16th GCC Closed Forum: ICH M10 implementation; NGS, qPCR/dPCR, flow cytometry validation; tissue biomarkers; IS response; immunogenicity harmonization; bioanalytical industry status.

The 16th GCC Closed Forum was held in Orlando, FL, USA, on 23 June 2023. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: IS response, flow cytometry, changes to the bioanalytical industry, NGS assays, biomarker assay for tissues, dPCR validation, immunogenicity harmonization and ICH M10 implementation. Conclusions and consensus from discussions of these topics are included in this article.

miR-146a is a critical target associated with multiple biological pathways of skin aging.

Introduction: The skin is the largest organ of the human body and fulfills protective, immune, and metabolic functions. Skin function and barrier integrity are actively regulated through circadian rhythm-associated genes and epigenetic mechanisms including DNA methylation/demethylation, histone acetylation/deacetylation, and microRNAs. MicroRNA-146a-5p (miR-146a) has been associated with immune activation and skin inflammation; however, the role of miR-146a in regulating skin aging is an open question. This study investigated the role of miR-146a in fibroblasts obtained from different donors in the context of aging, and a potential association of this miRNA with circadian rhythm. Methods: Normal human dermal fibroblasts (NHDFs) from 19y, 27y, 40y, and 62y old donors were used to analyze for miR-146a expression. Expression of miR-146a was downregulated with the hsa-mirVana miR-146a inhibitor, and upregulated with an extract from Adansonia digitata. Effects on markers of skin aging, including cell proliferation, production of Collagen-1 and inflammatory cytokines were assessed. Results: We show that the expression of miR-146a decreases with age in dermal fibroblasts and inhibition of miR-146a in 19y and 62y old NHDFs induced significant changes in essential clock genes indicating an association with circadian rhythm control. Furthermore, downregulation of miR-146a results in a reduction of cellular proliferation, Collagen-1 production, as well as an increase in DNA damage and pro-inflammatory markers. Activation of miR-146a with the Adansonia digitata extract reduced the deleterious effects seen during miR-146a inhibition and increased miR-146a transport through exosome transfer. Conclusion: miR-146a interacts with multiple biological pathways related to skin aging, including circadian rhythm machinery, cell-to-cell communication, cell damage repair, cell proliferation, and collagen production and represents a promising target to fight skin aging. Adansonia digitata extract can promote miR-146a expression and therefore support skin cells' health.

Histological Remission Placebo Rates in Ulcerative Colitis Trials: A Systematic Review and Meta-analysis.

BACKGROUND: High histologic remission rates have been reported with placebos in randomized controlled trials (RCTs) evaluating ulcerative colitis (UC) therapies and have varied based on trial designs. We performed a systematic review and meta-analysis to quantify placebo histological remission rates and identify factors influencing those rates. METHODS: MEDLINE, EMBASE, and the Cochrane library were searched from inception of the databases until December 2021. We included placebo-controlled RCTs of adult patients with UC treated with aminosalicylates, corticosteroids, immunosuppressives, biologics, and small molecules. We pooled estimates using a random-effects model and performed subgroup analysis and meta-regression to evaluate the effect of different covariates on placebo rates. RESULTS: Thirty-three studies (30 induction and 3 maintenance) were included. The overall placebo histological remission rate was 15.7% (95% confidence interval, 12.9%-19%) across all 33 studies. High heterogeneity was observed among studies with I2 = 62.10%. The pooled estimate of histological remission was 15.8% in induction studies and 14.5% in maintenance studies. Subgroup analysis revealed statistically significant differences in placebo rates when accounting for background medications, the intervention drug class, and disease severity (P = .041, .025, and .025, respectively). There was no statistical difference between induction vs maintenance studies or between different histological scales (P = .771, and .075, respectively). CONCLUSIONS: Placebo histological remission rates range from 13% to 19% in UC RCTs, but studies are highly heterogeneous. Factors found to influence placebo rates include presence of background medications, the drug used, and the disease severity. These observations inform future trial designs to minimize placebo rates and reduce heterogeneity.

