theoretically maximum number of exchangeable backbone amide hydrogens that could be replaced into each peptide in 90 % excess deuterium. Overlapping peptides covering the same region were used to increase the spatial resolution if their experimental back-exchange value were similar (i.e., difference below 10 %). The MEMHDX software (Hourdel et al., 2016) was used to visualize and statistically validate HDX-MS datasets (Wald test, false discovery rate of 5 %, biological threshold sets to 4 %).

## 3. Results

### 3.1. Production and characterization of EcoCRM(-Met)

EcoCRM®(-Met) was produced in the Fina "GorMet" *E. coli* strain, which contains an extra copy of the homologous methionine amino peptidase gene inserted into the Fina Δgor strain. As a result, EcoCRM®(-Met) is expected to have essentially no N-terminal methionine compared to EcoCRM® expressed in the Fina Δgor strain. Both EcoCRM and EcoCRM(-Met) were analyzed by SDS-PAGE. As shown in Fig. 1A, each sample displayed a main and intense band under non-reducing and reducing conditions with an electrophoretic migration consistent with the molecular mass of the CRM₁₉₇ monomer (~ 58.4 kDa). A small amount of protein species with higher (between 58 and 80 kDa) and lower (between 25 and 31 kDa) apparent molecular mass was detected in EcoCRM(-Met), probably due to residual traces of impurities that were not completely removed during the purification process.

Intact mass analysis was conducted to assess and compare the molecular composition of each protein (Fig. 1A). As expected, EcoCRM contained two species corresponding to the non-reduced CRM₁₉₇ protein with (58 539.21 Da) and without (58 408.33 Da) the N-terminal methionine. In contrast, EcoCRM(-Met) displayed a single population with a mass consistent with CRM₁₉₇ lacking the N-terminal methionine (58 408.31 Da). The additional methionine peptidase expressed in the Fina GorMet *E. coli* strain is therefore sufficient to completely remove essentially all the N-terminal methionine residue. Finally, the SEC-HPLC profiles of EcoCRM and EcoCRM(-Met) are similar, with a main peak at a retention volume around 8 min (Fig. 1B). Moreover, the main species of proteins in solution measured by mass spectrometry is the monomeric state of both EcoCRM and EcoCRM(-Met) (Fig. 1C and Table S2).

### 3.2. Comparative HDX-MS analysis of EcoCRM and EcoCRM(-Met)

Next, we compared the structure and dynamics of EcoCRM and EcoCRM(-Met) using Hydrogen/Deuterium eXchange (HDX) measured by Mass Spectrometry (MS). HDX-MS is a robust biophysical technique that probes the solvent accessibility of backbone amide hydrogens (NHs) against deuterium. The exchange rate of NHs is influenced by the structure, dynamics and conformation of proteins in solution (O'Brien 2015; O'Brien 2017; O'Brien 2019). Unstructured regions without stable hydrogen binding networks (e.g., disordered regions) exchange very rapidly, whereas folded elements (i.e., α-helices, β-stands) exchange at much slower rates due to hydrogen bonding (James et al., 2022; Oganesyan et al., 2018; Wales and Engen, 2006; Zheng et al., 2019).

