2023 White Paper on Recent Issues in Bioanalysis: ISR for ADA Assays, the Rise of dPCR vs qPCR, International Reference Standards for Vaccine Assays, Anti-AAV TAb Post-Dose Assessment, NanoString Validation, ELISpot as Gold Standard (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Biotherapeutics Immunogenicity & Risk Assessment; ADA/NAb Assay/Reporting Harmonization).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the 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 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) are published in volume 16 of Bioanalysis, issues 8 and 9 (2024), respectively.

Monitoring and reporting the US COVID-19 vaccination effort.

Immunizations are an important tool to reduce the burden of vaccine preventable diseases and improve population health.1 High-quality immunization data is essential to inform clinical and public health interventions and respond to outbreaks of vaccine-preventable diseases. To track COVID-19 vaccines and vaccinations, CDC established an integrated network that included vaccination provider systems, health information exchange systems, immunization information systems, pharmacy and dialysis systems, vaccine ordering systems, electronic health records, and tools to support mass vaccination clinics. All these systems reported data to CDC's COVID-19 response system (either directly or indirectly) where it was processed, analyzed, and disseminated. This unprecedented vaccine tracking effort provided essential information for public health officials that was used to monitor the COVID-19 response and guide decisions. This paper will describe systems, processes, and policies that enabled monitoring and reporting of COVID-19 vaccination efforts and share challenges and lessons learned for future public health emergency responses.

Pharmacokinetics of Alglucosidase Alfa Manufactured at the 4000-L Scale in Participants with Pompe Disease: A Phase 3/4 Open-Label Study.

Pompe disease is a rare, autosomal recessive, degenerative neuromuscular disease caused by deficiency of acid α-glucosidase, a lysosomal enzyme that degrades α-1,4 and α-1,6 linkages in glycogen. The objectives of this study (PAPAYA; NCT01410890) were to (1) characterize the pharmacokinetics of 20 mg/kg body weight alglucosidase alfa manufactured at the 4000-L scale following a single intravenous dose in participants aged less than 18 and 18 years or older with Pompe disease and (2) evaluate the relationship between anti-alglucosidase alfa antibody titers and the pharmacokinetics of alglucosidase alfa. Mean maximum plasma concentration and area under the concentration-time curve from time zero and extrapolated to infinite time were 204 µg/mL and 1110 µg â€¢ h/mL for participants aged less than 18 years (n = 10), respectively, and 307 µg/mL and 1890 µg â€¢ h/mL for participants aged 18 years or older (n = 10), respectively. Mean terminal half-life was 5.43 hours in participants aged less than 18 years with a high variability (70%) and 3.84 hours in participants aged 18 years or older with a low variability (21%). Mean maximum plasma concentration and area under the concentration-time curve from time zero and extrapolated to infinite time were 256 µg/mL and 1452 µg • h/mL, respectively, in anti-alglucosidase alfa-negative participants (n = 12) and 262 µg/mL and 1703 µg â€¢ h/mL, respectively, in anti-alglucosidase alfa-positive participants (n = 7). The study findings enrich available data from existing information on alglucosidase alfa without changing its known risks and benefits.

Failure to tolerate continuous subcutaneous treprostinil in pediatric pulmonary hypertension patients.

Continuous subcutaneous (SubQ) treprostinil is an effective therapy for pediatric patients diagnosed with pulmonary hypertension (PH). To date, the clinical characteristics and factors associated with failure to tolerate this therapy have not been described. The purpose was to describe patient-reported factors contributing to SubQ treprostinil intolerance in pediatric patients with PH. A retrospective descriptive study was performed at 11 participating sites in the United States and Canada for patients younger than 21 years of age diagnosed with PH who failed treatment to tolerate SubQ treprostinil between January 1, 2009, and December 31, 2019. All data were summarized using descriptive statistics. Forty-one patients met the inclusion criteria. The average age at SQ treprostinil initiation, and length of treatment, was 8.6 years and 22.6 months, respectively. The average maximum dose, concentration, and rate were 95.8 ng/kg/min, 6.06 mg/mL, and 0.040 mL/h, respectively. The reasons for failure to tolerate SubQ treprostinil included intractable site pain (73.2%), frequent site changes (56.1%), severe site reactions (53.7%), infections (26.8%), and noncompliance/depression/anxiety (17.1%). Thirty-nine (95.1%) patients transitioned to a prostacyclin therapy with 23 patients transitioning to intravenous prostacyclin, 5 to inhaled prostacyclin, 5 to oral prostacyclin, and 7 to a prostacyclin receptor agonist. A subset of pediatric PH patients failed to tolerate SubQ treprostinil infusions despite advances in SubQ site maintenance and pain management strategies. Intractable site pain, frequent SubQ site changes, and severe localized skin reactions were the most common reasons for failure.

