2023 White Paper on Recent Issues in Bioanalysis: EU IVDR 2017/746 Implementation/Impact, IVD/CDx/CLIA Approved Assays, High Dimensional Cytometry, Multiplexing Technologies, LBA Tissue Analysis, Vaccine Study Endpoints, Cell-Based Assays for Biomarkers, Cell Therapy and Vaccines (PART 2 - Recommendations on Development & Validation of Biomarkers, IVD, CDx, Cell-Based, Flow Cytometry, Ligand-Binding and Enzyme Assays; Advanced Critical Reagents Strategies).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on 19-23 June 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 2) covers the recommendations on Biomarkers, IVD/CDx, LBA and Cell-Based Assays. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 9 and 7 (2024), respectively.

Comparative study of cranial measurements between sexes from Brazil and The Netherlands: A cone-beam computed tomography study.

The objective of this study was to better understand human variation by comparing cone-beam computed tomography-based cranial measurements between both sexes of individuals from two distinct populations: Brazilian and Dutch. Cone-beam computed tomography volumes of 311 patients between 20 and 60 years from Brazil and The Netherlands were selected. Two radiologists performed 16 linear measurements in the maxillary sinuses and mandibular canal. Kruskall-Wallis test compared measurements of the two cranial structures between male and female for the two populations and four age ranges (20-30, 31-40, 41-50, 51-60). Mann-Whitney test compared individual measurements obtained from the cranial structures between male and female for each population, and between both populations for both sexes. Intra- and inter-observer reliability was assessed by intraclass correlation test (α = 0.05). No significant differences were found in the linear measurements among the experimental groups including sex, population and age group for both cranial structures (p > 0.05). Most of the cranial linear measurements were significantly higher for male than those for female irrespective of the population (p ≤ 0.05). When the populations were compared regardless of sex, Brazilians presented four significantly higher measurements, and Dutch presented seven significantly higher measurements (p ≤ 0.05). The assessed cranial structures did not differ between Brazilian and Dutch populations for both sexes and four age ranges. Multiple linear measurements differed between both populations with a predominance of larger dimensions for the Dutch population.

Galactosylsphingosine does not interfere with the quantitation of plasma glucosylsphingosine levels in Gaucher patients.

It has been shown that the plasma level of glucosylsphingosine (Lyso GL-1) is a useful biomarker for the diagnosis and monitoring of Gaucher disease. Potentially interfering with the quantitation of Lyso GL-1 is its isobaric structural isomer, galactosylsphingosine (psychosine). The contribution of psychosine is generally not accounted for in the determination of Lyso GL-1, due to the difficulty in separating these two isomers. Few methods have been presented in the literature to distinguish the two isomers, and those available tend to be tedious and time-consuming. Here, we developed a LC/MS/MS method able to chromatographically separate Lyso GL-1 and psychosine reproducibly and combine it with a simple, high-throughput sample preparation technique. We also show that the separation of these two isomers in the plasma of Gaucher patients is not necessary for the quantitation of Lyso GL-1 levels, as the relative psychosine level is <3% of Lyso GL-1.

DIMITRA paediatric skull phantoms: development of age-specific paediatric models for dentomaxillofacial radiology research.

OBJECTIVES: This report aims to describe the development of age-specific phantoms for use in paediatric dentomaxillofacial radiology research. These phantoms are denoted DIMITRA paediatric skull phantoms as these have been primarily developed and validated for the DIMITRA European research project (Dentomaxillofacial paediatric imaging: an investigation towards low-dose radiation induced risks). METHODS: To create the DIMITRA paediatric phantoms, six human paediatric skulls with estimated ages ranging between 4 and 10 years- old were selected, protected with non-radiopaque tape and immersed in melted Mix-D soft tissue equivalent material, by means of a careful procedure (layer-by-layer). Mandibles were immersed separately and a Mix-D tongue model was also created. For validation purposes, the resulting paediatric phantoms were scanned using a cone-beam CT unit with different exposure parameter settings. RESULTS: Preliminary images deriving from all scans were evaluated by two dentomaxillofacial radiologists, to check for air bubbles, artefacts and inhomogeneities of the Mix-D and a potential effect on the visualization of the jaw bone. Only skulls presenting perfect alignment of Mix-D surrounding the bone surfaces with adequate and realistic soft tissue thickness density were accepted. CONCLUSIONS: The DIMITRA anthropomorphic phantoms can yield clinically equivalent images for optimization studies in dentomaxillofacial research. In addition, the layer-by-layer technique proved to be practical and reproducible, as long as recommendations are carefully followed.

