High-resolution isotope-dilution mass spectrometry using metabolism of isotope-labeled compounds: application to drug metabolites.

RATIONALE: Herein we describe a generic quantitative method using high-resolution, isotope-dilution (HRID) metabolism of isotope-labeled compounds and apply it to the analysis of drug metabolites (DMs) in human plasma. Metabolites (drug) in Safety Testing (MIST) application was one goal. METHODS: Testosterone (T) and diclofenac (D) were chosen for mass defect characteristics. T, [(14)C]T, [(13)C(3)]T, D, [(14)C]D, and [(13)C(6)]D were metabolized separately in vitro to produce test metabolites. Liquid chromatography/radioactivity monitoring (LC/RAM) analysis was used to determine the concentration of the test metabolites in the incubates. The incubates containing 6ß-hydroxy-T (6ßHT), [(13)C(3)]6ßHT, 4'-hydroxy-D (4'HD) and [(13)C(6)]4'HD were used to make standard curves. Plasma samples were prepared by 'dilute-and-shoot' and analyzed by LC/MS using SCIEX 5000 and Thermo Orbitrap instrumentation. RESULTS: Human hepatic microsomes and the S9 fraction produced between 2-6 µM ß-hydroxy-T and 4'-hydroxy-D at 60 min starting with 10 µM parent drug as determined by LC/RAM. It was assumed that the amounts of [(13)C(3)]6ßHT and [(13)C(6)]4'HD produced were similar. Dilutions and standard curves were prepared in human plasma. Analysis of the DMs by LC/MS/MS and LC/HRMS exhibited linear responses over a useable range. CONCLUSIONS: HRID with metabolism of an isotope-labeled compound reduces the number of analytical variables considerably. Metabolism of the parent drug to DMs represents a simpler alternative quantitative method compared with traditional approaches. The method will have useful applications for evaluating MIST situations.

Recommendations on qPCR/ddPCR assay validation by GCC.

Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.

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.

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.

Toward a smoke-free Harlem: engaging families, agencies, and community-based programs.

The aim of this collaborative public health study was to engage families, agencies, and programs in reducing secondhand smoke exposure in Central Harlem, New York City. Baseline interviews (n=657) and focus groups (n=4) were conducted with adult members of households with children who had asthma and asthma-like symptoms in the Harlem Children's Zone Asthma Initiative. The interviews concerned the prevalence and determinants of exposure of enrolled children to secondhand smoke. Key findings were that participants: (1) were generally aware of the hazards of secondhand smoke; (2) used strategies to reduce exposure to secondhand smoke in their homes; (3) believed that outdoor pollutants are sometimes just as bad for the health of their children as secondhand smoke; and (4) used smoking to provide stress relief and help diffuse otherwise volatile situations in their homes. The Harlem Smoke-Free Home Campaign was launched in October 2007 based in part on these findings.

2019 White Paper On Recent Issues in Bioanalysis: FDA BMV Guidance, ICH M10 BMV Guideline and Regulatory Inputs (Part 2 - Recommendations on 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and Regulatory Agencies' Input on Bioanalysis, Biomarkers and Immunogenicity).

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA on 1-5 April 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 2) covers the recommendations on the 2018 FDA BMV guidance, 2019 ICH M10 BMV draft guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy. Part 1 (Innovation in small molecules and oligonucleotides and mass spectrometry method development strategies for large molecules bioanalysis) and Part 3 (New insights in biomarker assay validation, current and effective strategies for critical reagent management, flow cytometry validation in drug discovery and development and CLSI H62, interpretation of the 2019 FDA immunogenicity guidance and gene therapy bioanalytical challenges) are published in volume 10 of Bioanalysis, issues 22 and 24 (2019), respectively.

GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline.

The 13th GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5th, 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. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the ICH M10 Bioanalytical Method Validation Draft Guideline and to build unified comments to be provided to the ICH.

12th GCC Closed Forum: critical reagents; oligonucleotides; CoA; method transfer; HRMS; flow cytometry; regulatory findings; stability and immunogenicity.

The 12th GCC Closed Forum was held in Philadelphia, PA, USA, on 9 April 2018. 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: critical reagents; oligonucleotides; certificates of analysis; method transfer; high resolution mass spectrometry; flow cytometry; recent regulatory findings and case studies involving stability and nonclinical immunogenicity. Conclusions and consensus from discussions of these topics are included in this article.

