Charles Mille
email: charles.mille@ensam.eu

Olivier Christmann

Sylvain Fleury

Simon Richir

Effects of continuous and discontinuous non-relevant stimulus on creativity

Keywords: Creativity, cognitive load, virtual reality, stimulus, environment

Innovation is made of different tasks from strategic positioning to the first phases of production. One of the main activities is the generation of ideas through different creativity phases. These creativity activities are carried out in numerous conditions and with different tools having more or less context and interactions. This study focuses on the influence of different nonrelevant stimuli composing our environment on cognitive load and creativity. This experiment consists of two creativity tasks which consisted in providing answers to a given question using the visuo-spatial and audioverbal area, with three stimulus appearance conditions: without, continuous, discontinuous. For the visuo-spatial task, participants have to illustrate their answers using an immersive drawing software. For the audio-verbal task, participants have to answer a question with a simple text editor software. Regarding the drawing task, results indicate that the discontinuous stimulus had decreased the number of ideas per participant. For the writing task, the stimulus had increased the intrinsic and extraneous cognitive load. This study reveals our creativity is under the influence of the different elements of our environment, the tool used and the task.

Introduction & related work

It is said that it is better to have a rich and stimulating environment in order to be creative, and sometimes that, conversely, it is better to have a simple environment. In order to provide answers on this point, we need to better understand the phenomena at play in creative tasks when task-unrelated stimuli are present in the environment. Creativity is usually defined as the ability to generate new concepts in line with a context in which they are realized (T. [START_REF] Lubart | Psychologie de la créativité-2e édition[END_REF]. Creativity can be mobilized in problem-solving tasks [START_REF] Wang | Fostering Learner Creativity in the English L2 Classroom: Application of the Creative Problem-Solving Model[END_REF] as in tasks of ideas generations with specific constraints, such as the alternate uses test [START_REF] Guilford | The Nature of Human Intelligence[END_REF]. It is the result of complex cognitive activities influenced by various factors stemming from a sum of personal and social experiences [START_REF] Ward | Cognition and creativity[END_REF]. Amabile (1983b) has built a model gathering the cognitive factors affecting creativity. Even though the temporal, material and human components are essential [START_REF] Amabile | The Dynamic Componential Model of Creativity and Innovation in Organizations: Making Progress, Making Meaning[END_REF], some levers can regulate our creativity, for example, by affecting reasoning or problem-solving patterns (Amabile 1983a; T. I. [START_REF] Lubart | An investment approach to creativity: Theory and data[END_REF]. Several works have focused on the understanding of the construction of ideas based on knowledge and the effects of fixations [START_REF] Crilly | Fixation and Creativity in Concept Development: The Attitudes and Practices of Expert Designers[END_REF][START_REF] Cassotti | Fixation Effect in Creative Ideas Generation: Opposite Impacts of Example in Children and Adults[END_REF]. Thus, these studies demonstrate that creativity is influenced by personal factors (called intrinsic) and external factors arising from the environment (call extraneous). Among the first, personal motivation will encourage us to solve the task, and cultural and social background will engage us more or less in the task [START_REF] Zhang | Linking Empowering Leadership and Employee Creativity: The Influence of Psychological Empowerment, Intrinsic Motivation, and Creative Process Engagement[END_REF][START_REF] Binnewies | What Makes a Creative Day? A Diary Study on the Interplay Between Affect, Job Stressors, and Job Control[END_REF][START_REF] Schepers | Social Factors of Work-environment Creativity[END_REF]. However, the environment is also composed of elements, stimuli we do not control and which might be non-relevant for the creativity task. Several studies have contributed to build a taxonomy on how the environment and the stimuli it contains can impact on our creativity [START_REF] Jett | Work Interrupted: A Closer Look At the Role of Interruptions in Organizational Life[END_REF][START_REF] Mochi | Chapter 5 -Interruptions and Multitasking: Advantages and Disadvantages for Creativity at Work[END_REF]. Stimuli which are not linked to the creativity task can improve the idea generation by inspiring the participants, i.e. by let them associate their idea with the stimuli to generate new ideas [START_REF] Goldschmidt | Variances in the Impact of Visual Stimuli on Design Problem Solving Performance[END_REF][START_REF] Baird | Inspired by Distraction: Mind Wandering Facilitates Creative Incubation[END_REF][START_REF] Jett | Work Interrupted: A Closer Look At the Role of Interruptions in Organizational Life[END_REF]. Non-task stimuli can also be a distraction for the participants, forcing them to make an inhibition effort to concentrate on their task. Finally, these stimuli can interrupt the participants, i.e. force them to stop the creativity task at a certain point and resume it. Current work about environments, especially in open spaces, lists numerous elements being able to interrupt people when working [START_REF] Addas | The Many Faces of Information Technology Interruptions: a Taxonomy and Preliminary Investigation of Their Performance Effects[END_REF][START_REF] Tams | Selective Attention As a Protagonist in Contemporary Workplace Stress: Implications for the Interruption Age[END_REF]. In most cases, interruptions are detrimental [START_REF] Mochi | Chapter 5 -Interruptions and Multitasking: Advantages and Disadvantages for Creativity at Work[END_REF]. Some stimuli appear to be annoying during creativity and problem-solving tasks and contribute to reduced performance [START_REF] Kasof | Creativity and Breadth of Attention[END_REF]). On the one hand, an uncluttered, easy-to-understand environment can enhance creative performance [START_REF] Kosmadoudi | Engineering Design Using Game-enhanced Cad: The Potential to Augment the User Experience with Game Elements[END_REF][START_REF] Feeman | Exploration and Evaluation of Cad Modeling in Virtual Reality[END_REF]. On the other hand, some studies suggest that during some activities, a rich environment with many different stimuli may increase the amount of ideas produced, for example, in a task of sketching solutions for product design problems [START_REF] Goldschmidt | Variances in the Impact of Visual Stimuli on Design Problem Solving Performance[END_REF]. This paradox could be based on the fact that, depending on the characteristics of the irrelevant stimuli, it could have a positive inspirational effect, or a distracting effect, or it could interrupt the task. Studying the cognitive load attributed to the task versus that attributed to the environment and the use of tools could provide a better understanding of this phenomenon. Sweller began to link mental work load and the amount of production made by the participants of his experimentation. Although cognitive load measurement was initially used to understand and improve learning [START_REF] Sweller | Cognitive Load Theory, Learning Difficulty, and Instructional Design[END_REF], it is also used in studies focused on problem-solving tasks [START_REF] Sweller | Cognitive Load During Problem Solving: Effects on Learning[END_REF]) and creativity [START_REF] Sweller | Cognitive Bases of Human Creativity[END_REF]. The cognitive load can be split into three components [START_REF] Sweller | Cognitive Load During Problem Solving: Effects on Learning[END_REF][START_REF] Paas | Cognitive Load Measurement As a Means to Advance Cognitive Load Theory[END_REF]: intrinsic, extraneous and germane. Intrinsic cognitive load is related to the presentation of the task (whether it requires learning new knowledge or not) and the level of skills and habits to solve it. Extraneous cognitive load is linked to a misrepresentation of the task or to the efforts needed to apprehend the different elements appearing in the environment. The heavier or more comprehensive the tool and the environment, the higher this cognitive load. Last, germane cognitive load is related to the response to the task to create new mental links and thus to produce new ideas. Unlike the extraneous cognitive load, it is higher when tools are easier to use.

