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Adeno‐associated virus (AAV) vector‐based gene therapy is an innovative modality being increasingly investigated to treat diseases by modifying or replacing defective genes or expressing therapeutic entities. With its unique anatomic and physiological characteristics, the eye constitutes a very attractive target for gene therapy. Specifically, the ocular space is easily accessible and is generally considered "immune‐privileged" with a low risk of systemic side effects following local drug administration. As retina cells have limited cellular turnover, a one‐time gene delivery has the potential to provide long‐term transgene expression. Despite the initial success with voretigene neparvovec (Luxturna), the first approved retina gene therapy, there are still challenges to be overcome for successful clinical development of these products and scientific questions to be answered. The current review paper aims to integrate published experience learned thus far for AAV‐based retina gene therapy related to preclinical to clinical translation; first‐in‐human dose selection; relevant bioanalytical assays and strategies; clinical development considerations including trial design, biodistribution and vector shedding, immunogenicity, transgene expression, and pediatric populations; opportunities for model‐informed drug development; and regulatory perspectives. The information presented herein is intended to serve as a guide to inform the clinical development strategy for retina gene therapy with a focus on clinical pharmacology. [ABSTRACT FROM AUTHOR]

CAR‐T therapies have shown remarkable efficacy against hematological malignancies in the clinic over the last decade and new studies indicate that progress is being made to use these novel therapies to target solid tumors as well as treat autoimmune disease. Innovation in the field, including TCR‐T, allogeneic or "off the shelf" CAR‐T, and autoantigen/armored CAR‐Ts are likely to increase the efficacy and applications of these therapies. The unique aspects of these cell‐based therapeutics; patient‐derived cells, intracellular expression, in vivo expansion, and phenotypic changes provide unique bioanalytical challenges to develop pharmacokinetic and immunogenicity assessments. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) Translational and ADME Sciences Leadership Group (TALG) has brought together a group of industry experts to discuss and consider these challenges. In this white paper, we present the IQ consortium perspective on the best practices and considerations for bioanalytical and immunogenicity aspects toward the optimal development of CAR‐T and TCR‐T cell therapies. [ABSTRACT FROM AUTHOR]

With the promise of a potentially "single dose curative" paradigm, CAR‐T cell therapies have brought a paradigm shift in the treatment and management of hematological malignancies. Both CAR‐T and TCR‐T cell therapies have also made great progress toward the successful treatment of solid tumor indications. The field is rapidly evolving with recent advancements including the clinical development of "off‐the‐shelf" allogeneic CAR‐T therapies that can overcome the long and difficult "vein‐to‐vein" wait time seen with autologous CAR‐T therapies. There are unique clinical pharmacology, pharmacometric, bioanalytical, and immunogenicity considerations and challenges in the development of these CAR‐T and TCR‐T cell therapies. Hence, to help accelerate the development of these life‐saving therapies for the patients with cancer, experts in this field came together under the umbrella of International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) to form a joint working group between the Clinical Pharmacology Leadership Group (CPLG) and the Translational and ADME Sciences Leadership Group (TALG). In this white paper, we present the IQ consortium perspective on the best practices and considerations for clinical pharmacology and pharmacometric aspects toward the optimal development of CAR‐T and TCR‐T cell therapies. [ABSTRACT FROM AUTHOR]

JNJ‐64264681 is an irreversible covalent inhibitor of Bruton's tyrosine kinase. This phase 1, first‐in‐human, 2‐part (single‐ascending dose [SAD]; multiple‐ascending dose [MAD]) study evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD; Bruton's tyrosine kinase occupancy [BTKO]) of JNJ‐64264681 oral solution in healthy participants. For SAD (N = 78), 6 increasing doses of JNJ‐64264681 (4–400 mg) or placebo were evaluated in fasted males. The effects of sex, food, and a capsule formulation were evaluated in separate cohorts. For MAD (N = 27), sequential cohorts of male and female participants received 36/100/200 mg JNJ‐64264681 once daily for 10 days. JNJ‐64264681 exposure (peak concentration; area under the concentration‐time curve) was less than dose proportional from 4 mg to 36 mg. Dose‐normalized area under the concentration‐time curves following the 36 mg and 100 mg doses were generally similar. The mean terminal half‐life was 1.6–13.2 hours. With multiple doses, steady state was achieved by day 2. A semimechanistic PK/PD model was developed using the first 5 SAD cohorts' data to predict %BTKO in MAD cohorts. PK/PD model guided dose‐escalation, and all participants in the 200/400 mg single‐dose cohorts achieved ≥90% BTKO at 4 hours after dosing (peak) with prolonged occupancy. As BTKO data became available from MAD cohorts, it was found that observed BTKO data were consistent with model predictions. JNJ‐64264681 showed no safety signals of concern. Overall, safety, tolerability, PK, BTKO, and PK/PD modeling guided the rationale for dose selection for the subsequent first‐in‐patient lymphoma studies. [ABSTRACT FROM AUTHOR]