Recommendations for classification of commercial LBA kits for biomarkers in drug development from the GCC for bioanalysis.

Over the last decade, the use of biomarker data has become integral to drug development. Biomarkers are not only utilized for internal decision-making by sponsors; they are increasingly utilized to make critical decisions for drug safety and efficacy. As the regulatory agencies are routinely making decisions based on biomarker data, there has been significant scrutiny on the validation of biomarker methods. Contract research organizations regularly use commercially available immunoassay kits to validate biomarker methods. However, adaptation of such kits in a regulated environment presents significant challenges and was one of the key topics discussed during the 12th Global Contract Research Organization Council for Bioanalysis (GCC) meeting. This White Paper reports the GCC members' opinion on the challenges facing the industry and the GCC recommendations on the classification of commercial kits that can be a win-win for commercial kit vendors and end users.

11th GCC Closed Forum: cumulative stability; matrix stability; immunogenicity assays; laboratory manuals; biosimilars; chiral methods; hybrid LBA/LCMS assays; fit-for-purpose validation; China Food and Drug Administration bioanalytical method validation.

The 11th Global CRO Council Closed Forum was held in Universal City, CA, USA on 3 April 2017. Representatives from international CRO members offering bioanalytical services were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The second CRO-Pharma Scientific Interchange Meeting was held on 7 April 2017, which included Pharma representatives' sharing perspectives on the topics discussed earlier in the week with the CRO members. The issues discussed at the meetings included cumulative stability evaluations, matrix stability evaluations, the 2016 US FDA Immunogenicity Guidance and recent and unexpected FDA Form 483s on immunogenicity assays, the bioanalytical laboratory's role in writing PK sample collection instructions, biosimilars, CRO perspectives on the use of chiral versus achiral methods, hybrid LBA/LCMS assays, applications of fit-for-purpose validation and, at the Global CRO Council Closed Forum only, the status and trend of current regulated bioanalytical practice in China under CFDA's new BMV policy. Conclusions from discussions of these topics at both meetings are included in this report.

A sensitive liquid chromatography and mass spectrometry method for the determination of posaconazole in human plasma.

Posaconazole is a novel extended-spectrum triazole that has favorable in vitro, in vivo and clinical activity against a number of yeasts and moulds. Posaconazole is available as an oral suspension. The dosage found to result in monitored plasma levels that correlate with clinical evidence of good antifungal activity is 800 mg/day in divided doses. A liquid chromatographic/mass spectrometric method (LC-MS/MS) that can be used by clinicians wishing to quantitate, and thereby monitor, plasma levels of posaconazole in certain patients was validated. The method utilized semi-automated 96-well protein precipitation with gradient chromatographic separation of analytes using a Varian Polaris C-18A (2.0 mm x 50 mm, 5-microm particle size) column. The approximate retention time of posaconazole was 2.0 min. Analytes were detected by using tandem mass spectrometry. Sample introduction and ionization was performed by atmospheric pressure chemical ionization in the positive-ion mode. This method has been proven suitable for routine quantitation of posaconazole over the concentration range of 5.00-5000 ng/mL. Inter-run precision based on percent relative deviation for replicate quality controls was < or = 6.2%. Inter-run accuracy expressed as %DIFF was +/-4.0%. Posaconazole quality controls were stable in human plasma for up to five freeze-thaw cycles, when frozen at -20 degrees C for at least 105 days and when kept at room temperature for 24 h. The lower limit of quantitation was 5.00 ng/mL for a 100-microL sample aliquot. These data indicate that the LC-MS/MS method described is suitable for the rapid measurement of posaconazole over the concentration range of 5.00-5000 ng/mL.

Evaluation of automated micro solid phase extraction tips (micro-SPE) for the validation of a LC-MS/MS bioanalytical method.

Automated micro-SPE tips were successfully utilized for the determination of posaconazole in rat plasma. The bioanalytical method using micro-SPE tips was successfully qualified for routine quantitation of posaconazole over the concentration range of 10.0-10,000 ng/mL in rat EDTA plasma. Inter-assay precision, based on percent relative deviation for n=18 replicate quality controls, was < or =5.7%. Inter-assay accuracy based on n=18 replicate quality controls was +/-7.7%. Complete solid phase extraction using micro-SPE tips was demonstrated on a Tomtec liquid handler where >95% recovery for posaconazole was obtained. The micro-SPE tips had sufficient capacity to extract at least 100 microL plasma fortified with 10 microg/mL of posaconazole and the analyte could be efficiently eluted with as little as 60 microL of methanol. Of particular note is the unique ability of these micro-SPE tips to perform exhaustive solid phase extraction more commonly performed when using liquid/liquid extraction.