Validation study of a new method for sexual prediction based on CBCT analysis of maxillary sinus and mandibular canal.

OBJECTIVE: The aim of this study was to evaluate the accuracy of two craniometric methods for sexual prediction (SP) using cone-beam computed tomography (CBCT) in the Dutch population and to construct a formula for each method and then the two combined. DESIGN: One-hundred sixty CBCT images were selected from a Dutch database (80 males and 80 females). The images were analyzed by two examiners taking seven measurements in the maxillary sinus (MS) region (first method) and nine in the mandibular canal (MC) region (second method). The most predictive measurements in both methods were used to develop an equation to determine the accuracy of each method. RESULTS: All measurements showed statistical difference between genders. Logistic regression results showed two variables with greater SP index with 75% accuracy in the first method and four variables with 71.9% accuracy in the second. The two methods combined showed another four variables with 78.5% accuracy. CONCLUSION: All measurements showed statistically significant differences between sexes. The SP accuracy values were 75% for first 71.9% for the second method. When the two methods were combined, the accuracy increased to 78.5%. The formulas developed in this study can be applied as a complementary method for human identification in the Dutch population.

Predictive Value of Panoramic Radiography for Injury of Inferior Alveolar Nerve After Mandibular Third Molar Surgery.

PURPOSE: The purpose of the present systematic review was to assess the added value of panoramic radiography in predicting postoperative injury of the inferior alveolar nerve (IAN) in the decision-making before mandibular third molar (MM3) surgery. MATERIALS AND METHODS: MEDLINE and EMBASE were searched electronically to identify the diagnostic accuracy of studies that had assessed the predictive value of 7 panoramic radiographic signs, including root-related signs (darkening of the root, deflection of the root, narrowing of the root, and dark and bifid apex of the root) and canal-related signs (interruption of the white line of the canal, diversion of the canal, and narrowing of the canal) for IAN injury after MM3 surgery. RESULTS: A total of 8 studies qualified for the meta-analysis. The pooled sensitivity and specificity of the 7 signs ranged from 0.06 to 0.49 and 0.81 to 0.97, respectively. The area under the summary area under the receiver operating characteristic curve ranged from 0.42 to 0.89. The pooled positive predictive value (PPV) and negative predictive value (NPV) ranged from 7.5 to 26.6% and 95.9 to 97.7%, respectively. The added value of a positive sign for ruling in an IAN injury (PPV minus the prior probability) ranged from 3.4 to 22.2%. The added value of a negative sign for ruling out an IAN injury (NPV minus [1 minus the prior probability]) ranged from 0.1 to 2.2%. CONCLUSIONS: For all 7 signs, the added value of panoramic radiography is too low to consider it appropriate for ruling out postoperative IAN in the decision-making before MM3 surgery. The added value of panoramic radiography for determining the presence of diversion of the canal, interruption of the white line of the canal, and darkening of the root can be considered sufficient for ruling in the risk of postoperative IAN injury in the decision-making before MM3 surgery.

The value of thyroid shielding in intraoral radiography.

