Loss of fatty acid binding protein 3 ameliorates lipopolysaccharide-induced inflammation and endothelial dysfunction.

Circulating fatty acid-binding protein 3 (FABP3) is an effective biomarker of myocardial injury and peripheral artery disease (PAD). The endothelium, which forms the inner most layer of every blood vessel, is exposed to higher levels of FABP3 in PAD or following myocardial injury, but the pathophysiological role of endothelial FABP3, the effect of FABP3 exposure on endothelial cells, and related mechanisms are unknown. Here, we aimed to evaluate the pathophysiological role of endothelial FABP3 and related mechanisms in vitro. Our molecular and functional in vitro analyses show that (1) FABP3 is basally expressed in endothelial cells; (2) inflammatory stress in the form of lipopolysaccharide (LPS) upregulated endothelial FABP3 expression; (3) loss of endogenous FABP3 protected endothelial cells against LPS-induced endothelial dysfunction; however, exogenous FABP3 exposure exacerbated LPS-induced inflammation; (4) loss of endogenous FABP3 protected against LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling pathways. Together, these findings suggest that gain-of endothelial FABP3 exacerbates, whereas loss-of endothelial FABP3 inhibits LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling. We propose that an increased circulating FABP3 in myocardial injury or PAD patients may be detrimental to endothelial function, and therefore, therapies aimed at inhibiting FABP3 may improve endothelial function in diseased states.

When awareness is not a barrier to PrEP uptake among men who have sex with men who are eligible for PrEP in France.

Despite PrEP being available and free of charge in France, a gap remains between the estimated number of men who have sex with men (MSM) with high-risk exposure to HIV and the number of MSM PrEP users. The objective of this study is to identify factors associated with non-intention to use PrEP among PrEP-eligible and PrEP-aware MSM in France, "non-intenders".European MSM Internet Survey (EMIS)-2017 was a cross-sectional survey conducted among MSM concerning their HIV prevention needs. Logistic regression models were used to identify factors associated with "non-intenders".Compared to PrEP users, factors associated with non-intention to use PrEP were: age (aOR[95%CI] = 3.80[2.21;6.53]); not being vaccinated against hepatitis B (2.20[1.45;3.34]); self-efficacy (1.84[1.29;2.60]); lower knowledge about on-demand PrEP (11.48[7.37;17.87]) and daily PrEP (2.58[1.27;5.25]); not having a PrEP discussion at a hospital (12.39[8.90;17.27]) or at a community service/drop-in (4.93[3.48;6.97]); living in a department with few PrEP access points (1.70[1.10;2.63]).On-demand PrEP may meet the prevention needs of "non-intenders" who have lower HIV risk perception. Increasing communication from health providers and community health workers to all MSM is needed.

Rapid multi-attribute characterization of intact bispecific antibodies by a microfluidic chip-based integrated icIEF-MS technology.

Rapid, direct identification and quantitation of protein charge variants, and assessment of critical quality attributes with high sensitivity are important drivers required to accelerate the development of biotherapeutics. We describe the use of an enhanced microfluidic chip-based integrated imaged capillary isoelectric focusing-mass spectrometry (icIEF-MS) technology to assess multiple quality attributes of intact antibodies in a single run. Results demonstrate comprehensive detection of multiple charge variants of an aglycosylated knob-into-hole bispecific antibody. Upfront, on-chip separation by icIEF coupled to MS provides the orthogonal separation required to resolve and identify acidic posttranslational modifications including difficult-to-detect deamidation and glycation events at the intact protein level. In addition, on-chip UV detection enables pI determination and relative quantitation of charge isoforms. Six charge variant peaks were resolved by icIEF, mobilized toward the on-chip electrospray tip and directly identified by in-line icIEF-MS using a connected quadrupole time-of-flight mass spectrometer. In addition to acidic charge variants, basic variants were identified as C-terminal lysine, N-terminal cyclization, proline amidation, and the combination of modifications (not typically identified by other intact methods), including lysine and one or two hexose additions. Nonspecific chain cleavages were also resolved, along with their acidic charge variants, demonstrating highly sensitive and comprehensive intact antibody multi-attribute characterization within a 15-min run time.

NPAideS: a drug-checking study among 3-methylmethcathinone (3-MMC) users.

