Elsa Bodier-Montagutelli

Renaud Respaud

Gérald Perret

Linda Baptista

Philippe Duquenne

Nathalie Heuzé-Vourc'h

Laurent Vecellio

Protein stability during nebulization: mind the collection step!

Keywords: (5-10) Protein therapeutics, nebulization, aerosols, aerosol collection, stability studies, aggregation Ig, immunoglobulin, PP15, 15-mL polypropylene tube, PP2, 2-mL polypropylene tube, AGI, allglass impinger, BS, BioSampler, CYC, Cyclone-BC112, NGI, next-generation impactor, LDH, lactate dehydrogenase

Inhaled protein therapeutics meet growing interest for the treatment of respiratory diseases. In liquid aerosols, proteins face stresses that may generate instabilities, such as physicochemical denaturations, aggregation and loss of activity. Monitoring protein stability is thus crucial but implies to collect aerosol droplets before analysis. Many aerosol collection methods may be used, still their interference on protein stability is unknown. In this study, we compared the impact of six aerosol samplers on the stability of a model monoclonal antibody (Ig1), aerosolized with a mesh nebulizer. Ig1 stability was assessed for aggregation and biological activity. The six aerosol samplers generated distinct aggregation profiles for Ig1 at all size scales; counts of micron-sized particles varied by a factor of 100. The heterogeneity did not impact Ig1 activity, which was not significantly changed after nebulization. To extrapolate these results, we evaluated the impact of two samplers on three other proteins. Depending on the protein, samplers gave discordant aggregation and/or activity profiles, sometimes in the reverse trend as compared to Ig1. In conclusion, aerosol samplers interfere with protein stability; this impact depends both on the samplers and the protein, highlighting the importance of using the same collection device throughout the aerosol development process.

Introduction

Protein therapeutics are considered as promising opportunities in the field of respiratory medicine, bringing significant clinical benefit in several pathologies [START_REF] Sécher | Therapeutic antibodies : A new era in the treatment of respiratory diseases ?[END_REF]. While biotherapeutics are most frequently developed for systemic administration, topical lung delivery may offer a number of advantages. Indeed, inhalation has been used for decades for non-invasive pulmonary delivery to improve drug therapeutic index, by reaching optimized targeting of the diseased organ and limit systemic toxicity. This approach seems particularly attractive to take full advantage of protein therapeutics intended to operate within the respiratory tract, for which diffusion from the bloodstream into the lungs is hindered by the pulmonary alveolar capillary membrane [START_REF] Dall'acqua | Properties of Human IgG1s engineered for enhanced binding to the neonatal Fc Receptor (FcRn)[END_REF]. As a matter of fact, several preclinical studies have shown the relevance of inhalation for topical protein delivery, leading to market approval of dornase alpha and ongoing clinical evaluations for about 20 other protein therapeutics [START_REF] Bodier-Montagutelli | Designing inhaled protein therapeutics for topical lung delivery: what are the next steps?[END_REF].

Pulmonary delivery of protein therapeutics can be achieved using several devices such as nebulizers, pressurized metered dose inhalerssoft mist inhalers or dry powder inhalers [START_REF] Bodier-Montagutelli | Designing inhaled protein therapeutics for topical lung delivery: what are the next steps?[END_REF][START_REF] Hertel | Protein stability in pulmonary drug delivery via nebulization[END_REF].

Among them, nebulizers have been a common choice for first stage of inhaled protein development, as they can achieve large dose delivery, high pulmonary deposition while requiring limited formulation development (by avoiding drying steps, which might be of certain complexity for macromolecules) [START_REF] Martin | Nebulizers for drug delivery to the lungs[END_REF]. Various nebulization studies have been carried out on protein therapeutics, as nicely reviewed by Hertel et al. [START_REF] Hertel | Protein stability in pulmonary drug delivery via nebulization[END_REF]. They underline that nebulization generates stresses when converting bulk liquids into fine inhalable droplets; this includes massive exposition to air-liquid interface, solvent evaporation, heating or aerosol recirculation and recycling [START_REF] Phipps | Evaporation of aqueous aerosols produced by jet nebulizers: effects on particle size and concentration of solution in the droplets[END_REF][START_REF] Steckel | Factors affecting aerosol performance during nebulization with jet and ultrasonic nebulizers[END_REF][START_REF] Fängmark | Protein nebulization[END_REF]. These may result in partial to major protein degradation, resulting from physicochemical and conformational changes.

The main marker of protein instability during nebulization is aggregation, which extent depends on the nebulizer and the drug. This type of degradation was reported for various proteins (e.g. lactate dehydrogenase, dornase alpha, G-CSF or monoclonal antibodies), with different nebulization settings (i.e. jet, ultrasonic or vibrating-mesh nebulization) [START_REF] Niven | Protein nebulization: I. Stability of lactate dehydrogenase and recombinant granulocyte-colony stimulating factor to air-jet nebulization[END_REF][START_REF] Niven | Protein nebulization II. Stabilization of G-CSF to air-jet nebulization and the role of protectants[END_REF][START_REF] Cipolla | Assessment of Aerosol Delivery Systems for Recombinant Human Deoxyribonulcease[END_REF][START_REF] Maillet | Aerodynamical, immunological and pharmacological properties of the anticancer antibody cetuximab following nebulization[END_REF][START_REF] Respaud | Heuz??-VourC'H, Effect of formulation on the stability and aerosol performance of a nebulized antibody[END_REF][START_REF] Respaud | Development of a drug delivery system for efficient alveolar Mis en forme : Anglais (États-Unis) delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin[END_REF]. Besides, other nebulization-associated degradations have been reported, such as oxidation for IGF-I [START_REF] Germershaus | Insulin-like growth factor-I aerosol formulations for pulmonary delivery[END_REF]. For some proteins, such alterations may result in a loss of activity, as was the case for dornase alpha (up to 40% loss with ultrasonic nebulization) or LDH (up to 80% loss with jet nebulization) [START_REF] Cipolla | Assessment of Aerosol Delivery Systems for Recombinant Human Deoxyribonulcease[END_REF][START_REF] Niven | Some factors associated with the ultrasonic nebulization of proteins[END_REF]. To limit these instabilities and their potential impact on protein activity and immunogenicity, device and/or formulation optimization have been widely used as stability optimization tools. For instance, dornase alpha was successfully aerosolized with jet and vibrating-mesh nebulizers, allowing full retention of its biological activity [START_REF] Cipolla | Assessment of Aerosol Delivery Systems for Recombinant Human Deoxyribonulcease[END_REF][START_REF] Johnson | Aerosol delivery of recombinant human DNase I: in vitro comparison of a vibrating-mesh nebulizer with a jet nebulizer[END_REF][START_REF] Scherer | A technical feasibility study of dornase alfa delivery with eFlow® vibrating membrane nebulizers: Aerosol characteristics and physicochemical stability[END_REF]. Likewise, limiting heating by cooling the nebulizer allowed Hertel et al. to improve SM101 stability (a 20 kDa soluble Ig receptor) during vibrating-mesh nebulization [START_REF] Hertel | That's cool! -Nebulization of thermolabile proteins with a cooled vibrating mesh nebulizer[END_REF]. Encouraging results were also obtained with larger proteins: our group showed that careful selection of the nebulizer and formulation optimization (i.e. antibody concentration adjustment and addition of surfactants)

were key parameters to limit immunoglobulin (Ig) aggregation during nebulization [START_REF] Respaud | Heuz??-VourC'H, Effect of formulation on the stability and aerosol performance of a nebulized antibody[END_REF].

Thus, the development of nebulized protein therapeutics often requires parallel nebulizer and formulation optimizations. In this context, protein stability studies are the compass that drives the design of the final drug and device combination. Given the complex nature of proteins, their modes of degradation are multiple (changes in primary, secondary and/or tertiary structures) and require the combination of several complementary analytical techniques to achieve extensive characterization. Yet, studying protein stability directly in aerosol droplets is not feasible to date, as standard techniques require volumes in the range of microliters/milliliters. Consequently, aerosol collection is a mandatory step: aerosol droplets are pooled to re-create a bulk liquid for protein characterization.

Numerous devices were developed to collect aerosols, for various applications, mainly bioaerosol sampling and drug aerosol characterization. In such devices, droplet pooling relies on different physical collection mechanisms, such as filtration, liquid/solid impaction, condensation or electrostatic precipitation and have various aerosol collection efficiencies [START_REF] Grinshpun | Sampling for Airborne Microorganisms[END_REF].