OBJECTIVES: To evaluate the utility of the application of a thyroid shield in intraoral radiography when using rectangular collimation. METHODS: Experimental data were obtained by measuring the absorbed dose at the position of the thyroid gland in a RANDO(®) (The Phantom Laboratory, Salem, NY) male phantom with a dosemeter. Four protocols were tested: round collimation and rectangular collimation, both with and without thyroid shield. Five exposure positions were deployed: upper incisor (Isup), upper canine (Csup), upper premolar (Psup), upper molar (Msup) and posterior bitewing (BW). Exposures were made with 70 kV and 7 mA and were repeated 10 times. The exposure times were as recommended for the exposure positions for the respective collimator type by the manufacturer for digital imaging. The data were statistically analyzed with a three-way ANOVA test. Significance was set at p < 0.01. RESULTS: The ANOVA test revealed that the differences between mean doses of all protocols and geometries were statistically significant, p < 0.001. For the Isup, thyroid dose levels were comparable with both collimators at a level indicating primary beam exposure. Thyroid shield reduced this dose with circa 75%. For the Csup position, round collimation also revealed primary beam exposure, and thyroid shield yield was 70%. In Csup with rectangular collimation, the thyroid dose was reduced with a factor 4 compared with round collimation and thyroid shield yielded an additional 42% dose reduction. The thyroid dose levels for the Csup, Psup, Msup and BW exposures were lower with rectangular collimation without thyroid shield than with round collimation with thyroid shield. With rectangular collimation, the thyroid shield in Psup, Msup and BW reduced the dose 10% or less, where dose levels were already low, implying no clinical significance. CONCLUSIONS: For the exposures in the upper anterior region, thyroid shield results in an important dose reduction for the thyroid. For the other exposures, thyroid shield augments little to the reduction achieved by rectangular collimation. The use of thyroid shield is to be advised, when performing upper anterior radiography.

A mechanism-based model for the population pharmacokinetics of free and bound aflibercept in healthy subjects.

AIM: Aflibercept (VEGF-Trap), a novel anti-angiogenic agent that binds to VEGF, has been investigated for the treatment of cancer. The aim of this study was to develop a mechanism-based pharmacokinetic (PK) model for aflibercept to characterize its binding to VEGF and its PK properties in healthy subjects. METHODS: Data from two phase I clinical studies with aflibercept administered as a single intravenous infusion were included in the analysis. Free and bound aflibercept concentration-time data were analysed using a nonlinear mixed-effects modelling approach with MONOLIX 3.1. RESULTS: The best structural model involved two compartments for free aflibercept and one for bound aflibercept, with a Michaelis-Menten type binding of free aflibercept to VEGF from the peripheral compartment. The typical estimated clearances for free and bound aflibercept were 0.88 l day(-1) and 0.14 l day(-1), respectively. The central volume of distribution of free aflibercept was 4.94 l. The maximum binding capacity was 0.99 mg day(-1) and the concentration of aflibercept corresponding to half of maximum binding capacity was 2.91 µg ml(-1). Interindividual variability of model parameters was moderate, ranging from 13.6% (V(max) ) to 49.8% (Q). CONCLUSION: The present PK model for aflibercept adequately characterizes the underlying mechanism of disposition of aflibercept and its nonlinear binding to VEGF.

Challenges and opportunities in establishing scientific and regulatory standards for assuring therapeutic equivalence of modified release products: workshop summary report.

Modified release products are complex dosage forms designed to release drug in a controlled manner to achieve desired efficacy and safety. Inappropriate control of drug release from such products may result in reduced efficacy or increased toxicity. This workshop provided an opportunity for pharmaceutical scientists from academia, industry, and regulatory agencies to discuss current industry practices and regulatory expectations for demonstrating pharmaceutical equivalence and bioequivalence of MR products, further facilitating the establishment of regulatory standards for ensuring therapeutic equivalence of these products.

Challenges and opportunities in establishing scientific and regulatory standards for assuring therapeutic equivalence of modified-release products: workshop summary report.

Modified-release products are complex dosage forms designed to release drug in a controlled manner to achieve desired efficacy and safety. Inappropriate control of drug release from such products may result in reduced efficacy or increased toxicity. This workshop provided an opportunity for pharmaceutical scientists from academia, industry and regulatory agencies to discuss current regulatory expectations and industry practices for demonstrating pharmaceutical equivalence and bioequivalence of MR products, further facilitating the establishment of regulatory standards for ensuring therapeutic equivalence of these products.