Aims: To assess safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and immunogenicity of CNTO 7160, an anti‐interleukin‐33 receptor (IL‐33R) monoclonal antibody, in healthy subjects and patients with asthma or atopic dermatitis (AD). Methods: In Part 1 of this Phase I, randomized, double‐blind, placebo‐controlled study, healthy subjects (n = 68) received single ascending intravenous (IV) CNTO 7160 dose (0.001 to 10 mg/kg) or placebo. In Part 2, patients with mild asthma (n = 24) or mild AD (n = 15) received 3 biweekly IV CNTO 7160 doses (3 or 10 mg/kg) or placebo. Results: CNTO 7160 was generally well tolerated, with 1 serious adverse event of severe cellulitis reported (AD, CNTO 7160, 3 mg/kg). CNTO 7160 exhibited nonlinear PK (0.01–10 mg/kg). Mean clearance decreased with increasing dose (2.43 to 18.03 mL/d/kg). CNTO 7160 PK was similar between healthy subjects and patients with asthma or AD (3 or 10 mg/kg). Free sIL‐33R suppression was rapid and dose dependent. Ex vivo inhibition of p38 phosphorylation of basophils was dose‐dependent (1–10 mg/kg) and sustained inhibition (≥75%) was observed at higher doses (3 or 10 mg/kg). PK/PD modelling and simulation suggests that 1 mg/kg IV every 2 weeks provides adequate systemic drug exposure for sustained inhibition of p38 phosphorylation of basophils. Despite confirmation of target engagement, no apparent CNTO 7160 clinical activity was observed in patients (asthma or AD). Conclusion: This first‐in‐human study provides PK, PD and safety data, supporting further clinical investigation of CNTO 7160 in patients with asthma and AD. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study is to evaluate the pharmacokinetics, immunogenicity, safety, and tolerability of guselkumab, a human monoclonal antibody with high affinity and specificity for binding to interleukin-23. Methods: In this first-in-human, phase 1, randomized study, a single intravenous (IV; 0.03-10 mg/kg) or subcutaneous (SC; 10-300 mg) dose of guselkumab was administered to 47 healthy subjects, and a single SC dose (placebo, 10, 30, 100, 300 mg) was administered to 24 patients with moderate-to-severe psoriasis. Results: Mean maximum observed serum concentration and area under the zero-to-infinity serum concentration-time curve of guselkumab increased in an approximately dose-proportional manner over the dose range of 0.03-10 mg/kg following a single IV administration or 10-300 mg following a single SC administration. Mean clearance and volume of distribution ranged from 3.62-6.03 mL/day/kg and 99.38-123.22 mL/kg, respectively. Mean half-life ranged from 12 to 19 days in healthy subjects and patients with psoriasis. Among guselkumab-treated subjects/patients, 1/30 (3.3 %) healthy subjects in the IV group, 0/6 healthy subjects in the SC group, and 1/20 (5.0 %) patients with psoriasis tested positive for antibodies to guselkumab. No clinically significant adverse events were identified in this study. Conclusion: Guselkumab pharmacokinetic profiles were generally comparable between healthy subjects and patients with psoriasis. Guselkumab, administered as an IV infusion or SC injection, was well tolerated in healthy subjects and patients with psoriasis. [ABSTRACT FROM AUTHOR]

Gas reservoirs of Permian Jiamuhe Formation in Xinguang area, Jungger Basin are tight sandstone gas reservoirs. Based on present geological data by well and seismic, and previous geological knowledge about this area, the author made a systematic summary about the geological characteristics: (1)abundant source, close distance between source and reservoirs; (2)tight reservoirs with “sweet spots”; (3)the tight time of reservoir is matched with the accumulation time, and the gas charging is multiphased; (4)good cap— ping capability; (5)matched relationship between unconformity, faults and microcracks created good conditions for gas migration; (6)located in a wide and gentle slope area, gas—bearing universality, but local rich gas, and the gas water relationship is inverted; (7)abnormal pressure is widely distributed. Based on above geological characteristics, the author confirmed the main controlling factors of reservoir formation, and got the key evaluation parameters, then used these parameters to determine favorable exploration area by multiconditional analysis. Based on the actual data, the author optimized resource abundance analogy method, Monte Carlo stochastic simulation method and the rapid assessment method to estimate gas resources in favorable area, and the gas resources of tight sandstone in this area is about 1 468×108m3. [ABSTRACT FROM AUTHOR]