In the literature, a variety of collection methods has been employed to study protein stability during nebulization, such as glass impingers, laboratory impaction systems, next-generation impactors (NGIs) or filters [START_REF] Maillet | Aerodynamical, immunological and pharmacological properties of the anticancer antibody cetuximab following nebulization[END_REF][START_REF] Johnson | Aerosol delivery of recombinant human DNase I: in vitro comparison of a vibrating-mesh nebulizer with a jet nebulizer[END_REF][START_REF] Scherer | A technical feasibility study of dornase alfa delivery with eFlow® vibrating membrane nebulizers: Aerosol characteristics and physicochemical stability[END_REF][START_REF] Cipolla | Characterization of Aerosols of Human Recombinant Deoxyribonuclease I (rhDNase) Generated by Jet Nebulizers[END_REF]. Each device implying a specific collection mechanism, proteins may face different post-nebulization stresses depending on the collection process, in addition to general modifications induced by droplet pooling. Together, these factors may modify protein behavior between nebulization and analysis, thereby inducing variations in the results and conclusions of stability studies. Although many studies focused on the consequences of transforming protein solutions into aerosol droplets (during nebulization), data on the impact of re-converting protein aerosols into bulk liquids (during collection) to dissect protein stability are scarce, but critical to interpret nebulized protein stability. To the best of our knowledge, only two comparative studies have been carried out so far: one with dornase alpha, where no difference was observed between two laboratory collection devices [START_REF] Cipolla | Method for collection of nebulized proteins[END_REF]. The other one was carried out by Hertel et al. on two proteins, with a limited number of devices intended for aerosol collection, and showed different aggregation profiles depending on the collection method [START_REF] Hertel | Pulmonary Delivery of Pharmaceutical Proteins by Means of Vibrating Mesh Nebulization[END_REF]. Overall, knowledge about the impact of the aerosol collection system on protein therapeutics behavior remains poor.

Herein, we evaluated the impact of six commercialized aerosol collection systems on the physicochemical and biological stability of a nebulized monoclonal antibody (Ig1). To assess the applicability of our results to other protein therapeutics, we compared two samplers with different profiles on three additional proteins (two Igs and an enzyme). Protein aerosols were produced with mesh nebulizers, which are commonly used for inhaled protein development [START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF][START_REF] Pritchard | Mesh nebulizers have become the first choice for new nebulized pharmaceutical drug developments[END_REF]. After aerosol collection, protein stability was assessed in terms of aggregation (from oligomers to visible particles) and biological activity. Protein stability profiles were then established for each aerosol samplers.

Materials and methods

Materials

HEPES, CaCl2, MgCl2, bovine serum albumin (BSA), polysorbate 20, polysorbate 80, thimerosal, DNA-methyl green, citric acid, sodium chloride, glycine and phosphate buffered saline (PBS) solution were purchased from Sigma-Aldrich (Saint-Louis, MI). Flow cytometry antibodies were labeled with Alexa Fluor™ 488 (Thermo Fisher Scientific, Waltham, MA). Ig1 isotype-matched nonbinding antibody was purchased from Roche SAS (Boulogne-Billancourt, France). Minimum Essential Media (MEM) without phenol red was purchased from Thermo Fisher Scientific.

Protein solutions

Ig1 (an anti-EGFR IgG1), was supplied by Merck KGaA (Darmstadt, Germany) and diluted to a final concentration of 2 mg/mL in a formulation containing 10 mM citric acid, 100 mM sodium chloride, 0.1% polysorbate 80 and 100 mM glycine. Ig2 (anti-Pseudomonas aeruginosa IgG2b) was generated using a PTA-9180 hybridoma (LGC Standards, Molsheim, France) and supplied as a sterile, pyrogen-free solution in PBS, in accordance with good manufacturing practices (BioXCell, West Lebanon, NH); the final concentration was adjusted to 1.8 mg/mL. Ig3 (polyclonal Ig targeting the human Respiratory Syncytial Virus, hRSV) was provided by LFB Biotechnologies (Les Ulis, France) at a concentration of 0.2 mg/mL in a formulation containing mannitol, glycine, and polysorbate 80. Dornase alpha (Pulmozyme™) was purchased from Roche SAS (Boulogne-Billancourt, France) at a concentration of 1 mg/mL in its formulation containing sodium chloride and calcium chloride.

All protein solutions were passed through a 0.22 µm PVDF syringe filter (EMD Millipore, Burlington, MA) and stored at 4°C before nebulization.

Protein nebulization and aerosol collection

Generation of protein aerosols

Proteins were aerosolized with mesh nebulizers, which are both favorable to protein stability [START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF] and suitable with all the evaluated samplers. Briefly, mesh nebulizers produce aerosols by passing a liquid through a calibrated mesh; air supply is thus not needed for aerosol generation. All Igs were nebulized with the Aerogen® vibrating-mesh technology (Aerogen, Galway, Ireland): Aerogen® Solo for Ig1, Ig3 and Aerogen® Pro for Ig2 (this fully-autoclavable nebulizer was selected as Ig2 activity assays required sterile samples). The volume mean diameters (VMDs) were 4.9, 4.1 µm and 4.6 µm for Ig1, Ig2 and Ig3, respectively. For dornase alpha, nebulization was done with an eFlow® rapid (PARI GmbH, Starnberg, Germany), in accordance with the precautions for disposal (VMD = 3.9 µm) [START_REF]Pulmozyme -Summary of Product Characteristics[END_REF].

Except for Ig3, two milliliters of the protein solution were placed in the nebulizer reservoir, and nebulization was carried out. Five replicates were generated for each nebulization/collection condition. For Ig3, the available amount of material allowed us to do 1-milliliter nebulizations, in triplicate.

For each protein, control samples were generated: formulations alone were nebulized and collected following the same process than proteins.

Aerosol samplers and collection of protein aerosols

Six aerosol samplers were included in this comparative study, with . They display different characteristics regarding the original application, physical principle of collection, liquid addition and dilution volume, required air flow rate, and existing literature with proteins (see Table 1).

Experimental setups are illustrated in Figure 1; in all configurations, nebulizers were connected to aerosol samplers with a T-piece (Aerogen, Galway, Ireland). Aerosols were collected for the entire duration of nebulization, i.e. 4.5 to 9 minutes, depending on the considered protein.

15-mL conical polypropylene (PP) laboratory tubes (Corning Life Sciences, Tewksbury, MA) and 2-mL PP tubes (Dutscher, Brumath, France) were used as aerosol samplers. Empty tubes were connected to the nebulizer outlet through a T-piece in a hermetic setup (the unused Tpiece inlet was sealed). Aerosols were collected by condensation on tube walls. No air suction was used and no dilution was applied to samples after collection.

We also used theIn tThe AGI-4 Impinger (Ace Glass Inc., Vineland, NJ), a glass device designed for bioaerosol sampling. In this system, droplets are driven through the air flow to impact at high velocity on the bottom of a glass collection vessel [START_REF] Jensen | Sampling and characterization of bioaerosols, NIOSH Man[END_REF]. For ourthe experiments, the AGI-4 was used in a cooled setup [START_REF] Springorum | A temperature-controlled AGI-30 impinger for sampling of bioaerosols[END_REF], plunged in crushed ice; no collection fluid was added in the collection vessel to avoid sample dilution. The AGI-4 was connected to the nebulizer through a T-piece; air was suctioned at 12-12.5 L/min through an Anest-Guard respiratory filter (Teleflex Inc., Wayne, PA) placed on the distal inlet of the T-piece (see Figure 1).

The BioSampler (SKC Inc., Eighty Four, PA), a bioaerosol sampler, was also included in this comparison. This device is similar to the AGI-4, except that aerosol droplets are driven through curved nozzles, placed above a swirling collection fluid. The resulting tangential impaction reduces agitation and re-aerosolization [START_REF] Jensen | Sampling and characterization of bioaerosols, NIOSH Man[END_REF][START_REF] Riemenschneider | Characterization of reaerosolization from impingers in an effort to improve airborne virus sampling[END_REF]. In this study, the BioSampler was used with an air flow rate of 12-12.5 L/min, allowing a -500 mbar depression; incoming air was filtered as for the AGI-4 (see Figure 1). 5 mL PBS were placed in the collection vessel as collection liquid.

The last bioaerosol sampler included in this comparison was theThe Cyclone-BC112 (NIOSH, Washington, DC), a personal one-stage cyclone sampler, was also included in the study [START_REF] Chen | Development of a personal sampler for collecting fungal spores[END_REF][START_REF] Su | Evaluation of physical sampling efficiency for cyclone-based personal bioaerosol samplers in moving air environments[END_REF][START_REF] Duquenne | Performances of the BC-112 NIOSH cyclone for the measurement of endotoxins in bioaerosols: A study in laboratory conditions[END_REF][START_REF] Wang | Field evaluation of personal sampling methods for multiple bioaerosols[END_REF]. AIn this device, air flows through a 1.5 mL conical PP tube (Thermo Fisher Scientific) in a vortex-like trajectory, allowing aerosol droplets to impact on the tube walls. Air escapes the sampler after passing through a 3-piece filtration cassette. Air flowrate was set at 4 L/min, suctioned through a respiratory filter placed on the nebulizer T-piece. After nebulization, the collected liquid was recovered by rinsing the tube with 1 mL of protein formulation.

Finally, we included aaerosol was collected in a pharmaceutical aerosol sampler, the NGI (Copley Scientific Limited, Nottingham, UK), . It is a seven-stage impactor, in which aerosol droplets are collected after impaction on solid metallic plates. Briefly, each stage contains a series of nozzles of decreasing size. As the air flows through the stages, its velocity increases, leading to selective droplet impaction depending on their aerodynamic size [START_REF] Marple | Next generation pharmaceutical impactor (a new impactor for pharmaceutical inhaler testing). Part I: Design[END_REF]. We used theThe NGI was used with an air flowrate of 15 L/min [START_REF] Marple | Next generation pharmaceutical impactor: a new impactor for pharmaceutical inhaler testing. Part III. extension of archival calibration to 15 L/min[END_REF], filtered by a respiratory filter at the setup inlet. Before aerosol collection, the impactor was pre-cooled for 90 minutes at +4°C [START_REF] Dennis | Cooling the NGI-an approach to size a nebulised aerosol more accurately[END_REF].