High histological remission rates have been reported with placebos in ulcerative colitis randomized control trials. This review aims to quantify placebo histological remission rates and identify factors influencing those rates to improve future trial designs.

Conference report for the 10th China Bioanalysis Forum annual meeting.

The 10th China Bioanalysis Forum annual conference was held in Suzhou between 9 and 11 June 2023. This year a full range of bioanalytical topics were discussed such as new technology and bioanalytical approaches for biotherapeutics and biomarkers, particularly in the areas of gene and cell therapy. Another research area covered extensively at the conference was drug metabolism, including new drug metabolism and pharmacokinetic methods; absorption, distribution, metabolism and excretion of new modality drugs, recent regulatory guidance such as human mass balance study and preclinical study of antibody-drug conjugates and case studies of drug metabolism support to newly approved drugs.

Person-centered care for diverse women: Narrative review of foundational research.

Despite advocacy and recommendations to improve health care and health for persons who identify as women, women continue to face inequities in access to and quality of care. Person-centered care for women is one approach that could reduce gendered inequities. We conducted a series of studies to understand what constitutes person-centered care for women and how to achieve it. The overall aim of this article is to highlight the key findings of those studies that can inform policy, practice, and ongoing research. We conducted a narrative review of all studies related to person-centered care for women conducted in our group starting in 2018 over a 5-year period, which was general at the outset, and increasingly focused on racialized immigrant women who constitute a large proportion of the Canadian population. We organized study summaries by research phase: synthesis of person-centered care for women research, exploration of existing person-centered care for women guidance, consultation with key informants, consensus survey of key informants to prioritize strategies to achieve person-centered care for women, and consensus meeting with key informants to prioritize future research. We conducted the reported research in collaboration with an advisory group of diverse women and managers of community agencies. Our research revealed that little prior research had fully established what constitutes person-centered care for women, and in particular, how to achieve it. We also found little acknowledgment of person-centered care for women or strategies to support it in medical curriculum, clinical guidelines, or healthcare policies. We subsequently consulted women who differed by age, ethno-cultural group, health issue, education and geography, and clinicians of different specialties, who offered considerable insight on strategies to support person-centered care for women. Other diverse women, clinicians, healthcare managers, and researchers prioritized issues that warrant future research. We hope that by compiling a summary of our completed research, we draw attention to the need for person-centered care for women and motivate others to pursue it through policy, practice, and research.

Impacts of patient and family engagement in hospital planning and improvement: qualitative interviews with patient/family advisors and hospital staff.

BACKGROUND: Patient engagement (PE) in hospital planning and improvement is widespread, yet we lack evidence of its impact. We aimed to identify benefits and harms that could be used to assess the impact of hospital PE. METHODS: We interviewed hospital-affiliated persons involved in PE activities using a qualitative descriptive approach and inductive content analysis to derive themes. We interpreted themes by mapping to an existing framework of healthcare performance measures and reported themes with exemplar quotes. RESULTS: Participants included 38 patient/family advisors, PE managers and clinicians from 9 hospitals (2 < 100 beds, 4 100 + beds, 3 teaching). Benefits of PE activities included 9 impacts on the capacity of hospitals. PE activities involved patient/family advisors and clinicians/staff in developing and spreading new PE processes across hospital units or departments, and those involved became more adept and engaged. PE had beneficial effects on hospital structures/resources, clinician staff functions and processes, patient experience and patient outcomes. A total of 14 beneficial impacts of PE were identified across these domains. Few unintended or harmful impacts were identified: overextended patient/family advisors, patient/family advisor turnover and clinician frustration if PE slowed the pace of planning and improvement. CONCLUSIONS: The 23 self reported impacts were captured in a Framework of Impacts of Patient/Family Engagement on Hospital Planning and Improvement, which can be used by decision-makers to assess and allocate resources to hospital PE, and as the basis for ongoing research on the impacts of hospital PE and how to measure it.

Recommendations on ELISpot assay validation by the GCC.

