2020 White Paper on Recent Issues in Bioanalysis: BAV Guidance, CLSI H62, Biotherapeutics Stability, Parallelism Testing, CyTOF and Regulatory Feedback (Part 2A - Recommendations on Biotherapeutics Stability, PK LBA Regulated Bioanalysis, Biomarkers Assays, Cytometry Validation & Innovation Part 2B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.

Effectiveness of a normative nutrition intervention in Chilean pregnant women on maternal and neonatal outcomes: the CHiMINCs study.

BACKGROUND: Some nutritional interventions have shown their efficacy in reducing gestational weight gain (GWG); however, their applicability in routine care is limited. OBJECTIVE: We assessed the effectiveness of a low-intensity and high-coverage nutritional intervention on maternal and offspring outcomes; the intervention enhanced existing nutritional health care standards and practices at the primary health care level in Chile. METHODS: This study was a cluster-randomized controlled trial of 12 primary health care centers (PHCCs) from Santiago, Chile. PHCCs were randomly allocated to either nutritional intervention [intervention group (IG), n = 5] or routine care [control group (CG), n = 7]. A total of 4631 pregnant women were recruited (IG, n = 2565; and CG, n = 2066). Primary outcomes were adequate GWG and glycemic control in mothers and birth weight, birth length, macrosomia, and large for gestational age in neonates. The intervention consisted of 4 key actions: training of health care professionals on nutritional recommendations, counseling of pregnant women on diet and physical activity recommendations, offering a physical activity program implemented in the participating PHCCs, and adequate referral to dietitians. Women randomly assigned to the CG received routine antenatal care. RESULTS: At baseline, the mean age was 26.1 y; 45% of women were primipara and 24% were obese. No differences were found in the percentage of women achieving adequate GWG (IG: 30.3%, compared with CG: 31.3%; OR: 0.94; 95% CI: 0.81, 1.09), but women in the IG had lower GWG than those in the CG (11.3 compared with 11.9 kg; mean difference: -0.63 kg; 95% CI: -1.19, -0.08). Effects of the intervention were significantly higher in women with obesity at the begining of pregnancy (mean difference: -1.24 kg; 95% CI: -2.18, -0.30; P for interaction < 0.05). No differences were found between groups regarding maternal glycemic control or neonatal outcomes. CONCLUSIONS: Our findings demonstrate that a low-intensity, high-coverage intervention delivered through the Chilean public health care system under standard operating conditions reduces GWG and has the potential for successful scale-up. This trial was registered at clinicaltrials.gov as NCT01916603.

Development of a Highly Specific Anti-drug Antibody Assay in Support of a Nanoparticle-based Therapeutic.

PEGylated biotherapeutics can elicit anti-PEG (polyethylene glycol) immune responses in patients treated with this category of drugs. While anti-PEG antibody assays for this class of biotherapeutics have become a common element of the clinical immunogenicity testing strategy, the overall antibody incidence induced by the nanoparticle (NP) delivery system (such as ACCURINS®) has not been fully studied to date. To support the immunogenicity assessment of one of Pfizer's NP-based therapeutics, consisting of gedatolisib (GEDA) encapsulated in ACCURINS® (GEDA-NP), we developed an anti-GEDA-NP antibody (ADA) assay on the MSD platform for the detection of GEDA-NP induced ADA in human serum. The focus of our strategy was on developing a clinically relevant ADA assay and systematically addressing assay interference through rigorous assay optimization. Our efforts led to a fit-for-purpose assay for the detection of anti-GEDA-NP ADA in serum samples obtained from breast cancer patients. Results from method qualification indicated robust assay performance, as highlighted by inter and intra-assay precision within 25% CV for all controls, and reproducible response profiles across multiple runs during the assessment of assay cut points with breast cancer samples. The assay sensitivity was between 4.3 ng/mL and 123 ng/mL for surrogate positive controls of IgG and IgM isotypes, respectively. Additionally, assay interference from nonspecific matrix proteins and circulating drug was addressed, which ensured accurate assessment of ADA incidence that can be attributed to GEDA-NP.

The joint use of life cycle assessment and data envelopment analysis methodologies for eco-efficiency assessment: A critical review, taxonomy and future research.

