Romain Sonneville
email: romain.sonneville@aphp.fr

Etienne De Montmollin

Damien Contou

Ricard Ferrer

Mohan Gurjar

Kada Klouche

Benjamine Sarton

Sophie Demeret

Pierre Bailly

Daniel Da Silva

Etienne Escudier

Loic Le Guennec

Russel Chabanne

Laurent Argaud

Omar Ben

Hadj Salem

Aurelien Frerou

Guillaume Louis

Gennaro De Pascale

Janneke Horn

Raimund Helbok

Guillaume Geri

Fabrice Bruneel

Ignacio Martin-Loeches

Fabio Silvio Taccone

Jan J De Waele

Stephane Ruckly

Quentin Staiquly

Giuseppe Citerio

Jean- Francois Timsit

Clinical features, etiologies, and outcomes in adult patients with meningoencephalitis requiring intensive care (EURECA): an international prospective multicenter cohort study

Keywords: Meningoencephalitis, Encephalitis, Meningitis, Intensive care unit, Outcome

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

Meningoencephalitis is an inflammatory process involving the brain and meninges, most often produced by pathogens which invade the central nervous system, and occasionally by autoimmune disorders, toxins, and other conditions [START_REF] Venkatesan | Acute encephalitis in immunocompetent adults[END_REF]. Traditionally, patients with meningoencephalitis develop an acute-onset encephalopathy with a combination of fever, focal signs and/or new-onset seizures, together with cerebrospinal fluid (CSF) pleocytosis, brain imaging features of inflammation, and/or electroencephalography (EEG) alterations [START_REF] Venkatesan | Case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium[END_REF]. Epidemiological studies conducted in adult patients suggest that approximately one in two will require care in an intensive care unit (ICU) [START_REF] Glaser | Beyond viruses: clinical profiles and etiologies associated with encephalitis[END_REF][START_REF] Mailles | Infectious encephalitis in France in 2007: a national prospective study[END_REF][START_REF] Mailles | Changing profile of encephalitis: results of a 4year study in France[END_REF]. In those patients requiring ICU admission, meningoencephalitis is associated with a poor prognosis, including refractory seizures, prolonged hospital stay, neurological disability, and death [START_REF] Thakur | Predictors of outcome in acute encephalitis[END_REF][START_REF] Singh | The spectrum of acute encephalitis: causes, management, and predictors of outcome[END_REF][START_REF] Sonneville | Clinical spectrum and outcomes of patients with encephalitis requiring intensive care[END_REF][START_REF] Sonneville | Early-onset status epilepticus in patients with acute encephalitis[END_REF].

Herpes simplex encephalitis (HSE) remains the most common viral cause of sporadic meningoencephalitis in immunocompetent adults, and is associated with a poor functional outcome [START_REF] Mailles | Changing profile of encephalitis: results of a 4year study in France[END_REF][START_REF] Granerod | Causes of encephalitis and differences in their clinical presentations in England: a multicentre, population-based prospective study[END_REF][START_REF] Jaquet | Functional outcomes in adult patients with herpes simplex encephalitis admitted to the ICU: a multicenter cohort study[END_REF][START_REF] Singh | Predictors of outcome in HSV encephalitis[END_REF]. Patients with acute bacterial meningitis may also develop encephalitis symptoms, including coma, seizures, and/or focal signs which are, respectively, reported in 13-50%, 7-14%, and 14-22% of adult patients in multicenter cohort studies [START_REF] Auburtin | Detrimental role of delayed antibiotic administration and penicillin-nonsusceptible strains in adult intensive care unit patients with pneumococcal meningitis: the PNEUMOREA prospective multicenter study[END_REF][14][START_REF] Mcgill | Incidence, aetiology, and sequelae of viral meningitis in UK adults: a multicentre prospective observational cohort study[END_REF].

The etiological profile of meningoencephalitis has changed considerably in recent years, with the emergence of a wide variety of autoimmune causes (i.e., acute disseminated encephalomyelitis (ADEM), and anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis) that may require ICU care and urgent immunomodulatory therapies [START_REF] Sonneville | Acute disseminated encephalomyelitis in the intensive care unit: clinical features and outcome of 20 adults[END_REF][START_REF] De Montmollin | Anti-N-methyl-d-aspartate receptor encephalitis in adult patients requiring intensive care[END_REF][START_REF] Sonneville | Understanding autoimmune encephalitis in the ICU[END_REF]. The development of new diagnostic tools, such as multiplex polymerase chain reaction (PCR) CSF analysis and metagenomics now permits a better identification of uncommon infectious causes of meningoencephalitis [START_REF] Wilson | Clinical metagenomic sequencing for diagnosis of meningitis and encephalitis[END_REF][START_REF] Trujillo-Gomez | Biofire FilmArray Meningitis/Encephalitis panel for the aetiological diagnosis of central nervous system infections: a systematic review and diagnostic test accuracy meta-analysis[END_REF]. However, the proportion of cases with meningoencephalitis of unknown origin remains high, ranging from 30 to 48% [START_REF] Thakur | Predictors of outcome in acute encephalitis[END_REF][START_REF] Singh | The spectrum of acute encephalitis: causes, management, and predictors of outcome[END_REF][START_REF] Sonneville | Clinical spectrum and outcomes of patients with encephalitis requiring intensive care[END_REF].

Available data on the epidemiology and outcomes of ICU patients with all-cause acute meningoencephalitis are mainly based on single-center retrospective cohorts [START_REF] Thakur | Predictors of outcome in acute encephalitis[END_REF][START_REF] Singh | The spectrum of acute encephalitis: causes, management, and predictors of outcome[END_REF][START_REF] Sonneville | Clinical spectrum and outcomes of patients with encephalitis requiring intensive care[END_REF], whereas multicenter studies have been conducted in patients with specific infectious or autoimmune etiologies, such as HSE and anti-NMDAR meningoencephalitis [START_REF] Jaquet | Functional outcomes in adult patients with herpes simplex encephalitis admitted to the ICU: a multicenter cohort study[END_REF][START_REF] De Montmollin | Anti-N-methyl-d-aspartate receptor encephalitis in adult patients requiring intensive care[END_REF]. A previous prospective multicenter study conducted in patients with severe pneumococcal meningitis requiring ICU care reported high mortality rates and disability at 3 months in one-third of survivors [START_REF] Auburtin | Detrimental role of delayed antibiotic administration and penicillin-nonsusceptible strains in adult intensive care unit patients with pneumococcal meningitis: the PNEUMOREA prospective multicenter study[END_REF].

In the present study, we aimed to characterize the clinical presentation, etiologies, and outcomes in adult patients with severe meningoencephalitis requiring care in the ICU. Specifically, we aimed to identify baseline factors independently associated with an increased risk of poor functional outcome.

Methods

Study design and population

The EURopean study on Encephalitis in intensive Care (EURECA) is a prospective international multicenter cohort study endorsed by the European Society of Intensive Care Medicine (ESICM). The study is registered in Clinical Trials (NCT03144570). The entire protocol has been reviewed and approved by the Comité de protection des personnes sud méditerranée 2 (ID RCB 2017-A00139-44). National/local approvals at the international study sites were obtained by the national coordinators and local investigators according to the local regulations. The study is reported according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement guidelines. Participating centers were asked to recruit consecutive patients for a duration of at least 6 months. Eligible patients were adults admitted to the ICU with symptoms suggestive of meningoencephalitis, defined by an acute onset of encephalopathy (a score on the Glasgow coma scale (GCS) </= 13), a CSF pleocytosis >/= 5 cells/mm 3 , and at least two of the following criteria: (a) fever (≥ 38°C within 72 h before or after admission), (b) generalized or partial seizures non-attributable to pre-existing epilepsy, (c) new-onset focal neurological deficit, (d) parenchymal abnormalities on neuroimaging compatible with encephalitis, and (e) EEG alterations compatible with encephalitis. Therefore, all eligible patients met the Infectious Diseases Society of America 2013 diagnostic criteria for "probable" or "confirmed" encephalitis at inclusion [START_REF] Venkatesan | Case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium[END_REF]. Patients with missing core data and those lost-to-follow-up at 3 months were excluded.

Collected data

Epidemiological data were entered prospectively into a password-protected and secured webbased electronic case record form (CRF), including age, gender, premorbid score on the modified Rankin Scale (mRS), ethnicity, chronic immunodepression (i.e., HIV infection, solid organ transplantation recipient, hematology/cancer patient, and long-term use of steroids and/or immunosuppressant drugs), pre-existing neurologic disease and other comorbidities. Data collected within the first 24 h following ICU admission included the main reason for ICU admission, each component of the GCS [START_REF] Teasdale | Assessment of coma and impaired consciousness. A practical scale[END_REF], the Simplified Acute Physiology Score (SAPS) II [START_REF] Gall | A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study[END_REF], and of the sequential organ failure assessment (SOFA) score [START_REF] Vincent | The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine[END_REF]. Coma was defined by a score on the GCS < 8 [START_REF] Koelman | Pneumococcal meningitis in adults: a prospective nationwide cohort study over a 20-year period[END_REF], and fever was defined by a body temperature >/=38°C [START_REF] Venkatesan | Case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium[END_REF]. Acute respiratory failure and cardiovascular failure were defined as a respiratory and cardiovascular component of the SOFA score > 2, respectively [START_REF] Vincent | The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine[END_REF].