According to intrinsic and extraneous cognitive load, we notice that environment elements can have a negative effect on creativity because of distraction [START_REF] Paas | Cognitive-load Theory: Methods to Manage Working Memory Load in the Learning of Complex Tasks[END_REF]. On the opposite, the different elements of the environment can have inspiring and positive effects on creativity [START_REF] Goldschmidt | Variances in the Impact of Visual Stimuli on Design Problem Solving Performance[END_REF]. These elements have an influence on the cognitive load. That's why it is essential to use simple tools that offer the possibility to control the environment to be able to get inspiration without increasing too much cognitive load.

Virtual reality seems to be a great option by its ease of use [START_REF] Feeman | Exploration and Evaluation of Cad Modeling in Virtual Reality[END_REF][START_REF] Mille | Effects of Digital tools Feature on Creativity and Communicability of Ideas for Upstream Phase of Conception[END_REF] or the inspiring possibilities [START_REF] Yang | Can An Integrated System of Electroencephalography and Virtual Reality Further the Understanding of Relationships Between Attention, Meditation, Flow State, and Creativity?[END_REF][START_REF] Fleury | A Study of the Effects of a Natural Virtual Environment on Creativity During a Product Design Activity[END_REF]. In this regard, virtual reality appears to be a relevant tool to respond to the paradox above-mentioned paradox, as it allows the work environment to be controlled and thus adapted to the creative task. Thus, it can be used to assess the pertinence and impact of irrelevant stimuli in a creative task.

The reasons why some non-task stimuli have a positive impact on creativity and others have a negative impact are not clearly identified in the literature. As mentioned above, it seems that some stimuli can be inspiring, or necessitate mental workload to be inhibited, or other can interrupt the creative mental process. The main objective of the present study is to clarify the links between the stimuli characteristic and the consequences on creative activity. We assume that a stimulus, even subtle, can have an influence on personal experience and increase cognitive load. This increase will reduce creativity if we refer to [START_REF] Paas | Cognitive Load Measurement As a Means to Advance Cognitive Load Theory[END_REF]: the extra resources used to inhibit the environment will reduce the quantity and quality of ideas. We also think that the continuous or discontinuous nature of the stimulus will have an influence on creativity. A continuous stimulus will not interrupt the activity, while the scientific literature shows that a discontinuous stimulus breaks participant's thinking and reduce his creativity [START_REF] Kasof | Creativity and Breadth of Attention[END_REF][START_REF] Baird | Inspired by Distraction: Mind Wandering Facilitates Creative Incubation[END_REF]. We assume that the unpredictable nature the of the stimulus can strongly increase cognitive the load by making it more interruptive.

To study the impact of non-relevant stimuli and their nature on creativity and cognitive load, we designed two tasks using either audio-verbal or visual-spatial resources We have deliberately chosen two creativity tasks and types of stimuli that were as different as possible in order to arrive, to some extent, at a general discourse on the effects of irrelevant external stimuli on creativity. The two tasks were realized under three experimental conditions: without stimulus, continuous stimulus or discontinuous stimulus, interfering with the same resources [START_REF] Baddeley | Working Memory[END_REF]. We measured through questionnaires the evolution, according to the stimulus, of cognitive load.

Method

Participants

For this experiment, a sample group of 67 participants working or studying in the field of virtual reality was gathered. This group was composed of 20 women (30%) and 47 men (70%). The mean age was 25.31 years (sd = 6.59), for a maximum of 53 years old and a minimum of 18 years old.

Procedure

At the beginning, participants were invited to answer a first questionnaire collecting their age, sex and virtual reality skill level. During this experiment, two tasks were performed by the participants. Each task used different cognitive resources and was associated with a stimulus that interferes with the corresponding area [START_REF] Baddeley | Working Memory[END_REF]. We designed an audio-verbal task that we call 'writing task', and a visual-spatial task that we call '3D drawing task'. Each tack can be realized under three stimulus appearance conditions: continuous (the stimulus is always perceptible during the task), discontinuous (the stimulus is discontinuously perceptible during the task), without (no additional stimulus during the task). Participants had to execute one of the two tasks (drawing or writing) in one of the three conditions (without stimulus, discontinuous or continuous stimulus). Then, the questionnaire of [START_REF] Klepsch | Development and Validation of Two Instruments Measuring Intrinsic, Extraneous, and Germane Cognitive Load[END_REF] measuring cognitive load was administered. This sequence was repeated one more time but for the other task and in another stimulus condition. The order of presentation of the tasks and experimental conditions were counterbalanced. The objective of the study was not to compare the two types of task (audio-verbal and visuo-spatial), but to compare within these two tasks the three conditions of non-relevant stimuli (without, discontinuous, continuous). Before each creativity task, two minutes of training were given to participants to familiarize them with the tools. Each creativity task lasts for 5 min during participants are asked to suggest as many ideas as they can.

Writing task

The creative writing task was inspired by the work of Reiter-Palmon, Illies, and [START_REF] Reiter-Palmon | Conscientiousness is Not Always a Good Predictor of Performance: The Case of Creativity[END_REF]. For this task, participant had to answer the following question 'What will be the consequences if we suddenly can no longer use our arms and legs?'. A text writing software (see Figure 1), was given to participants to record their textual ideas. Creativity in this task is not problem-solving, but based on generating creative ideas from among many possible answers that respond to the task and are not the result of simple linear reasoning, as in the alternate uses test [START_REF] Guilford | The Nature of Human Intelligence[END_REF]. Participants usually give short answers, such as 'boxers would wear helmets instead of gloves and hit each other with their head'. The software allowed participants to formulate their answers using a mouse and a keyboard, on the left white part of the screen. After pressing Enter key or Validate button, the sentence was saved and appeared on the right grey part of the screen. It allowed participants to enter new answers until the end of the experiment. When the participants use this button, it counts for one idea. This task duration was 5 min. One minute before the end, a pop-up appeared to warn the participant there was only one minute left. Finally, at the end of the creative task, the software stopped by itself and saved the ongoing writing idea.