Eco-efficiency has become a cornerstone for any company that seeks to improve their environmental and economic aspects. In this context, the joint use of Life Cycle Assessment and Data Envelopment Analysis, known as LCA + DEA methodology, is an emerging and growing line of research. LCA estimates the environmental impacts of the products or services, while DEA evaluates their efficiency, providing targets and benchmarks for the inefficient ones. In this way, both the environmental and economic aspects are considered in the eco-efficiency assessment. Since LCA + DEA methodology is a novel research line, a literature review is necessary to depict its full scope and to support researchers and practitioners. This manuscript presents the first comprehensive and structured literature review of the joint use of LCA and DEA for eco-efficiency assessment. We propose a taxonomy for the reviewed articles based on the theoretical and practical issues of LCA + DEA methodology and classify them accordingly. This classification allows recognizing and discussing the main findings, which offer some managerial implications for professionals who want to start employing this methodology. In addition, a procedure for selecting a suitable method is proposed and the main limitations and research opportunities are identified. Finally, this review could be a starting point and a guide for systematically building knowledge about the in the joint use of LCA and DEA for eco-efficiency assessment.

Anti-drug Antibody Assay Conditions Significantly Impact Assay Screen and Confirmatory Cut-Points.

Assays for the detection and confirmation of anti-drug antibodies (ADA) are commonly used tools for assessing the immunogenicity of drug candidates in both clinical and nonclinical studies. During the development of such assays, it is typical to optimize the assay conditions based on factors such as sensitivity or signal/noise ratio (S/N) and is commonly done using an assay positive control (PC). However, even carefully optimized methods often suffer with problems due to low cut-point factors and failure to distinguish assay "noise" from a true biological response. In this paper, we describe an approach to assay development in which the impacts of assay conditions on the response and variability, both analytical and biological, of drug-naïve samples are tested by way of PC-independent assay condition optimization. Using two ADA methods as model systems, we examine the impact of minimum required dilution, assay reagent (labeled drug) concentrations, incubation time, assay, and wash buffer composition. We find that the choice of assay conditions, particularly the labeled drug concentration, can greatly affect the distribution of naïve sample responses and thus impact screening and confirmatory assay cut-points. In two case studies presented, screening assay cut-point (SCP) varied from 1.38 to 2.20 and 1.04 to 1.20 while the confirmatory assay cut-point (CCP) varied from 58.5 to 95.6% and 26.2 to 16.2% depending on the conditions tested. Some of the conditions produced unacceptably high CCP values. It is proposed that the degree of the observed impact of the assay conditions on SCP and CCP values depends on the compound nature and assay matrix composition and is likely connected with the diversity of interactions between drug protein and matrix components. Because it was also observed that higher assay SCP can associate with a loss of the PC-based assay sensitivity, additional assessment of the assay conditions would be required to determine an overall assay performance acceptability, including assay PC-based sensitivity, drug, and target tolerance characteristics. In conclusion, it is suggested that by assessing performance of treatment-naïve samples at various assay conditions, one can identify potential assay protocols that allow to avoid undesirably low screening (e.g., < 1.2) and confirmatory (e.g., < 25%) cut-points.

Comparing two CF+DEA methods for assessing eco-efficiency from theoretical and practical points of view.

Nowadays, sustainability has become of increasing concern in policy and the decision making of stakeholders. Companies have been increasing their attention to their environmental performance. In order to measure sustainability performance, different approaches have been proposed. The joint application of Life Cycle Assessment (LCA) and Data Envelopment Analysis (DEA), called LCA+DEA, is used to assess eco-efficiency, which is to produce more with less environmental impact and with fewer resources. In this manuscript, we compare two methods that implement the LCA+DEA approach, the five-step and four-step methods, focused on Carbon Footprint (CF), called CF+DEA. These methods have not been compared previously. This comparison will encompass the theoretical and practical points of view according to efficiency indices, best practices, and targets for the CF emissions. To perform this comparison, we use a case study of raspberry producers in Chile. From a practical point of view, we have observed that both methods have achieved the main objective of reducing CF. Moreover, results show similar eco-efficiency scores; the targets given by the five-step method are less demanding than those given by the four-step method. In this sense, the four-step method provides a higher average CF reduction. This is due to the inclusion of CF as an undesirable output in the DEA assessment and the use of an output-oriented DEA model. Additionally, in including the CF within the DEA assessment, the four-step method reflects better the definition of eco-efficiency. Following these results, it is advisable to implement the targets provided by the five-step method for a short term and then the targets provided by the four-step method for a mid-term or long term.