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 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 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

Characterization of the pharmacokinetics and biodistribution of therapeutic proteins (TPs) is a hot topic within the pharmaceutical industry, particularly with an ever-increasing catalog of novel modality TPs. Here, we review the current practices, and provide a summary of extensive cross-company discussions as well as a survey completed by International Consortium for Innovation and Quality members on this theme. A wide variety of in vitro, in vivo and in silico techniques are currently used to assess pharmacokinetics and biodistribution of TPs, and we discuss the relevance of these from an industry perspective, focusing on pharmacokinetic/pharmacodynamic understanding at the preclinical stage of development, and translation to human. We consider that the 'traditional in vivo biodistribution study' is becoming insufficient as a standalone tool, and thorough characterization of the interaction of the TP with its target(s), target biology, and off-target interactions at a microscopic scale are key to understand the overall biodistribution on a full-body scale. Our summary of the current challenges and our recommendations to address these issues could provide insight into the implementation of best practices in this area of drug development, and continued cross-company collaboration will be of tremendous value. SIGNIFICANCE STATEMENT: The Innovation and Quality Consortium Translational and ADME Sciences Leadership Group working group for the absorption, distribution, metabolism, and excretion of therapeutic proteins evaluates the current practices and challenges in characterizing the pharmacokinetics and biodistribution of therapeutic proteins during drug development, and proposes recommendations to address these issues. Incorporating the in vitro, in vivo and in silico approaches discussed herein may provide a pragmatic framework to increase early understanding of pharmacokinetic/pharmacodynamic relationships, and aid translational modeling for first-in-human dose predictions., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Therapeutic proteins (TPs) comprise a variety of modalities, including antibody-based drugs, coagulation factors, recombinant cytokines, enzymes, growth factors, and hormones. TPs usually cannot traverse cellular barriers and exert their pharmacological activity by interacting with targets on the exterior membrane of cells or with soluble ligands in the tissue interstitial fluid/blood. Due to their large size, lack of cellular permeability, variation in metabolic fate, and distinct physicochemical characteristics, TPs are subject to different absorption, distribution, metabolism, and excretion (ADME) processes as compared with small molecules. Limited regulatory guidance makes it challenging to determine the most relevant ADME data required for regulatory submissions. The TP ADME working group was sponsored by the Translational and ADME Sciences Leadership Group within the Innovation and Quality (IQ) consortium with objectives to: (1) better understand the current practices of ADME data generated for TPs across IQ member companies, (2) learn about their regulatory strategies and interaction experiences, and (3) provide recommendations on best practices for conducting ADME studies for TPs. To understand current ADME practices and regulatory strategies, an industry-wide survey was conducted within IQ member companies. In addition, ADME data submitted to the U.S. Food and Drug Administration was also collated by reviewing regulatory submission packages of TPs approved between 2011 and 2020. This article summarizes the key learnings from the survey and an overview of ADME data presented in biologics license applications along with future perspectives and recommendations for conducting ADME studies for internal decision-making as well as regulatory submissions for TPs. SIGNIFICANCE STATEMENT: This article provides comprehensive assessment of the current practices of absorption, distribution, metabolism, and excretion (ADME) data generated for therapeutic proteins (TPs) across the Innovation and Quality participating companies and the utility of the data in discovery, development, and regulatory submissions. The TP ADME working group also recommends the best practices for condu-cting ADME studies for internal decision-making and regulatory submissions., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "context of use" [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 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 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

The 2018 12 th 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.

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.

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event - A Full Immersion Bioanalytical Week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 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 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.