Gene therapy, cell therapy and vaccine research have led to an increased need to perform cellular immunity testing in a regulated environment to ensure the safety and efficacy of these treatments. The most common method for the measurement of cellular immunity has been Enzyme-Linked Immunospot assays. However, there is a lack of regulatory guidance available discussing the recommendations for developing and validating these types of assays. Hence, the Global CRO Council has issued this white paper to provide a consensus on the different validation parameters required to support Enzyme-Linked Immunospot assays and a harmonized and consistent approach to Enzyme-Linked Immunospot validation among contract research organizations.

How is patient-centred care conceptualized in obstetrical health? comparison of themes from concept analyses in obstetrical health- and patient-centred care.

BACKGROUND: Due to gender inequities that exist for women of childbearing age, there exists a need to deliver care tailored to their needs and preferences. Patient-centred care (PCC) can be used to meet these needs. This review aims to compare patient care delivery between PCC and obstetrical care. This can help us address how PCC should be delivered to women before, during and after pregnancy versus how it is delivered to patients regardless of sex. METHODS: A review of literature was conducted on MEDLINE, EMBASE, CINAHL and SCOPUS for English PCC and high-quality perinatal reviews published between 2010 and 2021. The data were analysed using a modified Walker and Avant framework. RESULTS: A total of 2138 unique studies were identified, with 11 PCC and 9 high-quality obstetrical care studies included. Common defining features between PCC and obstetrical care include respect and dignity, informed decision-making, therapeutic alliance, effective communication, social relationships and autonomy. PCC-specific features were holistic care, empowerment, individualized care, coordinated care and empathy. Unique high-quality obstetrical care themes included continuity of care, privacy and confidentiality, provider education and status, physical environment and equitable maternal care. CONCLUSIONS: There are shared defining attributes between PCC and obstetrical care, including respect and dignity, informed decision-making, the therapeutic alliance, effective communication, social relationships and autonomy. However, there remain unique defining attributes for high-quality obstetrical care and PCC. This highlights the need for a unique approach to obstetrical care. More research on care for different physiological conditions in women is needed to address patient care that addresses different parts of the lifespan and develop frameworks that can influence health policy, patient care and health system evaluation. PATIENT OR PUBLIC CONTRIBUTION: This study was one part of a larger, multicomponent study of how to implement PCC for women across the lifespan. While we did not specifically consult or involve women in this dual concept analysis, our larger study (content analysis of clinical guidelines and government policies, qualitative interviews with women and clinicians, Delphi study to prioritize consensus recommendations for achieving PCC for women) was guided by the experiences and input of a 50+ women advisory panel.

Development and Validation of a Simoa Assay for Determination of Recombinant Batroxobin in Human Serum.

Recombinant batroxobin (S3101) is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system. A literature survey showed no adequate method that could determine sufficient concentrations to evaluate pharmacokinetic parameters for phase I clinical studies. Therefore, a sensitive method is urgently needed to support the clinical pharmacokinetic evaluation of S3101. In this study, a sensitive bioanalytical method was developed and validated, using a Quanterix single molecular array (Simoa) assay. Moreover, to thoroughly assess the platform, enzyme-linked immunosorbent assay and electrochemiluminescence assay were also developed, and their performance was compared with that of this novel technology platform. The assay was validated in compliance with the current guidelines. Measurements with the Simoa assay were precise and accurate, presenting a valid assay range from 6.55 to 4000 pg/mL. The intra- and inter-run accuracy and precision were within -19.3% to 15.3% and 5.5% to 17.0%, respectively. S3101 was stable in human serum for 280 days at -20°C and -70°C, for 2 h prior to pre-treatment and 24 h post pre-treatment at room temperature (22°C-28°C), respectively, and after five and two freeze-thaw cycles at -70°C and -20°C, respectively. The Simoa assay also demonstrated sufficient dilution linearity, assay sensitivity, and parallelism for quantifying S3101 in human serum. The Simoa assay is a sensitive and adequate method for evaluating the pharmacokinetic parameters of S3101 in human serum.