After completing nebulization, each of the 8 plates was rinsed with 5 mL PBS. Samples recovered from the NGI were analyzed both separately and pooled (by mixing equal parts of each element's solution).

Next-Generation Impactor (NGI)

Therapeutic aerosols (European Pharmacopoeia) Impaction on solid surface 0.98 µm [START_REF]Quality Solutions for Inhaler Testing[END_REF] 15.0 40 mL (added after collection) [START_REF] Cortez-Jugo | Pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform[END_REF] N/D: not determined * To the best of the authors' knowledge Table 1. Characteristics of the compared aerosol samplers.

Protein characterization in collected aerosols

Protein concentration and collection efficiency

Each sampler was weighed before and after aerosol collection, to calculate the volume of aerosol collected. This method being unsuitable with the NGI, the volume of aerosol collected in each element was considered negligible compared to the PBS volume added. Protein concentration in the collected samples was measured using a BCA colorimetric assay (Thermo Fisher Scientific). The total amount of protein collected was obtained by multiplying the final protein concentration by the total volume. Each sampler's collection efficiency was expressed as the ratio: mass of protein collected/mass of protein loaded in the nebulizer.

Protein aggregation

Visible particles (visual inspection)

Samples were observed to assess the presence (+) or absence (-) of visible particles (larger than 500 µm).

Micron-sized particles (flow cell microscopy)

All protein samples were analyzed before and after nebulization with a FC200S-IPAC protein aggregates counting imager (Occhio SA, Liège, Belgium). Briefly, 240 µL of each sample were passed through an analysis flow cell, where particles between 2 and 100 µm were counted and analyzed in the Callisto® software. Raw results are expressed as the number of particles per mL of solution. When the sampler required to dilute the aerosol (i.e. BioSampler, Cyclone and NGI), particle concentration was adjusted with the dilution factor, and thus standardized with respect to protein concentration. Final results are expressed as the number of particles per mL of aerosol, at equal protein concentration.

For each protein, control samples (i.e. aerosols of formulation alone) were also analyzed to assess the "particle background" generated by the experimental setup itself. This background was then subtracted from particle counts to get net particle concentrations (i.e. protein particles only).

Submicronic aggregates (dynamic light scattering)

Dynamic light scattering (DLS) experiments were carried out at 25°C, with a Dynapro Nanostar® instrument (Wyatt Technology, Santa Barbara, CA), as described previously [START_REF] Respaud | Development of a drug delivery system for efficient alveolar Mis en forme : Anglais (États-Unis) delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin[END_REF].

Briefly, a 659 nm/100 mW laser and a 90° detection angle were used to determine the size distribution profile of protein species before and after nebulization. For each sample, we carried out 10 acquisitions of 7 s each. The results were evaluated using Dynamics® software;

samples giving fewer than 70% successful acquisitions were considered to be "not evaluable", as recommended by the manufacturer. The dynamic data filter was set as follows: baseline limit ±0.04 and maximum SOS 1000. For each sample, the global polydispersity index (Pd index corresponds to the distribution width divided by the mean and calculated from the cumulants analysis) was reported, as well as the percentage, in mass, of the monomer, and the percentage polydispersity of the monomer population.

When particle size distribution was multimodal, samples were filtered through a 0.45 µm membrane to remove large particles and re-analyzed.

DLS experiments could not be performed when the dilution factor was too important, i.e. for the BioSampler, Cyclone and NGI.

Oligomers and soluble aggregates (size-exclusion chromatography)

Size-exclusion chromatography (SEC) analyses were performed on an Ultimate™ 3000 UHPLC system equipped with an in-line. For each sample, 50 µg was injected by an Ultimate™ autosampler in an Agilent Technologies (Santa Clara, CA) Bio SEC-3 column (3 µm, 300 Å, 7.8 x 300mm), and separated at a flow rate of 1 mL/min. The elution buffer was PBS pH 7.2 and UV detection was performed at 280 nm with an Ultimate™ diode array detector (Thermo Fisher Scientific). Results are expressed as the percentages of monomers and higher molecular weight species (HMWS).

SEC experiments could not be carried out when the dilution factor was too important, i.e. for Cyclone and NGI.

Protein biological activity

Ig1binding to EGFR

Ig1 binding to its target antigen was assessed through indirect competition in a cell-based assay, as described previously [START_REF] Maillet | Aerodynamical, immunological and pharmacological properties of the anticancer antibody cetuximab following nebulization[END_REF]. Briefly, 1.5x10 5 A431 cells (human epidermoid carcinoma cells that express EGFR) were incubated with unlabeled native or nebulized Ig1 (1.10 -6 to 5.10 -3 mg/mL) for 30 min at 4°C. Then, cells were washed twice with PBS + 2% fetal calf serum (FCS) incubated for 30 min at 4°C with FITC-Ig1 (5 µg/mL) or with the isotype-matched nonbinding antibody FITC-Ig (5 µg/mL). After two washes with PBS + 2% FCS, cells were resuspended in 100 µL PBS + 2% FCS + 2 mM EDTA pH8. Flow cytometry analyses were performed using a MACSQuant analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and VenturiOne software (Applied Cytometry, Sheffield, UK). Inhibition curves were obtained by plotting mean fluorescence intensity (in % of maximum intensity) against the logarithmic concentration of mAb1; half maximal inhibitory concentration (IC50) was calculated for each sample using GraphPad Prism (GraphPad Software Inc., La Jolla, CA).

Ig2antibacterial activity

Ig2 targets a protein included in Pseudomonas aeruginosa type III secretion system, which is used by the bacterium to inject cytotoxic effectors into host cells [START_REF] Anantharajah | Targeting the Type Three Secretion System in Pseudomonas aeruginosa[END_REF]. Thus, we assessed Ig2 biological activity through a bacterial neutralization assay. Briefly, 2x10 4 A549 cells (human adenocarcinomic alveolar basal epithelial cells) were infected with 2x10 4 cfu of the PA103 WT-Luc P. aeruginosa strain, and native or nebulized Ig2 was added to the medium (100 µg/mL). After 6h incubation at 37C°, supernatants were collected to assay cytotoxicity with a colorimetric LDH release assay (Thermo Fisher Scientific). After 30 min incubation with the reaction mixture, stop solution was added and optical density was read at 490 nm (OD490). % cytotoxicity was determined as follows: % cytotoxicity = 100 x (Ig2-treated OD490spontaneous OD490) / (positive control OD490spontaneous OD490). Spontaneous OD490 was obtained from uninfected cells; positive controls were obtained from uninfected cells incubated with lysis buffer for 45 min. Loss of Ig2 activity was compared before and after nebulization, based on the increase in %cytotoxicity as compared to untreated cells.

Ig3antiviral activity

Ig3 antiviral activity was assessed by measuring its ability to inhibit hRSV infection of human epithelial cells. To do so, HEp-2 cells (CCL-23) were obtained from ATCC (Manassas, VA, USA) and cultivated in MEM containing 5% FCS and glutamine. Recombinant hRSV was derived from the Long strain and engineered by Marie-Anne Rameix-Welti (INRA UR892, Jouy-en-Josas, France) to express the mCherry red fluorescent protein [START_REF] Rameix-Welti | Visualizing the replication of respiratory syncytial virus in cells and in living mice[END_REF]. Cell infection assays were performed with the resulting hRSV(18)CHERRY 3-P4 virus library (2348 18 541).

Insertion of mCherry reporter gene between P and M genes had no major impact on the replicative capacities of the virus. MEM without phenol red was used for cell culture and virus dilutions.

The day before the test, HEp-2 cells were seeded at 5x10 4 with excitation and emission wavelengths of 580 and 620 nm, respectively. Non-infected HEp-2 cells were used as standards for fluorescence background levels; 100% infection controls consisted in HEp-2 cells incubated with diluted virus suspension only (no antibodies).

For each sample, % hRSV infection was plotted against Ig3 logarithmic concentration as a dose-response inhibition of HEp-2 infection; IC50 was determined using GraphPad Prism.

Dornase alphaenzymatic activity

The enzymatic activity of dornase alpha was assessed using a colorimetric assay that measures the release of methyl green from DNA upon digestion by dornase alpha [START_REF] Scherer | A technical feasibility study of dornase alfa delivery with eFlow® vibrating membrane nebulizers: Aerosol characteristics and physicochemical stability[END_REF]. Briefly, the DNA-methyl green complex was dissolved in assay diluent (25 mM HEPES, 4 mM CaCl2, 4 mM MgCl2, 0.1% BSA, 0.05% polysorbate 20 and 0.01% thimerosal, pH 7.5). Standards were prepared by diluting native dornase alpha in assay diluent to concentrations ranging from 10 -5 to 10 -2 mg/mL. Samples were diluted in assay diluent by factors of 1:200, 1:400 and 1:800.

Equal volumes of standards, controls and samples were each mixed with the same volume of DNA-methyl green solution and incubated 1h at 37°C under agitation. H2O2 (4 mM) diluted in assay diluent was added and samples were incubated 3h at 25°C under agitation. Finally, absorbances at 620 nm (methyl green absorption maximum) and 492 nm (reference wavelength) were recorded by a SpectraMax® microplate reader and SoftMax Pro software (Molecular Devices, San José, CA). The decrease in A620nm-A492nm was plotted against the logarithmic concentration of dornase alpha; the average activity from three sample dilutions was obtained as µg/mL and divided by total protein concentration to calculate the %activity.