The stimulus, for the writing task, was a radio program broadcasted through two speakers placed in front of the participants. The program was an interview of Francis Groux, one of the co-founders of the comic book Angoulême Festival. This program was chosen because the language spoken was the same as the participants' and the subject matter was far from the theme of creativity task. For the continuous condition, the program was broadcasted entirely and without any interruptions during the creative session. For the discontinuous one, the program was randomly cut five times for one to three seconds. For the 'without stimulus' condition, the radio was not running.

3D drawing task

Concerning the 3D drawing task, it was the same as Fleury et al. (2021) and quite similar to Goldschmidt and Smolkov (2006) (sketching ideas for improving a product) and [START_REF] Yang | Can An Integrated System of Electroencephalography and Virtual Reality Further the Understanding of Relationships Between Attention, Meditation, Flow State, and Creativity?[END_REF] (sketching innovative product in virtual reality).

Participants were asked to answer the following question: 'The student's bag is too heavy, can you give solutions to make it more transportable?'.

To do this, an immersive drawing application was given to participants (see Figure 2).

Thanks to the virtual reality headset HTC Vive, participants were immersed in virtual white room. At its centre, a 3D model of backpack was placed on a pedestal. The backpack could be moved from the pedestal, using the grips of the controllers. On the right of the pedestal, a save button and a colour palette were disposed. A press on the save button recorded participant's drawing and reset the area to allow the participant to start a new idea. When the participants used this button, it counted for one idea. The colour palette lets choose a colour, a thickness, saturation of the line drawn by a controller. Each controller can be used as an independent brush and thus have a particular colour. By pushing the trigger of the controllers, the user can start drawing lines. When the backpack moves, the draw follows the movement. The duration of this task was 5 min. In the condition with continuous stimuli, there was a blue light which moved in circles in front of the participant in the virtual environment. This stimulus was considered as continuous because it stayed in front of the participant and remained perceptible during all the tasks. In the condition with discontinuous stimulus, the blue light moved randomly in the virtual environment during the task. It was considered as a discontinuous stimulus because the light entered randomly on the visual field of the participants and left it in the same way during the activity. For the 'Without stimulus' condition, there was no blue light in the environment.

Measures

After each task, the cognitive load was measured through the questionnaire of [START_REF] Klepsch | Development and Validation of Two Instruments Measuring Intrinsic, Extraneous, and Germane Cognitive Load[END_REF]. This questionnaire is composed of nine questions, using Likert's scale from 1 to 7, to measure independently intrinsic (two items), extraneous (three items) and germane (two items) cognitive loads.

Creativity was measured quantitatively: for each participant, the number of ideas produced was counted.

Results

This section is divided into two parts presenting the results of each task: writing then drawing.

Writing task

First, we tested the homoscedasticity (also called 'homogeneity of variances', assesses whether the variances of the groups are sufficiently equivalent to allow parametric tests) and normality of our data sample. The homoscedasticity is confirmed for the number of ideas per participant (p = 0.588), extraneous cognitive load (p = 0.38) and germane cognitive load (p = 0.756) depending on the distraction. On the opposite, the heteroscedasticity is confirmed for the intrinsic cognitive load (p = 0.018), depending on the distraction.

Analysis of idea production

We begin by analysing the impact of the stimulus on the number of ideas per participant. An analysis of variances depending on the condition did not reveal any statistically significant difference (F(2; 60) = 0.371; p = 0.691).

Cognitive load analysis

Regarding the cognitive load induced by the stimulus, a Kruskal-Wallis non-parametric test revealed statistically significant difference for the intrinsic cognitive load (x 2 = 19.775;p , 0.001). A pairwise non-parametric comparison using Tukey and Kramer test revealed differences between the condition without stimulus and the other two (see Table 1). Figure 3 shows that the intrinsic cognitive load is higher when the stimulus is present in the environment.

An analysis of variances revealed statistically significant difference for the extraneous cognitive load (F(2; 60) = 13.79; p , 0.001). A pairwise comparison using T test revealed differences between the condition without stimulus and the other two (see Table 2). Figure 4 shows that the extraneous cognitive load is higher when the stimulus is present in the environment.

An analysis of variances did not reveal any statistically significant difference for the germane cognitive load (F(2; 60) = 2.417; p = 0.09).

Drawing task

To begin this analysis, we tested the homoscedasticity and normality of our data sample. The homoscedasticity is confirmed for the number of ideas per participant (p = 0.522), intrinsic (p = 0.469), extraneous (p = 0.119) and germane cognitive load (p = 0.412) depending of the distraction.

Analysis of idea production

First, we analysed the impact of the stimulus on the number of ideas per participant. An analysis of variances depending on the condition revealed a statistically significant difference (F(2; 64) = 4.311; p = 0.018). A pairwise comparison using T test revealed differences between the condition continuous stimulus and discontinuous stimulus (see Table 3). Figure 5 shows that the number of ideas per participant is lower when the stimulus is discontinuous in the environment.

Cognitive load analysis

Regarding the cognitive load induced by the stimulus, a Kruskal-Wallis test did not reveal any statistically significant differences for the intrinsic cognitive load (x 2 = 0.513; p = 0.773) and extraneous (x 2 = 1.527; p = 0.466). An analysis of variances for the germane cognitive load depending on the condition did not reveal any statistically significant difference (F(2; 64) = 0.048; p = 0.953).

Discussion

We measured the impact of external stimuli, which compose our work environment, on creativity and cognitive load. We relied on a writing and a drawing task, in which participants had to propose ideas to solve a given problem.

For the drawing task, we observe that a discontinuous stimulus caused a reduction in the number of ideas per participant. It seems that the different stimulus's appearance in the visual field requires an effort. When the stimulus is continuous, a constant effort, but probably lower, must be made to inhibit it. But, when the stimulus is discontinuous, appearance and disappearance demand to participants to reproduce the same effort to ignore it. The reflection necessary for the participants to plan their actions is therefore interrupted each time the stimulus appears. These observations are consistent with those of [START_REF] Kasof | Creativity and Breadth of Attention[END_REF] and [START_REF] Baird | Inspired by Distraction: Mind Wandering Facilitates Creative Incubation[END_REF], but allow to clarify that the interruptive nature of the stimuli seems to be a factor of being or not detrimental to creativity.