Meningoencephalitis etiology

Routine investigations (i.e., CSF and serum analyses, imaging, EEG, oriented samples in case of non-neurological manifestations) were suggested to investigators in patients without an obvious etiological diagnosis, in accordance with most recent Infectious Diseases Society of America encephalitis guidelines [START_REF] Venkatesan | Case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium[END_REF]. Additional investigations were suggested in other circumstances, such as immunocompromised status, specific clinical presentation (i.e., psychiatric signs/abnormal movements), or depending on topography of brain lesions on neuroimaging. Etiologies of meningoencephalitis were categorized into 5 groups: (1) acute bacterial meningitis; (2) infectious encephalitis of viral, bacterial, or fungal/parasitic origin;

(3) autoimmune encephalitis; (4) neoplastic/toxic encephalitis; (5) encephalitis of unknown origin. Definitions of these groups are provided in the Appendix (ESM Table 1).

Patients included in the study were treated according to local guidelines, depending on the identified etiology. Dose and duration of antimicrobials (i.e., intravenous acyclovir and antibiotics) and/or immunomodulating agents (i.e., intravenous steroids, intravenous immunoglobulins, and plasma exchange) were left to the discretion of local investigators.

Endpoints

Local investigators prospectively entered all study endpoints into the electronic CRF. The primary endpoint was poor functional outcome 3 months after ICU admission, defined by a score on the modified Rankin scale (mRS) of 3 to 6, indicating moderate-to-severe disability or death. Causes of death were divided into two categories, i.e., systemic causes (cardiovascular failure, multiple organ failure) or neurological causes (withdrawal of care because of diffuse neurological injury or brain death). Secondary endpoints included need for organ support (i.e., mechanical ventilation, tracheostomy, vasopressors, renal replacement therapy), neurosurgery (i.e., intracranial pressure monitoring, external ventricular drainage, brain biopsy), development of systemic complications (i.e., septic shock, nosocomial pneumonia, pulmonary embolism, overt gastrointestinal bleeding), neurological complications (i.e., status epilepticus, empyema/cerebral abscess, cerebral ischemia, intracranial bleeding, and brain death), and hospital outcomes (i.e., ICU and hospital lengths of stay).

Statistical analysis

Data management and statistical analysis were performed by independent statisticians (QS, SR, ICUREsearch). Data are presented as numbers (interquartile range) or numbers (percentage), as appropriate. Multivariable logistic regression analysis stratified on centers explored the association between baseline characteristics and the primary endpoint. Clinically relevant variables associated with the primary endpoint (p < 0.2) in univariate analysis were entered into the multivariable analysis. Two-by-two interactions and collinearity between selected variables were tested. Missing values were imputed to the median or mode for quantitative and qualitative variables, respectively. Statistics were performed using SAS 9.4 software (SAS Institute Inc.). A p value < 0.05 was considered significant. Based on a syndromic approach, sensitivity analyses were performed to investigate two a priori defined subgroups of patients, namely patients diagnosed with bacterial meningitis, and patients with primary encephalitis of any cause, after adjustment for main etiologies (e.g., infectious, autoimmune, and unknown origin).

Role of the funding source

The European Society of Intensive Care Medicine (ESICM) played no role in study design, data collection, data analysis, data interpretation, or in the writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Results

Study population

Between June 21, 2017, and April 6, 2020, we identified 599 eligible patients from 68 centers across 7 countries, with a median number of patients per center of 7 (IQR 5-11). Among them, 589 (98%) patients were included, and all completed the 3-month follow-up. The study flowchart is presented in the Appendix (ESM Fig. 1). Baseline characteristics of included patients are shown in Table 1. The median age of the study population was 66 years (IQR 44-71) and 346 (58.7%) were male. The most frequent comorbidities were diabetes (n = 111, 18.8%) and immunodepression (n = 137, 23.3%). The time from hospital admission to ICU transfer was 1 (IQR 1-3) day. At ICU admission, the SAPS II score was 42 (IQR 30-57), and 202 (34.3%) patients were comatose. Respiratory and cardiovascular failures were observed in 97 (16.5%) and 136 (23.1%) cases, respectively. Meningoencephalitis characteristics at ICU admission are shown in Table 2. Patients had a severe clinical presentation with a median score on the GCS of 9 (IQR 6-11), convulsive seizures (n = 217, 36.8%), and hemiparesis/ hemiplegia (n = 91, 15.4%). Altered verbal and motor responses on the GCS, and hemiparesis/hemiplegia were associated with poor outcome. The body temperature was 38.4°C (IQR 37.5-39.1), with fever present in 379 (64.5%) patients. A lumbar puncture was performed in all patients, at a median time from ICU admission of one (IQR 1-1) day. Neuroimaging was also performed in all patients, including CT scans in 495 (84%) cases and MRI in 365 (60.4%) cases. The time from ICU admission to the first CT scan and MRI were one (IQR 1-1) and one (IQR 0-7) days, respectively. A normal CT scan and MRI were observed in 317/495 (64%) and 94/365 (25.7%) cases, respectively, and associated with better outcomes in univariate analysis. EEG was performed in 404 (68.7%) cases, with a median time from ICU admission of two (IQR 1-2) days.

The 591 etiologies of meningoencephalitis diagnosed in the 589 included patients are presented in Table 3. The detailed causes of bacterial meningitis and infectious encephalitis are presented in the Appendix (ESM Tables 2,3). The bacterial meningitis category mainly consisted of community-acquired infections (n = 220/247, 89%). Intravenous acyclovir and third-generation cephalosporins were started at admission in 284/588 (48.3%) patients, and 324/588 (55.1%) patients, respectively. Acyclovir, and thirdgeneration cephalosporin prescriptions differed significantly between etiological groups (p < 0.01), being more frequently prescribed in infectious encephalitis and bacterial meningitis categories, respectively (Appendix, ESM Table 4).

Adjunctive steroids were given at admission in 202 (34.3%) patients (dexamethasone: n = 189; other steroids: n = 13). Specifically, 167/247 (67.6%) bacterial meningitis patients received adjunctive steroids (dexamethasone: n = 165; other steroids: n = 2). Intravenous methylprednisolone pulses were given during ICU stay in 90 (15.3%) patients, including 33/38 (86.8%) cases of autoimmune encephalitis, 26/247 (10.5%) cases of bacterial meningitis, 9/138 (6.5%) cases of infectious encephalitis, and 19/155 (12.3%) cases of encephalitis of unknown origin, and 3/11 (27.2%) cases of toxic/neoplastic encephalitis.

Study endpoints

Overall, 298 patients (50.5%, 95% CI 46.6-54.6%) had poor functional outcome at 3 months, including 152 deaths (25.8%). Causes of death included neurologic (102/152 (67.1%) deaths, including n = 29/152 (19.1%) brain deaths) and systemic causes (50/152 (32.8%) deaths). Decisions to withdraw care during ICU stay were reported in 103/152 (67.8%) deaths and were more frequent in neurologic causes of death than in systemic causes of death (71/102 (69.6%) deaths versus 15/50 (30%) deaths, p < 0.01). The distribution of mRS scores at 3 months in the entire cohort and in the different etiologic groups are presented in Fig. 1 (panel A). Patients with an autoimmune cause of encephalitis had the worst functional outcomes. The distribution of mRS score at 3 months in patients with bacterial meningitis and infectious encephalitis are presented in Fig. 1, panels B and C, respectively. Among patients with infectious encephalitis, those with fungal/parasitic causes had the worst outcomes.

Secondary endpoints are reported in Appendix (ESM Table 5). Overall, 426 (72.3%) required invasive mechanical ventilation during ICU stay, for a duration of eight (4-17) days, and 61 (10.4%) underwent tracheostomy. ICU and hospital lengths of stay were nine [START_REF] Mailles | Changing profile of encephalitis: results of a 4year study in France[END_REF][START_REF] Thakur | Predictors of outcome in acute encephalitis[END_REF][START_REF] Singh | The spectrum of acute encephalitis: causes, management, and predictors of outcome[END_REF][START_REF] Sonneville | Clinical spectrum and outcomes of patients with encephalitis requiring intensive care[END_REF][START_REF] Sonneville | Early-onset status epilepticus in patients with acute encephalitis[END_REF][START_REF] Granerod | Causes of encephalitis and differences in their clinical presentations in England: a multicentre, population-based prospective study[END_REF][START_REF] Jaquet | Functional outcomes in adult patients with herpes simplex encephalitis admitted to the ICU: a multicenter cohort study[END_REF][START_REF] Singh | Predictors of outcome in HSV encephalitis[END_REF][START_REF] Auburtin | Detrimental role of delayed antibiotic administration and penicillin-nonsusceptible strains in adult intensive care unit patients with pneumococcal meningitis: the PNEUMOREA prospective multicenter study[END_REF](14)[START_REF] Mcgill | Incidence, aetiology, and sequelae of viral meningitis in UK adults: a multicentre prospective observational cohort study[END_REF][START_REF] Sonneville | Acute disseminated encephalomyelitis in the intensive care unit: clinical features and outcome of 20 adults[END_REF][START_REF] De Montmollin | Anti-N-methyl-d-aspartate receptor encephalitis in adult patients requiring intensive care[END_REF][START_REF] Sonneville | Understanding autoimmune encephalitis in the ICU[END_REF][START_REF] Wilson | Clinical metagenomic sequencing for diagnosis of meningitis and encephalitis[END_REF] and 23 (14-46) days, respectively.