Development and validation of a triplex assay to study an antibody cocktail against Ebola virus in cynomolgus serum.

Background: To support the rapid development of an antibody cocktail against Ebola virus and avoid unnecessary exposure to infectious environments, an automatic and fast turnover triplex assay was developed using Simoa® (Quanterix Corporation, MA, USA). Materials & methods: A robust triplex assay was developed and validated for simultaneous quantification of the antibody cocktail against Ebola virus in cynomolgus serum. Results: The assay had a quantitation range of 78.1-5000 ng/ml. The intra- and interassay precisions (%CV) were within 11.4 and 13.9%, and the accuracies (%RE) were within -10.8 to 6.8%, respectively. Cross-reactivity was evaluated, and the results met the acceptance criteria. Conclusion: The assay was successfully applied to a pharmacokinetics study following a single-dose intravenous administration of 10 mg/kg the antibody cocktail against Ebola virus to cynomolgus monkeys.

Recommendations for the content and management of Certificates of Analysis for reference standards from the GCC for bioanalysis.

The 13th Global CRO Council (GCC) closed forum for bioanalysis was held in New Orleans, LA, USA on 5 April 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. While ICH M10 will cover requirements for reference standards, one of the biggest challenges facing the CRO community is the lack of consistency and completeness of Certificates of Analysis for reference standards used in regulated bioanalysis. Similar challenges exist with critical reagents (e.g., capture and detection antibodies) used for assays supporting biologics. The recommendations provided in this publication are the minimum requirements for the content that GCC members believe should be included in Certificates of Analysis for reference standards obtained from commercial vendors, sponsors and compendial suppliers, for use in regulated bioanalytical studies. In addition, recommendations for internal standards, metabolites and critical reagents are discussed.

New development and update on China Bioanalysis Forum.

This article provides an update on new development of China Bioanalysis Forum (CBF). CBF became a member association of Chinese Pharmaceutical Association (CPA) at the end of 2019. The official ceremony and first scientific symposium were held in Shanghai on 18 September 2020. The president of Chinese Pharmaceutical Association and representatives from industry, Contract Research Organization (CRO), hospitals and academic institutes attended the ceremony. Seven experts in the field gave presentations on various topics including Drug Metabolism and Pharmacokinetics (DMPK) and bioanalytical support in drug discovery and development as well as experience in Traditional Chinese Medicine research. With the continuous growth of research and development in China, it is well acknowledged that bioanalysis provides critical support for new innovative medicines and generic drug development in the region.

Simple and Rapid LC-MS/MS Methods for Quantifying Catabolites of Antibody-Drug Conjugates with SMCC Linker.

The stability and exposure of toxin-related catabolites in system circulation contributes to the evaluation of the stability, targeted delivery and off-target toxicity for antibody-drug conjugates (ADC) at different stages during drug development. In this study, simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for determination catabolites of Mertansine (DM1), MCC-DM1 and Lys-MCC-DM1 in cynomolgus serum have been developed. The serum samples are processed by protein precipitation. The LC-MS/MS methods are applied on a Phenomenex C8 column (50 × 2.0 mm, 5 µm) with gradient elution with water-formic acid 0.1% (A) and acetonitrile-formic acid 0.1% (B) at a flow rate of 0.5 mL/min. The analytical run time is only 4.0 min and the calibration ranges of the standard curve are 0.500-200 ng/mL for DM1, 1.00-500 ng/mL for MCC-DM1 and 2.00-1000 ng/mL for Lys-MCC-DM1. Intra- and inter-day precision of low, middle and high quality controls was <15%, and accuracy was 99.2-110.9%. The methods were successfully applied to evaluate three catabolites of novel ADCs with N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate linker in vitro and in vivo studies.

Simultaneous quantitative determination of liraglutide and insulin degludec in rat plasma by liquid chromatography-tandem mass spectrometry method and its application.

