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.

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.

Mass balance and metabolism of the antimalarial pyronaridine in healthy volunteers.

This was a single dose mass balance and metabolite characterization study of the antimalarial agent pyronaridine. Six healthy male adults were administered a single oral dose of 720 mg pyronaridine tetraphosphate with 800 nCi of radiolabeled (14)C-pyronaridine. Urine and feces were continuously collected through 168 h post-dose, with intermittent 48 h collection periods thereafter through 2064 h post-dose. Drug recovery was computed for analyzed samples and interpolated for intervening time periods in which collection did not occur. Blood samples were obtained to evaluate the pharmacokinetics of total radioactivity and of the parent compound. Total radioactivity in urine, feces, and blood samples was determined by accelerator mass spectrometry (AMS); parent concentrations in blood were determined with LC/MS. Metabolite identification based on blood, urine, and feces samples was conducted using a combination of LC + AMS for identifying radiopeaks, followed by LC/MS/MS for identity confirmation/elucidation. The mean cumulative drug recovery in the urine and feces was 23.7 and 47.8 %, respectively, with an average total recovery of 71.5 %. Total radioactivity was slowly eliminated from blood, with a mean half-life of 33.5 days, substantially longer than the mean parent compound half-life of 5.03 days. Total radioactivity remained detectable in urine and feces collected in the final sampling period, suggesting ongoing elimination. Nine primary and four secondary metabolites of pyronaridine were identified. This study revealed that pyronaridine and its metabolites are eliminated by both the urinary and fecal routes over an extended period of time, and that multiple, varied pathways characterize pyronaridine metabolism.

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.

Ultrasensitive and automated 1 pg/ml fluticasone propionate assay in human plasma using LC-MS/MS.

BACKGROUND: To develop and validate an ultrasensitive bioanalytical assay for quantitation of fluticasone propionate in human plasma, aliquots of 0.6 ml of K(3)EDTA human plasma were treated with zinc sulfate solution and loaded onto a preconditioned SPE plate. The sample solutions were washed, eluted, dried and reconstituted. The extracted sample was injected onto a LC-MS/MS system and separated by a reverse-phase HPLC column with a 5 min gradient program, and detected by MS/MS for fluticasone propionate quantitation. RESULTS: Linearity was from 1 to 200 pg/ml. The intra- and inter-day accuracy and precision of the assay met validation acceptance criteria. Various stabilities were established and interference drug assessment was evaluated. The assay has been used for clinical studies. CONCLUSION: This ultrasensitive method has been successfully validated using LC-MS/MS for determination of fluticasone propionate in human plasma at low pg/ml level.

Bioanalysis of drug in tissue: current status and challenges.

Distribution of drugs into tissues is an important determinant of the overall PK and PD profile. Thus, bioanalysis of drugs and their metabolites in tissues can play an important role in understanding the pharmacological and toxicological properties of new drug candidates. Unlike liquid matrices, bioanalysis in tissues offers unique challenges such as proper tissue sampling, appropriate tissue sample preparation, efficient extraction of the analytes from the tissue homogenates, and demonstration of stability and recovery of analytes in intact tissues. This article provides a systematic review of tissue sample analysis for small molecules using LC-MS/MS. The authors provide rationale for tissue sample analysis, and discuss strategies for method development, method qualification or validation, and sample analysis. Unique aspects of method development and qualification/validation are highlighted based on authors' direct experiences and literature summary. Analysis using intact tissue samples such as MALDI imaging is also briefly discussed.

4th Global CRO Council for Bioanalysis: coadministered drugs stability, EMA/US FDA guidelines, 483s and carryover.

The Global CRO Council for Bioanalysis (GCC) was formed in September 2010. Since then, the representatives of the member companies come together periodically to openly discuss bioanalysis and the regulatory challenges unique to the outsourcing industry. The 4th GCC Closed Forum brought together experts from bioanalytical CROs to share and discuss recent issues in regulated bioanalysis, such as the impact of coadministered drugs on stability, some differences between European Medicines Agency and US FDA bioanalytical guidance documents and lessons learned following recent Untitled Letters. Recent 483s and agency findings, as well as issues on method carryover, were also part of the topics discussed.

LC-MS/MS approach for quantification of therapeutic proteins in plasma using a protein internal standard and 2D-solid-phase extraction cleanup.

The bioanalysis of plasma samples generated from in vivo studies of therapeutic proteins is of increasing interesting in the biopharmaceutical industry. The conventional ELISA approach has a long assay development time which can limit use in the early discovery and development of protein-based drugs. In this study, an LC-MS/MS bioassay was developed for the quantification of somatropin and a therapeutic human monoclonal antibody. The assay used bovine fetuin as an internal standard and a two-dimensional solid-phase extraction for the cleanup of the plasma digest. Sample extracts were resolved on an analytical size column using a 6 min LC gradient and analyzed using a triple-quadruple mass spectrometer. The linearity of the assay for somatropin was established from 1 to 1000 microg/mL with accuracy and precision within 15%. This LC-MS approach was also applied to a rat pharmacokinetic study of the therapeutic monoclonal antibody with a lower quantitation limit of 0.5 microg/mL. The LC-MS assay had improved accuracy and precision, and the results from analysis of in vivo study samples showed good agreement with the data obtained with an ELISA. The results from this study indicate that the LC-MS bioassay is a simple and feasible approach for the bioanalysis of therapeutic proteins to support in vivo studies during early drug discovery and development.

Purification and characterization of an immunomodulatory Peptide from bovine placenta water-soluble extract.

An immunomodulatory peptide was isolated from bovine placenta water-soluble extract and purified by consecutive chromatography on DEAE Sepharose CL-6B, Sephadex G-25, and Sephasil C18 column using lymphocyte proliferation assay to identify the active fractions. The immunomodulatory peptide showed a dose-dependent stimulating effect on lymphocyte proliferation. The isoelectric point of the immunodulatory peptide was determined to be 3.82 by capillary isoelectric focusing electrophoresis. The molecular mass of the immunomodulatory peptide was determined to be 2133.52 Da by mass spectrometry. The first 10 amino acid sequence of the immunomodulatory peptide was Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr. This immunomodulatory peptide showed no significant homology with other immunomodulatory peptides. Additionally, it was thermostable, retaining about 60% of immune activity after incubating at 80 degrees C for 30 min.