Workshop Report: AAPS Workshop on Method Development, Validation, and Troubleshooting of Ligand-Binding Assays in the Regulated Environment.

A novel format was introduced at the recent AAPS NBC Workshop on Method Development, Validation and Troubleshooting in San Diego on 18th May 2014. The workshop format was initiated by Binodh De Silva; Marie Rock and Sherri Dudal joined the initiative to develop and chair the workshop. Questions were solicited by a variety of avenues, including a Linked-In Discussion Group. Once collated and clarified, the topics covered assay development, validation, and analysis of PK, Immunogenicity, and Biomarkers with an additional topic on alternative bioanalytical technologies. A panel of experts (workshop report co-authors) was assigned to each topic to bring forward thought-provoking aspects of each topic. The format of the workshop was developed to target the needs of bioanalytical scientists with intermediate to advanced experience in the field ranging to enable robust discussion and to delve deeper into the current bioanalytical hot topics. While the new format allowed for an interactive session with the topical discussion driven by the audience members, it did not foster equal discussion time for all of the proposed topics, especially Biomarkers and alternative LBA technologies.

ISR: background, evolution and implementation, with specific consideration for ligand-binding assays.

ISR was highlighted as a topic of major interest to the US FDA in 2006, having been previously required, then discontinued, by Canadian regulatory authorities. Following an FDA focus on ISR, this topic has also been emphasized by regulatory agencies in Europe, Asia and Latin America. Extensive discussions on proper implementation of programs have taken place in multiple settings, including pharmaceutical companies, regulatory agencies, professional associations and CROs. These efforts have led to recommendations for ISR conduct that are now included in a final guideline on bioanalytical method validation from the European Medicines Agency, a draft validation guidance from the Ministry of Health, Labor and Welfare in Japan and a revised draft validation guidance from the FDA. In this Review we look at the background, evolution and implementation of ISR for all assays, while including some specific considerations on this topic for ligand-binding assays.

Recommendations from the Global Bioanalysis Consortium Team A8: documentation.

The A8 Global Harmonization Team focused on the documentation needed to support both small and large molecule bioanalysis. Current regulatory requirements were compiled and compared. The scope of the team's discussions included the validation report, the bioanalytical report, study plans, raw data, and bioanalytical summaries for the common technical document (CTD). A common high-level table of contents for method validation and sample analysis reports is proposed. Suggestions have been made as to how the CTD can be standardized to improve usability and review. Additional comments have been made on reports of failure investigations, study plans, and raw data documentation. The recommendation is that no prescriptive guidelines are required in these areas but should be led by good scientific practices subject to particular circumstances.

Large molecule run acceptance: Recommendation for best practices and harmonization from the Global Bioanalysis Consortium Harmonization Team.

The L1 Global Harmonization Team provides recommendations specifically for run acceptance of ligand binding methods used in bioanalysis of macromolecules in support of pharmacokinetics. The team focused on standard curve calibrators and quality controls for use in both pre-study validation and in-study sample analysis, including their preparation and acceptance criteria. The team also considered standard curve editing and the concept of total error.

Large molecule specific assay operation: recommendation for best practices and harmonization from the global bioanalysis consortium harmonization team.

The L2 Global Harmonization Team on large molecule specific assay operation for protein bioanalysis in support of pharmacokinetics focused on the following topics: setting up a balanced validation design, specificity testing, selectivity testing, dilutional linearity, hook effect, parallelism, and testing of robustness and ruggedness. The team additionally considered the impact of lipemia, hemolysis, and the presence of endogenous analyte on selectivity assessments as well as the occurrence of hook effect in study samples when no hook effect had been observed during pre-study validation.