Statistical analysis

For Ig1, aggregation data (i.e. concentrations in micronic aggregates, monomer and HMWS contents and monomer polydispersity) were compared through a 1-way ANOVA, Kruskal-Wallis multiple comparison test. Regarding biological activity, mAb1 inhibition curves were compared before and after nebulization-collection with a 1-way ANOVA, Friedman's test. For the three other proteins, aggregation data and biological activities (i.e. % cytotoxicity for Ig2, % hRSV infection for Ig3 and % initial activity for dornase alpha) were compared between PP2

and AGI samples with the Mann-Whitney test. All statistical analyses were done under GraphPad Prism. P-values below 0.05 were considered for statistical significance.

Results

Effects of the aerosol collection method on nebulized mAb1

Aerosol collection performances

For each device, we quantified the proportion of protein loaded in the nebulizer that was recovered in the aerosol sampler after nebulization, named collection efficiency. As exposed in Table 2, all devices had collection efficiencies comprised between 60 and 75%, except CYC which was under 10%. Indeed, after 40-60 seconds of nebulization, this sampler was saturated: droplet impaction stopped, and the aerosol deposited either in the terminal filter of the device, or in the connection between the nebulizer and the CYC.

Effect of the aerosol samplers on Ig1 aggregation

Mis en forme : Police :Non Gras

Aggregation is one of the main markers of protein instability. It results from the clustering of several native or denatured protein molecules, generating structures ranging from soluble dimers (several nanometers) to micron-sized insoluble particles [START_REF] Wang | Protein aggregation-Pathways and influencing factors[END_REF][START_REF] Philo | Mechanisms of Protein Aggregation[END_REF]. Thus, protein aggregation is commonly assessed by a combination of complementary analytical techniques, with different principles of analysis and particle size ranges [START_REF] Mahler | Protein aggregation: Pathways, induction factors and analysis[END_REF][START_REF] Den Engelsman | Strategies for the assessment of protein aggregates in pharmaceutical biotech product development[END_REF]. Herein, we used visual inspection for particles above 500 µm, flow cell microscopy for micron-sized particles, DLS for submicronic aggregates and SEC for protein monomer and oligomers.

Aggregation results were heterogeneous among the tested aerosol samplers. Visible particles were constantly observed in PP15 and PP2 samples, but not for AGI, BS or CYC (see Figure 2A). For the NGI, this particle population was observed only on certain impaction plates, namely the ones that collect droplets < 1.36 µm, despite lower amounts of deposited protein (see Figure S1). However, these results were not predictive of smaller-scale aggregation, as evidenced by concentrations in micron-sized aggregates (Figure 2B). Indeed, PP15 and PP2 concentrations were within the same order of magnitude than BS and CYC (which presented no visible particles), roughly 10 times higher than AGI and 10 times lower than NGI samples.

Overall, the range of concentration in micronic aggregates varied amongst aerosol samplers by a factor of 100, with a significant difference between AGI and NGI. In the submicronic range (Figure 2C), all DLS profiles were modified after nebulization, as evidenced by global Pd indexes. Again, the aerosol samplers led to different results: while AGI samples had increased Pd indexes, low polydispersities and a monomer content similar to the initial solution (99.4 vs 100.0%), aerosol collection in PP2 led to a significant decrease in monomer proportion (-6.5%).

Besides, the global Pd index was not measurable in this condition, indicating a multimodal distribution of submicronic protein particles. Interestingly, PP15 samples presented a singular profile: even though the decrease in monomer content was limited (-3.4%), the polydispersity was significantly higher than for AGI samples (37.7 vs 11.1%, respectively), suggesting the presence of dimers or oligomers. Finally, SEC results were concordant, indicating a higher loss in monomer for PP2 than PP15 and AGI samples (-3.2%, -0.9% and -0.4%, respectively), originating from a significant enrichment in higher molecular weight species. DLS and SEC data could not be obtained when final protein concentrations were too low (BS, CYC and NGI for DLS, CYC and NGI for SEC).

Overall, Ig1 aggregation was profile modified after nebulization, each sampler affecting specific particle/aggregate populations. While AGI collection seemed to raise essentially micron-sized particles, PP2 induced marked protein aggregation, associating significant monomer loss, micron-sized and visible particles. Surprisingly, PP15 and PP2 profiles were very close for micronic particles, but not for monomers and oligomers; BS, CYC and NGI characterization, although incomplete, showed different behaviors regarding particles over 1 µm. Overall, as protein aggregation cannot be assessed in the aerosol, it is difficult to conclude on the specific bias generated by each aerosol sampler. Results are presentedexpressed as means  one standard deviationSD betweenof the five replicates.

Before neb: antibody solution before nebulization; Pd: polydispersity; HMWS: higher molecular weight species; ND: no data available.

Effect of the aerosol samplers on Ig1 biological activity

To assess Ig1 biological activity, we compared its affinity for its target (EGFR), pre-and postnebulization. To do so, we carried out an indirect competition assay on EGFR-expressing cells,

Applicability to other protein therapeutics

In a second part, we investigated whether the observed trends may be generalized to other protein therapeutics. To do so, we selected two of the six aerosol samplers and tested their impact on the physicochemical and biological stability of three nebulized proteins. Selection criteria included: distinct mechanism of aerosol collection, low/no sample dilution (to be suitable with all analyses) and distinct profiles regarding Ig1 stability. Based on these criteria, we selected PP2 and AGI for this part, because they did not dilute samples and gave different aggregation patterns for Ig1.

Aerosol collection performances

As previously done with Ig1, we compared the aerosol samplers in terms of aerosol collection performance (see Table 4). Depending on the considered protein, collection efficiencies were variable regarding protein concentration, volume and final collection efficiency. Overall, AGI samples contained higher protein concentrations (76-112% of the initial solution concentration) than PP2 samples (54 to 94% of the initial solution concentration). Compared with Ig1, where PP2 and AGI concentrations were respectively 90% and 97% of the initial concentration, greater variations were observed with these 3 proteins, resulting in some cases in substantial protein up-concentration or dilution. Regarding volumes, the two samplers behaved in a similar way, except with Ig2 which was associated with lower collected volumes for the AGI (56%) than the PP2 (72%). Resulting collection efficiencies were variable and heterogeneous between the three proteins, with slightly higher efficiencies for the AGI than PP2. Globally, PP2

and AGI gave different results in terms of collection characteristics, depending on the considered protein. µm and nebulization output rates of [0.31 -0.50] mL/min.

Comparison of PP2 and AGI on nebulized Ig2 stability

Ig2 aerosols were produced from a 1.8 mg/mL solution, nebulized with an Aerogen® Pro mesh nebulizer. As for Ig1, nebulized Ig2 physicochemical stability was monitored through aggregation, by combining 4 orthogonal analytical techniques. In the range of large aggregates, both PP2 and AGI produced visible particles, but with distinct profiles: PP2 produced larger particles than AGI, but in fewer quantity (see Figure 3A). This difference was not retrieved for micronic aggregates, with very similar concentrations for the two aerosol samplers (see Figure 3B). Accordingly, at the submicron scale, PP2 and AGI displayed similar behaviors. In both cases, 0.45 m pre-filtration was required for DLS analysis, and the global Pd index was not measurable, suggesting a multimodal profile of particle populations. Besides, monomer loss (-4-6%) and polydispersity (15-19%) were comparable (see Figure 3C). In contrast, AGI samples contained significantly higher proportions of HMWS, as indicated by SEC data (see Figure 3D). These results seem to indicate that, in spite of close profiles at the micrometer scale, PP2 and AGI produced distinct populations of aggregates in the ranges of visible particles and oligomers.

Finally, nebulized Ig2 activity was quantified through a bacterial neutralization assay. Herein, we assessed the ability of Ig2 to reduce P. aeruginosa-induced cytotoxicity on lung epithelial cells after 6h incubation. As shown in Figure 3E, Ig2 aerosols collected with AGI were associated with significantly higher levels of cytotoxicity than those collected with PP2 (14.1% vs 8.0%, respectively), indicating a lower biological activity. Interestingly, Ig2 biological activity was unchanged by nebulization and collection with PP2.

Pd: polydispersity; HMWS: higher molecular weight species Mis en forme : Police :Non Gras

Comparison of PP2 and AGI on nebulized Ig3 stability

Ig3, a polyclonal antiviral Ig, was nebulized at a concentration of 0.2 mg/mL using an Aerogen® Solo nebulizer. Ig3 aggregation profile was very comparable between PP2 and AGI at all size scales, with no visible particle and low concentrations in micronic aggregates (see Figures 4A and4B). Even though DLS could not be performed due to low protein concentration, SEC data also suggest similar profiles in terms of monomer and HMWS contents (see Figures 4C and4D). Interestingly, AGI samples contained slightly higher proportions in monomer (and lower amounts of HMWS) than the non-nebulized antibody solution, but these differences were not significant.

Finally, Ig3 antiviral activity was measured as its ability to neutralize hRSV and thus reduce HEp-2 cell infection in vitro. Dose-response inhibition curves were obtained from incubation with various Ig3 concentrations and led to IC50 determination for each condition. As shown in Figure 4E, AGI-collected samples had a slight (but not significant) difference in IC50 value as compared to non-nebulized and PP2 samples. Besides, higher variabilities in inhibition curves were observed for AGI samples (data not shown).