A Canadian, retrospective, multicenter experience with selexipag for a heterogeneous group of pediatric pulmonary hypertension patients.

Introduction: Selexipag, an oral nonprostanoid prostaglandin receptor agonist, has led to reduced morbidity and mortality in adults with pulmonary arterial hypertension (PAH). While the adult literature has been extrapolated to suggest selexipag as an oral treatment for severe pediatric pulmonary hypertension (PH), longitudinal, multicenter data on the benefits of selexipag in this population are lacking. The purpose of this study is to present a longitudinal, multicentre experience with selexipag in a relatively large cohort of pediatric PH patients and add to the existing selexipag literature. Materials and methods: We performed a retrospective, multicenter review describing the clinical outcomes of pediatric PH patients receiving selexipag in addition to standard oral pulmonary vasodilator therapy across three Canadian centers between January 2005 and June 2021. Results: Twenty-four pediatric patients (fifteen female) with a mean age of 9.7 (range 2.0-15.5) years were included. Of this cohort, eighteen (75.0%) were in group 1, one (4.2%) was in group 2, four (16.7%) were in group 3, and one (4.2%) was in group 4. Twenty-two (91.7%) patients were on dual PH therapy after six months. Dosing was targeted to achieve 20-30 mcg/kg/dose orally every twelve hours. Median dose after twelve months was 30 mcg/kg/dose. Twelve months following selexipag initiation, median decreases of 0.2 cm in tricuspid annular plane systolic excursion, 3.5 mmHg in right-ventricular systolic pressure, and 6.1 mmHg in mean pulmonary arterial pressure were observed; none of these changes were statistically significant. Three patients died, one clinically deteriorated and required admission to a pediatric intensive care unit, ten had gastrointestinal symptoms, and three had flushing. Conclusion: Selexipag appears to be a safe and effective adjunctive therapy for pediatric PH patients and has a tolerable adverse effect profile aside from gastrointestinal disturbances. Additional prospective studies of changes in hemodynamics and functional classification over a longer period and with a larger sample are needed. Future research should aim to identify subgroups that stand to benefit from the addition of selexipag as well as optimal timing and dosing for the pediatric population.

Multicenter review of a tadalafil suspension formulation for infants and children with pulmonary hypertension: A North American experience.

Introduction: Phosphodiesterase type 5 (PDE5) inhibitors, with sildenafil the earliest among them, are widely used in the management of pediatric pulmonary arterial hypertension (PAH). Tadalafil is a PDE5 inhibitor with a long half life (16 h), stable pharmacokinetics and pharmacodynamics, and minimal adverse effects. However, the utility of tadalafil suspensions in this setting has not been widely explored due to a lack of clinical experience. We present a multicenter experience that details the safety and tolerability of a tadalafil suspension, either alone or in combination with another vasodilator, for the management of pediatric pulmonary hypertension (PH). Methods and materials: This is a retrospective chart review of infants and children at Children's Wisconsin and the Stollery Children's Hospital enrolled in pediatric PH programs between December 2013 and April 2022 managed with a tadalafil suspension. Patients aged six years of age and under who were treated with a tadalafil suspension were included. Demographics, clinical information, echocardiographic and hemodynamic measurements, and laboratory data were collected before and six months after tadalafil initiation. Results: Over the study period, 154 children with a median age of 1.0 (range 0.0-6.9) years were treated with tadalafil therapy. Of these, 39 (25.3%) were in group 1 (PAH), 79 (51.3%) were in group 3 (lung disease), and 33 (21.4%) were in group 5 (pulmonary hypertensive vascular disease). The median initial dose of tadalafil was 1.0 mg/kg once daily. Eleven (7.1%) patients in the cohort were established on tadalafil therapy de novo. The suspension formulation was necessary for 103 (66.9%) patients due to an inability to take enteral tablets and for 49 (31.8%) due to a need for feeding via gastric or jejunal tubes. We observed a statistically significant increase in tricuspid annular plane systolic excursion as well as significant decreases in right-ventricular systolic pressure and NT-proBNP. Tadalafil therapy was well tolerated over the six-month period: at six months, no adverse effects were reported aside from gastrointestinal disturbances by 2 (1.3%) patients. Conclusion: Tadalafil, a long-acting PDE5 inhibitor, when administered in a suspension formulation, has a safe and tolerable adverse effect profile. Following six months of therapy, our cohort showed improvements in clinical parameters, echocardiographic measurements, and laboratory results. Patient compliance was good and adverse effects were rare, minor, and manageable with nonpharmacological means.