Orthogonal extraction/chromatography and UPLC, two powerful new techniques for bioanalytical quantitation of desloratadine and 3-hydroxydesloratadine at 25 pg/mL.

Validation of the bioanalytical method for determination of desloratadine and 3-hydroxydesloratadine was conducted using ultra high pressure liquid chromatography (UPLC) in conjunction with mix mode solid phase extraction. The dynamic range of the assay was from 0.025 ng/mL to 10 ng/mL using 96-well solid phase extraction. On an UPLC system, the inter-run accuracy was better than 94.7% for desloratadine (n = 18) and 94.0% for 3-hydroxydesloratadine (n = 18). The between-run precision (%CV) ranged from 2.6% to 9.8% for desloratadine (n = 18) and 3.1% to 11.1% for 3-hydroxydesloratadine (n = 18). The limit of quantitation represented 0.478 pg and 0.525 pg of extracted material injected on-column for desloratadine and 3-hydroxydesloratadine, respectively. The total run time was slightly over 2 min per sample. The approach of orthogonal extraction/chromatography and UPLC significantly improves assay performance while also increasing sample throughput for drug development studies.

9th GCC closed forum: CAPA in regulated bioanalysis; method robustness, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, regulatory audit experiences and electronic laboratory notebooks.

The 9th GCCClosed Forum was held just prior to the 2015 Workshop on Recent Issues in Bioanalysis (WRIB) in Miami, FL, USA on 13 April 2015. In attendance were 58 senior-level participants, from eight countries, representing 38 CRO companies offering bioanalytical services. The objective of this meeting was for CRO bioanalytical representatives to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues selected at this year's closed forum include CAPA, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, and ELNs. A summary of the industry's best practices and the conclusions from the discussion of these topics is included in this meeting report.

Minimization of ion suppression in LC-MS/MS analysis through the application of strong cation exchange solid-phase extraction (SCX-SPE).

Ion suppression of drug response is a major source of imprecision for bioanalytical analysis using LC-MS/MS. Endogenous phospholipids cause ion suppression in both positive ESI and negative ESI modes and must be removed or resolved chromatographically. Three types of ion-exchange solid-phase extraction mediums were evaluated to determine their abilities to remove phospholipids. It was determined that although mixed mode phases fulfills the requirements of retaining both analytes and diverse metabolites, reverse phase retention mechanisms are detrimental in eliminating ion suppression caused by late eluting phospholipids. If an analyte and its metabolites can be retained using an ion-exchange mechanism alone, mixed mode extraction phases should be avoided.

2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 1 - small molecules by LCMS).

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of over 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. It is 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, LBA approaches including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations that emerged 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 advance scientific excellence, improve quality and deliver better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 1 covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (hybrid LBA/LCMS and regulatory agencies' inputs) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will also be published in volume 7 of Bioanalysis, issues 23 and 24, respectively.

Recommendations on incurred sample stability (ISS) by GCC.

The topic of incurred sample stability (ISS) has generated considerable discussion within the bioanalytical community in recent years. The subject was an integral part of the seventh annual Workshop on Recent Issues in Bioanalysis (WRIB) held in Long Beach, CA, USA, in April 2013, and at the Global CRO Council for Bioanalysis (GCC) meeting preceding it. Discussion at both events focused on the use of incurred samples for ISS purposes in light of results from a recent GCC survey completed by member companies. This paper reports the consensus resulting from these discussions and serves as a useful reference for depicting ISS issues and concerns, summarizing the GCC survey results and providing helpful recommendations on ISS in the context of bioanalytical method development and application.

2014 White Paper on recent issues in bioanalysis: a full immersion in bioanalysis (Part 3 - LBA and immunogenicity).

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years' editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for Large molecules bioanalysis using LBA and Immunogenicity. Part 1 (Small molecules bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' Input) were published in the Bioanalysis issues 6(22) and 6(23), respectively.