Out-of-specification (OOS) results were reported by a contract lab in the in vitro adventitious agent assay (AVA) for two products manufactured using mouse myeloma cells in perfusion bioreactors. Cytopathic effect observed for test article-inoculated MRC-5 monolayers resembled foci seen in tissue culture cells infected with transforming viruses. All reasonable known technologies, including highly sensitive, state-of-the-art methodologies and multiple, redundant, and orthogonal methods, were deployed to screen broadly for potential viral and microbial contaminants. Due to the appearance of apparent foci, testing for murine, bovine, and human polyomavirus contamination was heavily represented in the analytical investigation. The results obtained in this extensive screening provided convincing evidence for the lack of an infectious viral or other biological agent. Although the initial investigation produced no reason to invalidate AVA yielding OOS results or to suspect an assay artifact, an extended evaluation revealed several irregularities at the contract test lab reporting the OOS results. The extended investigation also included attempts to reproduce OOS results at alternate contract testing labs and an inter-laboratory study in which methodological differences in the AVA at the three different contract labs were investigated. Only the contract lab initially reporting the OOS results reported foci during this extended evaluation. The results of the inter-laboratory study suggested that the foci artifact might be attributed to the prolonged exposure of the MRC-5 monolayer to cell debris present in the test article. Confocal immunofluorescence microscopy and transmission electron microscopy were subsequently used to provide convincing evidence that the foci observed in test article-inoculated AVA wells were composed of a core of degraded myeloma cell debris covered by one or more layers of MRC-5 cells. The observation that the foci were detected in the AVA at a contract lab where the MRC-5 monolayer is exposed to production cell line debris for a prolonged period strongly suggests that these foci form when MRC-5 grow over the cell debris present in the test article. The cumulative results of the investigation supported the conclusion that the OOS results were artifacts of the AVA test system and not a result of contamination with a virus or other biological agent. Testing was discontinued at the contract lab generating the OOS results and validated at a second contract lab. Manufacturing resumed in consultation with health authorities. The lots were retested following a standard operating procedure (SOP) already in place and ultimately dispositioned for use in normal distribution channels.

In this study we used adenovirus vector-mediated transduction of either the p53 gene (rAd-p53) or the p21(WAF1/CIP1) gene (rAd-p21) to mimic both p53-dependent and -independent up-regulation of p21(WAF1/CIP1) within a human ovarian cancer cell line, 2774, and the derivative cell lines, 2774qw1 and 2774qw2. We observed that rAd-p53 can induce apoptosis in both 2774 and 2774qw1 cells but not in 2774qw2 cells. Surprisingly, overexpression of p21(WAF1/CIP1) also triggered apoptosis within these two cell lines. Quantitative reverse transcription-PCR analysis revealed that the differential expression of BAX, BCL2, and caspase 3 genes, specific in rAd-p53-induced apoptotic cells, was not altered in rAd-p21-induced apoptotic cells, suggesting p21(WAF1/CIP1)-induced apoptosis through a pathway distinguishable from p53-induced apoptosis. Expression analysis of 2774qw1 cells infected with rAd-p21 on 60,000 cDNA microarrays identified 159 genes in response to p21(WAF1/CIP1) expression in at least one time point with 2.5-fold change as a cutoff. Integration of the data with the parallel microarray experiments with rAd-p53 infection allowed us to extract 66 genes downstream of both p53 and p21(WAF1/CIP1) and 93 genes in response to p21(WAF1/CIP1) expression in a p53-independent pathway. The genes in the former set may play a dual role in both p53-dependent and p53-independent pathways, and the genes in the latter set gave a mechanistic molecular explanation for p53-independent p21(WAF1/CIP1)-induced apoptosis. Furthermore, promoter sequence analysis suggested that transcription factor E2F family is partially responsible for the differential expression of genes following p21(WAF1/CIP1). This study has profound significance toward understanding the role of p21(WAF1/CIP1) in p53-independent apoptosis.

A field emission scanning electron microscopy (FESEM) method was developed to assess the stability of a recombinant adenovirus (rAd). This method was designed to simultaneously sort, count, and size the total number of rAd viral species observed within an image field. To test the method, a preparation of p53 transgene-expressing recombinant adenovirus (rAd/p53) was incubated at 37 degrees C and the viral particles were evaluated by number, structure, and degree of aggregation as a function of time. Transmission electron microscopy (TEM) was also used to obtain ultrastructural detail. In addition, the infectious activity of the incubated rAd/p53 samples was determined using flow cytometry. FESEM image-analysis revealed that incubation at 37 degrees C resulted in a time-dependent decrease in the total number of detectable single rAd/p53 virus particles and an increase in apparent aggregates composed of more than three adenovirus particles. There was also an observed decrease in both the diameter and perimeter of the single rAd/p53 viral particles. TEM further revealed the accumulation of damaged single particles with time at 37 degrees C. The results of this study demonstrate that FESEM, coupled with sophisticated image analysis, may be an important tool in quantifying the distribution of aggregated species and assessing the overall stability of rAd samples.