For the writing task, the presence of the stimulus increased intrinsic and extraneous cognitive loads. This increase indicates that taking the stimulus into account required extra effort. However, this extra effort did not result in a decrease of the number of ideas. This increase can also indicate that participants tried to use the stimulus as inspiration sources [START_REF] Goldschmidt | Variances in the Impact of Visual Stimuli on Design Problem Solving Performance[END_REF]. The radio broadcast was in the native language and could be easily interpreted by participants. Some of them have made a reference to the Angoulême Festival in their idea.

We can explain these differences by several factors linked to the task and also to the stimulus. Without stimulus, the average number of ideas per participant is 5.52 for the drawing task and 10.86 for the writing task. The idea's realization time is quite different between the two tasks. So, we think the tools have an impact on the time to plan the actions needed to realize a new idea. The participants have to maintain their idea while they sketch it in virtual reality, but they do not have to maintain it in writing activity (or during a very short time) because writing an idea was very quick. Indeed, we believe that the appearance of the stimulus will briefly interrupt the participants' thinking for the planning of their actions, which is more disadvantageous for a tool that requires a longer planning time (i.e. the drawing tool). For the writing task, we think the habit of using text editing tools, especially in disrupted environments (e.g. transportation), allowed participants to remain focused on the creative task. In future research, it would be useful to explore the effects of other types of stimuli, such as music, which is likely to have an additional emotional impact. In addition, the virtual reality idea generation situation makes it possible to test an on-demand inspiration feature. Putting inspirational elements in the environment can potentially be distracting for the user, especially if these elements are animated. However, if users trigger the inspirational elements when they want to, then perhaps they can benefit from the positive effects without being disturbed. This will need to be investigated.

Conclusion

This experiment shows that the different stimuli composing the environment have an impact on cognitive load and also on creativity. The tools, tasks or a simple stimulus (e.g. a bug, or a flicker) have more or less desirable consequences on creativity. For the drawing task, the appearance and disappearance of the stimulus required an additional effort that slowed down participants' creativity. Regarding to the cognitive load for the writing task, we notice that participants have in fact listened to the radio and tried to get inspired by it. We think the understandable nature of a stimulus has an impact on the way participants deal with it. In our experimentation, the stimuli have different levels of interpretation. The stimulus of the drawing task leaves some room for interpretation and is perceived more as a disruptive element of the environment. Quite the opposite, by its intelligible characteristic, the stimulus of the writing task was used to bring new knowledge. However, this input of new knowledge in the environment increased extraneous cognitive load. In order to design efficient tools or environments dedicated to creativity, we need to understand how stimuli bring new knowledge and/or break creativity.

One of the difficulties revealed during the notation of ideas is linked to the perception of ideas realized in an immersive environment. This difficulty has resulted in a lack of understanding of some ideas. Due to the threedimensional environment, the visual perspective of the author's idea is not always found during the evaluation and leads to different interpretations. It is essential to investigate in future works which tools and media could help to communicate the ideas represented in an immersive environment.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Notes on contributors

Figure 1 .

 1 Figure 1. Screenshot of the tool used for the writing task.

Figure 2 .

 2 Figure 2. Screenshot of the 3D drawing tool.

Figure 3 .

 3 Figure 3. Boxplot of intrinsic cognitive load score depending on the condition for the writing task.

Figure 4 .

 4 Figure 4. Boxplot of extraneous cognitive load score depending on the condition for the writing task.

Charles

  Mille is PhD Student at Arts et Metiers Institute of Technology, HESAM Université. His research focuses on the uses of virtual reality to improve creativity. Olivier Christmann is assistant professor at Arts et Metiers Institute of Technology, HESAM Université. His research focuses on human-machine interactions in the use of immersive technologies. Sylvain Fleury is a research engineer at Arts et Metiers Institute of Technology, HESAM Université. His research focuses on the uses of virtual reality for product design, but also on the cognitive processes involved in design activities. Simon Richir is Professor at Arts et Metiers Institute of Technology, HESAM Université and co-editor in chief of IJVR.eu. His research focuses on the uses of virtual and augmented reality for product design, management training and ideation.

Table 1 .

 1 Matrix of the pairwise comparisons for the intrinsic cognitive load of the writing task.

		Continuous	Discontinuous
	Discontinuous Without	0.48 0.006 **	-,0.001 ***
	* p<0.05.

** p<0.01. *** p<0.001.

Table 2 .

 2 Matrix of the pairwise comparisons for the extraneous cognitive load of the writing task. * p<0.01. *** p<0.001.

		Continuous	Discontinuous
	Discontinuous Without	0.215 ,0.001 ***	-,0.001 ***
	* p<0.05.

*

Table 3 .

 3 Matrix of the pairwise comparisons for the number of ideas for the drawing task. p<0.05; ** p<0.01; *** p<0.001.

		Continuous	Discontinuous
	Discontinuous	0.016 *	-
	Without	0.351	0.129

*


Alexie Mayor

Béatrice Thibert

Sylvain Huille

Renaud Respaud

Héloïse Audat

Nathalie Heuzé-Vourc'
email: nathalie.vourch@med.univ-tours.fr

Nathalie Heuzé- Vourc'h

Inhaled antibodies: formulations require specific development to overcome instability due to nebulization

Keywords: (4-6): monoclonal antibody, inhalation, respiratory tract infections, formulation development, meshnebulization Declarations

Introduction

Acute respiratory infections remain a major health issue, as recently highlighted by the SARS-CoV2 pandemic.

Overall, acute respiratory infections are the world's fourth leading cause of death in human of all ages and the first one among children under 5 year-old [START_REF]Why Amnesty opposes the death penalty without exception[END_REF][START_REF] Ritchie | Causes of Death. Our World in Data[END_REF]. Although treatments, such as vaccines and antibiotics have been developed, respiratory infections are not under control, mainly because of increasing occurrence of antibioticresistant bacteria and emerging viral pathogens. For instance, SARS-CoV2 infection, which can lead to severe pneumonia and respiratory distress syndrome, has already caused more than 2,500,000 deaths around the world, as of March 2021 [3]. Facing this major threat of public health, innovative anti-infective approaches are urgently needed. Among them, therapeutic antibodies (Ab) is a growing class of anti-infective agents, with 3 monoclonal Ab marketed and indicated in respiratory infections, and several molecules targeting respiratory pathogensincluding 90 anti-SARS-CoV2 Abs -in development [START_REF] Sécher | Therapeutic antibodies: A new era in the treatment of respiratory diseases?[END_REF][START_REF] Sécher | Inhalation of Immuno-Therapeutics/-Prophylactics to Fight Respiratory Tract Infections: An Appropriate Drug at the Right Place![END_REF][START_REF]COVID-19 Antibody Therapeutics Tracker -Chinese Antibody Society[END_REF]. Anti-infective Ab are mostly full-length IgG, acting directly by neutralizing the pathogens and/or stimulating immune responses. As exemplified by the SARS-CoV2 pandemic, therapeutic Ab, that do not target the pathogens and are already approved in non-communicable diseases, may also be relevant to prevent respiratory infection-mediated uncontrolled inflammation or abnormal coagulation.