Variables independently associated with the primary endpoint were age > 60 years (OR 1.75, 95% CI 1.22-2.51), immunodepression (OR 1.98, 95% CI 1.27-3.08), time between hospital and ICU admission > 1 day (OR 2.02, 95% CI 1.44-2.99), a motor component on the GCS</=3 (OR 2.23, 95% CI 1.49-3.45), hemiparesis/hemiplegia (OR 2.48, 95% CI 1.47-4.18), respiratory failure (OR 1.76, 95% CI 1.05-2.94), and cardiovascular failure (OR 1.72, 95% CI 1.07-2.75). In contrast, administration of a third-generation cephalosporin (OR 0.54, 95% CI 0.37-0.78) and acyclovir (OR 0.55, 95% CI 0.38-0.80) on ICU admission were protective (Fig. 2,panel A).

Predefined subgroup analyses are presented in Fig. 2 (panel B andC). Among acute bacterial meningitis patients, a motor response on the GCS </= 3 (OR 2.56, 95% CI 1.32-4.97), hemiparesis/hemiplegia (OR 3.53, 95% CI 1.43-8.70), and third-generation cephalosporin on day one of ICU admission (OR 0.39, 95% CI 0.19-0.79) remained independently associated with the primary endpoint (Fig. 2,panel B). Among patients with encephalitis, age > 60 years (OR 2.15, 95% CI 1.29-3.60), immunodepression (OR 2.16, 95% CI 1.14-4.10), hospital to ICU admission > 1 day (OR 2.23, 95% CI 1.30-3.82), a motor response on the GCS</= 3 (OR 1.88, 95% CI 1.03-3.41), acyclovir on day one of ICU admission (OR 0.47, 95% CI 0.27-0.83), and autoimmune etiology (OR 3.53, 95% CI 1.31-9.49) remained independently associated with the primary endpoint (Fig. 2,panel C).

Kaplan Meier survival curves are presented in Fig. 3 (panel A andB).

Characteristics of patients according to meningoencephalitis etiologies (post hoc analyses) are presented in the Appendix (ESM Tables 4, 6, 7). Characteristics of patients with delayed ICU admission (post hoc analysis) are presented in the Appendix (ESM Table 8).

Post hoc analyses investigating the association between adjunctive steroids on day 1 and outcomes are presented in the Appendix (ESM Table 9).

Discussion

EURECA is the largest prospective international multicenter cohort study to investigate outcomes of adult patients with meningoencephalitis requiring intensive care. We observed a poor functional outcome at 3 months in about half of patients, in the form of moderate-tosevere functional disability or death. We identified patient characteristics, time to ICU admission, neurologic presentation, antimicrobial therapy, and respiratory and cardiovascular complications at admission as independent indicators of outcome.

Patients with meningoencephalitis often require ICU admission to ensure oxygenation, airway protection, circulatory support, and prevention and treatment of secondary complications that might impact outcomes. These include cerebral edema [START_REF] Thakur | Predictors of outcome in acute encephalitis[END_REF], seizures/status epilepticus [START_REF] Thakur | Predictors of outcome in acute encephalitis[END_REF][START_REF] Sonneville | Early-onset status epilepticus in patients with acute encephalitis[END_REF], and systemic complications, such as fever [START_REF] Jaquet | Functional outcomes in adult patients with herpes simplex encephalitis admitted to the ICU: a multicenter cohort study[END_REF], aspiration pneumonia [START_REF] Sonneville | Clinical spectrum and outcomes of patients with encephalitis requiring intensive care[END_REF], respiratory failure requiring mechanical ventilation [START_REF] Thakur | Predictors of outcome in acute encephalitis[END_REF][START_REF] Singh | The spectrum of acute encephalitis: causes, management, and predictors of outcome[END_REF][START_REF] Jaquet | Functional outcomes in adult patients with herpes simplex encephalitis admitted to the ICU: a multicenter cohort study[END_REF], and thrombocytopenia/disseminated intravascular coagulopathy [START_REF] Thakur | Predictors of outcome in acute encephalitis[END_REF][START_REF] Singh | The spectrum of acute encephalitis: causes, management, and predictors of outcome[END_REF]. Due to the lack of prospective studies, there is currently no high-quality evidence suggesting that prevention of secondary complications is associated with improved outcomes. However, early ICU admission for monitoring and prevention of secondary insults has been proposed in encephalitis patients with altered mental status, seizures and/or nonneurologic organ failure(s) [START_REF] Stahl | Guidelines on the management of infectious encephalitis in adults[END_REF].

The prognostic variables identified in EURECA are critical to delineate goals of care at ICU admission and to identify patients who may benefit from prolonged aggressive care in the ICU and rehabilitation. We identified older age, and immunodepression as non-modifiable factors associated with a poor functional outcome. Of note, atypical presentations of HSV encephalitis have been described in these populations, which may result in delays in diagnosis and appropriate treatment, and worse outcomes [START_REF] Raschilas | Outcome of and prognostic factors for herpes simplex encephalitis in adult patients: results of a multicenter study[END_REF][START_REF] Poissy | Factors associated with delay to acyclovir administration in 184 patients with herpes simplex virus encephalitis[END_REF][START_REF] Tan | Atypical manifestations and poor outcome of herpes simplex encephalitis in the immunocompromised[END_REF]. We also identified actionable factors for which improvement could be made in future interventional studies, including time from hospital to ICU admission, time to antimicrobial therapy, and the presence of secondary complications at admission. These findings reinforce the fact that in patients who eventually required intensive care, a delayed ICU admission was an independent marker of poor prognosis [START_REF] Sonneville | Clinical spectrum and outcomes of patients with encephalitis requiring intensive care[END_REF][START_REF] Jaquet | Functional outcomes in adult patients with herpes simplex encephalitis admitted to the ICU: a multicenter cohort study[END_REF]. However, decisions of ICU admission depend on many factors and may significantly differ among centers. Although this remains speculative, delayed ICU admission might reflect inappropriate triage at hospital admission or early deterioration because of secondary complications. As such, respiratory failure identified in the multivariable analysis might be a consequence of aspiration pneumonia, frequently observed in patients with acute meningoencephalitis [START_REF] Sonneville | Clinical spectrum and outcomes of patients with encephalitis requiring intensive care[END_REF], and cardiovascular failure might reflect other complications such as septic shock, stunned myocardium or myocarditis, and dysautonomia. Use of early antimicrobial treatment (i.e., within 1 day of ICU admission) is also strongly associated with a better outcome, consistent with previously published literature for both community-acquired bacterial meningitis [START_REF] Auburtin | Detrimental role of delayed antibiotic administration and penicillin-nonsusceptible strains in adult intensive care unit patients with pneumococcal meningitis: the PNEUMOREA prospective multicenter study[END_REF][START_REF] Eisen | Longer than 2 hours to antibiotics is associated with doubling of mortality in a multinational community-acquired bacterial meningitis cohort[END_REF], and herpes simplex encephalitis [START_REF] Raschilas | Outcome of and prognostic factors for herpes simplex encephalitis in adult patients: results of a multicenter study[END_REF][START_REF] De Montmollin | Herpes simplex virus encephalitis with initial negative polymerase chain reaction in the cerebrospinal fluid: prevalence, associated factors, and clinical impact[END_REF]. While time to adequate treatment is paramount in patients with allcause meningoencephalitis, it is interesting to note that early antibacterial and antiviral treatment are associated with better outcomes in the total population, including patients with etiologies not requiring such treatments. Early antimicrobial treatment could be considered as a proxy for early identification of meningoencephalitis in the ICU, leading to an implementation of an adequate bundle of care, including an aggressive diagnostic workup and prevention of secondary brain insults.

We observed a relatively low rate of prescription of adjunctive steroids in patients with bacterial meningitis, reflecting uncertainty around their utility in the ICU setting, and a poor compliance to current guidelines [START_REF] Van De Beek | ESCMID guideline: diagnosis and treatment of acute bacterial meningitis[END_REF]. Although we could not find an independent association between adjunctive steroids and outcome in our post hoc analyses, ancillary studies using appropriate matching methods would be of interest, especially in bacterial meningitis patients.

We detected substantial heterogeneity among etiologies, with the worst outcomes observed in patients with encephalitis of autoimmune origin, compared to patients with acute bacterial meningitis and encephalitis of infectious or unknown origin. These results emphasize that an early etiological diagnosis may not only have important therapeutic implications but may also represent key information for appropriate prognostication [START_REF] Thy | Critical care management of meningitis and encephalitis: an update[END_REF]. In EURECA, the distribution of etiologies of bacterial meningitis and infectious encephalitis was comparable to current epidemiology in the general population. The prognosis of patients with bacterial meningitis appeared relatively similar to that observed in a recent large nationwide study conducted on patients with pneumococcal meningitis [START_REF] Koelman | Pneumococcal meningitis in adults: a prospective nationwide cohort study over a 20-year period[END_REF]. By contrast, we observed a more severe prognosis in patients with infectious encephalitis, as compared to a recent multicenter cohort study which reported a much lower mortality rate < 10% [START_REF] Mailles | Changing profile of encephalitis: results of a 4year study in France[END_REF]. Previous single-center retrospective studies conducted in adult ICU patients with all-cause encephalitis, reported poor outcomes in 25 to 50% of cases, with mortality rates ranging from 6 to 17%, depending on the etiology [START_REF] Thakur | Predictors of outcome in acute encephalitis[END_REF][START_REF] Singh | The spectrum of acute encephalitis: causes, management, and predictors of outcome[END_REF][START_REF] Sonneville | Clinical spectrum and outcomes of patients with encephalitis requiring intensive care[END_REF]. We observed the worst prognosis in patients with autoimmune encephalitis, with higher morbidity but similar mortality compared to other etiological groups. Of note, meaningful delayed post ICU-recovery has been observed in these patients, and it is possible that the 90-day outcome chosen in EURECA may not reflect the final functional outcome in this etiological category [START_REF] De Montmollin | Anti-N-methyl-d-aspartate receptor encephalitis in adult patients requiring intensive care[END_REF].