BACKGROUND: 3-methylmethcathinone (3-MMC) has been available on the European drug market for several years, but an increase in its availability seems to have occurred around 2020, associated with reports of harm and death. We aimed to analyze the composition of the supposed 3-MMC samples purchased and its concordance with the assumed composition of the drug. METHODS: A prospective multicenter (n = 6) study was conducted between February 2021 and September 2021 in Auvergne-Rhone-Alpes, France. The inclusion criteria were: 3-MMC users over 18 years of age in contact with a community-based organization (CBO) called AIDES. Consumption was evaluated with an anonymized questionnaire and samples of 3-MMC powder were analyzed with a combination of qualitative (GC-MS) and quantitative methods (UPLC-MS/MS), to compare the assumed and real compositions of the products purchased. RESULTS: We studied 45 samples provided by 33 users. The study population was predominantly male (91%), with a median age of 40 years, most were university graduates and regular users of 3-MMC. Intravenous drug use was reported by 15.2% of the population. Most of the users bought their 3-MMC online via the Clear Web. Drug testing was requested by 86% of the users, highlighting the need for this type of harm reduction strategy. The purity of the 3-MMC powder samples tested ranged from 21 to 98%. Other NPS drugs, such as 4-CEC (4-chloroethcathinone), 4-MMC, and 2-fluorodeschloroketamine (2-FDCK), supplied as methoxphenidine (MXP), were also detected. CONCLUSION: This prospective study shows that 3-MMC purity and dose vary considerably. It also describes the characteristics of 3-MMC users and their expectations of a drug-checking program. Our data suggest that drug-checking services may be useful in this population. Health associations and laboratories should work together to help increase access to such programs.

Non-disclosure of drug injection practices as a barrier to HCV testing: results from the PrebupIV community-based research study.

BACKGROUND: Hepatitis C virus (HCV) infection prevalence is particularly high in people who inject drugs (PWID), a population that faces many barriers to HCV testing and care. A better understanding of the determinants of access to HCV testing is needed to improve their engagement in the HCV care cascade. We used data from a cross-sectional survey of people who inject drugs, mainly opioids, to identify factors associated with recent HCV testing. METHODS: Self-reported data on HCV antibody testing were analyzed for 550 of the 557 PWID enrolled in PrebupIV, a French cross-sectional community-based survey which assessed PWID acceptability of injectable buprenorphine as a treatment. Factors associated with recent (i.e., in the previous six months) HCV antibody testing were identified performing multivariable logistic regression. RESULTS: Among the study sample, 79% were men and 31% reported recent HCV antibody testing. Multivariable analysis found that PWID who did not disclose their injection practices to anyone (aOR [95% CI] 0.31 [0.12,0.82], p = 0.018), older PWID (aOR [95% CI] 0.97 [0.95,1.00], p = 0.030) and employed respondents (aOR [95% CI] 0.58 [0.37,0.92], p = 0.019) were all less likely to report recent HCV testing. No association was found between opioid agonist therapy and HCV testing. CONCLUSIONS: Our findings suggest that non-disclosure of injection practices, employment and age were all barriers to HCV antibody testing. Preventing stigma around injection practices, developing the HCV testing offer in primary care and addiction care services, and training healthcare providers in HCV care management could improve HCV testing and therefore, the HCV care cascade in PWID.

[Unsafe practices fostering cutaneous abscesses in people who inject substances : Results from the ANRS-OUTSIDER study]. / Des pratiques à risque favorisant la survenue d'abcès cutanés chez les personnes qui injectent des substances : résultats de l'étude ANRS-OUTSIDER.

INTRODUCTION: Cutaneous abscesses are a common complication of intravenous substance use. Although these skin and soft tissue infections represent one of the main causes of emergency room visits and hospitalizations in people who inject drugs (PWID), data on their prevalence and causes are scarce. The present study was part of the larger ANRS-OUTSIDER project and aimed to study the factors associated with skin abscesses in PWID, focusing in particular on the different stages of injection. METHOD: Analyses were based on data of the 164 persons who regularly injected psychoactive substances participating in the French ANRS-OUTSIDER project. A face-to-face questionnaire collected data on sociodemographics, substance use, injection practices, and experience of skin abscesses in the previous six months. Factors associated with skin abscesses were studied using a logistic regression model. RESULTS: Of the 140 participants for whom abscess data were recorded at inclusion, 35% reported having a skin abscess in the previous six months. Factors associated with skin abscesses were: benefiting from universal health coverage (PUMA/CMU/AME) (Odd ratio (OR) = 0.28, confidence interval (CI) à 95%=0.08-0.99), finding the vein to inject by touch (OR=3.44, 95% CI=1.41-8.43) and licking the needle before injection (OR=5.16, 95% CI=1.10-24.30). CONCLUSION: Our results highlight that skin abscesses were very frequent among the complications observed in our sample of French PWID, and that certain injection practices fostered their occurrence. These data will provide stakeholders with useful information to improve prevention and harm reduction messages for PWID.

Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins.

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.

Impact of the early 2020 COVID-19 crisis and lockdown on PrEP use among men who have sex with men (MSM) in France.

OBJECTIVE: To describe changes in pre-exposure prophylaxis (PrEP) use during and following the COVID-19 lockdown in France (March-May 2020) and identify the factors associated with PrEP discontinuation among men who have sex with men (MSM) after the lockdown. METHODS: Data from the, an anonymous, cross-sectional internet survey among MSM in July 2020, were analysed. Among respondents who were using PrEP prior to the lockdown, a binary logistic regression model was used to compare participants who were still taking PrEP (current PrEP users) with those who were not taking PrEP at the time of the survey (former PrEP users). RESULTS: Among 8345 respondents, 946 were PrEP users before the lockdown, of whom 58.8% (n=556) reported stopping PrEP during the lockdown and 15.4% (n=146) were not using PrEP at the time of the survey. Among the 556 who stopped PrEP during lockdown, 86.5% (n=481) reported no sexual activity; 76.8% (n=427) restarted PrEP after lockdown. Former PrEP users were more likely to be younger, not living with a stable male sexual partner, report moderate anxiety, report increased psychoactive drug use during the lockdown, and report not having tested for HIV or STI since the end of the lockdown because they did not know where to go, preferred to wait or for another reason. Reporting fewer male sexual partners in the last 6 months was also significantly associated with being a former PrEP user. CONCLUSIONS: MSM adapted PrEP use to their sexual activity during and after the French lockdown. After the lockdown, discontinued PrEP occurred more often among MSM who had fewer sexual partners and had mental health vulnerabilities. These factors could also be predictive of PrEP discontinuation in a more general context. PrEP users should be informed on how to safely stop/start PrEP and on the use of other prevention tools to reduce potential risk exposure during PrEP discontinuation.

Étude qualitative de l'acceptabilité de la téléconsultation pour le traitement préventif du VIH.

Introduction : In France, the incidence of HIV infections remains at 6,000 infections a year. Pre-Exposure Prophylaxis (PrEP) could reduce this number. Despite available reimbursement since 2016 to exposed populations, the recipients remain mostly men who have sex with men (MSM) living in large cities. In Center-Val de Loire, where newly HIV diagnoses and difficulties of access to care are tremendous, community tele-support and dedicated teleconsultations have been launched to promote access, particularly in rural areas. This study aimed to identify the interests and limits of these teleconsultations for PreP users.Purpose of research : E-PrEP qualitative study conducted by semi-structured interviews with MSM PrEP users allowing the thematic analysis of eight interviews lasting an average of one hour, conducted with MSM taking at least one teleconsultation.Results : The teleconsultation met user expectations with good acceptability. It integrated naturally into the associated preventive care pathway where applicable. The description of a more impersonal face-to–face exchange is balanced by establishing a trusting relationship. Teleconsultation has sometimes eased talking about sexuality. An enhanced confidentiality could represent a specific motif to choose teleconsultation. Access to the specialist seems to be an asset compared to a GP follow-up hardly chosen for this type of consultation. Fears of lack of discretion or judgmental behavior from these unspecialized healthcare professionals were pronounced..Conclusions : The deployment of teleconsultations can be encouraged to facilitate access to PrEP with vigilance on the associated health path.

Factors associated with fear of COVID-19 infection in people living with HIV / Facteurs associés à la crainte de l'infection par la COVID-19 chez des personnes vivant avec le VIH

INTRODUCTION: People living with HIV (PLHIV) who may have experienced biographical disruptions in their life trajectory may have a vulnerability to risk that differs from the general population, particularly in the context of an infectious health crisis. This study aimed to understand the factors associated with concerns about being infected with COVID-19 among PLHIV during the first period of the health crisis. METHODS: This was an online cross-sectional study using an online self-administered questionnaire in the context of the COVID-19 epidemic in France among a population of PLHIV. The recruitment was done via social networks and through various actors in the fight against HIV. The self-questionnaire was available from July 2020 to September 2020. RESULTS: The ACOVIH study collected 249 responses, 202 men and 47 women, with a mean age of 46.6 ± 12.9 years. The most represented socio-professional categories were employees (n=73.29%), followed by managers, professionals and artists (n=59.24%). The PLHIV most worried about being infected by COVID-19 had a level of education lower than or equal to the baccalaureate, family difficulties related to HIV and a deterioration in the relationship of trust with the HIV medical team. CONCLUSION: Feelings of anxiety can have a health and psychosocial impact on PLHIV. It is necessary to consider these negative factors by proposing adapted support and by carrying out preventive actions aiming in particular at improving the literacy of the PLHIV.