2013 White Paper on recent issues in bioanalysis: 'hybrid'--the best of LBA and LCMS.

The 2013 7th Workshop on Recent Issues in Bioanalysis was held in Long Beach, California, USA, where close to 500 professionals from pharmaceutical and biopharmaceutical companies, CROs and regulatory agencies convened to discuss current topics of interest in bioanalysis. These 'hot' topics, which covered both small and large molecules, were the starting point for fruitful exchanges of knowledge, and sharing of ideas among speakers, panelists and attendees. The discussions led to specific recommendations pertinent to bioanalytical science. Such as the previous editions, this 2013 White Paper addresses important bioanalytical issues and provides practical answers to the topics presented, discussed and agreed upon by the global bioanalytical community attending the 7th Workshop on Recent Issues in Bioanalysis.

Theoretical considerations and practical approaches to address the effect of anti-drug antibody (ADA) on quantification of biotherapeutics in circulation.

Continuous improvement in bioanalytical method development is desired in order to ensure the quality of the data and to better support pharmacokinetic (PK) and safety studies of biotherapeutics. One area that has been getting increasing attention recently is in the assessment of "free" and "total" analyte and the impact of the assay format on those assessments. To compliment these considerations, the authors provide a critical review of available literature and prospectively explore methods to mitigate the potential impact of anti-drug antibody on PK assay measurement. This challenge is of particular interest and importance since biotherapeutic drugs often elicit an immune response, and thus may have a direct impact on quantification of the drug for its PK and safety evaluations.

2012 white paper on recent issues in bioanalysis and alignment of multiple guidelines.

Over 400 professionals representing pharmaceutical companies, CROs, and multiple regulatory agencies participated in the 6th Workshop on Recent Issues in Bioanalysis (WRIB). Like the previous sessions, this event was in the format of a practical, focused, highly interactive and informative workshop aiming for high-quality, improved regulatory compliance and scientific excellence. Numerous 'hot' topics in bioanalysis of both small and large molecules were shared and discussed, leading to consensus and recommendations among panelists and attendees representing the bioanalytical community. The major outcome of this year's workshop was the noticeable alignment of multiple bioanalytical guidance/guidelines from different regulatory agencies. This represents a concrete step forward in the global harmonization of bioanalytical activities. The present 2012 White Paper acts as a practical and useful reference document that provides key information and solutions on several topics and issues in the constantly evolving world of bioanalysis.

Bioanalytical approaches to quantify "total" and "free" therapeutic antibodies and their targets: technical challenges and PK/PD applications over the course of drug development.

The predominant driver of bioanalysis in supporting drug development is the intended use of the data. Ligand-binding assays (LBA) are widely used for the analysis of protein biotherapeutics and target ligands (L) to support pharmacokinetics/pharmacodynamics (PK/PD) and safety assessments. For monoclonal antibody drugs (mAb), in particular, which non-covalently bind to L, multiple forms of mAb and L can exist in vivo, including free mAb, free L, and mono- and/or bivalent complexes of mAb and L. Given the complexity of the dynamic binding equilibrium occurring in the body after dosing and multiple sources of perturbation of the equilibrium during bioanalysis, it is clear that ex vivo quantification of the forms of interest (free, bound, or total mAb and L) may differ from the actual ones in vivo. LBA reagents and assay formats can be designed in principle to measure the total or free forms of mAb and L. However, confirmation of the forms being measured under the specified conditions can be technically challenging. The assay forms and issues must be clearly communicated and understood appropriately by all stakeholders as the program proceeds through the development process. This paper focuses on monoclonal antibody biotherapeutics and their circulatory L that are either secreted as soluble forms or shed from membrane receptors. It presents an investigation into the theoretical and practical considerations for total/free analyte assessment to increase awareness in the scientific community and offer bioanalytical approaches to provide appropriate PK/PD information required at specific phases of drug development.

Quality assessment of bioanalytical quantification of monoclonal antibody drugs.