The main strengths of EURECA include a large sample of consecutive patients from 68 centers in 7 countries. Patients were included prospectively based on an internationally accepted definition for probable or confirmed encephalitis. We recorded prospectively major complications and interventions during ICU stay, and we evaluated functional outcomes at 3 months with a validated functional scale.

EURECA also has limitations, including the study's observational nature, with a possible heterogeneity in patient management among centers, notably for antimicrobial therapies, adjunctive therapies, and indication and timing of decisions to withhold/withdraw care in patients with an expected unfavorable outcome. Second, because we did not use a standardized diagnostic protocol among participating centers, it is possible that some patients from the unknown etiology encephalitis group were in fact culture-negative bacterial meningitis patients. Third, many physicians would not consider a mRS score of three at 3 months as an unfavorable outcome, given the high severity of illness at admission. It is not known to what extent moderately to severely disabled patients eventually improved and regained functional independence in the long term. Fourth, we mainly identified clinical indicators of outcome and the additional value of neuroimaging or EEG variables should be investigated [START_REF] Sarton | Assessment of magnetic resonance imaging changes and functional outcomes among adults with severe herpes simplex encephalitis[END_REF][START_REF] Jeantin | Electroencephalography for prognostication of outcome in adults with severe herpes simplex encephalitis[END_REF]. Finally, other important patient-centered endpoints, such as cognitive impairment, psychological problems and post-encephalitic epilepsy were not recorded.

In conclusion, we observed that half of patients with meningoencephalitis requiring intensive care had poor functional outcomes at 3 months, in the form of moderate-to-severe disability or death. We identified several admission variables and the etiology of meningoencephalitis as independent indicators of outcome. While further research aiming at a better characterization of 1-year outcomes in these patients is currently ongoing (Clinical-Trials.gov Identifier: NCT02906631), the findings from this study are critical for better identification of patients who might benefit from prolonged aggressive care in the ICU and rehabilitation.

Fig. 1

 1 Fig. 1 Distribution of scores on the modified Rankin scale (mRS) at 3 months. Panel A shows the distribution of mRS scores in the total study population and in the principal different etiological categories. Two patients with more than one infectious etiology are not shown. Panel B shows the distribution of mRS scores in patients with bacterial meningitis overall and according to the main bacterial causes. Panel C shows the distribution of mRS score in patients with infectious encephalitis overall and according to the main viral causes. Two patients with more than one infectious etiology are not shown

Fig. 2

 2 Fig. 2 Multivariable logistic regression of factors associated with poor neurological outcome at 3 months. Panel A shows results in the total population. Panel B shows results in patients with bacterial meningitis. Panel C shows results in patients with encephalitis, of infectious, immune-mediated, or unknown origin. Immunodepression: HIV infection, solid organ transplant recipient, hematology/cancer patient, and long-term use of steroids and/or immunosuppressant drugs. Respiratory failure: respiration SOFA score 3-4. Cardiovascular failure: cardiovascular SOFA score 3-4

Acknowledgements

The authors wish to thank the European Society of Intensive Care Medicine office for the help in the conduct of the study. RS wishes to thank Mary Pellegrin for her help in the writing of the manuscript. EURECA investigator study group (collaborators): Manuel Santafe, Roland Smonig, Damien Roux, Guillaume Voiriot, Bertrand Souweine, Keyvan Razazi, Thibault Ducrocq, Patricia Boronat, Nadia Aissaoui, Danielle Reuter, Alain Cariou, Philippe Mateu, Barabara Balandin Moreno, Paula Vera, Estela Val Jordan, Francois Barbier, Mickael Landais, Jeremy Bourenne, Antoine Marchalot, Mathilde Perrin, Benjamin Sztrympf, Carole Schwebel, Shakti Bedanta Mishra, Patrick Chillet, Maelle Martin, Hugues Georges, Jean-Claude Lacherade, Romaric Larcher, Gregory Papin, David Schnell, Sulekha Saxena, Frank Chemouni, Juliette Audibert, Eric Mariotte, Shidasp Siami, Italo Calamai, Cedric Bruel, Alexandre Massri, Jesus Priego, Xavier Souloy, Pascal Beuret, Bikram Kumar Gupta, Thomas Ritzenthaler, Sami Hraiech, Aguila Radjou, and M. K. Renuka.

Funding

European Society of Intensive Care Medicine. GC was supported by the Italian MUR Dipartimenti di Eccellenza 2023-2027 (l. 232/2016, art. 1, commi 314-337).

Data availability

The data supporting the findings of the study are available upon reasonable request after approval of a proposal from the corresponding author (RS). Related documents will also be made available, including the study protocol, and the statistical analysis plan.

Author contributions

RS: conception of the work (PI), funding application, enrollment of participating centers, supervision of the data collection, participation in data analysis, verification of the data and interpretation, drafting the manuscript, critical revision of the manuscript, final approval of the version. RS is the guarantor of the entire manuscript and responsible for the decision to submit the manuscript. EdM: enrollment of participating centers, drafting the manuscript, including tables and figures, participation in data interpretation, critical revision of the manuscript, final approval of the version to be published. JH, RH, IM-L, FST, JJdW, GC: participation in the definition of the protocol, critical revision of the manuscript, and final approval of the version to be published. SR, QS: data management, and statistical analysis. JFT: conception of the work, funding application, supervision of the data collection, analysis and interpretation, critical revision of the manuscript, final approval of the version. Other authors: data collection, interpretation of data, critical revision of the manuscript, final approval of the version. The EURECA investigators (listed in the EURECA investigator group), who participated in the data collection are non-author contributors.

Declarations

Conflicts of interest

RS reports a grant from the European Society of Intensive Care Medicine (ESICM) for the current work and grants from the French Ministry of Health, outside the submitted work. Other authors report no competing interest.

Ethical approval

The entire protocol has been reviewed and approved by the Comite de protection des personnes sud-méditérranée 2 (ID RCB 2017-A00139-44). National/ local approvals at the international study sites were obtained by the national coordinators and local investigators according to the local regulations. Since meningoencephalitis patients could not be informed at the time of study recruitment, each center referred to local/national law on the issue of lack of capacity. If the patients regained capacity before 3 months, they were asked either to consent to the use of the acute and follow-up data or to refuse to participate in the research.


Annamaria Lapenna

Camille Dagallier

Sylvain Huille

Christophe Tribet
email: christophe.tribet@ens.psl.eu

Poly(glutamic acid)-based viscosity reducers for concentrated formulations of a monoclonal IgG antibody

Keywords: antibody, viscosity, poly(amino acid), coacervate, polyanion:polycation complexes

Above a concentration threshold the viscosity of solutions of proteins increases abruptly, which hampers injectability of therapeutic formulations. Concentrations above 200 g/L is an ideal goal for subcutaneous application of antibodies. Molecular additives, such as amino acids (ex. Arg) help to decrease viscosity, but they are used at concentrations as high a about 200 mmol/L. We addressed the question of whether poly(amino acids) could be more efficient than small molecular additives. We observed marked fluidification of a model therapeutic mAb solution by poly (D,L-glutamic acid) and poly(L-glutamic acid) derivatives added at < 6.5 g/L (i.e., 2 mAb/polymer chain molar ratio between 4:1 and 1:1 mol/mol). The bare poly(glutamate) parent chains were compared with polyethyleneglycol-grafted chains as PEGylation is a common way to enhance stability. Viscosity could be lowered down to ~ 20 mPa.s as compared to values of ~100 mPa.s in the absence of polymers at 200 g/L mAb. Formation of complexes between the mAb and the polyglutamates was characterized by capillary electrophoresis analysis in dilute solutions (1g/L mAb) and by observation of phase separation at higher concentration, suggesting tight association at about 2:1 mol/mol mAb/polymer. Altogether these results show that polyglutamate derivatives hold an untapped potential as excipient for fluidification of concentrated protein solutions. concentrated mAb solutions, calibration data of FACCE method, supplementary viscosity data, and a photograph of concentrated mAb:PLE(L) mixture with/out phase separation.

Introduction.