Presently, most anti-infective Ab are administered intravenously (IV) [START_REF] Sécher | Therapeutic antibodies: A new era in the treatment of respiratory diseases?[END_REF], but the inhalation route may be more appropriate to improve Ab therapeutic index in respiratory infections, matching the delivery route with the pathogen one, limiting systemic passage and associated risk of systemic toxicity, along with possibly reducing the dose to administer. Inhalation is the mainstay route for drug delivery to treat pulmonary diseases, such as asthma and chronic obstructive pulmonary diseases (COPD) [START_REF]Global Initiative for Asthma -GINA[END_REF][START_REF]Global Initiative for Chronic Obstructive Lung Disease -GOLD[END_REF]. Inhalation consists in delivering a drug as an aerosola suspension of 1 to 5 µm-solid particles or -liquid droplets in a gas-directly into the respiratory tract. Most inhaled drugs are small-molecules, like corticosteroids, beta-sympathomimetics, muscarinic antagonists, and antibiotics.

The inhalation route remains underexploited for biotherapeutics, with only one protein therapeutics approved so far, Pulmozyme® [START_REF] Bodier-Montagutelli | Designing inhaled protein therapeutics for topical lung delivery: what are the next steps?[END_REF]. Despite inhalation of interferon-β (SNG001, Synairgen) recently achieved positive results in phase 2 clinical trial in hospitalized COVID-19 patients (NCT04385095) [10], the inhalation route is often dismissed/neglected for protein therapeutics because of the lack of supportive clinical data demonstrating its benefit and the challenges associated to inhaled protein development [START_REF] Sécher | In a murine model of acute lung infection, airway administration of a therapeutic antibody confers greater protection than parenteral administration[END_REF][START_REF] Guillon | Insights on animal models to investigate inhalation therapy: Relevance for biotherapeutics[END_REF][START_REF] Maillet | The Airways, a Novel Route for Delivering Monoclonal Antibodies to Treat Lung Tumors[END_REF]. Better understanding the behaviour of inhaled Ab during aerosolization and after deposition into the respiratory tract is critical to support development of appropriate and successful anti-infective Ab products.

Pharmaceutical development aims to design a high-quality product and its associated manufacturing process ensuring an efficacious and safe treatment along the life of the product [START_REF] Falconer | Advances in liquid formulations of parenteral therapeutic proteins[END_REF]. The formulation scientists play a critical role to produce Ab, with an adequate formulation to ensure shelf life stability and appropriate quality.

During formulation development, quality by design principles and ICH guidelines are applied, in particular for biotherapeutics ICH Q1A(R2), Q6B and Q8 (R2) [START_REF]Anonymous. ICH Q1A (R2) Stability testing of new drug substances and products[END_REF][START_REF]Anonymous. ICH Q6B Specifications: test procedures and acceptance criteria for biotechnological/biological products[END_REF][START_REF]ICH Q8 (R2) Pharmaceutical development[END_REF]. Usually, parenteral (IV or subcutaneous) Ab formulations are developed for storage at 5 °C ± 3 °C for at least two years and should protect the Ab against stress, such as temperature changes, light exposure or shearing associated to shaking during transport and administration to patients. For inhalation, most protein therapeutics in clinical trial are developed as a liquid aerosol, intended to be delivered by nebulizationtransforming solutions in micron droplets [START_REF] Bodier-Montagutelli | Designing inhaled protein therapeutics for topical lung delivery: what are the next steps?[END_REF]. During nebulization, proteins are subjected to multiple stress, like temperature rise, exposure to interfaces (liquid-solid or liquid-gas), ultrasound and mechanical shearing [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][START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF]. Ultimately, nebulization can result in Ab aggregation, which may be associated with impaired biological activity and/or unexpected immune responses. As shown in recent published studies, mesh-nebulization is suitable to administer efficiently high dose of proteins into the lungs and limit Ab aggregation [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][START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF].

Herein, we hypothesized that liquid formulations developed for intravenous delivery may be appropriate for inhalation delivery, which can facilitate a quick product development to assess the interest of the inhaled route for anti-infectious Ab or repurpose easily relevant IgG molecules during health crisis. In this study, we evaluated the stability of three pharmaceutical Ab formulations during mesh-nebulization in comparison to a vigorous mechanical shaking stress.

Material and Methods

Antibodies

Three Ab were supplied by Sanofi in their pharmaceutical formulations, compatible for intravenous (IV) administration. They are all full-length IgG1. mAb1 was formulated at 20 mg/mL in histidine buffer at pH 6.0, mAb2 was formulated at 10 mg/mL in histidine buffer at pH 5.8 and mAb3 was formulated at 30 mg/mL in phosphate buffer at pH 6.0. The formulation had the following characteristics: mAb1 2Cp (20°C) and 360 mOsm/kg, mAb2 1.3Cp (20°C) and 307 mOsm/kg and mAb3 2.4Cp (20°C) and 306 mOSm/kg. All formulations contained polysorbate 80. Before nebulization, all Ab were filtered to eliminate residual particles (0.22 µm PES syringe filter, Sartorius).

Antibody nebulization and aerosol collection

Two types of vibrating-mesh nebulizers, which are known to limit Ab instability, were used in this study [START_REF] Maillet | Immunological and Pharmacological Properties of the Anticancer Antibody Cetuximab Following Nebulization[END_REF]. The Device 1 was a commercial vibrating-mesh nebulizer and the Device 2 was a customized vibrating-mesh nebulizer.

Between each nebulization, nebulizers were washed in a bath of hot water (45-55°C) with a detergent product compatible for non-invasive medical devices (Surfanios premium) and rinsed with purified water. Then, 2 mL of purified water were nebulized to remove residual particles and detergent. For Ab nebulization, 2 mL of Ab solutions were introduced in the nebulizer reservoir. The aerosols were collected in a 15-mL sterile conical tube (Corning Life Sciences) by condensation. As recently described, the collection efficiency is approximately 60% [START_REF] Bodier-Montagutelli | Protein stability during nebulization: Mind the collection step![END_REF]. Each Ab was nebulized in triplicate on each device. The nebulized and collected Ab was immediately analysed for aggregation.