Introduction: Les personnes vivant avec le VIH (PVVIH) ayant déjà vécu des perturbations biographiques dans leur trajectoire de vie peuvent avoir une vulnérabilité accrue aux risques, particulièrement dans le cadre d'une crise sanitaire infectieuse. La présente étude (« Adaptations à la COVID-19 lorsque l'on vit avec le VIH ¼ [ACOVIH]) vise à appréhender les facteurs associés à l'inquiétude d'être infecté par la COVID-19 chez les PVVIH durant la première période de la crise sanitaire. Méthodes: Il s'agissait d'une étude transversale en ligne par questionnaire autoadministré conduite dans le contexte épidémique de la COVID-19 sur le territoire français auprès d'une population de PVVIH. Les participants ont été recrutés via les réseaux sociaux et par le biais de divers acteur·trice·s de la lutte contre le VIH. L'autoquestionnaire était disponible de juillet 2020 à septembre 2020. Résultats: L'étude ACOVIH a recueilli 249 réponses, 202 hommes et 47 femmes, d'un âge moyen de 46,6 ± 12,9 ans. Les catégories socioprofessionnelles les plus représentées étaient les employés (n = 73, 29 %), suivis des cadres et des professions intellectuelles et artistiques (n = 59, 24 %). Les PVVIH les plus inquiètes à l'idée d'être infectées par la COVID-19 ont un niveau d'études inférieur ou égal au baccalauréat, des difficultés familiales en lien avec le VIH et une dégradation de la relation de confiance vis-à-vis de l'équipe médicale VIH. Conclusion: Le sentiment d'inquiétude peut avoir un impact sanitaire et psychosocial chez les PVVIH. Il est nécessaire de considérer ces facteurs négatifs en proposant un accompagnement adapté et en menant des actions préventives visant notamment à améliorer la littératie des PVVIH.

La recherche communautaire en temps de pandémie : retour sur l'étude multi-pays EPIC.

In 2020, the COVID-19 health crisis affected all regions of the world, not sparing people already vulnerable to other viral epidemics such as HIV or HCV and/or those in precarious or socially marginalized situations. This is particularly the case for drug users or sex workers.Coalition PLUS, a network of associations fighting against HIV and viral hepatitis which defends and promotes the community-based approach, and its partners, have set up a multi-country community-based research project aimed at documenting the impact of the health crisis on the fight against HIV and viral hepatitis (key populations and community workers/activists), as well as the community responses put in place (EPIC survey).The objective of this paper is to reflect on the implementation of this community-based research study during the COVID-19 health crisis, and in particular the unforeseen difficulties to which the community-based research process had to confront and adapt. The goal is to draw lessons on what worked (and what did not work) in order to capitalize on community-based research practices during this pandemic and subsequently, facilitate the implementation of new research projects in similar contexts.

Interaction and conflict between outreach workers and research officers in implementing collaborative action research in the field of harm reduction: a qualitative study.

BACKGROUND: The effectiveness of collaborative approaches in health interventions is underlined in the literature. Given the serious challenges to adequately managing the HCV epidemic in people who inject drugs (PWID), and the need to improve existing harm reduction (HR) interventions in this population, it seems important to investigate how collaboration between stakeholders is ensured in action research interventions. The present study aimed to explore interactions between outreach workers and research officers collaborating in the implementation of an action research project for PWID entitled OUTSIDER. METHODS: Using three focus groups, we studied the views of 24 outreach workers involved in the implementation and evaluation of a harm reduction educational intervention to help PWID inject more safely in off-site settings. RESULTS: The analysis of participants' discourses highlighted the mixed perceptions they had about OUTSIDER. Several limitations to collaboration emerged. Epistemological (theoretical vs. practical knowledge), methodological (science vs. intervention), axiological (standardised vs. adapted approach), and material (mobilised vs. available resources) issues all placed a burden on the outreach worker-research officer relationship. Outreach workers' acceptance of the project's intervention dimension but rejection of its scientific dimension highlights a lack of contractualisation between the stakeholders involved, and a more general problematisation of the role of outreach workers in implementing action research in HR. How collaboration was perceived and practised by outreach workers participating in OUTSIDER can be considered a reflection of the current challenges to implementing action research in HR. CONCLUSION: This study of the interaction between the research and implementation dimensions of an action research project explored the tensions between different intervention stakeholders that must work together. Equitable participation and integration of the expertise, practices, and knowledge of all stakeholders involved is essential for successful action research. Given current HCV epidemiological challenges, new forms of cooperation are needed when developing healthcare services and when strengthening collaborative approaches.