Combined with aggregation profiles, these results indicate that nebulized Ig3 behaves similarly when collected with AGI or PP2. However, we may not rule out differences since the low initial protein concentration resulted in incomplete sample characterization (DLS could not be performed) and low particle counts. Results are presentedexpressed as means  one standard deviation betweenSD of the three replicates.

Comparison of PP2 and AGI on nebulized dornase alpha stability

Finally, we chose to include dornase alpha in this comparison, given its clinical relevance in the context of aerosoltherapy. After nebulization with an eFlow® rapid nebulizer at a concentration of 1 mg/mL, samples were collected with PP2 or AGI and characterized for aggregation and biological activity. As shown in Figures 5A and5B, in spite of the absence of visible particles, a significant difference appeared for micronic aggregate concentrations, with a factor 4.7 between AGI and PP2 (18319 vs 3917 particles/mL, respectively). This discordance was also retrieved in DLS analyses (see Figure 5C): unlike PP2, AGI samples could not be analyzed without 0.45 µm pre-filtration, indicating multimodal distributions (which was confirmed by the global Pd index). After filtration, AGI samples had slightly lower monomer contents than PP2 samples. Even though their polydispersity tended to be reduced, these results suggest a greater loss of integrity for AGI samples. SEC analyses, performed after 0.2 µm filtration, showed quasi-identical profiles for the two aerosol samplers in terms of monomer and HMWS contents (see Figure 5D). For this protein, aerosol samplers had an impact on aggregation at the micrometric and nanometric scales. From a biological point of view, we measured dornase alpha DNase activity by a colorimetric assay. Briefly, it quantifies the release of the dye methyl green from DNA upon enzymatic digestion by dornase alpha. Herein, the activity of the nebulized samples was expressed as a percentage of the activity of the initial solution. Results displayed in Figure 5E show that, even though nebulization caused a moderate decrease in enzymatic activity (-16%), the aerosol sampler had no impact on this parameter.

Pd: polydispersity; HMWS: higher molecular weight species

Synthesis: effect of the aerosol sampler on nebulized protein stability parameters

Table 5 summarizes the results obtained with each of the four studied protein therapeutics, in terms of concordance of aerosol samplers on protein stability parameters. For each parameter, we identified the sampler with the lowest impact on protein stability (i.e. with lower levels of aggregation or better preservation of biological activity). These data indicate a variable behavior of aerosol samplers, depending both on the protein and the stability parameter. For instance, concordance was observed for Ig3 on all parameters, whereas it happened only on biological activity for Ig1. Likewise, the impact of aerosol samplers on aggregation was heterogeneous, with inconstant concordance of the devices for the different particle populations. Finally, in discordant situations, the aerosol sampler with the lowest impact varied depending on the considered protein.

Discussion

When assessing nebulized protein stability, aerosol collection is a mandatory pre-analytical step; still, its interference on protein stability remains unknown. In the present study, we compared the effect of different aerosol samplers on the stability of four nebulized protein therapeutics.

First, we compared the devices in terms of aerosol collection. For Ig1, all devices gave similar collection efficiencies (60-75% of total Ig1 loaded in the nebulizer), except CYC, due to early saturation. However, PP2 and AGI displayed different collection performances depending on the protein therapeutics. On one hand, the concentration of Ig2 was notably affected by PP2 collection, with an apparent 2-fold dilution, which might result from non-specific protein adsorption on the plastic surfaces of the collection setup. On the other hand, AGI was associated with lower collection volumes for Ig2, maybe due to higher aerosol evaporation. In the end, when tested on four different proteins, these devices displayed variable collection performances, in terms of volume, protein concentration and protein amount. As aerosol characteristics were similar (regarding VMDs and nebulization output rates), these differences in aerosol collection might originate from specificities of protein molecules themselves, as could be the case with any "conventional" (non-biological) drug.

On the physicochemical side, protein aggregation was strongly affected by the aerosol collection device, with heterogeneous results for the four proteins. With Ig1, the six samplers gave six distinct aggregation profiles: micron-sized particle counts varied by a factor of 100, associated with variable monomer loss and inconstant presence of visible particles. For Ig2, the aerosol sampler rather impacted visible particles and oligomers, but not micronic particle counts. Conversely, after collection, dornase alpha displayed differences in micron-sized particles independently from similar profiles for oligomers and visible particles. The results for Ig3 are less conclusive because of the low initial protein concentration but tend to show similar aggregation profiles independently of the collection device. This heterogeneity of biotherapeutics aggregation due to the aerosol sampler is in agreement with Hertel, who observed that SM101 and IgG1 aggregation varied depending on the aerosol collection process [START_REF] Hertel | Pulmonary Delivery of Pharmaceutical Proteins by Means of Vibrating Mesh Nebulization[END_REF]. Interestingly, in their previous study on dornase alpha aerosols, Cipolla and Gonda showed no difference in monomer loss after collection with two devices [START_REF] Cipolla | Method for collection of nebulized proteins[END_REF]. This discrepancy with our results may originate from differences in experimental conditions (the nebulizer and collection systems were different from ours) and incomplete characterization of protein aggregation.

Overall, the discordance in stability profiles might result from the different stresses applied to proteins during the collection process, originating from the underlying mechanisms of aerosol droplet recovery (i.e. condensation, impaction on solid or in liquid, vortex impaction). So far, it does not seem possible to conclude on a "more relevant" collection procedure, as heterogeneities appeared for similar recovery mechanisms. For instance, AGI and NGI (which both imply impaction on a solid surface) yielded very different counts in micron-sized particles; likewise, PP15 and PP2, although very similar, gave distinct monomer/oligomer repartition.

Other factors might be implied in these differences, such as air flowrate, material of the impaction surface or kinetic of droplet deposition.

Besides, in spite of similar collection efficiencies, the size range of collected droplets is not always known for each device. Although Ig1 distribution followed aerosol droplets distribution, indicating a homogeneous repartition in aerosol droplets (verified in a preliminary experiment, data not shown), one could imagine that some aerosol particles might be associated with higher levels of stresses. This hypothesis is supported by Ig1 aggregation results obtained with the NGI, for which the last plates (corresponding to smaller droplets) contained higher levels of visible and micron-sized particles than the others. This population of droplets has a higher specific surface area [START_REF] Hertel | Protein stability in pulmonary drug delivery via nebulization[END_REF]; this implies that, for equal protein concentrations, a higher number of protein molecules is exposed to the deleterious air-liquid interface, as compared to larger droplets. Thus, fine droplets might be associated with more pronounced protein degradation, independent from collection stresses. In this case, large aggregates might probably appear after droplet deposition and coalescence, arising from interactions between highly altered protein species and/or nucleation of native monomers on the surface of aggregation nuclei [START_REF] Philo | Mechanisms of Protein Aggregation[END_REF].

In addition to aggregation, we also assessed the impact of the aerosol collection process on protein biological activity, which may change as a result of denaturation. Overall, aggregation was inconstantly associated with variations in protein biological activity. In spite of different aggregate populations obtained with AGI and PP2 collection, dornase alpha residual enzymatic activity was similar and well-conserved after nebulization, which is consistent with previous literature data [START_REF] Cipolla | Method for collection of nebulized proteins[END_REF]. Likewise, the binding of Ig1 to its target was not significantly affected by the nebulization/collection process; neither was Ig3 antiviral activity, in spite of higher variabilities observed with AGI. This is in agreement with current data on Ig nebulization, which was rarely reported to cause defects in target binding or cellular activity [START_REF] Maillet | Aerodynamical, immunological and pharmacological properties of the anticancer antibody cetuximab following nebulization[END_REF][START_REF] Respaud | Development of a drug delivery system for efficient alveolar Mis en forme : Anglais (États-Unis) delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin[END_REF][START_REF] Cortez-Jugo | Pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform[END_REF].

Interestingly, Ig2 behaved differently: its antibacterial activity was impacted by the collection system, as AGI samples were associated with higher rates of cytotoxicity. This effect seems to be linked rather to monomer-scale aggregation than to the absolute number of aggregates, as the amounts of micron-sized particles was similar for both devices. Additionally, these discrepancies in Ig functionality after nebulization might be explained by different aggregation patterns. Indeed, target recognition and binding (which were assessed for the three Igs) rely on small regions of Ig Fab portions. Thus, we could argue that for Ig1 and Ig3, aggregation affected other regions than the Fab, and hence was not critical for target binding (within the limit of sensitivity of the assays). Conversely, Ig2 aggregation might directly involve target-binding regions, either through denaturation mechanisms or by simple entrapment and hiding of the antigen-recognizing site inside protein aggregates. Besides, for these Igs, aggregation might also impair Fc-dependent functions (such as FcγR binding or complement recruitment), which were not explored herein.

Taken together, these results highlight the variable impact of aerosol collection on protein stability. However, it seems difficult to discern general trends and identify the "most relevant" device for protein aerosols. Indeed, Ig1 aggregation and activity results tend to designate the AGI as the least deleterious sampler (with low levels of aggregation and no alteration of biological activity), while PP2 tubes were associated with higher levels of protein denaturation.