Monoclonal antibody biotherapeutics are developed to bind to a specific target to affect the target's biological effect. Reliable measurements of monoclonal antibodies in biological fluids using ligand-binding assays are vital for understanding the pharmacokinetic and pharmacodynamic relationships for efficacy/safety evaluations and dose-regimen selection. The method should be properly characterized and demonstrate adequate assay performance to generate credible data for the right decision making at each specific stage, with considerations on the constraints of timeline, reagent availability and assay caveats. Quality assessment of the assay performance should be based on whether the method is 'fit-for-use' to meet the objectives of the study. The basic industrial requirements and recent trends in method and data quality of ligand-binding assays for drug exposure studies at various development stages are discussed.

Workshop report and follow-up--AAPS Workshop on current topics in GLP bioanalysis: Assay reproducibility for incurred samples--implications of Crystal City recommendations.

The Conference Report of the 3rd AAPS/FDA Bioanalytical Workshop (Crystal City III) endorsed the concept that assay methods supporting bioanalytical data in submissions must demonstrate assay reproducibility by using incurred samples. The present Workshop was convened to provide a forum for discussion and consensus building about incurred sample assay reproducibility for both nonclinical and clinical studies. Information about current regulatory perspectives on incurred sample reanalysis (ISR) was presented, implications of ISR for both large and small molecules were discussed, and the steering committee put forth recommendations for performing ISR. These recommendations from the Workshop, along with the subsequent evolution of approaches leading to a robust ISR program, may be used by scientists performing bioanalytical assays for regulated studies to provide additional confirmation of assay reproducibility for incurred samples.

Key elements of bioanalytical method validation for macromolecules.

The Third American Association of Pharmaceutical Scientists/US Food and Drug Administration (FDA) Bioanalytical Workshop, which was held May 1 and 2, 2006, in Arlington, VA, addressed bioanalytical assays that are being used for the quantification of therapeutic candidates in support of pharmacokinetic evaluations. One of the main goals of this workshop was to discuss best practices used in bioanalysis regardless of the size of the therapeutic candidates. Since the last bioanalytical workshop, technological advancements in the field and in the statistical understanding of the validation issues have generated a variety of interpretations to clarify and understand the practicality of using the current FDA guidance for assaying macromolecular therapeutics. This article addresses some of the key elements that are essential to the validation of macromolecular therapeutics using ligand binding assays. Because of the nature of ligand binding assays, attempts have been made within the scientific community to use statistical approaches to interpret the acceptance criteria that are aligned with the prestudy validation and in-study validation (sample analysis) processes. We discuss, among other topics, using the total error criterion or confidence interval approaches for acceptance of assays and using anchor calibrators to fit the nonlinear regression models.

Recommendations for the design, optimization, and qualification of cell-based assays used for the detection of neutralizing antibody responses elicited to biological therapeutics.

The administration of biological therapeutics can evoke some level of immune response to the drug product in the receiving subjects. An immune response comprised of neutralizing antibodies can lead to loss of efficacy or potentially more serious clinical sequelae. Therefore, it is important to monitor the immunogenicity of biological therapeutics throughout the drug product development cycle. Immunoassays are typically used to screen for the presence and development of anti-drug product antibodies. However, in-vitro cell-based assays prove extremely useful for the characterization of immunoassay-positive samples to determine if the detected antibodies have neutralizing properties. This document provides scientific recommendations based on the experience of the authors for the development of cell-based assays for the detection of neutralizing antibodies in non-clinical and clinical studies.

The detection of anti-erythropoietin antibodies in human serum and plasma: part II. Validation of a semi-quantitative 3H-thymidine uptake assay for neutralizing antibodies.