Effectiveness on maternal and offspring metabolic control of a home-based dietary counseling intervention and DHA supplementation in obese/overweight pregnant women (MIGHT study): A randomized controlled trial-Study protocol.

BACKGROUND: Lifestyle interventions are the primary prevention strategy for gestational diabetes (GDM) in obese/overweight women; however, these interventions have shown limited effectiveness. Omega-3 polyunsaturated fatty acids (PUFAs) intake has shown beneficial effects on glucose metabolism, lipid fractions and inflammatory factors in women who already have GDM. Combining PUFAs supplementation with a lifestyle intervention could achieve lower increase of glucose levels by improving insulin sensitivity. Our aim is to assess two prenatal nutritional interventions (home-based dietary counseling and/or docosahexaenoic acid (DHA) supplementation) delivered to obese/overweight women during pregnancy for them and their offspring to achieve better metabolic control. METHODS/DESIGN: Randomized controlled trial, 2 × 2 factorial design. Eligible pregnant women will be randomly allocated to one of the four parallel arms: 1) Home-based dietary counseling +800 mg/day DHA supplementation (n = 250); 2) 800 mg/day DHA (n = 250); 3) Home-based dietary counseling +200 mg/day DHA (n = 250); 4) 200 mg/day DHA (n = 250). Primary outcomes are: GDM; macrosomia; and neonatal insulin resistance. Data analyses will be done on an intention-to-treat basis. DISCUSSION: We expect the present study to contribute to the understanding of the potential effectiveness of an omega-3 supplementation on the risk of developing GDM in overweight/obese pregnant women. We will also test if the combination of having better dietary habits alongside with omega 3 supplementation will improve insulin sensitivity and as consequence, a lower elevation of glucose levels could be achieved. TRIAL REGISTRATION: NCT02574767.

Agenesis of the corpus callosum, developmental delay, autism spectrum disorder, facial dysmorphism, and posterior polymorphous corneal dystrophy associated with ZEB1 gene deletion.

We report on a girl diagnosed prenatally with agenesis of the corpus callosum (ACC) on fetal ultrasound and MRI. On postnatal follow-up she was noted to have developmental delay, facial dysmorphism, autism spectrum disorder, and posterior polymorphous corneal dystrophy (PPD). Array-comparative genomic hybridization analysis (Array-CGH) showed a 2.05 Mb de novo interstitial deletion at 10p11.23p11.22. The deleted region overlaps 1 OMIM Morbid Map gene, ZEB1 (the zinc finger E-box binding homeobox transcription factor 1), previously associated with posterior polymorphous corneal dystrophy type 3 (PPCD3). To our best knowledge this is the first reported case with a deletion of the ZEB1 gene in an individual with ACC and PPD, showing that the haploinsufficiency of the ZEB1 is likely the cause of our patient's phenotype.

Association between prepregnancy obesity and metabolic risk in Chilean premenopausal women 10 y postpartum.

OBJECTIVES: One of every four pregnant women in Chile is obese. Gestational obesity is associated with maternal metabolic complications in pregnancy (e.g., gestational diabetes, preeclampsia), but to our knowledge, there is little evidence on relationships with future metabolic risk. The aim of this study was to evaluate the association between prepregnancy obesity (prepregnancy body mass index ≥30 kg/m2) or excessive gestational weight gain (GWG; according to the 2009 recommendations from the Institute of Medicine), and maternal metabolic complications 10 y postpartum in premenopausal Chilean women. METHODS: A prospective study was conducted. In 2006, 1067 Chilean mothers of children born in 2002-participants of the GOCS (Growth and Obesity Cohort Study)-were recruited. Mothers completed a questionnaire concerning sociodemographic, anthropometric, and pregnancy characteristics. Of the sample, 402 women were randomly selected to participate in a study related to the determinants of breast cancer risk in 2012. At follow-up, anthropometry, blood pressure, and fasting labs were measured. Complete data was available for 366 women. RESULTS: Thirty-two percent of mothers had prepregnancy overweight/obesity and 39.1% had excessive GWG. In adjusted models, prepregnancy obesity was positively associated with increased insulin resistance (odds ratio [OR], 18; 95% confidence interval [CI], 5.2-62.7), metabolic syndrome (OR, 3.3; 95% CI, 1.3-8.3), and hyperglycemia (OR, 3; 95% CI, 1.1-8.6). Prepregnancy overweight/obesity was associated with increased risk for insulin resistance, metabolic syndrome, abdominal obesity, low high-density lipoprotein cholesterol, and hypertriglyceridemia (P < 0.05). Excessive GWG was not associated with metabolic risk in the main model but was found to be positively associated in models with correction of weight by possible recall bias. CONCLUSIONS: Gestational obesity was associated with maternal metabolic alterations 10 y postpartum. Prevention strategies for chronic diseases should consider prepregnancy obesity as a modifiable risk factor for future metabolic health.