Fast, Robust, and Sensitive Identification of Residual Host Cell Proteins in Recombinant Monoclonal Antibodies Using Sodium Deoxycholate Assisted Digestion.

Residual host cell proteins (HCPs) present in biotherapeutics can pose potential safety risks for patients or affect product stability, thus prompting a critical need to monitor HCPs in drug substance or product to ensure product safety and quality. Current approaches for robust HCP identification at or above 10 ppm levels require either concatenated peptide fractionation or enrichment via antibody depletion, which challenges the direct quantitation of HCPs. This paper describes a simple, fast sample preparation method without the need for sample fractionation or enrichment; instead, we utilize trypsin-friendly sodium deoxycholate (SDC) as an advantageous denaturant that can be effectively removed following acidification at the end of sample digestion. This new approach enables the end-to-end one-dimensional liquid chromatography-tandem mass spectrometry (1D LC-MS/MS) workflow (i.e., from sample preparation to HCP identification) to be completed in 7-8 h while demonstrating the ability to consistently identify HCPs across a broad molecular weight range at 10 ppm or above.

Probing the Impact of the Knob-into-Hole Mutations on the Structure and Function of a Therapeutic Antibody.

Bispecific antibodies (BsAbs) have drawn increasing interest in the biopharmaceutical industry due to their advantage to bind two distinct antigens simultaneously. The knob-into-hole approach is an effective way to produce bispecific antibodies by driving heterodimerization with mutations in the CH3 domain of each half antibody. To better understand the conformational impact by the knob and hole mutations, we combined size-exclusion chromatography (SEC), differential scanning calorimetry (DSC), and hydrogen-deuterium exchange mass spectrometry (H/D exchange MS), to characterize the global and peptide-level conformational changes. We found no significant alteration in structure or conformational dynamics induced by the knob-into-hole framework, and the conformational stability is similar to the wild-type (WT) IgG4 molecules (except for some small difference in the CH3 domain) expressed in E. coli. Functional studies including antigen-binding and neonatal fragment crystallizable (Fc) receptor (FcRn) binding demonstrated no difference between the knob-into-hole and WT IgG4 molecules in E. coli.

Are PrEP services in France reaching all those exposed to HIV who want to take PrEP? MSM respondents who are eligible but not using PrEP (EMIS 2017).

Pre-exposure prophylaxis (PrEP) is fully reimbursed by the French health insurance system since 2016. However, uptake of PrEP is slower than expected and little is known about men who have sex with men (MSM) who are eligible for PrEP according to French guidelines, but not using it. This study aims to (1) assess and describe MSM that are eligible to PrEP but not using it, and (2) identify potential individual and structural barriers of PrEP uptake among eligible MSM who are aware and intend to take PrEP. Data from EMIS-2017, a cross-sectional internet survey among gay, bisexual, and other MSM, were used. Among 7965 respondents without diagnosed HIV, 9.2% were PrEP users. Among 7231 non-PrEP users, 35.2% were eligible to PrEP and 15.2% were eligible, aware and intended to take PrEP. Eligible MSM who are not using PrEP are mostly younger, students, less "out", living in small cities, using condoms more frequently but still with low self-efficacy regarding safe sex and more distant from preventive health care and information than PrEP users. Despite free PrEP availability in France, results suggest that PrEP is not fully accessible and that there is a need to increase PrEP demand and decentralize PrEP delivery.

A 2D LC-MS/MS Strategy for Reliable Detection of 10-ppm Level Residual Host Cell Proteins in Therapeutic Antibodies.