The exchange behaviors of EcoCRM and EcoCRM(-Met) are similar and characteristic of well-folded proteins (Fig. 2A). Dynamic events (i.e.,
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Production and characterization of recombinant EcoCRM® and EcoCRM®(-Met). (A) SDS-PAGE and intact mass analysis of purified EcoCRM and EcoCRM(-Met). SDS-PAGE was performed under reducing and non-reducing conditions. Intact mass measurement was carried out under non-reducing conditions only. Two species corresponding to CRM₁₉₇ with (58 539.21 Da, expected mass = 58 539.21 Da [Δm = 0 Da/0 ppm]) and without (58 408.33 Da, expected mass = 58 408.02 Da [Δm = −0.31 Da/−5.31 ppm]) the first N-terminal methionine were detected by LC-MS in the EcoCRM sample. Only one unique population lacking the first N-terminal methionine was present in the EcoCRM(-Met) sample (58 408.31 Da, expected mass = 58 408.02 Da [Δm = −0.29 Da/−4.96 ppm]). (B) SEC-HPLC profiles of EcoCRM and EcoCRM(-Met). (C) Mass photometry profiles of EcoCRM and EcoCRM(-Met).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Comparison of the exchange behaviors of EcoCRM® and EcoCRM®(−Met). (A) Exchange profiles of EcoCRM (upper panel) and EcoCRM(−Met) (lower panel) measured at pD 7.4 and 23 °C. Relative fractional uptake values were calculated for each peptide and labelling time point, and plotted as a function of peptide position in the protein sequence. Each dot corresponds to the average value of three independent technical replicates. (B) Fractional Uptake Difference plot between EcoCRM and EcoCRM(−Met). Both proteins share the same exchange behavior with no significance difference in uptake.</figcaption></figure>

EX2 regime characterized by an increase in deuterium uptake with time), indicative of stable secondary structures, were observed throughout both proteins, including the catalytic domain (C, residues 1–187), the translocation domain (T, residues 200–377), and the receptor binding domain (R, residues 378–535) (Murphy, 2011). Two regions in the catalytic domain (residues 13–49 and 108–116) appeared fully accessible with no “dynamic” HDX behavior, i.e., exhibit the hallmarks of intrinsically disordered regions. The first region includes a surface loop near the active site (residues 30–33) and the active-site loop (residues 39–49), both absent in the crystal structure due to disorder. The second region contains a surface loop near the active site flanked by two short helices. In addition, 6 peptides from the translocation domain (278–284, 298–304, 328–344, 328–335, 360–367, and 361–377) showed resistance to deuteration and mainly correspond to internal α-helices, buried in the DT structure.

The difference in relative fractional uptake provides a more quantitative and visual assessment of the differences and/or similitudes between EcoCRM and EcoCRM(−Met). A positive value is indicative of a lower solvent accessibility in EcoCRM(−Met) compared to EcoCRM, whereas a negative value indicates a higher solvent accessibility (Fig. 2B). No change in HDX behavior was detected (difference equal to 0), hence confirming that the two proteins share similar structure and dynamics in solution. These results were statistically validated using MEMHDX (Fig. S3B).

### 3.3. Comparative HDX-MS analysis of EcoCRM and DT wild-type

DT wild-type and CRM₁₉₇ share the same fold and three-dimensional structure. The only notable difference observed by molecular dynamics lies in the increased flexibility of the active-site loop in CRM₁₉₇ that covers the NAD binding pocket (Malito et al., 2012).

To investigate the effects of G52E on the structure and dynamic of CRM₁₉₇, we analyzed and compared the “in solution” HDX behaviors of DT wild-type and CRM₁₉₇. Fig. 3A shows the differences in uptake between EcoCRM and DT wild-type measured at pD 7.4 and 23 °C (global HDX-MS behaviors are shown in Fig. S4). Our HDX results clearly demonstrate that G52E modifies the overall dynamics of the catalytic domain. Significant increases in deuterium uptake were detected in three segments of EcoCRM, including residues 1–12 (Cα1), residues 92–122 (Cα4-Cα5-Cα6-Cβ7), and residues 149–160 (Cβ7-Cα7-Cβ8). A slight reduction in uptake was also detected in region 65–88 (Cβ4-Cβ5). In addition, G52E affects the exchange behavior of the receptor-binding domain. Notably, an increase in solvent accessibility was observed in two peptides covering region 460–470 (Rβ9-Rβ10). These results were statistically validated using MEMHDX (Fig. S3B). Taken together, our HDX-MS data provides direct evidence that the G52E mutation locally destabilizes C and induces allosteric effects in R. The other segments of the protein, including the entire T domain and the active-site loop (residues 38–50), showed no difference in accessibility.