A simple, fast and high-throughput LC-tandem mass spectrometry method was developed and validated to simultaneously measure liraglutide and insulin degludec in rat plasma. After protein precipitation, plasma samples were subjected to gradient elution using an InertSustain Bio C18 column with 1000/20/1 water/acetonitrile/formic acid (v/v/v) and 1000/1 acetonitrile/formic acid (v/v) as the mobile phase. The method was validated from 1.00 to 500 ng/mL of liraglutide and insulin degludec. Further, the extraction recovery from the plasma was 41.8%-49.2% for liraglutide and 56.5%-69.7% for insulin degludec. Intra- and inter-day precision of liraglutide was 3.5%-9.4% and 8.4%-9.8%, respectively, whereas its accuracy was between -12.6% and -1.3%. Intra- and inter-day precision of insulin degludec was 5.2%-13.6% and 11.8%-19.1%, respectively, showing an accuracy between -3.0% and 9.9%. As a result, the method was successfully applied to a pharmacokinetics study of liraglutide and insulin degludec following a single-dose subcutaneous administration to rats.

Pre-column derivatization method for determining phenylephrine in human plasma and its application in a pharmacokinetic study.

In the present study, a rapid derivatization liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to evaluate phenylephrine in human plasma. The plasma samples were processed to precipitate the proteins, followed by derivatization of the phenylephrine in the plasma with dansyl-chloride solution and extraction with methyl tert-butyl ether-n-hexane (2:1, v/v). The treated samples were analyzed on a Gemini C18 column with 3 min gradient elution, and sensitive detection was achieved with a Waters TQ-s. The method gave linear results over a concentration range from 0.020 to 10.0 ng/ml. The lower limit of quantification was 0.020 ng/ml. Intra- and inter-day precision was <15%, and accuracy was 95.0-105.3%. The validated LC-MS/MS method was successfully applied in the pharmacokinetic analysis of phenylephrine in Chinese subjects with common cold after a single-dose administration of 5, 10 or 20 mg phenylephrine. This pre-column derivatization method may also be applied for the analysis of endogenous hormones such as norepinephrine and adrenaline in a biological matrix.

Age Associated Decrease of MT-1 Melatonin Receptor in Human Dermal Skin Fibroblasts Impairs Protection Against UV-Induced DNA Damage.

The human body follows a physiological rhythm in response to the day/night cycle which is synchronized with the circadian rhythm through internal clocks. Most cells in the human body, including skin cells, express autonomous clocks and the genes responsible for running those clocks. Melatonin, a ubiquitous small molecular weight hormone, is critical in regulating the sleep cycle and other functions in the body. Melatonin is present in the skin and, in this study, we showed that it has the ability to dose-dependently stimulate PER1 clock gene expression in normal human dermal fibroblasts and normal human epidermal keratinocytes. Then we further evaluated the role of MT-1 melatonin receptor in mediating melatonin actions on human skin using fibroblasts derived from young and old subjects. Using immunocytochemistry, Western blotting and RT-PCR, we confirmed the expression of MT-1 receptor in human skin fibroblasts and demonstrated a dramatic age-dependent decrease in its level in mature fibroblasts. We used siRNA technology to transiently knockdown MT-1 receptor in fibroblasts. In these MT-1 knockdown cells, UV-dependent oxidative stress (H2O2 production) was enhanced and DNA damage was also increased, suggesting a critical role of MT-1 receptor in protecting skin cells from UV-induced DNA damage. These studies demonstrate that the melatonin pathway plays a pivotal role in skin aging and damage. Moreover, its correlation with skin circadian rhythm may offer new approaches for decelerating skin aging by modulating the expression of melatonin receptors in human skin.

The 7th China Bioanalysis Forum Annual Meeting.

This article summarizes the highlights from the 7th China Bioanalysis Forum (CBF) Annual Conference in June 2019. The annual CBF conference provided a great platform for the Chinese bioanalytical community to discuss the current bioanalytical regulations, share challenges and experiences. With rapid growth of the pharma industry in China in the past decade and advancement in regulatory reform, the overall science and quality of regulated bioanalysis have now reached international standards. The CBF continues its efforts in promoting scientific excellence, quality and regulatory compliance in China and facilitating close collaboration with the international bioanalysis community.