Still, when tested on other proteins, AGI and PP2 gave opposite trends, PP2 being more favorable than the AGI for two (out of three) proteins, both from aggregation and activity prospects. Accordingly, Hertel and collaborators showed that PP2 tubes were associated with very low levels of particles and monomer loss of SM101, whereas the twin-stage impinger (which is close to the AGI) led to significant protein aggregation [START_REF] Hertel | Pulmonary Delivery of Pharmaceutical Proteins by Means of Vibrating Mesh Nebulization[END_REF]. Therefore, the behavior of one protein with one sampler cannot be simply applied to others. Actually, this could be explained by the fact that proteins have different aggregation patterns, conditioned by their sequence, higher order structures, environment (formulation, concentration) and the stresses they face (e.g. nebulization stresses). Thus, depending on the considered protein, the "most relevant" aerosol sampler (i.e. the least deleterious one) varies, making it difficult to identify a universal collection method.

Besides, our findings raise the question of the relevance of such collection methods towards real-life considerations. Indeed, in vivo, droplet deposition sets up at 37°C, with 100% relative humidity, multidirectional air movements, on a large fluid-covered epithelial surface, and mainly implies a combination of inertial impaction, gravitational sedimentation and Brownian diffusion [START_REF] Darquenne | Aerosol Deposition in Health and Disease[END_REF]. These conditions are very far from current in vitro aerosol collection processes, which probably induce significant biases on protein stability. For instance, with Ig2, both PP2 and AGI produced high amounts of particles > 25 µm, and even visible particles (> 500 µm). These species being larger than the biggest aerosol droplets (measured by laser diffraction), they were most probably generated after bulking of aerosol droplets, thereby favoring interaction between proteins from different droplets, regardless of the considered device.

Finally, this study allowed us to identify some of the criteria that would define an "ideal aerosol sampler" for protein therapeutics, namely suitability with any nebulization setup, representativeness of aerosol deposition in the lungs after inhalation and absence of interference on nebulized protein stability. Given that none of the tested devices fulfilled all these criteria, there is probably room for innovation in the field of aerosol collection for the development of inhaled protein therapeutics.

Conclusions

This study highlights the fact that the aerosol collection step, which is mandatory to characterize nebulized proteins, has an impact on stability studies. More importantly, this impact depends on both the collection device and the protein, and it is thus hardly predictable for one given protein. To date, we did not identify a "universal" aerosol sampler, optimal for the stability of any protein therapeutic. Thus, careful selection and exclusive use of an appropriate aerosol sampler is crucial in the development of nebulized proteins, to limit pre-analytical variations and obtain comparable datasets. Until analytical characterization of proteins becomes possible in aerosol droplets, further optimization studies might allow to identify or develop a more relevant, maybe more physiological protein aerosol sampler.

Figure 1 .

 1 Figure 1. Experimental setups of the evaluated aerosol collection devices.In each setup, the Tpiece was positioned in such a way that aerosol droplet trajectory was the same before reaching the collection device (i.e. with a 90° bend).

Figure 2 .

 2 Figure 2. Effects of aerosol samplers on Ig1 aggregation. A. Presence/absence of visible particles in Ig1 before/after nebulization. (-): absence / (+): presence / (+/-): inconstant presence (depending on the NGI plate). B. Concentrations in aggregates ≥ 1µm obtained by flow cell microscopy. For each condition, the number of particles in blank aerosols (i.e. formulation alone) was subtracted. C. SEC determination of the content in protein monomer, expressed as % of all detected species. Due to low final protein concentrations, CYC and NGI samples could not be analyzed. D. DLS determination of the content in protein monomer, expressed as % of all detected species. Due to low final protein concentrations, BS, CYC and NGI samples could not be analyzed.

  ability of Ig1 to displace the interaction between EGFR and fluorescent-labeled Ig1. Incubation of different Ig1 concentrations allowed us to build dose-inhibition curves for FITC-Ig1 binding and calculate the IC50 of each sample. As shown in

Figure 3 .

 3 Figure 3. Ig2 aggregation and antibacterial activity after nebulization and collection with PP2 and AGI. Results are presentedexpressed as means  one standard deviation betweenSD of the five replicates.

Figure 4 .

 4 Figure 4. Ig3 aggregation and antiviral activity after nebulization and collection with PP2 and AGI.

Figure 5 .

 5 Figure 5. Dornase alpha aggregation and enzymatic activity after nebulization and collection with PP2 and AGI. Results are presentedexpressed as means  one standard deviation betweenSD of the five replicates.

Table 2 . Aerosol collection performances measured for the 6 samplers with 2mL of Ig1 loaded in the nebulizer

 2 . mAbIg1 aerosols had a VMD of 4.9 µm.

		Volume of aerosol		Final protein	Collection
		collected (mL)	Volume of	concentration	efficiency
		Mean ± SD	diluent (mL)	(mg/mL)	% protein collected
				Mean ± SD	Mean ± SD
	Before nebulization	/	/	2.09 ± 0.17	/
	PP15	1.37 ± 0.13	0.0	1.90 ± 0.21	59.9 ± 5.6%
	PP2	1.43 ± 0.20	0.0	1.89 ± 0.18	65.5 ± 5.2%
	AGI	1.48 ± 0.10	0.0	2.02 ± 0.11	75.1 ± 6.4%
	BS	1.46 ± 0.12	5.0	0.52 ± 0.01	73.2 ± 6.1%
	CYC	0.16 ± 0.02	1.0	0.31 ± 0.05	8.5 ± 1.5%
	NGI	N/D	50.0	0.07 ± 0.01	72.7 ± 5.7%

Table 3 ,

 3 IC50 were very similar before and after nebulization, regardless of the collection method. Thus, nebulization and collection did not seem to have any impact on Ig1 biological activity.

		FITC-Ig1 binding
		IC50 (mg/mL)
	Before nebulization	2.2 10 -4
	PP15	1.8 10 -4
	PP2	2.0 10 -4
	AGI	1.7 10 -4
	BS	1.8 10 -4
	CYC	1.9 10 -4
	NGI	2.1 10 -4

Table 3 . Effect of aerosol samplers on Ig1 affinity for its target after nebulization.

 3

Table 4 . Aerosol collection performances depending on protein therapeutics

 4

			Final protein	Volume	Collection
	Aerosol sampler	Protein therapeutic	concentration (% initial)	collected (% loaded)	efficiency % protein collected
			Mean ± SD	Mean ± SD	Mean ± SD
		Ig1	90.4 ± 8.6	71.5 ± 10.0	65.5 ± 5.2
	PP2	Ig2 Ig3	54.4 ± 24.7 79.1 ± 5.1	72.0 ± 3.7 82.7 ± 4.0	39.4 ± 18.6 65.5 ± 7.2
		Dornase alpha	93.8 ± 5.4	77.5 ± 7.7	72.8 ± 9.4
		Ig1	96.7 ± 5.3	74.0 ± 5.0	75.1 ± 6.4
	AGI	Ig2 Ig3	97.1 ± 12.7 75.7 ± 8.3	56.0 ± 7.2 90.0 ± 8.7	53.7 ± 1.2 67.9 ± 6.6
		Dornase alpha	112.1 ± 4.3	72.2 ± 7.1	80.8 ± 5.9

. Aerosol collection performances were measured for PP2 and AGI with Ig2, Ig3 and dornase alpha; data obtained with Ig1 are recalled for each device. Aerosol characteristics were similar for all proteins, with VMDs of

[3.9 -4.9]

Acknowledgements

The authors wish to thank Thomas Sécher and Florent Creusat for their support on flow cytometry experiments and analysis. They also would like to thank William Lindsley from the NIOSH, who kindly provided the BC-112 cyclones. This work was funded by a government grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (reference: ANR-10-LABX-53-01).

Concordance between aerosol samplers?

(If not, sampler with the lowest impact on protein stability*)

Stability parameter

Ig1 ** For Ig1, results of device concordance were the same whether the comparison included 6 or 2 aerosol samplers.

Table 5. Concordance between aerosol samplers on protein stability parameters. This analysis included the 6 devices for Ig1, and two samplers (PP2 and AGI) for the other proteins. In case of discordance between aerosol samplers, the device with the lowest impact on protein stability is indicated in brackets.

Supplementary data


B05-0829 -(Invited) Synthesis and Optical

Properties of Rod-Shaped Graphene Nanoparticles

Abstract

The scientific community has given much interest to graphene in recent years because of its good mechanical, thermal and electrical properties. Nonetheless, applications in optics and optoelectronics can be limited due to the absence of a bandgap. In order to engineer a bandgap, we can reduce the size of graphene into graphene quantum dots (GQDs). GQDs synthesis is primarily described via top-down methods (lithography, hydrothermal and electrochemical approaches). [START_REF] Yang | Characterization and Applications of Graphene Quantum Dots in Recent Trends in Nanomaterials: Synthesis and Properties[END_REF] Though effective, these methods do not allow accurate control of the size, shape and edges of the GQDs. On the other hand, bottom-up graphene materials have developed exponentially for the last decade with the synthesis of highly controlled graphene nanoribbon structures and moderately soluble graphene quantum dots. [START_REF] Narita | New advances in nanographene chemistry[END_REF][START_REF] Wang | Polycyclic aromatic hydrocarbons in the graphene era[END_REF] Our group demonstrated that bottom-up GQDs could act as singlephoton emitters exhibiting high brightness and stability. [START_REF] Zhao | Single photon emission from graphene quantum dots at room temperature[END_REF] In order to investigate the structure-property relationship, we designed a series of rodshaped graphene nanoparticles that differ only by their length, keeping the same morphology, symmetry and edge states (see figure). These nanoparticles are fully soluble, which facilitate their purification and individualization in solution and give rise to welldefined absorption spectra. Here we report on the synthesis of these GQDs, and we will present results on advanced characterization of their optical properties. [5]


Modelling NMR Spectroscopy of Oxide Glasses with Machine Learning

Thibault Charpentier 1 Université Paris-Saclay, CEA, CNRS, NIMBE, CEA Paris-Saclay, Gif-sur-Yvette, 91191, France, thibault.charpentier@cea.fr Solid-State NMR has now become a key spectroscopy in numerous fields of material sciences. The local environment of each atom is characterized by a NMR fingerprint that can be acquired independently of the crystalline, disordered or vitreous form of the studied material. With recent advances in DFT computations, NMR can now be combined with Molecular Dynamics (MD) simulations to help the interpretation of experimental data. However, such calculations are severely limited in system size by the high-computational cost of DFT computations.