The differences in uptake between EcoCRM and DT wild-type were averaged and mapped onto the three-dimensional structure of monomeric EcoCRM (Gallagher et al., 2023). As illustrated in Fig. 3B and 4, the regions impacted by G52E in the C domain are located near the mutation site and include a portion of the hydrophobic NAD-binding pocket, specifically segment 149–160 (Cβ8-Cα7), which contains residue Trp153 involved in the flexibility of the active-site loop. The region affected in the R domain (residues 460–470) is part of the toxin cell-receptor binding site and contains residues in direct contact with the cell receptor, namely residues I464, V468 and F470 (Louie et al., 1997).
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Comparison of the exchange behaviors of DT wild-type and EcoCRM®. (A) Fractional Uptake Difference plot showing the difference in uptake measured between EcoCRM and DT wild-type. A positive difference indicates a higher solvent accessibility in EcoCRM compared to DT wild-type, while a negative difference is indicative of reduced solvent accessibility. Statistically significant peptides (Wald test, *p* < 0.05, Fig. S3B) are highlighted in gray. (B) Cartoon representation of monomeric CRM₁₉₇ (PDB entry 7rrw) showing the position of the different elements affected by G52E (black sphere) in EcoCRM. Fractional Uptake Differences (ΔDeuteration) calculated for each peptide at each time point were averaged and plotted on the crystal structure. Average ΔDeuteration values are colored from blue (reduced) to red (increased solvent accessibility).</figcaption></figure>

## 4. Discussion

We and others have recently compared the biophysical and immuno-
nological properties of CRM₁₉₇ proteins from diverse sources and
expression systems (Bröker et al., 2011; Goffin et al., 2017; Hickey et al.,
2018). Our findings indicate that recombinant CRM₁₉₇ proteins
expressed in heterologous systems, such as EcoCRM®, were equivalent
to those produced with the traditional C. diphtheriae expression system,
offering a cost-effective alternative for polysaccharide conjugate vaccine
production. However, a fraction of EcoCRM® expressed in E. coli BL21
Δgor cells contained non-native N-terminus due to insufficient methio-
nine aminopeptidase activity. In this study, we have compared
EcoCRM® produced in E. coli Fina Δgor strain to EcoCRM®(-Met)
expressed in a second generation of Δgor strain with an extra copy of a
methionine aminopeptidase gene, the Fina GorMet strain. We found the
two CRM₁₉₇ proteins to be highly similar, with no changes in exchange

behavior detected by HDX-MS, confirming their structural dynamics
similarities. In addition, MP showed that EcoCRM and EcoCRM(-Met) are
mainly monomeric, in agreement with previous works (Gallagher et al.,
2023). Changes in the oligomerization state would also affect the pro-
tein's solvent accessibility, resulting in significant differences in deute-
rium uptake between the two proteins. However, such variations were
not detected by HDX-MS when comparing the exchange profiles of both
proteins. Together, these observations confirm that EcoCRM and
EcoCRM(-Met) share the same structural dynamics in solution.

Two minor differences were noted between EcoCRM and EcoCRM<sub>(-Met)</sub>. First, some impurities were observed in EcoCRM<sub>(-Met)</sub> under non-reducing and reducing conditions. The nature of these impurities was not determined, but they likely corresponded to residual traces of contaminants that were not eliminated during the purification of EcoCRM<sub>(-Met)</sub>. Moving the purification process to GMP standards would significantly improve the final purity of EcoCRM<sub>(-Met)</sub>, as evidence by the
EcoCRM protein used in this study. Second, EcoCRM~(·-Met)~ was expressed to homogeneity with no non-native N-terminal methionine residue present, as shown in the present work by intact mass analysis. The homogeneity of the N-terminal was a result of the *GorMet* strain possessing additional methionyl aminopeptidase activity and confirmed by mass spectrometry in the present work.