Joint carbon footprint assessment and data envelopment analysis for the reduction of greenhouse gas emissions in agriculture production.

Operations management tools are critical in the process of evaluating and implementing action towards a low carbon production. Currently, a sustainable production implies both an efficient resource use and the obligation to meet targets for reducing greenhouse gas (GHG) emissions. The carbon footprint (CF) tool allows estimating the overall amount of GHG emissions associated with a product or activity throughout its life cycle. In this paper, we propose a four-step method for a joint use of CF assessment and Data Envelopment Analysis (DEA). Following the eco-efficiency definition, which is the delivery of goods using fewer resources and with decreasing environmental impact, we use an output oriented DEA model to maximize production and reduce CF, taking into account simultaneously the economic and ecological perspectives. In another step, we stablish targets for the contributing CF factors in order to achieve CF reduction. The proposed method was applied to assess the eco-efficiency of five organic blueberry orchards throughout three growing seasons. The results show that this method is a practical tool for determining eco-efficiency and reducing GHG emissions.

Effectiveness of a normative nutrition intervention (diet, physical activity and breastfeeding) on maternal nutrition and offspring growth: the Chilean maternal and infant nutrition cohort study (CHiMINCs).

BACKGROUND: Maternal obesity before and during pregnancy predicts maternal and infant risks of obesity and its associated metabolic conditions. Dietary and physical activity recommendations during pregnancy as well as weight monitoring are currently available in the Chilean primary health care system. However some of these recommendations are not updated and most of them are poorly implemented. We seek to assess the effectiveness of an intervention that enhances the implementation of updated nutrition health care standards (diet, physical activity, and breastfeeding promotion) during pregnancy on maternal weight gain and infant growth. DESIGN & SETTING: Cluster randomized controlled trial. The cluster units will be 12 primary health care centers from two counties (La Florida and Puente Alto) from the South-East Area of Santiago randomly allocated to: 1) enhanced nutrition health care standards (intervention group) or 2) routine care (control group). PARTICIPANTS: Women seeking prenatal care before 15 weeks of gestation, residing within a catchment area of selected health centers, and who express that they are not planning to change residence will be invited to participate in the study. Pregnant women classified as high risk according to the Chilean norms (i.e age <16 or >40 years, multiple gestation, pre-gestational medical conditions, previous pregnancy-related issues) and/or underweight will be excluded. INTERVENTION: Pregnant women who attend intervened health care centers starting at their first prenatal visit will receive advice regarding optimal weight gain during pregnancy and diet and physical activity counseling-support. Pregnant women who attend control health clinics will receive routine antenatal care according to national guidelines. We plan to recruit 200 women in each health center. Assuming a 20% loss to follow up, we expect to include 960 women per arm. MAIN OUTCOME MEASURES: 1) Achievement of adequate weight gain based on IOM 2009 recommendations and adequate glycaemic control at 24-28 weeks of pregnancy according to ADA 2011, and 2) healthy infant growth during the first year of age based on WHO standards. DISCUSSION: We expect that the intervention will benefit the participants in achieving adequate weight gain & metabolic control during pregnancy as well as adequate infant growth as a result of an increased impact of standard nutrition and health care practices. Gathered information should contribute to a better understanding of how to develop effective interventions to halt the maternal obesity epidemic and its associated co-morbidities in the Chilean population. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT01916603.

Cyclic strabismus in adults.

Cyclic strabismus is a rare condition that usually occurs in children and is characterized by alternating intervals of straight and strabismic eyes. In adults with the condition, strabismus surgery often eliminates the cycles. We report a case of adult-onset cyclic esotropia that was converted into a cyclic exotropia.

Monitoring cellular accumulation of 3'-deoxy-3'-fluorothymidine (FLT) and its monophosphate metabolite (FLT-MP) by LC-MS/MS as a measure of cell proliferation in vitro.