Challenges and opportunities in establishing scientific and regulatory standards for determining therapeutic equivalence of modified-release products: Workshop summary report.

BACKGROUND: Modified-release (MR) products are complex dosage forms designed to release drug in a controlled manner to achieve the desired efficacy and safety profiles. Inappropriate control of drug release from such products may result in reduced efficacy or increased toxicity. OBJECTIVE: This paper is a summary report of the American Association of Pharmaceutical Scientists, International Pharmaceutical Federation, and Product Quality Research Institute workshop titled "Challenges and Opportunities in Establishing Scientific and Regulatory Standards for Assuring Therapeutic Equivalence of Modified Release Products", held October 1-2, 2009, in Baltimore, Maryland. METHODS: The workshop provided an opportunity for pharmaceutical scientists from academia, industry, and regulatory agencies to discuss current regulatory expectations and industry practices for evaluating the pharmaceutical equivalence and bioequivalence of oral MR products. RESULTS: In the case of conventional monophasic MR formulations, the current regulatory approaches and criteria for bioequivalence evaluation were considered adequate for the assessment of therapeutic equivalence and inter-changeability of drug products. Additional measures may occasionally be needed to determine the bioequivalence of multiphasic MR products. The metric of partial AUC proposed by the US Food and Drug Administration received broad support as an additional measure for evaluating bioequivalence of multiphasic MR products designed to have a rapid onset of drug action followed by sustained response. The cutoff for partial AUCs may be based on the pharmacokinetic/pharmacodynamic or pharmacokinetic/ response characteristics of the products under examination. If the new metric is highly variable, the bioequivalence limits may be set based on the known within-subject variability for the reference product. CONCLUSIONS: The current regulatory approaches and criteria for bioequivalence evaluation were considered adequate for the assessment of therapeutic equivalence and interchangeability of conventional monophasic MR products. Additional measures may occasionally be needed to establish the bioequivalence of multiphasic MR products, and development of such measures is an important objective. The metric of partial AUC was proposed for products designed to have a rapid drug action followed by sustained response.

Accuracy of three-dimensional measurements obtained from cone beam computed tomography surface-rendered images for cephalometric analysis: influence of patient scanning position.

The aims of this study were to assess the accuracy of linear measurements on three-dimensional (3D) surface-rendered images generated from cone beam computed tomography (CBCT) in comparison with two-dimensional (2D) slices and 2D lateral and postero-anterior (PA) cephalometric projections, and to investigate the influence of patient head position in the scanner on measurement accuracy. Eight dry human skulls were scanned twice using NewTom 3G CBCT in an ideal and a rotated position and the resulting datasets were used to create 3D surface-rendered images, 2D tomographic slices, and 2D lateral and PA projections. Ten linear distances were defined for cephalometric measurements. The physical and radiographic measurements were repeated twice by three independent observers and were compared using repeated measures analysis of variance (P=0.05). The radiographic measurements were also compared between the ideal and the rotated scan positions. The radiographic measurements of the 3D images were closer to the physical measurements than the 2D slices and 2D projection images. No statistically significant difference was found between the ideal and the rotated scan measurements for the 3D images and the 2D tomographic slices. A statistically significant difference (P<0.001) was observed between the ideal and rotated scan positions for the 2D projection images. The findings indicate that measurements based on 3D CBCT surface images are accurate and that small variations in the patient's head position do not influence measurement accuracy.

Subjective image quality of digital panoramic radiographs displayed on monitor and printed on various hardcopy media.

The subjective image quality of panoramic radiographs shown on a diagnostic computer monitor were compared with professional direct thermal prints and with common inkjet prints on different paper qualities. Indirect digital panoramic radiographs were obtained from 15 patients. The images were printed with a direct thermal printer in their original format. Afterwards, these were loaded in an imaging software programme (Microsoft Photo Editor) and assessed both on computer monitor and inkjet prints on transparency, glossy, satin and regular paper. Five observers assessed subjective image quality for different regions and anatomical landmarks on a 5-point rating scale. Data were statistically analysed and inter- and intra-observer performances were calculated. Best image quality was obtained with direct thermal prints, followed in descending order by panoramic images viewed on the monitor, inkjet prints on transparencies, glossy paper, satin paper and finally regular paper. The differences were significant except for monitor images versus direct thermal prints, inkjet-transparencies and inkjet-glossy images and inkjet-satin versus inkjet-glossy images. The subjective image quality of indirect digital panoramic images is different for images shown on the computer monitor and for printed images depending on both the printer and paper type used.