Since the commercialization of the first therapeutic monoclonal antibody (mAb) in 1986, this class of proteins has grown to become dominant in the drug development pipeline. [START_REF] Lu | Development of therapeutic antibodies for the treatment of diseases[END_REF] mAbs solutions combined with (bio)polymers are now a common chassis for developments of new therapeutic approaches, for immunotherapies (ex: cell-targeted controlled delivery), [START_REF] Miura | Antigen Delivery to Antigen-Presenting Cells for Adaptive Immune Response by Self-Assembled Anionic Polysaccharide Nanogel Vaccines[END_REF] intracellular targeting, [START_REF] Davis | Protein Binding and Release by Polymeric Cell-Penetrating Peptide Mimics[END_REF] oral or occular delivery. [START_REF] Nikkhah | Copolyelectrolyte-Based Nanocapsules for Oral Monoclonal Antibody Therapy: A Mesoscale Modeling Survey[END_REF][START_REF] Ilochonwu | Hyaluronic Acid-PEG-Based Diels-Alder In Situ Forming Hydrogels for Sustained Intraocular Delivery of Bevacizumab[END_REF] Their use is however hampered by obstacles, especially due to difficulties to control stability (protein aggregation), and solution viscosity. In practice, fluidification of highly concentrated mAb solutions (e.g., > 150 g/L) is in demand because of requirement for (i) syringeability (injection through usual syringe needs viscosity below ~50 mPa.s), [START_REF] Deokar | Comparison of Strategies in Development and Manufacturing of Low Viscosity, Ultra-High Concentration Formulation for IgG1 Antibody[END_REF] and (ii) lowering injected volumes, specifically in the case of subcutaneous implementation (typically targeting < 2 mL). The viscosity issue and thickening effect observed in concentrated solutions of proteins is a key general question that has been discussed theoretically, with models coming from colloid science. [START_REF] Stradner | Potential and limits of a colloid approach to protein solutions[END_REF][START_REF] Xu | Studying Excipient Modulated Physical Stability and Viscosity of Monoclonal Antibody Formulations Using Small-Angle Scattering[END_REF] Viscosity-concentration relationships are now reliably used for small globular proteins. [START_REF] Von Bulow | Dynamic cluster formation determines viscosity and diffusion in dense protein solutions[END_REF] But it is still challenging to predict the viscosity pattern of non-spherical, flexible proteins such as mAbs. The complexity of viscosification mechanism of mAb and specifically a high sensitivity to concentration is presumably due to contributions of mAb-mAb contact points [START_REF] Arora | Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody[END_REF][START_REF] Yadav | The Influence of Charge Distribution on Self-Association and Viscosity Behavior of Monoclonal Antibody Solutions[END_REF][START_REF] Yadav | Establishing a Link Between Amino Acid Sequences and Self-Associating and Viscoelastic Behavior of Two Closely Related Monoclonal Antibodies[END_REF] or formation of transient interprotein clusters. [START_REF] Johnston | Concentrated Dispersions of Equilibrium Protein Nanoclusters That Reversibly Dissociate into Active Monomers[END_REF][START_REF] Skar-Gislinge | Using Cluster Theory to Calculate the Experimental Structure Factors of Antibody Solutions[END_REF] In other words, a pronounced viscosity increase is generally associated with the presence of attractive interactions between mAbs, as suggested by experimental investigations on the correlation between viscosity and interaction parameters [START_REF] Roche | Towards an improved prediction of concentrated antibody solution viscosity using the Huggins coefficient[END_REF][START_REF] Kingsbury | A single molecular descriptor to predict solution behavior of therapeutic antibodies[END_REF][START_REF] Neergaard | Viscosity of high concentration protein formulations of monoclonal antibodies of the IgG1 and IgG4 subclass -Prediction of viscosity through protein-protein interaction measurements[END_REF] ), or indications on the formation of interprotein reversible complexes at high concentrations. [START_REF] Zidar | Control of viscosity in biopharmaceutical protein formulations[END_REF][START_REF] Gong | Ultra-sub-stoichiometric "Dynamic" Bioconjugation Reduces Viscosity by Disrupting Immunoglobulin Oligomerization[END_REF][START_REF] Skar-Gislinge | Using Cluster Theory to Calculate the Experimental Structure Factors of Antibody Solutions[END_REF][START_REF] Godfrin | Effect of Hierarchical Cluster Formation on the Viscosity of Concentrated Monoclonal Antibody Formulations Studied by Neutron Scattering[END_REF] But discrepancies with this simple rule are also reported. [START_REF] Xu | Studying Excipient Modulated Physical Stability and Viscosity of Monoclonal Antibody Formulations Using Small-Angle Scattering[END_REF] Fluidifying formulations of mAb aims to modulate these inter-protein interactions, via an introduction of molecular additives (excipients). [START_REF] Wang | Viscosity-Lowering Effect of Amino Acids and Salts on Highly Concentrated Solutions of Two IgG1 Monoclonal Antibodies[END_REF] Conventional additives can be amino acids [START_REF] Dear | Contrasting the Influence of Cationic Amino Acids on the Viscosity and Stability of a Highly Concentrated Monoclonal Antibody[END_REF][START_REF] Dear | Enhancing Stability and Reducing Viscosity of a Monoclonal Antibody With Cosolutes by Weakening Protein-Protein Interactions[END_REF][START_REF] Inoue | Specific Decrease in Solution Viscosity of Antibodies by Arginine for Therapeutic Formulations[END_REF] , or surfactants such as polysorbates. [START_REF] Xu | Studying Excipient Modulated Physical Stability and Viscosity of Monoclonal Antibody Formulations Using Small-Angle Scattering[END_REF] These small molecules typically provide weak and labile perturbations of the interactions, either by affecting ionic strength or by screening specific patches onto mAbs. In situ PEGylation of IgGs has similarly been reported to improve fluidity. [START_REF] Gong | Ultra-sub-stoichiometric "Dynamic" Bioconjugation Reduces Viscosity by Disrupting Immunoglobulin Oligomerization[END_REF] In other words, protein-protein attractions may be discouraged, by using transient (dynamic) competitive binding with additives. In particular charged amino-acids reduces viscosity, which was tentatively explained by their ability to shield coulombic interactions and to disrupt mAb-mAb associations.

Arginine is the most prevalent amino acid additive in literature, [START_REF] Dear | Contrasting the Influence of Cationic Amino Acids on the Viscosity and Stability of a Highly Concentrated Monoclonal Antibody[END_REF] although various cationic or anionic amino-acids impact viscosity as well. [START_REF] Wang | Viscosity-Lowering Effect of Amino Acids and Salts on Highly Concentrated Solutions of Two IgG1 Monoclonal Antibodies[END_REF] But high concentrations of up to 0.1 -1 mol/L of these small molecular additives are required, making these formulations not always suitable for clinical trials or commercialization.. Molar amounts of amino acids makes it complicated to adjust pH, osmolarity and may introduce practical problems of cost or toxicity.

We hypothesized that tighter interactions could enhance the efficiency of viscosity-reducing agents. Here, we addressed the question of whether polymers of amino-acids, that afford a multiple binding pattern, offer more stable binding onto mAb while preserving the favorable effect of their monomeric amino-acids precursors. Short, ionized poly(amino acids) can bind onto mAbs of unlike charge. [START_REF] Izaki | Feasibility of Antibody-Poly(Glutamic Acid) Complexes: Preparation of High-Concentration Antibody Formulations and Their Pharmaceutical Properties[END_REF] A few studies are reported in literature, in which poly(glutamic acid) (PLE) and poly (-glutamic acid) bind onto mAbs. Association with PLE was used to trigger phase separation of native mAbs. [START_REF] Matsuda | Liquid Droplet of Protein-Polyelectrolyte Complex for High-Concentration Formulations[END_REF] Enhancement of thermal stability of antibodies was achieved using poly (glutamic acid), [START_REF] Lee | Enhancement of enzyme activity and stability by poly(γ-glutamic acid)[END_REF] or poly(glutamic acid)-b-polylysine diblocks. [START_REF] Li | Efficient synthesis of amino acid polymers for protein stabilization[END_REF] . The present work aims to interrogate the use of PLEs additives as viscosity reducers. As our goal was to establish a proof of principle, we included in the study both modified and unmodifed poly(glutamates) whose G.R.A.S. status (generally recognized as safe) may not be established. Specifically, in this work PLE parent chains were grafted with polyethylene glycol (PEG) strands i.e., a common enhancer of colloidal stability. We evaluated poly(glutamic acid) derivatives of either short (45 monomers) or longer (90 monomers) chains. We studied (i) the impact of PLE derivatives on viscosity of a model mAb and (ii) their binding to the mAb in dilute conditions. Viscosity was measured by capillary viscosimetry and by using tracer beads together with light scattering. Capillary electrophoresis enabled to characterize binding equilibrium.

Materials & Methods.

Preparation of mAb solutions.

For viscosity measurements, samples of concentrated mAbs were prepared by dialysis and reconcentration of stock solutions obtained from SANOFI (i.e., a 93 g/L IsatuximAb in 20mM Histidine-HCl buffer 10% sucrose pH 6.3, or the formulated 140 g/L mAb in the same buffer but with no sucrose and with excipients). Aliquots of 1-3 mL were dialyzed overnight (slide-A-Lyzer device 7-20 kDa MWCO, from Pierce, ThermoFisher Scientific), at 8°C against either 5mM Histidine-HCl + 10% sucrose (pH values of either 5.2 or 4.8) or 5 mM NaH2PO4-Na2HPO4 pH 5.2. An upshift in pH, compared to the dialyzing buffer, was systematically observed due to Donnan effect: the pH measured after dialysis in the sample was of 6.2-6.3 (resp. 5.9) when dialyzed against pH 5.2 (resp. 4.8). To prepare stock solutions at 180 g/L, the sample was kept in the slide-A-lyzer cassette that was deposited on an aluminium foil and covered by dry powder of poly(oxyethylene) (PEO 200 000 g/mol, Sigma) for 30 minutes. To prepare mAb solutions at ca. 250 mg/mL, the dialyzed samples were ultrafiltrated on Amicon Ultra 0.5 mL 10 kDa Centrifugal filters (from ThermoFisher, 30 min. at 12 000 g, 10°C). The final concentration of the mAb was determined from the absorbance at 280 nm (Nanodrop One, Thermo scientific) on undiluted samples, using eq. 1:

[mAb] (g/L) = 1.65  [absorbance(280nm) -absorbance (600)] eq.1

Where the coefficient 1.65 was obtained from a calibration curve measured on the used spectrometer.