Comparative strategies for stem cell biodistribution in a preclinical study.

Stem cell therapy represents the potential alternative effective strategy for some diseases that lack effective treatment currently. Correspondingly, it is crucial to establish high-sensitive and reliable quantification assay for tracing exogenous cell migration. In the present study, we first used both bioluminescence imaging (BLI) indirect labeling (human norepinephrine transporter-luciferase reporter system) and 89zirconium (89Zr)-hNSCs direct labeling combined with positron emission tomography/computer tomography (PET/CT) system for tracking human neural stem cells (hNSCs) migration into the brain via nasal administration in preclinical study. But the above two methods failed to give the biodistribution profile due to their low sensitivity. Considering its superior sensitivity and absolute quantitation capability, we developed and validated the droplet digital PCR (ddPCR) targeting species-specific gene in frozen and paraffin sections, slices, and whole blood with the sensitivity of 100-200 hNSCs. Accurate and high throughput quantification could be performed using ddPCR with the coefficient of variation (CVs) of lower quality control (LQC) below 30%. In combination with immunohistochemistry and ddPCR, we confirmed the migration of hNSCs into the brain via nasal administration, which supported the efficacy of hNSCs in MPTP-treated mice, an animal model of Parkinson's disease. In conclusion, the present study is the first to report the application of ddPCR in the pharmacokinetics profile description of tracking of hNSCs in preclinical studies.

Genetic Changes in Experimental Populations of a Hybrid in the Cryptococcus neoformans Species Complex.

Hybrids between Cryptococcus neoformans and Cryptococcus deneoformans are commonly found in patients and the environment. However, the genetic stability of these hybrids remains largely unknown. Here, we established mutation accumulation lines of a diploid C. neoformans × C. deneoformans laboratory hybrid and analyzed the genotypes at 33 markers distributed across all 14 chromosomes. Our analyses found that under standard culture conditions, heterozygosity at most loci was maintained over 800 mitotic generations, with an estimated 6.44 × 10-5 loss-of-heterozygosity (LoH) event per mitotic division. However, under fluconazole stress, the observed LoH frequency increased by > 50 folds for the two markers on Chromosome 1, all due to the loss of the fluconazole susceptible allele on this chromosome. Flow cytometry analyses showed that after the 40th transfer (120 days), 19 of the 20 lines maintained the original ploidy level (2N), while one line was between 2N and 3N. The combined flow cytometry, genotyping at 33 markers, and quantitative PCR analyses showed the allelic loss was compensated for by amplification of the resistant ERG11 allele in eight of the ten fluconazole-stress lines. Our results suggest that hybrids in C. neoformans species complex are generally stable but that they can undergo rapid adaptation to environmental stresses through LoH and gene duplication.

Perspectives on the draft ICH M10 guidance: an interview with Kelly Dong.

Kelly Dong, PhD, Chief Executive Officer, United-Power Pharma Tech Co., Ltd Kelly Dong obtained her PhD degree from McGill University, Canada. Kelly has nearly 25 years of multinational industry experience working for pharmaceutical companies and CROs in Canada, the UK and China. Her scientific expertise encompasses drug metabolism and pharmacokinetics (DMPK) in drug discovery and regulated bioanalysis for preclinical and clinical development. After 20-year overseas experience, she joined GlaxoSmithKline R&D China in August 2009. She was the Director of DMPK for CNS drug discovery and Head of Bioanalysis, Immunogenicity and Biomarkers, overseeing more than 40 preclinical and clinical studies across different therapeutic areas. She joined United-Power Pharma as the Chief Executive Officer in February 2018. She is also a research fellow at the National Engineering Research Center of Protein Drugs. She is one of the founders and a steering committee member of China Bioanalysis Forum. She is also an active contributor to the scientific community, with numerous scientific publications, invited presentations and organizing scientific conferences. This interview was conducted by Sankeetha Nadarajah, Managing Commissioning Editor of Bioanalysis.