Nonlinear accumulation in the brain of the new taxoid TXD258 following saturation of P-glycoprotein at the blood-brain barrier in mice and rats.

1. TXD258, a new taxoid antitumor agent, is a poor substrate for the P-glycoprotein (P-gp) in Caco-2 cells. In this study, we investigated the amount of drug accumulating in the brains of rats and mice under a variety of conditions (dose and infusion time, species and plasma concentration) using conventional in vivo pharmacokinetic techniques and in situ brain perfusion. 2. Mice were infused with radiolabeled TXD258 at 15, 30, 45 and 90 mg m(-2) for 45 s or 1 h and rats were infused with 15 and 60 mg m(-2) over 2.3 min. The radioactivity in the plasma and brains was measured. The brain concentrations of TXD258 in mice and rats were maximal from 2 min to 1 h postinfusion and radioactivity was still detectable at 168 h. While the plasma concentration of TXD258 increased linearly in mice with the infused dose, the brain content increased more than proportionally with the dose between 15 and 90 mg m(-2). This nonlinear uptake of TXD258 also occurred in the plasma and brain of the rat. 3. These findings suggest that the protein-mediated efflux across the blood-brain barrier (BBB) becomes saturated. In situ brain perfusion studies confirmed that TXD258 is a P-gp substrate at the BBB of mice and rats. The P-gp of both species was saturated at the half-inhibitory concentration ( approximately 13 micro M) produced by i.v. infusion. 4 Thus, the observed nonlinear accumulation of TXD258 in the brain seems to occur by saturation of the P-gp at the rodent BBB. This saturation could have several advantages, such as overcoming a P-gp-mediated efflux, but the nonlinear pharmacokinetics could increase the risk of toxicity.

Influence of patient characteristics and renal function on factor Xa inhibition pharmacokinetics and pharmacodynamics after enoxaparin administration in non-ST-segment elevation acute coronary syndromes.

BACKGROUND: The current standard of care for patients with non-ST-segment elevation acute coronary syndromes (ACS) includes antithrombotic therapy with aspirin and heparin. Although emerging data suggest that low-molecular weight preparations offer distinct advantages over unfractionated heparin, limited information on patient-related factors that may influence dosing, safety, and efficacy is available. PURPOSE: The purpose of our study was the determination of the impact of patient age, sex, body weight, and renal function on factor Xa inhibition pharmacokinetics and pharmacodynamics after enoxaparin administration in patients with ACS. METHODS AND RESULTS: Patients enrolled in the TIMI 11A trial received a full complement of antiischemic therapy, aspirin, and enoxaparin (30 mg intravenously, followed by weight-adjusted doses of either 1 mg/kg or 1.25 mg/kg subcutaneously every 12 hours). Before and after the third and last doses, blood samples were obtained from 445 patients for measurement of anti-Xa activity. The mean apparent clearance, distribution volume, and plasma half-life were 0.733 L/h, 5.24 L, and 5 hours, respectively. Among a wide range of clinical and laboratory covariates, creatinine clearance emerged as the most influential factor on apparent clearance, area under the curve, and anti-Xa activity. Patients with marked renal impairment (creatinine clearance <40 mL/min) had higher trough and peak anti-Xa activity compared with those with normal renal function and were more likely to have major hemorrhagic events. CONCLUSION: The pharmacokinetic and pharmacodynamic profiles after enoxaparin administration are consistent across a broad range of patients with ACS. Dose adjustments or anti-Xa coagulation monitoring or both will be necessary rarely in routine clinical practice, with the exception of patients with severe renal insufficiency.