The performances of the devices were characterized by nebulizing 0.5 mL of NaCl 0.9% or 2.0 mL of Ab formulations and laser diffraction. According to the manufacturer's instructions and our observations, the residual volumes were low (<0.1mL) after nebulization, with any devices and formulations. The concentration of each Ab was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher), at 280 nm, before and after nebulization to evaluate any change during the nebulization process. During each nebulization, the duration was recorded to determine the flow rate. Each experiment was done in triplicate.

Shaking conditions

Ab solutions (2 mL) were introduced in a 15-mL conical tube (Corning Life Sciences) and agitated with a rotator PTR 60 (Grant bio). The shaking cycle corresponds to successive alternated 360° vertical rotations for 15 seconds at 100 rpm and reciprocal rotations for 45 seconds, for a total of 10 minutes. The rotator was placed in an Ecotron (Infors HT) incubator at 37°C during the entire shaking period. Each experiment was done in triplicate.

Distribution of aerosol droplets by laser diffraction

The volume median diameter (VMD) of aerosol droplets was determined by laser diffraction with a Spraytec (Malvern) equipped with a horizontal inhalation cell (Malvern). The Device 1 was used vertically (normal position) and was connected to the inhalation cell with a T piece. The Device 2 was used horizontally (normal position) and was directly connected to the inhalation cell. The aerosol was aspirated into the inhalation cell with vacuum pump at a flow rate of 15 L/min. The results were expressed as VMD and percentage of droplets with a diameter between 1 and 5 µm.

Visual inspection of visible particles

The presence or absence of particles larger than 500 µm was assessed by visual inspection. The samples were placed in a glass vial and illuminated with an MLC-150 cold light source (Motic) on a black background.

Subvisible particles measured by flow-cell microscopy (FCM)

Flow-cell microscopy (FCM) was used to detect particles in the range of 1 µm to 100 µm. All Ab solutions were analyzed before and after nebulization with a particle counting imager Flowcell FC200-IPAC (Occhio) instrument.

A volume of 200 µL of each sample was passed through an analysis flow cell and particles were counted and analyzed with the Callisto® software (Occhio). The results were reported as the number of particles per milliter (particles/mL) and expressed as the concentration of all particles (all particles/mL), particles larger than 2µm (> 2µm/mL), particles larger than 10µm (> 10 µm/mL) and particles larger than 25µm (> 25µm/mL).

Submicron particles measured by dynamic light scattering (DLS)

Dynamic light scattering (DLS) was used to analyze particles from 50 nm to 2 µm. The measurements were carried out at 25°C, with a DynaPro NanoStar (Wyatt Technology) instrument using a 659 nm laser wavelength and 90° detection angle. Each sample was introduced in a plastic disposable cuvette (Uvette, Eppendorf) and carried out 10 acquisitions of 7 seconds. The data were analyzed with Dynamics 7.1.9 software (Wyatt Technology). Samples with more than 30% of the acquisitions rejected were considered non-exploitable, as recommended by the manufacturer. The acceptance criteria were an autocorrelation curve baseline limited at 1 ± 0.01 and a maximum SOS (Sum-Of-Squares error from the correlation function fit) of 100. The results were displayed as the correlogram curves, the monomer radius (nm), percentage of polydispersity of monomer population (%pd), percentage in intensity (% intensity) and in mass (% mass) of the monomer, which resulted from the regularization analysis. The polydispersity index (PDI) resulting from Cumulant analysis and corresponding to the distribution width divided by the mean, was also reported.

Oligomers measured by size-exclusion chromatography (SEC)

Size exclusion chromatography (SEC) was used to analyze oligomers. The measurements were performed on a series 200 (Perkin Elmer) high performance liquid chromatography (HPLC) system. The samples were filtered through a PES 0.22 μm syringe filter into glass vials with reducers. Samples were maintained at 4°C in the storage system before injection. For each sample, 50 μg of Ab was injected by an autosampler on an Advanced BioSEC 300 Å, 2.7 μm, 7.8 x 300 mm (Agilent) column. The elution phase was PBS 1X pH 7.2 with 0.03% (w/v) of NaN3 perfused at a constant flow rate of 1mL/min at a temperature of 25°C. UV detection was performed at 280 nm with diode array detector. The data were recorded and processed with TotalChrom software (Perkin Elmer). Results were expressed as the percentage of high molecular weights species (% HMW).

Biological activity of mAb1

The measurement of activity is based on the measurement of complement-dependent cytotoxicity (CDC) in the presence of mAb1. mAb1 before and after stress (=samples) and mAb1 reference were diluted at different concentrations and incubated with the antigen carrier cells in the wells of a 96-well plate before adding human complement. Living cells remaining in the wells were visualized with a tretrazolium salt and quantified with a plate reader at 450 nm. The absorbance measured by well is inversely proportional to mAb1 cytotoxicity, which allows to determine a dose-response curve for the samples and the reference. The results are presented as a percentage of CDC activity calculated as a ratio of EC50 value of mAb1 samples and mAb1 reference.

Results

Characterization of aerosols when nebulizing Ab formulations for IV

Pulmonary delivery of Ab can be achieved using several types of devices and, in theory, liquid IV Ab formulations may be directly aerosolized using nebulizers. Herein, we used two vibrating mesh-nebulizers, as this type of devices has been shown to maintain better the stability of IgG during nebulization and can achieve large dose delivery with a high pulmonary deposition [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][START_REF] Maillet | Immunological and Pharmacological Properties of the Anticancer Antibody Cetuximab Following Nebulization[END_REF]. The performances of the devices with saline solutions and Ab formulations were assessed by measuring the VMD of the aerosols and the flow rates. As reported in Table 1, the flow rates of the two devices with the saline solution were less than 0.5 mL/min and the Device 2 was slightly faster. The flow rates for the two devices decreased in the presence of Ab formulations and the drop was more important with Device 1. The reduction depends on the formulations, with the lowest flow rate observed with mAb3 (30 mg/mL, 2.4Cp (20°C)) essentially with device 1.

The VMD of Device 1 and Device 2 with saline solutions were 4.1 µm and 3.5 µm and correspond to a respiratory fraction (particles between 1-5 µm) of 64.4 and 71%, respectively. The VMD was not affected by the nebulization of Ab formulations, except for mAb3 for which the VMD was notably smaller with Device 1, thereby increasing the fine particle fraction (Table 1).