Neutralizing antibodies to erythropoietin (EPO) can cause a loss of response to recombinant human EPO (rHuEPO) and lead to rare cases of sudden, unexplained, severe anemia in chronic renal failure patients treated with rHuEPO. An assay for neutralizing anti-EPO antibodies has been validated that is based on the inhibition of proliferation of human UT-7/EPO cells, an immortalized cell line, by neutralizing antibodies in serum test samples using 3H-thymidine as a marker for proliferation. The dependence of the human cell line on EPO for growth and proliferation in a concentration-dependent manner enabled the validation of a rHuEPO standard curve for cell proliferation that can be used to determine the presence of neutralizing anti-EPO antibodies in serum samples. Proliferation of the cells increases with increasing concentrations of EPO, forming an S-shaped standard curve, which is fit with a 4-parameter logistic model, between 2.5 and 50 mU/mL rHuEPO, with a percent coefficient of variation (% CV) from 8.7% to 22.1% and a % accuracy of 103.5% to 109.5%. Anti-EPO antibodies and serum with anti-EPO antibodies neutralize UT-7/EPO proliferation by 10 mU/mL rHuEPO in a concentration- or dilution-dependent manner with < or = 25% CV. Percent neutralization is calculated by determining the amount of EPO recovered from the original 10 mU/mL added using the formula [((10-concentration recovered)/10)x100%]. Stem cell factor (SCF) stimulated cell proliferation, but not as effectively as rHuEPO. Antibodies to SCF were not able to inhibit the proliferative response induced by EPO and vice versa, confirming the specificity of the assay for antibodies to EPO. High EPO levels can impact both the radioimmunoprecipitation and neutralization assays to produce a false negative result. However, the impact can be mitigated by the large dilutions used in the neutralization assay.

The pharmacokinetics of erythropoietin in the cerebrospinal fluid after intravenous administration of recombinant human erythropoietin.

OBJECTIVES: Erythropoietin (EPO) was originally described as a regulator of erythropoiesis. Recently, synthesis of EPO and expression of the EPO receptor (EPO-R) have been reported for the central nervous system (CNS). The potential use of EPO to prevent or reduce CNS injury and the paucity of information regarding its entry into the human CNS led us to examine the pharmacokinetics (PK) of recombinant human EPO (r-HuEPO) in the serum and cerebrospinal fluid (CSF). METHODS: Four patients with Ommaya reservoirs were enrolled to facilitate serial CSF sampling. R-HuEPO was given intravenously (IV) in single doses of 40,000 IU or 1,500 IU/kg and in multiple doses of 40,000 IU daily for 3 days. RESULTS: The EPO concentrations in the CSF increased after a period of slow equilibration. Linear first-order distribution kinetics were observed for serum and CSF. The concentration of EPO in the CSF was proportional to the serum concentration of EPO and the permeability of the blood-brain barrier (BBB), as determined by the albumin quotient (QA=[albumin] CSF/[albumin] serum). A rise in the CSF concentration was seen as early as 3 h after IV administration. Peak levels (C(max)) were reached between 9 h and 24 h. After a single dose of 1,500 IU/kg, the Cmax in the CSF ranged from 11 mIU/ml to 40 mIU/ml, and the ratios of CSF/serum Cmax ranged from 3.6x10-4 to 10.2x10-4. The terminal half-life (t1/2) values of EPO in serum and CSF were similar. The t(1/2) of r-HuEPO in the CSF ranged from 25.6 h to 35.5 h after a single dose of 1,500 IU/l. Using these parameters a PK model was generated that predicts the concentration-time profile of EPO in the CSF. CONCLUSIONS: We report that r-HuEPO can cross the human BBB and describe for the first time the PK of EPO in the CSF after IV administration. Our data suggest that the concentration-time profile of EPO in the CSF can be predicted for individual patients if the serum concentration of EPO and the Q(A) are known. This information may be useful in the design of clinical trials to explore the potential therapeutic effects of EPO during CNS injury.

Development and validation of ELISA for herceptin detection in human serum.