In the last years, machine learning (ML) approaches have emerged as powerful mehods for accelerating MD and computing materials properties with an accuracy and an efficiency that are close to that of DFT methods and classical MD, respectively. In the specific field of solid state NMR, few approaches have been recently proposed that can be applied to oxide glasses which are complex materials which structural features that are still debated.

We describe here an approach based on the concept of atomic-centered descriptors (such as SOAP) combined with Kernel Ridge Regression (KRR) or Artificial Neural Network (ANN) techniques. This enables the prediction of NMR properties for structural models of thousands of atoms. An optimal scheme based on the Least-Square Support Vector Regression algorithm will be presented with applications to simple borate and silicate glasses.

sciencesconf.org:gdr-iamat:408065

mai -1 juin -Paris https://gdr-iamat.sciencesconf.org


Noémie Cartier

Adélaïde Chesnay

Camille Thorey

Marion Diama N'diaye

Olivier Ferreira

Éric Haillot

Guillaume Bailly

Desoubeaux

Diama N'diaye

Marion Ferreira

Olivier Haillot

Éric Bailly

Guillaume Desoubeaux
email: guillaume.desoubeaux@univ-tours.fr

Candida nivariensis: Identification strategy in mycological laboratories

Keywords: Candida nivariensis, Candida glabrata, candidemia, candiduria, Candida bracariensis, antifungal resistance

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Candidiasis is worldwide the most frequent deep-seated fungal infection. For example in France, 15,500 new cases occurred during the 2001-10 period, which represented 43.4% of all the recorded cases of fungal infection [START_REF] Bitar | Population-Based Analysis of Invasive Fungal Infections, France, 2001-2010[END_REF]. With the emergence of new antifungal drugs and the extensive practice of prophylaxis and empirical treatments, the epidemiology of candidiasis has been slightly changing. While Candida albicans remains the major species, minor or new species have progressively emerged and have become isolated more and more frequently in medical laboratories [START_REF] Yapar | Epidemiology and risk factors for invasive candidiasis[END_REF]. For instance in a French cohort focusing on candidemia (i.e. cases of bloodstream infection due to Candida species), the proportion of C. krusei moved from 3 to 8%, and this of C. glabrata from 18 to 29%, following pre-exposure to fluconazole [START_REF] Lortholary | Recent Exposure to Caspofungin or Fluconazole Influences the Epidemiology of Candidemia: a Prospective Multicenter Study Involving 2,441 Patients[END_REF].

However, such results should be interpreted with caution, because of possible species misidentifications in former studies. Through the recent advent of DNA sequencing techniques and MALDI-TOF mass spectrometry, a few new species have been described within large species complexes. These species are referred to as cryptic species because they do exhibit phenotypic features that are similar to those of the major species of the complex. Therefore, Candida nivariensis, which is very close to C. glabrata, was genetically identified as a new distinct species in 2005. C. nivariensis was first described in bronchial-alveolar lavage fluid, blood and urine from three distinct patients in a Spanish hospital [START_REF] Alcoba-Florez | Phenotypic and Molecular Characterization of Candida nivariensis sp. nov., a Possible New Opportunistic Fungus[END_REF].

Regarding the choice of the adequate antifungal drug, correct species identification of C. nivariensis is needed in order to thereafter decide to conduct antifungal susceptibility tests. Indeed, it was showed that C. nivariensis frequently expresses high minimal inhibitory concentrations (MICs) to azole drugs, like C. glabrata [START_REF] Borman | Candida nivariensis, an Emerging Pathogenic Fungus with Multidrug Resistance to Antifungal Agents[END_REF].

The present article describes a rare case of C. nivariensis candidemia occurring in a cancer patient of a French university hospital. It also underscores the technical limitations for its identification in mycological laboratories. Moreover, an updated overview of the literature about C. nivariensis is presented.

CASE REPORT

A 77-year-old man was hospitalized on day 0 for cysto-prostatectomy with Bricker trans-ileal bypass in a context of urothelial carcinoma relapse.

The patient also suffered from a primary pulmonary adenocarcinoma with lymph node involvement that was treated with pembrolizumab. On day 5, the patient was febrile at 38.5°C. He was empirically given an antibacterial treatment based on piperacillin/tazobactam, and blood cultures (BD Bactec TM Plus Aerobic/F vial, BD, Franklin Lakes, United States) were collected from his central catheter. One culture vial was detected positive after four days and six hours in the incubator. Its direct microscopic examination showed small budding yeasts that could rightfully evocate C. glabrata. After 48 hours of incubation at 35°C, subculture onto chromID ® Candida agar plates (BioMérieux, Marcy-l'Etoile, France) exhibited small white smooth and shiny colonies looking like C. glabrata. The Glabrata R.T.T. Fumouze® test (Biosynex, Illkirch-Graffenstaden, France), based on the ability of C. glabrata to hydrolyze trehalose but not maltose [START_REF] Freydiere | Rapid Identification of Candida glabrata with a New Commercial Test, GLABRATA RTT[END_REF], was revealed positive after eight minutes.

An Api 20C® auxanogram gallery (BioMérieux, Marcy-l'Etoile, France) suggested C. glabrata species with an identification score of 99.4%.

The MALDI-TOF mass spectrometry (Bruker Daltonics, Bremen, Germany) identified C. nivariensis with a Log-score of 1.72, although the profile was very close to C. glabrata (the software MBT Compass IVD® v.4.2 was used with the database MSP 7171, year 2018). Strain identification was finally confirmed as C. nivariensis by sequencing the D1/D2 region of the 28S rDNA (GenBank accession number No. MN306567) [START_REF] Gomez | Performance of Targeted Fungal Sequencing for Culture-Independent Diagnosis of Invasive Fungal Disease[END_REF]. The antifungal susceptibility of the isolate showed elevated MICs to fluconazole, but not to other azoles, neither to echinocandins nor to amphotericin B (Table 1).

Thereafter, a second blood culture collected from the central catheter on day 6 was detected as positive for C. nivariensis, after four days and 14 hours in the incubator. The urines collected on Bricker's system on day 8 were also positive for C. nivariensis. The central catheter was removed on day 9, but its in vitro culture remained sterile after 48 hours of incubation. On day 12, the concentration of β-D-glucan antigen was measured at 218 pg/mL in blood, with the Fungitell assay® serum test (Cape Cod, East Falmouth, United States). Candida antigen was negative with mannan concentration measured at 40 pg/mL in blood (BioRad, Hercules, United States).

On day 9, piperacillin/tazobactam antibiotics were withdrawn, and antifungal therapy was initiated with caspofungin. Fever then decreased, and all subsequent blood cultures remained sterile thereafter. Ultrasound examination didn't show any sign suggestive for endocarditis, and the dilated fundus exam was normal. After two weeks of treatment, caspofungin was switched to oral voriconazole for one month in order to complete the expected overall treatment duration for six weeks (as recommended for the intravascular infection, as a partial thrombosis was herein highlighted by a Doppler ultrasound examination in the right internal jugular vein after the removal of the catheter) [START_REF] Pappas | Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America[END_REF]. After one year of follow up, the patient was still alive.