Methodologies employing LC-MS/MS have been increasingly used for characterization and identification of residual host cell proteins (HCPs) in biopharmaceutical products to ensure their consistent product quality and safety for patients. To improve the sensitivity and reliability for HCP detection, we developed a high pH-low pH two-dimensional reversed phase LC-MS/MS approach in conjunction with offline fraction concatenation. Proof-of -concept was established using a model in which seven proteins spanning a size range of 29-78 kDa are spiked into a purified antibody product to simulate the presence of low-level HCPs. By incorporating a tandem column configuration and a shallow gradient through the second-dimension, all seven proteins were consistently identified at 10 ppm with 100% success rate following LC-MS/MS analysis of six concatenated fractions across multiple analysts, column lots and injection loads. Using the more complex Universal Proteomic Standard 1 (UPS-1) as an HCP model, positive identification was consistently achieved for 19 of the 22 proteins in 8-12 ppm range (10 ppm ±20%). For the first time, we demonstrate an effective LC-MS/MS strategy that not only has high sensitivity but also high reliability for HCP detection. The method performance has high impact on pharmaceutical company practices in using advanced LC-MS/MS technology to ensure product quality and patient safety.

Multi-site N-Glycan mapping study 2: UHPLC.

In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N-glycosylation analysis using capillary electrophoresis of APTS-labeled N-glycans were presented. The corresponding results from ultra-high performance liquid chromatography (UHPLC) with fluorescence detection are presented here from 12 participating sites. All participants used the same lot of samples, reagents, and columns to perform the assays. Elution time, peak area and peak area percent values were determined for all peaks ≥0.1% peak area, and statistical analysis was performed following ISO 5725-2 guideline principles. The results demonstrated adequate reproducibility, within any given site as well across all sites, indicating that standard UHPLC-based N-glycan analysis platforms are appropriate for general use.

Absolute Quantitation of Intact Recombinant Antibody Product Variants Using Mass Spectrometry.

Accurate and precise quantitative measurement of product-related variants of a therapeutic antibody is essential for product development and testing. Bispecific antibodies (bsAbs) are Abs composed of two different half antibody arms, each of which recognizes a distinct target, and recently they have attracted substantial therapeutic interest. Because of the increased complexity of its structure and its production process, as compared to a conventional monoclonal antibody, additional product-related variants, including covalent and noncovalent homodimers of half antibodies (hAbs), may be present in the bsAb product. Sufficient separation and reliable quantitation of these bsAb homodimers using liquid chromatography (LC) or capillary electrophoresis-based methods is challenging because these homodimer species and the bsAb often have similar physicochemical properties. Formation of noncovalent homodimers and heterodimers can also occur. In addition, since homodimers share common sequences with their corresponding halves and bsAb, it is not suitable to use peptides as surrogates for their quantitation. To tackle these analytical challenges, we developed a mass spectrometry-based quantitation method. Chip-based nanoflow LC-time-of-flight mass spectrometry coupled with a standard addition approach provided unbiased absolute quantitation of these drug-product-related variants. Two methods for the addition of known levels of standard (multi- or single-addition) were evaluated. Both methods demonstrated accurate and reproducible quantitation of homodimers at the 0.2% (w/w) level, with the single-addition method having the promise of higher analytical throughput.

Interlaboratory study to evaluate the robustness of capillary electrophoresis-mass spectrometry for peptide mapping.

A collaborative study on the robustness and portability of a capillary electrophoresis-mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent laboratories from academia and industry. All participants used the same batch of samples, reagents and coated capillaries to run their assays, whereas they utilized the capillary electrophoresis-mass spectrometry equipment available in their laboratories. The equipment used varied in model, type and instrument manufacturer. Furthermore, different types of sheath-flow capillary electrophoresis-mass spectrometry interfaces were used. Migration time, peak height and peak area of ten representative target peptides of trypsin-digested bovine serum albumin were determined by every laboratory on two consecutive days. The data were critically evaluated to identify outliers and final values for means, repeatability (precision within a laboratory) and reproducibility (precision between laboratories) were established. For relative migration time the repeatability was between 0.05 and 0.18% RSD and the reproducibility between 0.14 and 1.3% RSD. For relative peak area repeatability and reproducibility values obtained were 3-12 and 9-29% RSD, respectively. These results demonstrate that capillary electrophoresis-mass spectrometry is robust enough to allow a method transfer across multiple laboratories and should promote a more widespread use of peptide mapping and other capillary electrophoresis-mass spectrometry applications in biopharmaceutical analysis and related fields.