For binding isotherm measurements by electrophoresis or to prepare coacervates at 20 g/L mAb, we used a stock solution at 20 g/L in 20mM Histidine, 10% sucrose, pH 6.3 that was dialyzed in similar condition as above, followed by pH adjustment with a solution of 5mM Histidine + 10% Sucrose (resp. phosphate buffer), and eventually concentration by ultrafiltration in Amicon Ultra 0.5 mL 100 kDa Centrifugal filters (15 min. at 15000 g).

Preparation of polymer:mAb mixed solutions.

If not specified, mixtures were prepared by adding aliquots of concentrated polyglutamate stock solutions (NaCl 2M, polyglutamate derivatives of 10 g/L to 120 g/L) in a concentrated mAb solution and dilution with the ad hoc buffer at the same pH as the mAb solution. The pH of polyglutamate stock solutions in water was adjusted using 0.1 M HCl or NaOH to the final pH (6.3 or 5.9) before mixing.

Capillary electrophoresis

Measurements were performed using a Beckman P/ACE system MDQ instrument equipped with a diode array UV/visible detector (Beckman Instruments, Fullerton, CA), operating NaH2PO4-Na2HPO4 pH 6.3) for 5min. 2 psi. Frontal analysis (FACCE) consisted of running the separation with the inlet plunged in the sample, and the capillary outlet and initial filling being the analyte-free buffer. A voltage of 20 kV was applied in addition to a small pressure of 0.1 psi used to push the solution toward the detection window. In conditions of "positive" voltage (anode at the inlet), migration of the cationic mAb was the fastest. In condition of "negative" voltage (cathode at the inlet), the anionic polyglutamates were detected first. Possibly a small zone of neutral marker (0.1 g/L mesityloxide in the run buffer) was injected (1 psi, 5s.) prior to running the separation.

The elution time of this zone, teo was used to calculate the effective electrophoretic mobility μ of analytes from eq 2, where tm is the sample migration time, Lt is the total length of the capillary (31 cm), Ld is the length to the detector (21cm), and E the electric field. ) eq. 2

Capillary viscosimetry

Viscosity measurements were performed at 25 °C, using the Beckman P/ACE system MDQ instrument as for electrophoresis. The capillary was first conditioned by rinsing with water, 0.1 M HCl, and 0.1 M NaOH at 2 psi (2 min. each), then filled with the sample (injection at 5 psi for 5-10 minutes). A small zone of buffer was formed by injecting a solution of buffer (15 s., 1 psi). Then a constant injection pressure ∆Pa of 1 psi or 5 psi was applied, with the inlet plunged in the sample, to push the zone toward the detection window. Absorbance at 280nm or 200 nm enabled to detect the time, tb, of elution of this zone of buffer as a negative peak. The viscosity, ƞ, is calculated using the Hagen-Poiseuille's law (with D the capillary inner diameter) [START_REF] Allmendinger | Highthroughput viscosity measurement using capillary electrophoresis instrumentation and its application to protein formulation[END_REF] :

ƞ = ∆Pa * D 2
32 .𝐿 𝑡 .𝐿 𝑑 . tb eq. 3

Calibration was done by measurements of Sucrose solutions in water (40% -60%).

Tracer beads microviscosity by DLS

The protocol was adapted from ref [START_REF] He | Highthroughput dynamic light scattering method for measuring viscosity of concentrated protein solutions[END_REF] SI.1 in SI). In contrast, near quantitative grafting yield were obtained for the 10 mol% PEGylation and the fraction of ungrafted chains were low in that case. To evaluate the effect of PEGylation, we studied here the 10 mol% derivatives of 45PLE, 90PLE and 45PLEL chains that are named below as 45PLE-10PEG3, 90PLE-10PEG3 and 45PLEL-10PEG3.

Evidence for mAb:polyglutamate association

In dilute conditions, formation of mAb:polymer complexes was assessed by capillary electrophoresis. The (free/unbound) mAb was cationic in the acidic pH conditions used here, whereas the PLEs were anionic. Coulombic mAb:polymer complexes are expected to display a more balanced charge. Accordingly, the polyglutamate, polyglutamate:mAb complexes, and the free mAb shall have very distinctive electrophoretic migration rates. To avoid perturbation of association equilibria during the analysis, we implemented a continuous injection scheme (method called frontal analysis by continuous injection electrophoresis, FACCE [START_REF] Seyrek | Ionic strength dependence of proteinpolyelectrolyte interactions[END_REF] , see method section).

The continuous injection and migration of analytes resulted in adjacent zones detected as plateaus of absorbance. The first zone contained the fastest analyte (here mAb free or resp. polymer free in positive, resp. negative field conditions). Absorbance at the plateaus varied in proportion to concentrations (cf calibration curves in Figure SI.4 in SI). We analyzed samples prepared at a fixed total mAb concentration of 1 g/L (6.67 M) and varying polymer concentration. Representative electropherograms of mixtures are shown in Fig. 1. The fast drop in Fig. 1A of plateau height upon addition of micromolar concentration of 45PLE suggests that binding was very effective in these dilute conditions. Namely, above 10 micromolar 45PLE, about 80% of the 1 g/L mAb was "captured" under the form of complexes with polyglutamate chains. When represented as a function of electrophoretic mobilities (eq. 2 in "Materials & Methods" section) electropherograms confirmed that at pH 6.3 the mAb was weakly cationic (mobility at half plateau height of +3.3 10 - 5 cm 2 .V -1 .s -1 in Fig. 1C) and polyglutamate was anionic (mobility of ca. -35 10 -5 cm 2 .V -1 .s -1 ).

Interestingly, detection of 45PLE(L):mAb complexes started under the negative field conditions and of unbound polymer in the samples (Fig. 2B). In the experimental window (total mAb of 1g/L and polymer varying between 0.01 g/L and 1 g/L), R typically varied from 0.5 to ~ 3 mol/mol).

The smaller ratios were measured at the lower polymer concentrations. But when the amount of free polyglutamate was close or below the sensitivity of the technique, apparent R values were with no surprise close to the initial ratio of the mixture (dotted line in Fig. 2B). In mixtures containing reliably measured amounts of both free PLEs and free mAb, the data suggest that equilibrium corresponded to a minimum of R of 0.4-0.7 with 45PLEL, and of 0. A similar fit of the 45PLEL data however did not converge. Assuming a 2:1 ratio, and accordingly

K'=[45PLEL]free.
[mAb] 2 free/[mAb]bound , enabled a suitable fit (Fig. 2A). The matching between simple stoechiometric models and data are however likely incidental, due to a focus into a limited and specific range of concentrations (corresponding here to 0 -80% bound protein). Upon increasing the polymer concentration, a gradual and significant increase of R was always observed.

13

R of up to 3 mol/mol was reached with 45PLE (ex: 2.6 in 0.5 g/L 45PLE + 1 g/L mAb, or 2.8 mol/mol in 0.2 g/L mAB + 0.04 g/L 45PLE). Accordingly, R < 1 and mAb:polyglutamate ratio of 2:1 -1:1 may be a situation only observed in a limited range of composition. In the presence of excess free polymer, the mAb could accommodate several polyglutamate chains, a possibility that presumably corresponded to binding with lower affinities (ex: contribution of association of polymer chains onto weaker binding sites). In summary, all polymer chains (45PLE, 45PLE(L)-10PEG3, 45PLEL) qualitatively bind mAb with a similar association pattern: a fast capture of up to 80% of the mAb at [polymer chain] < 10 M, with a mean binding ratio increasing from R=0.5-1 mol/mol at low polymer concentration to 3-4 mol/mol in the presence of excess polymer. At higher mAb concentrations, mixed mAb:polyglutamate solutions underwent associative liquid-liquid phase separation which is an additional evidence for the formation of complexes.

Complex coacervation of protein:polyelectrolyte of unlike charge is well documented. [START_REF] Mctigue | Effect of polyelectrolyte structure on protein-polyelectrolyte coacervates: Coacervates of bovine serum albumin with poly(diallyidimethylammonium chloride) versus chitosan[END_REF] It has been reported recently by Shiraki and coll on a similar example of a monoclonal mAb with a polyglutamate chain. [START_REF] Mimura | Effect of additives on liquid droplet of protein-polyelectrolyte complex for high-concentration formulations[END_REF][START_REF] Tsumura | Lowering the viscosity of a high-concentration antibody solution by protein-polyelectrolyte complex[END_REF] We observed this phase separation with all the polymers studied here, including the PEGylated ones, both at pH 6.3 and 5.9 (5mM buffers). To quantify the amount of mAb involved in the dense phase, samples were prepared at 20 g/L mAb and varying polymer concentrations between 0.1 g/L and 1 g/L. These conditions corresponded to ratios of (total) 45PLE / mAb in the mixture varying between 0.1 and 5.5 mol/mol. Samples were incubated for 1 hour and ultracentrifuged (Airfuge 78 000 rpm, 10min.) to separate the two fairly transparent phases. Measurements of the volume of the phases and absorbance at 280 nm of the upper, more dilute phase enabled to calculate the compositions reported in Table 1. The mAb concentration in the dense phase increased with increasing the polyglutamate (60 g/L -177 g/L mAb), while the supernatant concentration decreased (20 -2 g/L mAb). The maximum fraction of mAb entering in the dense phase (up to 93% with 45PLEL, or 73% with 45PLE) was observed at a similar molar concentration of polymer for both 45PLE and 45PLEL, corresponding to about 0.5-0.7 mol of polyglutamate chains per mol of mAb (total amount in the mixture). Addition of polymer beyond this optimum increased the fraction of mAb in supernatant. An excess of polyglutamate presumably favors formation of soluble complexes having a significant anionic charge because of excess PLE, thus creating repulsion between the complexes. [START_REF] Mimura | Effect of additives on liquid droplet of protein-polyelectrolyte complex for high-concentration formulations[END_REF] Finally, addition of 50 mM NaCl in phase-separated samples enabled to recover a single phase, transparent solution. Altogether, these observations are consistent with the formation of coulombic complexes between the mAb and polyglutamate chains at pH 6.3, the tighter association being observed near a 2:1 mAb/polyglutamate molar ratio, which is expected to reflect a situation close to charge cancellation in the complexes. Viscosity.