Characterization of Ab aggregation when nebulizing pharmaceutical Ab formulations

As previously reported, proteins, including IgG, are highly sensitive to nebulization stress, and the main marker of protein instability during nebulization is aggregation [START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF][START_REF] Depreter | Inhaled proteins: Challenges and perspectives[END_REF]. The extent of aggregation depends on the protein nature, the nebulizer and the formulation. Here, we investigated the ability of liquid pharmaceutical formulations compatible for IV administration, which were developed to prevent Ab instability during the product lifespan and to certain environmental stress, to protect Ab from degradation during mesh-nebulization. A vigorous shaking method was used as a control. Aggregation was monitored using orthogonal methods allowing to cover the large range of aggregate populations that can be generated. Not surprisingly, aggregation was dependent on Ab properties and the applied stress. All Ab tolerated the vigorous shaking relatively well. No major generation of visible, subvisible (Figure 1) or submicron (Table 2) aggregates and oligomers (Table 3) has been detected. After nebulization, no visible particle was observed in the samples for the 3 Abs. However, small subvisible particles (<10 µm) were observed by FCM (Figure 1 and Table 2) in samples after nebulization, mostly with Device 2. Although the number of particles was relatively heterogeneous between nebulization runs, device 2 induced a higher amount of aggregates than device 1 (Figure 1 and Table 2).

The DLS profiles (Figure 2) displayed slight changes after nebulization for mAb1 and mAb2 as evidenced by the autocorrelation curves. Additionally, except for mAb3 with Device 1, PDI was increased after nebulization (see Table 2) for the 3 Ab, indicating the presence of submicron aggregates. This hypothesis is further supported by the decrease in intensity of the monomer content. However, the lack of decrease of the monomer content in mass suggest that the submicron particles generated during mesh-nebulization represented a very small quantity of particles. SEC results did not highlight important formation of oligomers, except for the nebulized mAb 3, which had a tendency to produce HMW species with Device 2. mAb 1 was one of the most sensitive IgG to nebulization as exemplified by the amount of subvisible particles, %Pd and PDI index observed after nebulization. Because the instability of protein during nebulization may result in some cases in a loss of activity, we analyzed some features of mAb1 activity after nebulization. As shown in Supplemental Table S2, the complement-dependent cytotoxicity of mAb1 was not markedly affected after nebulization.

Discussion

Therapeutic Ab, which mainly comprise monoclonal IgG, are one of the most important class of therapeutics and are gaining importance in infectious diseases [START_REF] Sécher | Therapeutic antibodies: A new era in the treatment of respiratory diseases?[END_REF]. Neutralizing the pathogens, inducing anti-infective immune responses and/or preventing excessive inflammation, Ab offer new opportunities for the prevention and treatment of respiratory infections, which still represent unmet medical needs. Several preclinical studies demonstrated the benefit to deliver anti-infective Ab topically to the lungs in viral and bacterial models of respiratory infections [START_REF] Sécher | In a murine model of acute lung infection, airway administration of a therapeutic antibody confers greater protection than parenteral administration[END_REF][START_REF] Mora | Delivery of ALX-0171 by inhalation greatly reduces respiratory syncytial virus disease in newborn lambs[END_REF][START_REF] Leyva-Grado | Direct Administration in the Respiratory Tract Improves Efficacy of Broadly Neutralizing Anti-Influenza Virus Monoclonal Antibodies[END_REF] . But the advantage of the inhalation route has not materialized yet in the clinic, thereby emphasizing the challenges to overcome during inhaled Ab development [START_REF] Sécher | Inhalation of Immuno-Therapeutics/-Prophylactics to Fight Respiratory Tract Infections: An Appropriate Drug at the Right Place![END_REF]. Among them, the instability of Ab during aerosolizationleading to Ab aggregation-raises both pharmacological and safety issues. The characterization of the best formulation, to ensure the stability of the Ab along its lifespan as a pharmaceutical product, is also a challenge that is faced during intravenous Ab development [START_REF] Brovč | Rational design to biologics development: The polysorbates point of view[END_REF][START_REF] Basle | Physicochemical Stability of Monoclonal Antibodies: A Review[END_REF]. As a result, formulation scientists select the formulations and explore the Ab stability to various stresses, some of them encountered during aerosolization.

Herein, we evaluated the potential use of pharmaceutical formulations, intended for IV delivery, for expanded applications for inhalation.

Parenteral protein formulations, including Ab formulations, are being increasingly developed as liquid dosage forms to make them ready to use and to ease use in clinics [START_REF] Falconer | Advances in liquid formulations of parenteral therapeutic proteins[END_REF][START_REF] Gervasi | Parenteral protein formulations: An overview of approved products within the European Union[END_REF]. Thus, numerous IV Ab formulations can be potentially tested in nebulizing systems for inhalation. Previous studies showed that mesh-nebulization was less deleterious on IgG as compared to jet-or ultrasonic-nebulization, most probably because mesh-nebulizers usually display low change in temperature over the nebulization period and no recycling [START_REF] Hertel | Protein stability in pulmonary drug delivery via nebulization[END_REF][START_REF] Maillet | Immunological and Pharmacological Properties of the Anticancer Antibody Cetuximab Following Nebulization[END_REF][START_REF] Carvalho | The function and performance of aqueous aerosol devices for inhalation therapy[END_REF]. However, drug formulations affect mesh-nebulizer performances [START_REF] Carvalho | The function and performance of aqueous aerosol devices for inhalation therapy[END_REF][START_REF] Ghazanfari | The influence of fluid physicochemical properties on vibrating-mesh nebulization[END_REF]. In particular, ion concentration and increased viscosity were associated with a decrease in droplet size and moderately viscous (>5cP) solutions were not suitable for mesh-nebulization. Herein, the viscosity of Ab formulations was sufficiently low (<5 cP) to enable mesh-nebulization and overall aerosolization was mostly unaffected by formulation characteristics for the two devices.

To a lesser extent, mesh-nebulizer performances may also be influenced by surface tension, which depends on formulation, in particular addition of surfactants [START_REF] Ghazanfari | The influence of fluid physicochemical properties on vibrating-mesh nebulization[END_REF]. For each device, the proportion of respirable droplets were in the same range, which is in agreement with aerodynamic diameter being similar to the ones obtained with saline solutions. Despite a decrease in flow rate, nebulization times remained within an acceptable time range for administration to spontaneously-breathing patients (<15 min for 2mL solution), with the exception of mAb3 with the device 1. Overall, this means that the 3 IV formulations were compatible with aerosol deposition into the respiratory tract.