Herceptin, a humanized anti-human epidermal growth factor receptor-2 (HER2) monoclonal antibody, is used for treatment of metastatic breast cancer patients overexpressing HER2 on tumor cells, and is being studied in clinical trials for therapy of other types of cancer. In the present work, we developed a Herceptin enzyme-linked immunosorbent assay (ELISA) from commercially available reagents to meet the growing needs of clinical studies. In this immunoassay, a mixture of monoclonal antibodies specific for the cytoplasmic domain of human HER2 (676-1255 amino acids) is adsorbed onto a microtiter plate, followed by addition of full-length HER2 protein, which is captured by the antibodies. Herceptin in the serum that is added to the microwells binds to the extracellular domain (ECD) of the captured HER2. The Herceptin bound to HER2 is detected by an antihuman IgG-horse radish peroxidase (HRP) conjugate and a (3, 5, 3', 5')-tetramethylenbenzidine (TMB) substrate. The calibration range of the assay is 5-100 ng/mL after 1:2000 sample dilution corresponding to 10-200 microg/mL Herceptin in undiluted serum. The intra- and interassay CVs ranged from 4.56% to 13.3% and from 9.9% to 18.9%, respectively. The assay shows dilutional linearity and specificity. Soluble p105 HER2, which can be shed into serum does not interfere with the assay. The analytical performance of the Herceptin ELISA indicates that this assay can be used for monitoring concentration levels of Herceptin in human serum.

Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules.

PURPOSE: With this publication a subcommittee of the AAPS Ligand Binding Assay Bioanalytical Focus Group (LBABFG) makes recommendations for the development, validation, and implementation of ligand binding assays (LBAs) that are intended to support pharmacokinetic and toxicokinetic assessments of macromolecules. METHODS: This subcommittee was comprised of 10 members representing Pharmaceutical, Biotechnology, and the contract research organization industries from the United States, Canada, and Europe. Each section of this consensus document addresses a specific analytical performance characteristic or aspect for validation of a LBA. Within each section the topics are organized by an assay's life cycle, the development phase, pre-study validation, and in-study validation. Because unique issues often accompany bioanalytical assays for macromolecules, this document should be viewed as a guide for designing and conducting the validation of ligand binding assays. RESULTS: Values of +/- 20% (25% at the lower limit of quantification [LLOQ]) are recommended as default acceptance criteria for accuracy (% relative error [RE], mean bias) and interbatch precision (%coefficient of variation [CV]). In addition, we propose as secondary criteria for method acceptance that the sum of the interbatch precision (%CV) and the absolute value of the mean bias (%RE) be less than or equal to 30%. This added criterion is recommended to help ensure that in-study runs of test samples will meet the proposed run acceptance criteria of 4-6-30. Exceptions to the proposed process and acceptance criteria are appropriate when accompanied by a sound scientific rationale. CONCLUSIONS: In this consensus document, we attempt to make recommendations that are based on bioanalytical best practices and statistical thinking for development and validation of LBAs.

The detection of anti-erythropoietin antibodies in human serum and plasma. Part I. Validation of the protocol for a radioimmunoprecipitation assay.

Rare cases of unexplained sudden severe anemia or red cell aplasia and resistance to recombinant human erythropoietin (rHuEPO) in patients with chronic renal failure (CRF) have been attributed to the development of anti-EPO antibodies. The development and validation of a radioimmunoprecipitation (RIP) assay to detect human anti-EPO antibodies in serum or plasma has been hampered by the lack of purified antibody to fully characterize and validate the assay. We have prepared an affinity-purified human antibody to EPO and used the antibody to characterize and validate a sensitive and reproducible RIP assay that can qualitatively measure anti-EPO antibody in serum or plasma samples. The lower limit of detection of the assay is 8 ng/ml of purified antibody. The threshold for detecting antibody is > or =0.9% cpm bound. The precision of the assay using purified antibody standards ranges from 5.8% to 15.3% and the precision of the assay using dilutions of the positive control ranges from 15.9% to 18.7%. EPO in the samples did not interfere with detection of the anti-EPO antibody except at high concentrations.