Viscosity in the absence of polyglutamates was determined as a function of mAb concentration either by capillary viscosimetry (CV) or dynamic light scattering with polystyrene tracer beads of known diameter (DLS, see method section). A rapid increase of viscosity with increasing concentration was observed by both methods (Fig. 3A) in the concentration window of 40-200 g/L (in 5mM Histidine + 10% sucrose buffer). However, the DLS method systematically returned a viscosity 1.5-1.6 times higher than CV measurements (Table SI.2 in SI). The origin of a constant shift between the two methods is presumably the use of an inappropriate reference relaxation time (i.e., incorrect bead's reference diameter). The reference relaxation time used in eq. 4 was here measured in water and in the histidine buffer and corresponded to an effective hydrodynamic radius of 185 ± 10 nm. It matched with the expected diameter of 180 nm claimed by the supplier.

In addition, calibration using sucrose solutions (50 wt% -60 wt% in the range of viscosities of 12 mPa.s to 44 mPa.s) returned the same values of the viscosity by either DLS or CV, suggesting that the reference diameter was correctly introduced in eq. 4. The difference observed in the presence of mAb, together with the constant ratio between effective "DLS" and "CV" viscosities (Table SI.2 in SI) suggest accordingly that the tracer beads were covered by adsorbed mAbs, which increased their effective diameter up to 270 nm. Adsorption of cationic mAb molecules onto the negatively charged beads is not surprising. Of note, effective viscosities as determined by DLS were stable for hours. And the same ratio of 1.5-1.6 between CV and DLS methods were also observed on mixtures of mAb with polyglutamate additives (Fig. SI.5 in SI). For the sake of consistency, we nevertheless show below the viscosity calculated from the reference bead diameter of 185 nm, that thus overestimate actual values. In the absence of polyglutamate, a rapid increase of viscosity was observed above 80 g/L (cf break in Fig. 3A), which is typical of molecular attraction and/or clusterization of the antibody molecules. [START_REF] Chari | Long-and Short-Range Electrostatic Interactions Affect the Rheology of Highly Concentrated Antibody Solutions[END_REF] , 36 , 14 At 200 g/L mAb, the viscosity was above the practical injectability threshold of 50 mPa.s, that was crossed near/above 140 g/L mAb. Viscosity also increased with decreasing pH (Fig. 4) making Histidine buffer at pH 6.3 (i.e., pH of the « as prepared » mAb stock solutions) slightly more fluid than preparations at pH 5.9 (pH adjusted after dialysis and concentration, see method section). Gradual dilution in the buffer was used to obtain data in Figure 3B, showing a roughly exponential increase of viscosity with mAb concentration. Linear variations in the semi-log representation of Fig. 3B characterizes the effect of concentration variation, irrespective of the presence or absence of additives (NaCl, polyglutamates). This suggests that measurements at one reference concentration can be representative of the role of polymers in a larger, practically relevant range of 100 -250 g/L mAb. To assess the effect of polyglutamates, we compared the viscosity at 140 g/L mAb (Fig. 4). A mixture of 3.5 g/L 45PLE with 140 g/L mAb corresponds to an average mol/mol ratio of ~ 2 mAb monomer per polymer chain (R=0.5). Irrespective of the polymer used, supplementation of the mAb solution with 3.5 g/L or 6.5 g/L polymer markedly fluidified the solutions and decreased viscosity by up to three fold (Fig. 4A and Table 2). Viscosity of polyglutamate solutions (no mAb) was negligibly low in this concentration range (viscosity of 40 g/L polymer in water with no mAb < 2 mPa.s, Table SI.3 in SI). Increasing the ionic strength by addition of NaCl further decreased the viscosity, suggesting the importance of coulombic contribution to the thickening effect observed in mAb solutions without additive. To assess the role of small ions having similar acidbase properties as the glutamic acid unit, we measured the decrease of viscosity achieved by addition of a stock solution of acetic acid, adjusted at pH 6.3 with NaOH. The equivalent concentration of glutamate units in 3.5 g/L (resp. 6.5 g/L) 45PLE was of about 20 mM (resp. ~ 40 mM). Of note, the ionic strength in 45PLE solution is expected to be lower than the one achieved in sodium acetate at the same molar concentration, because of counter ion condensation onto the polyelectrolyte chains. In the absence of NaCl, 45PLE decreased the viscosity slightly more than what was observed with molar equivalent sodium acetate addition (Table 2). This suggests that the contribution of the 45PLE chains cannot solely be explained by variation of the ionic strength. The PEGylated polyglutamates (45PLE-10PEG3 or 90PLE-10PEG3) have a molar mass about 3 time higher than their parent chains. Namely, a 6.5 g/L solution of 45PLE-10PEG corresponds to 14.5 mM glutamate units (resp 7.7 mM at 3.5 g/L). In comparison to similar concentration of sodium acetate, the PEGylated chains are shown in Table 2 to be either slightly more effective or of similar fluidification efficiency, though they were not as effective as 45PLE. Finally, comparison of polyglutamate derivatives having 45 or 90 monomer units (Fig. 4) suggested a higher efficiency of shorter polymers : in the presence of NaCl viscosity of samples containing 90PLE and 90PLE-10PEG3 was significantly higher than without polymer, or with shorter polymers (45PLE and 45PLE-10PEG3). However no obvious sedimentation neither phase separation took place with PLE during 24h (Fig.

SI.6. in SI)

. At pH 5.9, fast demixing was generally hampering stable measurements of samples containing either 45PLE or 45PLEL. In contrast, the PEGylated polymers were forming transparent mixtures at either pHs, with or without NaCl, enabling viscosity measurements on single-phase systems. Finally, to assess the effect of all polymers in homogeneous, one-phase, stable samples, we added NaCl in all mixtures (Fig. 4 and Table 2). At pH of 5.95, increasing the polymer concentration from 3.5 to 6.5 g/L decreased the viscosity (Fig. 4B). However, at pH 6.3 (Table 2), the presence of polymers at 6.5 g/L did not allow better fluidification in the presence of NaCl than compared to 3.5 g/L polymer. The viscosity of polymer-containing samples with NaCl at pH 6.3 may be higher or lower than the viscosity of reference samples containing both NaCl and sodium acetate (Table 2). As pointed in the paragraph on mAb:polyglutamate association, the coulombic complexes are sensitive to ionic strength and are likely dissociated by increasing the concentration of NaCl, which may counteract their fluidification effect. In summary, the results above point to a capacity of 45PLE to fluidify mAb solutions of low ionic strength beyond the effect due to variation of ionic strength. The other short polymers (i.e., 45 monomer units) also significantly decreased viscosity, though with magnitudes that were closer to that observed upon addition of sodium acetate at similar (molar) concentration of glutamate units. Because all polymers were associated with mAb at low ionic strength, the mechanism of viscosity reduction by polymer may however differ and mAb-mAb interactions shall be affected differently upon binding of poly(glutamate). The association with PEGylated chains could in this line contribute to adding repulsive interaction between the complexes. Our results points in addition that cautious adjustment of the ionic strength shall be considered to avoid losing the coulombic complexation between the polyglutamate chains and the antibody. Eventually, we prepared samples at 200 g/L mAb to assess whether injectable formulations (< 50 mPa.s) could be obtained. Figure 5 shows measurements of microviscosity (DLS) as obtained by mixing concentrated stock solutions (mAb ~ 250 g/L) with polymers and diluting in buffer down to 200 g/L mAb. The 5 g/L polymer concentration corresponds here to the same mAb:polyglutamate ratio as in 140 g/L mAb + 3.5 g/L polymers. All polymer additives among 45PLE, 90PLE, and 45PLEL-10PEG3 were capable to decrease the viscosity of the mAb below the practical threshold of 50 mPa.s. Of note, the addition of 45PLE-10PEG3 without NaCl (11 mM eq glutamate units) also provided a fluidification near 50mPa.s, more effective than NaCl 55mM with no polyglutamate. As PEGylated copolymers may in addition contribute to stability (in contrast to the use of NaCl that can impair stability by enabling sticky collisions between mAbs and faster irreversible aggregation rate [START_REF] Gong | Ultra-sub-stoichiometric "Dynamic" Bioconjugation Reduces Viscosity by Disrupting Immunoglobulin Oligomerization[END_REF] ) the trends observed suggest the interest of further studies of the ageing and formulations using PLEs and/or PEGylated PLE derivatives. In this line, the use of buffers of low ionic strength (in term of simple salt concentration) has been documented to provide better long term stability of highly concentrated solution of mAbs. 37 45PLE and its PEGylated forms when used at low concentrations deserve accordingly to be considered as their impact on ionic strength shall be lower that simple salts.

Conclusion.