A North American, single-center experience implanting fenestrated atrial devices and atrial flow regulators into a heterogeneous group of pediatric pulmonary hypertension patients.

Introduction: The clinical deterioration commonly experienced by pediatric patients with pulmonary arterial hypertension (PAH) has motivated a shift in the treatment of pulmonary hypertension (PH) through innovations in surgical salvage interventions. The Occlutech fenestrated atrial septal defect (FASD) Occluder and the atrial flow regulator (AFR), which provides a protective, atrial-level shunt during hypertensive crises, have found an important role in treating pediatric patients with PAH. Other groups of pediatric patients with PH may also benefit from a similar protective physiology. The primary aim of this work is to present a single center's experience with AFR and FASD devices for managing a heterogeneous group of pediatric PH patients. A secondary goal is to identify hemodynamic changes and complications following device implantation. Materials and Methods: We performed a retrospective review of all pediatric PH patients who, after being found suitable, either successfully or unsuccessfully received an FASD or AFR device between January 2015 and December 2021 at the Stollery Children's Hospital in Edmonton, Canada. Results: Fourteen patients (eight female) with a median age of 4.6 (range 0.3-17.9) years and a median body mass index of 15.1 (Q1 = 13.8, Q3 = 16.8) kg/m2 underwent device implantation: five received FASDs, eight received AFRs, and one was ultimately unable to receive an implant due to thrombosed iliac vessels and required surgical intervention. Of the fourteen patients, seven were in group 1 (PAH), one was in group 3 (lung disease), and six were in group 5 (primarily pulmonary hypertension vascular disease) under the World Symposium PH classification. All patients were on mono-, dual-, or triple-drug PH therapy. Device stabilization was not possible for two patients, who then required a repeat catheterization. Of the group 1 patients, three AFR and three FASD implants were successful, while one FASD implant was unsuccessful due to thrombosed vessels. At a six-month clinical assessment, all group 1 patients had patent devices and improved WHO FCs. Conclusion: This work presents a single center's experience with AFR and FASD implants in a heterogeneous group of fourteen pediatric patients with severe PH. This treatment strategy is novel in the pediatric population and so this work provides momentum for future studies of interventional cardiac catheterization procedures for pediatric patients with PH. Further collaborations are required to develop criteria to identify ideal pediatric candidates and optimally time interventions in order to maximize the benefits of this treatment.

A rare homozygous missense GDF2 (BMP9) mutation causing PAH in siblings: Does BMP10 status contribute?

Pulmonary arterial hypertension (PAH) is a disease characterized by pathological remodeling of the pulmonary vasculature causing elevated pulmonary artery pressures and ultimately, right ventricular failure from chronic pressure overload. Heterozygous pathogenic GDF2 (encoding bone morphogenetic protein 9 (BMP9)) variants account for some (>1%) adult PAH cases. Only three pediatric PAH cases, harboring homozygous or compound heterozygous variants, are reported to date. Ultra-rare pathogenic GDF2 variants are reported in hereditary hemorrhagic telangiectasia and overlapping disorders characterized by telangiectasias and arteriovenous malformations (AVMs). Here, we present two siblings with PAH homozygous for a GDF2 mutation that impairs BMP9 proprotein processing and reduces growth factor domain availability. We confirm an absence of measurable plasma BMP9 whereas BMP10 levels are detectable and serum-dependent endothelial BMP activity is evident. This contrasts with the absence of activity which we reported in two children with homozygous pathogenic GDF2 nonsense variants, one with PAH and one with pulmonary AVMs, both with telangiectasias, suggesting loss of BMP10 and endothelial BMP activity in the latter may precipitate telangiectasia development. An absence of phenotype in related heterozygous GDF2 variant carriers suggests incomplete penetrance in PAH and AVM-related diseases, indicating that additional somatic and/or genetic modifiers may be necessary for disease precipitation.

Unbiased approach to identify and assess efficacy of human SARS-CoV-2 neutralizing antibodies.