DISCUSSION

Candida nivariensis is an emergent yeast belonging to the Candida glabrata complex. Since its first description, 181 human cases (ranging from simple colonization to true infection) have been reported (Table 2 and Suppl. table 1): 112 in Europe, 51 in Asia, 11 in Oceania, six in America and one in Africa. The actual prevalence of C. nivariensis infection is thought to be probably underestimated in view of the theoretical worldwide distribution of this yeast species. This hypothesis has been raised from several previous studies [START_REF] Lockhart | Identification of Candida nivariensis and Candida bracarensis in a Large Global Collection of Candida glabrata Isolates: Comparison to the Literature[END_REF]- [START_REF] Tay | Occurrence and Characterization of Candida nivariensis from a Culture Collection of Candida glabrata Clinical Isolates in Malaysia[END_REF], because at that times laboratories didn't have access to modern diagnostics tools for distinguishing between C. nivariensis and C. glabrata (e.g. auxanogram galleries, like the API® yeast identification, didn't permit the identification of cryptic species like C. nivariensis and both are able to assimilate glucose and trehalose [START_REF] Alcoba-Florez | Phenotypic and Molecular Characterization of Candida nivariensis sp. nov., a Possible New Opportunistic Fungus[END_REF], [START_REF] Swoboda-Kopeć | Candida nivariensis in comparison to different phenotypes of Candida glabrata[END_REF], that can mislead the interpretation of the Glabrata R.T.T. Fumouze® test). In our mycology laboratory, it was the first time that C. nivariensis was unambiguously isolated, whereas the annual incidence of C. glabrata has been meanwhile estimated at 41.1 per 1,000 samples for the last ten years. There are however big needs to accurately identify C. nivariensis, mostly for epidemiological purposes. Until recently, genomic analysis was the only reliable method for C. nivariensis characterization. Many PCR methods have been developed to distinguish between C. nivariensis and C. glabrata, and some have showed good results [START_REF] Alcoba-Florez | PCR Protocol for Specific Identification of Candida nivariensis, a Recently Described Pathogenic Yeast[END_REF], [START_REF] Dudiuk | Multiplex PCR designed to differentiate species within the Candida glabrata complex[END_REF]. Most target the internal transcribed spacer (ITS) sequences. A PCR-based assay using a single primer pair targeting the RPL31 gene allowed correct discrimination between C. glabrata, C. nivariensis and C. bracarensis (i.e. the latter is another cryptic species belonging to the C. glabrata complex), according to the size of the amplicon [START_REF] Enache-Angoulvant | Rapid Discrimination between Candida glabrata, Candida nivariensis, and Candida bracarensis by Use of a Singleplex PCR[END_REF]. In contrast, a home-made multiplexed PCR specifically-developed for identification of clinically-important yeast species didn't permit the identification of C. nivariensis [START_REF] Arastehfar | YEAST PANEL multiplex PCR for identification of clinically important yeast species: stepwise diagnostic strategy, useful for developing countries[END_REF]. Unfortunately, no marketed commercial kit for such a purpose is available for now. Hopefully for a few years, MALDI-TOF technology has been introduced into diagnostic laboratories and now enables the reliable identification of various microorganisms, including some cryptic fungal species. As mass spectrometry databases of spectra libraries are continuously improving, MALDI-TOF-based identification has become the first-line technique in routine practice for most of the microbiological laboratories. Many articles showed very good results for the identification of C. nivariensis and C. bracarensis, particularly with recent Bruker MS® Biotyper databases [START_REF] Arastehfar | Molecular characterization and antifungal susceptibility testing of Candida nivariensis from blood samplesan Iranian multicentre study and a review of the literature[END_REF], [START_REF] Aznar-Marin | Candida nivariensis as a New Emergent Agent of Vulvovaginal Candidiasis: Description of Cases and Review of Published Studies[END_REF], [START_REF] Gorton | Candida nivariensis isolated from a renal transplant patient with persistent candiduria-Molecular identification using ITS PCR and MALDI-TOF[END_REF], or with the user-made MSI platform [START_REF] Normand | Optimization of MALDI-ToF mass spectrometry for yeast identification: a multicenter study[END_REF]. For example, in the updated library Biotyper® MSP 7171, six reference spectra are available for C. nivariensis. In contrast in a recent study focusing on the identification of Candida sp from blood culture, the BioMérieux Vitek MS® system failed to identify the two isolates of Candida nivariensis [START_REF] Hou | Identification and Antifungal Susceptibility Profiles of Candida nivariensis and Candida bracarensis in a Multi-Center Chinese Collection of Yeasts[END_REF], [START_REF] Li | Oligonucleotide Array and VITEK Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Species Identification of Blood Yeast Isolates[END_REF]. Thus, the BioMérieux Vitek MS® v3.2 library has been incremented in 2018 with C. nivariensis spectra. In the present case, the Bruker MS® system (Bruker, Billerica, United States) correctly identified C. nivariensis in 100% of isolates tested (two from blood cultures, one from urines). The mass spectrometry Log-score for C. nivariensis identification was 1.72 for the first blood culture isolate, 1.85 for the urine isolate and 1.93 for the second blood culture isolate (when considering only the score of the top-ranked proposition for "C. nivariensis" amongst the whole listing of suggestions for species names). As the three isolates obviously corresponded to a unique strain, the difference between the three scores was probably due to slight insignificant technical variations (e.g. quality of the deposit on the MALDI-TOF plate). Noteworthy, the spectra of C. nivariensis and C. glabrata remained very similar, and it's the reason why caution is still warranted to interpret the results (Suppl. figure 1). Similar diagnostic challenges can be encountered with other emergent yeasts: for instance, some diagnostic hindrance has been reported with Candida auris for which identification issues could complicate the management of infection [START_REF] Desoubeaux | Candida auris in contemporary mycology labs: A few practical tricks to identify it reliably according to one recent French experience[END_REF].

A correct identification of the yeast species name is useful for the management of patients infected with Candida sp., especially because of the potentially high MICs to azole drugs for C. nivariensis [START_REF] Borman | Candida nivariensis, an Emerging Pathogenic Fungus with Multidrug Resistance to Antifungal Agents[END_REF], [START_REF] Figueiredo-Carvalho | First description of Candida nivariensis in Brazil: antifungal susceptibility profile and potential virulence attributes[END_REF], [START_REF] Fujita | Catheter-Related Fungemia Due to Fluconazole-Resistant Candida nivariensis[END_REF]. Most reports indicate variable profiles of antifungal susceptibility which is quite similar to C. glabrata. When summarizing all the cases that were reported in the literature, fluconazole MICs ranged from 0.06 µg/mL to > 256 µg/mL (Table 2 and Suppl. table 1), so that 17.2% C. nivariensis strains (5/ 29 tested) should be probably considered as resistant [START_REF] Eucast | Antifungal Agents -Breakpoint tables for interpretation of MICs[END_REF]. The variation in fluconazole MIC values for C. nivariensis (as well as for C. glabrata) warrants antifungal susceptibility testing, as species identification alone cannot predict resistance profile in this particular context. Since invasive candidiasis are globally known to generate high mortality rate about 30% [START_REF] Jia | Clinical characteristics and predictors of mortality in patients with candidemia: a six-year retrospective study[END_REF], [START_REF] Andes | Impact of Treatment Strategy on Outcomes in Patients with Candidemia and Other Forms of Invasive Candidiasis: A Patient-Level Quantitative Review of Randomized Trials[END_REF], [START_REF] Pfaller | Epidemiology of Invasive Candidiasis: a Persistent Public Health Problem[END_REF], ignoring potential resistance could be particularly pejorative for the clinical outcome, worsening consequently the overall mortality. By collating all the data of reported cases (Table 2 and Suppl. table 1), the mortality rate of invasive C. nivariensis candidiasis is estimated at 42%. In the near future, more information will be needed to adjudicate the true clinical virulence of C.

nivariensis.

Considering the frequent misidentification of C. nivariensis, it therefore seems essential to optimize the techniques available for the in vitro diagnosis of C. nivariensis infections. For this purpose, identification by MALDI-TOF mass spectrometry appears to be the best option available to date, combining speed, simplicity and reliable results. We also largely encourage systematic antifungal susceptibility testing when isolating such strains.

Table 1 . Minimal inhibition concentrations (MICs) for the Candida nivariensis strain isolated in this study

 1

	MIC (µg/mL)	Amphotericin	Micafungin Fluconazole Voriconazole Posaconazole
		B
	E-test method ♦ ,	0.750	0.012	6	0.047	0.380
	RPMI culture
	medium
	Broth	0.06	≤ 0.008	4	0.25	0.125
	microdilution
	method*

♦ According to the manufacturer's recommendations (BioMérieux, Marcy-l'Etoile, France) *According to EUCAST

[START_REF] Eucast | Antifungal Agents -Breakpoint tables for interpretation of MICs[END_REF]

Table 2 . Overview of the published reports about Candida nivariensis isolates (2014-2020)

 2 The previous cases have already been reviewed by[START_REF] Angoulvant | Old and new pathogenic Nakaseomyces species: epidemiology, biology, identification, pathogenicity and antifungal resistance[END_REF] [START_REF] Angoulvant | Old and new pathogenic Nakaseomyces species: epidemiology, biology, identification, pathogenicity and antifungal resistance[END_REF]. See supplementary table 1.

ACKNOWLEDGMENTS

The authors thank Prof. Jacques Chandenier for his valuable scientific opinion. They also thank CNRMA (Centre National de Référence des Mycoses invasives et Antifongiques, Pasteur Institute Paris) for his help in the measure of MICs through the broth microdilution method. A native English speaker reviewed the English syntax and the editing of the manuscript.

The patient did not emit any opposition to the publication.

FUNDING

This work was supported by none. The authors did not receive any specific research funding for this study.

DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The authors declare no conflict of interest. They have submitted the ICMJE form. The data was obtained as a part of routine work at the University Hospital in Tours, France.

analysis. Noteworthy, the mass spectra are quite close to each other between C. nivariensis and C. glabrata, as they both belong to the same 267 species complex. In comparison, spectral differences appear largely more obvious with C. albicans.


Amélie Deglaire

Francoise Nau

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Raphaël Chacon

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Introduction de protéines végétales dans les laits infantiles : opportunités et verrous fonctionnels et nutritionnels

Introduction de protéines végétales dans les laits infantiles : opportunités et verrous fonctionnels et nutritionnels

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Nutrition