The main result of the present work is that a commercial poly(glutamate), PLE, and PEGylated derivatives of PLE can decrease the viscosity of a model therapeutic mAb, Isatuximab.

At the highest concentration assessed (200 g/L) the viscosity was decreased to values that compared with a conventionally formulated sample of mAb, making the mixed solutions compatible with syringe injection in terms of viscosity. A very low amount of PLE additive was required: we explored polymer/mAb ratio of the order of 2.5/100 -5/100 wt/wt, which corresponds to polymer/mAb of ½ -3/1 mol/mol (depending of polymer's Mw). As compared to molecular amino acids such as Arginine, often used at >100 mM concentration, PLE was here of 23 -43 mM eq. glutamate units (resp 7.7 -14 mM for PEGylated derivatives). We determined in addition using capillary electrophoresis that association took place at millimolar concentration of the polyglutamates in dilute conditions (mAb of 1 g/L). At low ionic strength and in a range of PLE/mAb mol/mol ratios of 0.5 -1, a predominant fraction of the mAb formed complexes with the polyglutamate additives.

A likely origin of the impact of PLEs on viscosity is a change of interactions developed between the mAbs as compared to interaction between polymer-bound mAbs (ex: perturbation of inter-mAb clusterization). Because coulombic effects significantly contribute both to viscosification of the bare mAb in solutions without PLE and to PLE:mAb association, it was however difficult to decipher the exact origin of fluidification. Present results point nevertheless to examples were fluidification was higher than what could be achieved by using a simple 1:1 salt such as sodium acetate or NaCl. It opens a yet poorly explored field of using poly(aminoacids) instead of small molecular excipients to modulate inter-mAb interaction in concentrated conditions. In formulation of mAbs, stability issues encountered with addition of simple salts, including irreversible mAb aggregation, shall not be overlooked. This motivates the interest for a diversity of viscocityreducing additives, and we accordingly believe that polyglutamate derivatives deserve studies along this line.

Supporting Information. Supporting Information is available free of charge at https:// $$. It contains a description of the PEGylation procedure of polyglutamates and characterization by 1H NMR and electrophoresis , representative autocorrelation functions from light scattering of

at 25 °C

 25 , and fitted with bare silica capillaries of either 50 μm or 75 μm internal diameter (Polymicro Technologies CM Scientific). The capillary was flushed daily with 0.1 M NaOH (5 min., 10 psi), followed by a water rinse. Bare silica capillary was used to analyze PLE derivatives in the absence of mAb. To analyze mAb-containing samples, the capillary was coated by flushing (5min. 2 psi) 1.5 g/L PLL(20)-g[3.5]-PEG5 (a comb-like PEGylated poly(lysine) of 20 kDa from Susos Dübendorf Switzerland) before being conditioned by flushing the running buffer (5mM

  photomultiplier detection. Effective relaxation time (bead) was calculated by fitting the field correlation functions to a biexponential decay model (using nonlinear fitting of y = A1*exp(-x/t1) + A2*exp(-x/t2) + y0 in the software OriginPro 2022 from Origin Lab Corp.). The slower decay mode was ascribed to beads, whereas the faster one was ascribed to the mAb. The effective viscosity then reads:

Figure 1 .

 1 Figure 1. Representative electropherograms of mAb:polyglutamate mixtures at pH 6.3, 1 g/L mAb and concentration of polyglutamate varied between 0.05 g/L and 0.5 g/L. A, B) 50 mm ID capillary and 5mM Histidine + 10% Sucrose buffer, application of +/-12kV, A) anode at the outlet, i.e. free mAb migrating toward the detector. The solid blue line corresponds to the mAb solution without 45PLE. The dashed dotted lines are mAb + 45PLE mixtures as quoted. B) cathode at the outlet, i.e., free 45PLE migrating toward the detector. The solid lines correspond to the polymer with no mAb in the solution. The dashed dotted lines are mixed samples as quoted. C,D) 75 mm ID capillary and 5mM Na-phosphate buffer, application of +/-20kV + 0.1 psi pressure and x-axis calculated as the electrophoretic mobility.

Figure

  Figure 2A compares the binding efficacy of 45PLE and 45PLEL in 5mM phosphate buffer pH

  8-1.2 in the case of 45PLE. Similarly, the lowest R values were in the window of 0.75-1.25 with 45PLE-10PEG3 or 45PLEL-10PEG3 suggesting a mAb/polymer ratio of 2:1 -1:1 mol/mol association window in equilibrium with measurable unbound mAb. Interestingly, the 45PLE isotherm in Fig 2A was fitted to a conventional binding equilibrium with a binding constant K=[45PLE]free.[mAb]free/[mAb]bound.

Figure 2 .

 2 Figure 2. Association of mAb with 45PLE or 45PLEL as measured by FACCE in mixtures of 1g/L mAb, in 5mM phosphate buffer pH 6.3 and varying polymer concentration between 0.01 g/L and 0.5 g/L. (A) Free (unbound) mAb as a function of total (bound and unbound) polyglutamate chains. Dashed line shows a fit by a model of 1:1 complex with binding constant K = 4.06 M , dotted line shows a fit by a model of 2:1 complex with binding constant K' = 1.27 M 2 (see text); (B) Binding ratio R of bound polymer / bound mAb vs the total concentration polymer in the same samples.

Figure 3 .

 3 Figure 3. Viscosity of mAb in Histidine-HCl buffer pH 6.3 + 10% sucrose at 25°C (A) 20mM Histidine

Figure 4 .

 4 Figure 4. Viscosity of 140 g/L mAb solutions as measured by DLS method in 5 mM Histidine-HCl

Figure 5 .

 5 Figure 5. Viscosity measured by DLS of 200 g/L mAb solutions in 5 mM Histidine-HCl pH

Table 1 .

 1 Compositions of two-phases mixtures formed by addition of 45PLE or 45PLEL in a 20 g/L mAb solution in 5 mM phosphate-NaOH buffer pH 6.3.

			45PLE			45PLEL
	Polyglutamate	% mAb	[mAb]supernatant	[mAb]	% mAb	[mAb]supernatant	[mAb]
	total	dense phase	g/L	dense phase	dense phase	g/L	dense phase
	g/L			g/L			g/L
	0.1	18%	17.7±0.1	49±4	23%	16.6±0.2	65±5
	0.2	31%	15.3±0.3	62±4	58%	9.3±0.2	105±7
	0.35	64%	8.3±0.3	83±5	93%	1.8±0.1	108±8
	0.5	73%	5.9±0.2	177±16	92%	2.0±0.1	128±10
	1	59%	8.7±0.3	177±17	66%	7.4±0.2	156±15

Table 2 .

 2 Viscosity in the presence of sodium acetate or PLE additives of 140g/L mAb in 10% sucrose, 5mM Histidine buffer pH 6.3 for (a) commercial PLE and (b) PEG-modified PLE. For reference viscosity with no additives and no NaCl = 20.2 mPa.s, or 50mM NaCl = 9.0 mPa.s. n.d.Liquid-liquid phase separation as observed at 20 g/L mAb could also occur at 140 g/L mAb. At pH 6.3, this was the case of the mixture with 3.5 g/L 45PLEL (no NaCl) that formed a dense phase of high viscosity (Table2, Fig. SI.6. in SI) dispersed in a very fluid (1.6 mPa.s) "light" phase. The dispersion was not stable enough to enable viscosity measurement of the full mixture. But as suggested by Shiraki and coll.[START_REF] Tsumura | Lowering the viscosity of a high-concentration antibody solution by protein-polyelectrolyte complex[END_REF] , emulsion-like dispersions of droplets of a dense phase of mAbs can fulfill practical requirements of injectability. The 45PLE at 3.5 g/L + mAb mixture at pH 6.3 was opalescent, suggesting the onset of clusterization of mAbs with polyglutamate chains.

	= not determined.
	(a)
		Weight	Charge		Viscosity
		concentration	concentration of		(mPa.s)
		of PLE/PLE derivative (g/L)	PLE or sodium (mM) acetate	acetate Sodium	45PLE	45PLEL
	Without	3.5	21	11.0	8.4	2 phases
	NaCl					1.6 / 24.2
		6.5	40	8.0	6.6	9.0
	With 50 mM	3.5	21	9.2	7.2	6.6
	NaCl	6.5	40	8.2	9.0	10.5
	(b)
		Weight	Charge		Viscosity
		concentration	concentration of		(mPa.s)
		of PLE/PLE derivative (g/L)	PLE or sodium acetate (mM)	Sodium acetate	45PLE-10PEG3	45PLEL-10PEG3
	Without	3.5	7.7	16.7	12.6	16.0
	NaCl	6.5	14.5	15.4	15.1	13.6
	With 50 mM	3.5	7.7	n.d.	10.7	10.4
	NaCl	6.5	14.5	n.d.	9.8	nd

near zero mobility suggesting sligthly negative complexes (ex: mobilities between 0 and -5 10 -5 cm 2 .V -1 .s -1 in Fig.1D). The jump of absorbance associated to the detection of the mAb:PLE complexes was close to the time of electrosmotic flow, and broad enough to reach the zero axis.Accordingly, complexes between mAb and 45PLE(L) likely have compositions near charge cancellation.

Funding Sources

The work was supported by SANOFI research grant iTech award to C. Tribet, ANR Labex "Dynamo" ANR-11-LABX-0011-01, and ANR Fliposomes 20 CE06-007. ABBREVIATIONS FACCE, frontal analysis continuous capillary electrophoresis ; CV, capillary viscosimetry; DLS, dynamic light scattering ;

.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.