Coronavirus disease 2019 (COVID-19) continues to significantly impact the global population, thus countermeasure platforms that enable rapid development of therapeutics against variants of SARS-CoV-2 are essential. We report use of a phage display human antibody library approach to rapidly identify neutralizing antibodies (nAbs) against SARS-CoV-2. We demonstrate the binding and neutralization capability of two nAbs, STI-2020 and STI-5041, against the SARS-CoV-2 WA-1 strain as well as the Alpha and Beta variants. STI-2020 and STI-5041 were protective when administered intravenously or intranasally in the golden (Syrian) hamster model of COVID-19 challenged with the WA-1 strain or Beta variant. The ability to administer nAbs intravenously and intranasally may have important therapeutic implications and Phase 1 healthy subjects clinical trials are ongoing.

Anti-drug Antibody Validation Testing and Reporting Harmonization.

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting.

European Immunogenicity Platform 11th Open Scientific Symposium on immunogenicity of biopharmaceuticals.

Given the expanding number of complex therapeutic protein drugs and advanced therapy medicinal products that are being developed, improving our ability to assess the potential immunogenicity of biologics is critical to ensuring treatment efficacy and patient safety. In this context, the European Immunogenicity Platform annual meeting provides opportunities for experts from industry and academia, regulators and clinicians to convene and discuss immunogenicity assessment methods and tools. This report summarizes the key messages on immunogenicity testing, prediction, clinical relevance and advanced therapy medicinal products discussed at the 11th Open Scientific European Immunogenicity Platform Symposium on Immunogenicity of Biopharmaceuticals, Lisbon, Portugal, 17-19 February 2020.

At three years, patients with acute lymphoblastic leukaemia are still at risk for relapse. Results of the international MRC UKALLXII/ECOG E2993 trial.

Late relapse [>3 years from complete remission (CR)] in acute lymphoblastic leukaemia (ALL), is unusual. Data from the MRC UKALLXII/ECOG E2993 trial are presented to evaluate the incidence and characteristics of late relapse in adult ALL. Of 1,909 patients, 1,752 (92%) achieved CR and among these 757 (43·2%) relapsed; 691 (91·3%) within three years and 66 (8·7%) beyond. Among these 66 patients, median time to relapse was 47 (37-144) months. Relapse beyond three years occurred in 3·8% of all who achieved CR. The cumulative risk of relapse was 40%, 43% and 45% at three, five and ten years respectively. Out of the 1 752 patients who achieved CR, 11·7% underwent autologous and 40·6% allogeneic transplant, while in CR1. Of the autologous patients, 43·2% relapsed early and 3·4% relapsed late. However, among the allogeneic patients, 13·2% relapsed early and only 1·3% late. The five-year overall survival from relapse was 5·8% and 20% in the early and late relapse patients respectively. In conclusion, late relapse in adults with ALL is not uncommon, and is associated with better outcome after relapse compared to early relapse.

2019 White Paper on Recent Issues in Bioanalysis: FDA Immunogenicity Guidance, Gene Therapy, Critical Reagents, Biomarkers and Flow Cytometry Validation (Part 3 - Recommendations on 2019 FDA Immunogenicity Guidance, Gene Therapy Bioanalytical Challenges, Strategies for Critical Reagent Management, Biomarker Assay Validation, Flow Cytometry Validation & CLSI H62).

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the 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 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of Bioanalysis, issues 22 and 23 (2019), respectively.

Impaired Recognition of Metrical and Syntactic Boundaries in Children with Developmental Language Disorders.

In oral language, syntactic structure is cued in part by phrasal metrical hierarchies of acoustic stress patterns. For example, many children's texts use prosodic phrasing comprising tightly integrated hierarchies of metre and syntax to highlight the phonological and syntactic structure of language. Children with developmental language disorders (DLDs) are relatively insensitive to acoustic stress. Here, we disrupted the coincidence of metrical and syntactic boundaries as cued by stress patterns in children's texts so that metrical and/or syntactic phrasing conflicted. We tested three groups of children: children with DLD, age-matched typically developing controls (AMC) and younger language-matched controls (YLC). Children with DLDs and younger, language-matched controls were poor at spotting both metrical and syntactic disruptions. The data are interpreted within a prosodic phrasing hypothesis of DLD based on impaired acoustic processing of speech rhythm.

An immune tolerance approach using transient low-dose methotrexate in the ERT-naïve setting of patients treated with a therapeutic protein: experience in infantile-onset Pompe disease.