Nebulization-mediated aggregation is a serious issue to consider for inhaled Ab. Indeed, aggregation can result in a loss of Ab activity, as the tertiary structure may be impaired, and can elicit, in vivo, antidrug-antibody (ADA) production that may neutralize Ab and lead to side effects, such as hypersensitivity responses and anaphylactic reaction. Accordingly, the presence and levels of Ab aggregates along upstream and downstream processes is highly documented for each product. Aggregation covers a broad range of sizes and the European (Eur. Ph. 703) and United States (USP788) pharmacopeias require to evaluate visible particles and subvisible particles over 10 and 25 µm for drug product release [START_REF] Zölls | Particles in Therapeutic Protein Formulations, Part 1: Overview of Analytical Methods[END_REF]. In addition, the regulatory agencies recommend to monitor smaller sized aggregates, including subvisible particles (2-10 µm) and submicronic particles (0.1-2 µm), for full protein product characterization as they may pose a clinical risk [START_REF]FDA Guidance for Industry -Immunogenicity Assessment for Therapeutic Protein Products[END_REF]. In this study, pharmaceutical Ab formulations subjected to mesh-nebulization resulted in a slight to moderate increase of subvisible particles, mostly those <10 µm. The aggregation profile, as characterized by orthogonal methods, was dependent on the mAb and the mesh-nebulizer.

The difference of stability observed with the two mesh-nebulizers may be attributable to heating, shear and mechanical stress inherent to each device or the aerosol size, increasing the air-liquid interface [START_REF] Hertel | Protein stability in pulmonary drug delivery via nebulization[END_REF][START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF]. Although IV Ab development takes into account Ab stability to environment stress, such as temperature rise and shearing, pharmaceutical formulations did not seem appropriate for inhalation. Combination of shear stress and local thermal stress appears unique for nebulizers and is likely not enough accurately mimicked by the shaking and thermal stresses as applied for IV formulation development. Our findings are in agreement with those obtained for other parenteral protein formulations [START_REF] Hertel | Prediction of protein degradation during vibrating mesh nebulization via a high throughput screening method[END_REF].

Aggregation may be associated to a loss of activity. Thus, one may question the impact of the aggregates produced during mesh-nebulization on Ab activity. As for mAb1, its biological activity was unaffected by mesh-nebulization (see supplemental Table S1). However, it would be difficult to extrapolate this finding to other Ab, since nebulization-mediated aggregation was inconsistently associated with altered Ab activity [START_REF] Bodier-Montagutelli | Protein stability during nebulization: Mind the collection step![END_REF]. Aggregation is also associated to Ab-related immunogenicity and immunogenicity depends for a part on the route of administration.

To the best of our knowledge, the impact of aggregates generated during mesh-nebulization and after inhalation on immune responses has not been investigated yet. But, small-sized subvisible particles (2-10 µm), which correspond to the aggregates mostly produced during mesh-nebulization, have been shown to enhance immune response and are expected to be the most immunogenic [START_REF] Carpenter | Overlooking Subvisible Particles in Therapeutic Protein Products: Gaps That May Compromise Product Quality[END_REF][START_REF] Xiang | Pathogen recognition and development of particulate vaccines: does size matter?[END_REF].

It is noteworthy that a vigorous mechanical shaking at elevated temperature was unable to reproduce the combined shear thermal stresses applied in mesh-nebulizer. Overall, the 3 Abs displayed remarkable stability towards strong mechanical shaking. Our results do not match those of [START_REF] Hertel | Prediction of protein degradation during vibrating mesh nebulization via a high throughput screening method[END_REF], who defined a shaking method as a surrogate of nebulization stress for protein therapeutics, using one protein nebulized with one device as a study model [START_REF] Hertel | Prediction of protein degradation during vibrating mesh nebulization via a high throughput screening method[END_REF]. In addition to the slight differences between the methods of "shaking at elevated temperature", the discrepancy may be attributable to the protein nature and the device. As for us, vigorous shaking at elevated temperature may mimic in some but not all cases, mesh-nebulization. This may be explained by the difficulty to reproduce some nebulization stress by shaking, in particular the huge air-liquid interface generated by pumping the liquid through the mesh and, by definition, in the aerosol droplets. Moreover, local and transient temperature rise in the reservoir of the nebulizer may contribute to additional aggregation. From a formulation scientist's perspective, it means that the vigorous shaking applied in the present study is not suitable to accelerate inhaled Ab development and the study of the stress induced by the device intended for human use has to be performed early in the development process.

Parenteral Ab formulations and particularly intravenous injectable dosage forms require pH and osmolality characteristics basically compatible for inhalation as the lungs tolerate inhaled drug products with osmolality ranging 150-549 mOsm/kg -even if isotonicity has been recommended-and pH ranging 3.5 to 8.0 [START_REF] Beasley | Adverse reactions to the non-drug constituents of nebuliser solutions[END_REF]. Beyond the pH and osmolality, the excipients (nature and dose) in the IV Ab formulations may not be adapted to inhalation and if not used in inhaled drug products they would require toxicity investigations by the inhalation route.

Overall, our findings indicate that Ab formulations developed for IV delivery may not easily be repurposed for inhalation delivery and point to the requirement of a specific formulation development for inhaled Ab. Formulation scientists may select carefully the dose and excipients to be added in the formulation to stabilize Ab during meshnebulization, taking into account the paucity of toxicity data on inhaled excipients and their potential impact on formulation properties, and thereby device performances.

Acknowledgements

This work was supported by the French National Research Agency as part of the "Investissements d'Avenir" program (LabEx MAbImprove, ANR-10-LABX-53-01) and SANOFI. AM was funded by a CIFRE thesis partnership between the CEPR-Inserm U1100 and Sanofi.

We would like to thank Ronan Crepin (Sanofi) for his technical assistance.

This work was supported by the French National Research Agency as part of the "Investissements d'Avenir" program (LabEx MAbImprove, ANR-10-LABX-53-01) and SANOFI. AM was funded by a CIFRE thesis partnership between the CEPR-Inserm U1100 and Sanofi.

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Conflict of interests/competing interests

NHV is co-founder and scientific expert for Cynbiose Respiratory. In the past 3 years, she received consultancy fees from Argenx, Eli Lilly and research support from Signia Therapeutics, Sanofi and Aerogen Ltd.

-Ethics approval: not applicable -

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.