We also took advantage of the HDX-MS technology to gain new structural insights into the mechanism of CRM₁₉₇ inactivation. Indeed, HDX-MS is a powerful approach to decipher structural dynamic changes of proteins upon ligand binding, protein-protein interactions or interaction with various biomolecules (Brier et al., 2021, 2006, 2004; Canul-Tec et al., 2017; Chalmers et al., 2011; Grauslund et al., 2024; Toporowska et al., 2024). CRM₁₉₇ and DT wild-type crystal structures are nearly identical with no detectable conformational changes. The only difference is in the conformation of the flexible active-site loop that is well ordered in the nucleotide-free DT wild-type protein and completely

disordered in CRM₁₉₇ regardless of the presence or absence of nucleotide. The enhanced flexibility of the active-site loop was shown to be directly influenced by the G52E substitution using molecular dynamic simulations (Salmas et al., 2016). Notably, the mutation decreases the frequency of hydrogen bond formation between the active-site loop and residue W153, which in turn reduces the affinity for the substrate and influences the overall stability of the protein. Our HDX-MS data confirm that G52E affects the interaction of residue W153 with the active-site loop. The solvent accessibility of region 149–160 containing residue W153 increases by ~ 30 % in CRM₁₉₇, supporting the hypothesis of hydrogen bond disruption (Fig. 4). In addition, no changes in solvent accessibility were detected in the active-site loop, which remains in a similar solvent-exposed environment in DT wild-type and CRM₁₉₇. Our HDX-MS data further reveals that, besides the G52E mutation, the substitution also induces the destabilization of a significant portion of the catalytic domain in CRM₁₉₇ (Figs. 3 and 4).

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Visualization of the different elements affected by G52E in the DT-NAD-HBEGF complex. Residues involved in the NAD and HBEGF binding interfaces and located in regions with modified solvent accessibility in CRM₁₉₇ are shown in sticks and labelled. NAD (yellow) and HBEGH (cyan) are shown in surface representation. EcoCRM® is colored as in Fig. 3B. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
An additional effect of G52E, which was not revealed by crystallography, is the change in solvent accessibility of the receptor-binding domain in response to the mutation (Gallagher et al., 2023). This result highlights the communication between the catalytic and the receptor binding domains of the toxin and sheds light on the mechanism of altered binding properties of CRM197 to the cell receptor (Mitamura et al., 1997). Importantly, we showed that the G52E mutation specifically increases the solvent accessibility of strands β9 and β10 in the DT R domain containing residues in direct contact with the cell receptor (Fig. 4). The difference in R binding property between DT and CRM197 originates from a change in dynamics within the receptor-binding domain induced by an allosteric effect of the mutation in the C domain. Taken together, the altered binding properties of CRM197 to the cell receptor is due to a local increase of structural flexibility of strands β9 and β10, which are involved in the interaction site of the DT R domain with HB-EGF.

In conclusion we showed that EcoCRM shares the same structural dynamics that EcoCRM(-Met) and possesses therefore the same properties for vaccine applications. The use of advanced HDX-MS allows a better understanding of the mechanism of toxin inactivation. It provides for the first time an explanation for the altered cell-receptor binding properties of CRM197.

### CRediT authorship contribution statement

**Mathilde Briday:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. **Nicolas Carvalho:** Methodology, Investigation. **Natalia Oganesyan:** Writing – original draft, Validation, Resources, Investigation, Formal analysis. **Min-Ju Chang:** Investigation, Formal analysis. **Andrew Lees:** Writing – original draft, Validation, Resources, Project administration, Funding acquisition, Formal analysis. **Sébastien Brier:** Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Alexandre Chenal:** Writing – original draft, Validation, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Chenal reports financial support was provided by Institut Pasteur. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

N.C. was supported by Institut Pasteur (DARRI-Emergence S-PI15006-12B) and ANR (ANR 21 CE11 0014 01 3DTransCyaA). The HDX-MS platform was funded by the CACSICE Equipex ANR-11-EQPX-0008. We also acknowledge funding from the Agence Nationale de la Recherche (ANR 21 CE11 0014 01 3DTransCyaA), the CNRS (UMR 3528), and the Institut Pasteur (PTR 166-19, DARRI-Emergence S-PI15006-12B).

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpharm.2025.125535.

### Data availability

The mass spectrometry data have been deposited to the

ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2022) partner repository with the dataset identifier PXD057388.

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