PURPOSE: To investigate immune tolerance induction with transient low-dose methotrexate (TLD-MTX) initiated with recombinant human acid α-glucosidase (rhGAA), in treatment-naïve cross-reactive immunologic material (CRIM)-positive infantile-onset Pompe disease (IOPD) patients. METHODS: Newly diagnosed IOPD patients received subcutaneous or oral 0.4 mg/kg TLD-MTX for 3 cycles (3 doses/cycle) with the first 3 rhGAA infusions. Anti-rhGAA IgG titers, classified as high-sustained (HSAT; ≥51,200, ≥2 times after 6 months), sustained intermediate (SIT; ≥12,800 and <51,200 within 12 months), or low (LT; ≤6400 within 12 months), were compared with those of 37 CRIM-positive IOPD historic comparators receiving rhGAA alone. RESULTS: Fourteen IOPD TLD-MTX recipients at the median age of 3.8 months (range, 0.7-13.5 months) had a median last titer of 150 (range, 0-51,200) at median rhGAA duration ~83 weeks (range, 36-122 weeks). One IOPD patient (7.1%) developed titers in the SIT range and one patient (7.1%) developed titers in the HSAT range. Twelve of the 14 patients (85.7%) that received TLD-MTX remained LT, versus 5/37 HSAT (peak 51,200-409,600), 7/37 SIT (12,800-51,000), and 23/37 LT (200-12,800) among comparators. CONCLUSION: Results of TLD-MTX coinitiated with rhGAA are encouraging and merit a larger longitudinal study.

2018 White Paper on Recent Issues in Bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 - LBA/cell-based assays: immunogenicity, biomarkers and PK assays).

The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the 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 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis, issues 22 and 23 (2018), respectively.

2017 White Paper on recent issues in bioanalysis: a global perspective on immunogenicity guidelines & biomarker assay performance (Part 3 - LBA: immunogenicity, biomarkers and PK assays).

The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule analysis involving LC-MS, hybrid ligand-binding assay (LBA)/LC-MS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the 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 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC-MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.

Intravitreous injection of AAV2-sFLT01 in patients with advanced neovascular age-related macular degeneration: a phase 1, open-label trial.

BACKGROUND: Long-term intraocular injections of vascular endothelial growth factor (VEGF)-neutralising proteins can preserve central vision in many patients with neovascular age-related macular degeneration. We tested the safety and tolerability of a single intravitreous injection of an AAV2 vector expressing the VEGF-neutralising protein sFLT01 in patients with advanced neovascular age-related macular degeneration. METHODS: This was a phase 1, open-label, dose-escalating study done at four outpatient retina clinics in the USA. Patients were assigned to each cohort in order of enrolment, with the first three patients being assigned to and completing the first cohort before filling positions in the following treatment groups. Patients aged 50 years or older with neovascular age-related macular degeneration and a baseline best-corrected visual acuity score of 20/100 or less in the study eye were enrolled in four dose-ranging cohorts (cohort 1, 2 × 108 vector genomes (vg); cohort 2, 2 × 109 vg; cohort 3, 6 × 109 vg; and cohort 4, 2 × 1010 vg, n=3 per cohort) and one maximum tolerated dose cohort (cohort 5, 2 × 1010 vg, n=7) and followed up for 52 weeks. The primary objective of the study was to assess the safety and tolerability of a single intravitreous injection of AAV2-sFLT01, through the measurement of eye-related adverse events. This trial is registered with ClinicalTrials.gov, number NCT01024998. FINDINGS: 19 patients with advanced neovascular age-related macular degeneration were enrolled in the study between May 18, 2010, and July 14, 2014. All patients completed the 52-week trial period. Two patients in cohort 4 (2 × 1010 vg) experienced adverse events that were possibly study-drug related: pyrexia and intraocular inflammation that resolved with a topical steroid. Five of ten patients who received 2 × 1010 vg had aqueous humour concentrations of sFLT01 that peaked at 32·7-112·0 ng/mL (mean 73·7 ng/mL, SD 30·5) by week 26 with a slight decrease to a mean of 53·2 ng/mL at week 52 (SD 17·1). At baseline, four of these five patients were negative for anti-AAV2 serum antibodies and the fifth had a very low titre (1:100) of anti-AAV2 antibodies, whereas four of the five non-expressers of sFLT01 had titres of 1:400 or greater. In 11 of 19 patients with intraretinal or subretinal fluid at baseline judged to be reversible, six showed substantial fluid reduction and improvement in vision, whereas five showed no fluid reduction. One patient in cohort 5 showed a large decrease in vision between weeks 26 and 52 that was not thought to be vector-related. INTERPRETATION: Intravitreous injection of AAV2-sFLT01 seemed to be safe and well tolerated at all doses. Additional studies are needed to identify sources of variability in expression and anti-permeability activity, including the potential effect of baseline anti-AAV2 serum antibodies. FUNDING: Sanofi Genzyme, Framingham, MA, USA.