Accurate measurement of in vitro cell growth is critical for oncology drug development, but cell counting and the most accurate indirect proliferation assays are impractical. Here, we describe a robust alternative method that monitors proliferating cell thymidine kinase 1 (TK1) activity via LC-MS/MS quantification of 3'-deoxy-3'-fluorothymidine (FLT) and its monophosphate metabolite FLT-MP. LNCaP prostate cancer cells were cultured at four densities (20,000; 10,000; 5000; and 500 cells/well) and incubated with 2000 ng/mL FLT in multi-well plates. Internal standards were FLT-d3 for FLT and d4-thymidine for FLT-MP. In culture medium, peak area ratios of FLT to FLT-d3 and FLT-MP to d4-thymidine were linear over the range 0.25-100 ng/mL (r(2)≥0.998). Accuracy for quality controls was between -7.3% and 6.3% for FLT, and from -3.3% to 1.7% for FLT-MP. Quality control precision was from 2.4% to 5.7% for FLT and 3.2% to 7.5% for FLT-MP. The limit of quantification was 0.25 ng/mL, with good control results (precision of 9.6% for FLT and 14.8% for FLT-MP). FLT-MP formation was linearly proportional to cell number from 500 to 20,000 cells/well 1 h after FLT addition. FLT-MP and ATP generation were comparable in LNCaP cells exposed to cell cycle inhibitor drugs (Spearman r=0.925, p<0.0001), demonstrating assay suitability for drug screening. This fit for purpose method is amenable to analysis of tumor tissue extracts, and should enable direct assessment of in vitro-in vivo relationships in animal models of cancer.

Validación del Cuestionario de Salud del Paciente como herramienta de tamizaje para trastornos por somatización / Validation of the Patient Health Questionnaire like screening tool for somatization disorders

El objetivo del trabajo realizado fue evaluar la utilidad del “Cuestionario de Salud del Paciente” como posible instrumento de tamizaje para Trastornos por somatización. La metodología consistió en la validación de lenguaje del instrumento, mediante su traducción doble ciego. Posteriormente se realizó su validación de contenido con un panel de expertos. El cuestionario fue aplicado en una muestra aleatoria de100 personas en la atención primaria de salud del Hospital El Pino. Se aplicó de forma anónima y se excluyeron los síntomas de los pacientes producidos por enfermedades médicas ya diagnosticadas previamente. Al analizar los resultados se constató una sospecha de un 34,15 por ciento para trastornos por somatización según el “cuestionario de salud del paciente”, la cual sería 12 veces mayor a la indicada con los criterios del CIE-10 y casi 4 veces mayor que la indicada por los criterios de Escobar. Esto podría explicarse porque los síntomas pesquisados por el cuestionario se deban a otras enfermedades orgánicas o psiquiátricas, que no se discriminan bien por esta herramienta, o a errores en su aplicación.

The objective of the work performed was to evaluate the usefulness of the “Patient Health Questionnaire” as a possible somatization disorders screening tool. The methodology consisted in validating the language of the instrument, using a double blind translation. Then the questionnaire was presented to health professionals who gave us their impressions. The questionnaire was applied to a random sample of 100 people at the primary attention health service of Hospital El Pino. It was applied anonymously, and the symptoms of patients caused by medical diseases previously diagnosed were excluded. When the results were analyzed , a suspicion of 34.15 percent in somatization disorders was found according to the “patient health questionnaire”, which would be 12 times greater than that indicated in the criteria of the CIE-10, and almost 4 times greater than the one indicated by the criteria of Escobar. This could be explained if the symptoms investigated were caused by other organic or psychiatric diseases, that had not been discriminated by this tool or errors in its application.

A system dynamics model for the environmental management of the Sepetiba Bay Watershed, Brazil.

In the recent past, the Sepetiba Bay watershed, located in the Rio de Janeiro State, Brazil has experienced rapid industrial development and population growth, as well as an increase in water pollution and environmental degradation. To analyze the complex interrelationships among the agents affecting the Sepetibza Bay environment, a system dynamics model was developed. The model builds on extensive studies conducted for the watershed, and simulates different hypotheses of economic growth and of demographic expansion. Thus, it can be used as a decision support tool for the identification of investment priorities and policy analyses under various scenarios. In order to provide a comprehensive approach to the environmental management of the Sepetiba Bay watershed, the model had to consider only the most relevant aspects of the behavior and the key interactions among agents operating in the watershed. In this article, the model's structure is presented together with some of its main results.