Determination of label efficiency and label degree of critical reagents by LC-MS and native MS.

Degree of labeling and label efficiency are key factors for optimal characterization of critical reagents that are used in ligand binding assays. Here, three case studies are shown demonstrating how liquid chromatography-mass spectrometry (LC-MS) was utilized to characterize critical reagents using three unique methodologies. Critical reagent batches were prepared for LC-MS analysis by use of: 20 mM dithiothreitol (DTT) (Case 1), rapid PNGaseF (Case 2), and a mobile phase diluent (Case 3). LC-MS was run at three different MS method conditions in each troubleshooting case specific for reduced IgG, intact IgG, and native LC-MS, respectively. Specified LC-MS methods based on sample type and configuration elucidated clear MS profiles, allowing for degree of labeling and label efficiencies to be calculated. Ultimately the LC-MS analyses were fine-tuned for critical reagent characterization, and practices for analyzing similar reagents in the future can be established.

Best practices on critical reagent characterization, qualification, and life cycle management for HCP immunoassays.

The performance of immunoassays for the detection and quantification of host-cell proteins (HCPs) in biopharmaceuticals depends on the quality of the critical assay reagents. Not only their preparation, but also a stringent life-cycle management, including reagent qualification, requalification, and replacement, plays a crucial role in ensuring consistent and reliable results. To provide a cross-industry perspective on HCP reagent management, we conducted a survey on common practices among several pharmaceutical and biotech companies. Based on its outcome, as well as informed by a corresponding roundtable session ("Managing critical reagents over time") at the BioPharmaceutical Emerging Best Practices Association HCP conference in 2019, this study presents specific recommendations and proven concepts to support immunoassay reagent management for monitoring HCPs.

Long term investigation of formulation buffers to mitigate stability issues of conjugated critical reagents.

Stability of conjugated critical reagents supporting ligand binding assays to enable biotherapeutic drug development is a universal concern. Formulation buffer employed for long-term cold storage may be key to mitigate protein aggregation issues. We investigated biophysical and functional attributes of murine mAb and human multispecific drug labeled with biotin, ruthenium, and Alexa fluor 647 frozen at -80 °C in PBS or a protein storage buffer for 3-15 months. Aggregation was observed at 4 months in mAb A-Ru (11.2%) and -Alexa (10%) in PBS followed by precipitation and reduced biological binding at 15 months. Increased aggregation in drug Ru (11.7%, 6 months) and Alexa (6.9%, 15 months) were noted but without impact on performance. There were no observations with biotin labeled reagents.

Critical reagent considerations for immunogenicity assay development for bispecific biotherapeutic candidates.

Aim: To demonstrate the importance of critical reagent characterization for immunogenicity assay development for multi-specific drugs using two case studies.Methods: Bridging anti-drug antibody (ADA) assay with acid-dissociated samples were used for both cases.Results: In the first case study, the unexpected interference in an ADA assay from clinical samples was identified; a model was created to replicate the issue, and an anti-target antibody was identified to mitigate the target interference. In the second case study, an issue due to non-specific binding of a domain-specific confirmatory reagent was identified, and various mitigation techniques were evaluated.Conclusion: A thorough characterization of the critical reagents helped identify the issues with these ADA case studies and provided strategies for resolving them.

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Unique challenges required reassessment and alterations to critical reagents to rescue a neutralizing antibody assay.

Aim: To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC).Materials & methods: Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target.Results & conclusion: A larger dynamic range, high inhibition and higher drug tolerance were achieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.

Many of the drugs available today are produced by a living organism and these are called biologics. Biologics are larger than chemical drugs and the human body can detect them as foreign and create antibodies against them. This is called immunogenicity. When the antibodies created against the biologic blocks the drug's ability to work correctly, they are called neutralizing antibodies (NAbs). Testing for NAbs is one of the requirements of regulatory agencies for biologics. Here we describe challenges encountered developing an assay to test for NAbs against a biologic.

Simple method to determine the concentration and incorporation ratio of ruthenium-labeled antibodies.

Aim: Ruthenium-labeled antibodies are commonly used detection reagents in bioanalysis assays and must be characterized to ensure quality. The aim of this work was to develop a method to determine the concentration and incorporation ratio (the degree of labeling [DOL]) of ruthenium-labeled antibodies by UV/VIS spectroscopy. Materials & methods: Free SULFO-TAG compound was scanned using UV/VIS and showed an absorbance peak at 292 nm. In contrast, antibodies demonstrate UV absorbance at 280 nm. After experimentally determining the extinction coefficients at 280 and 292 nm of free ruthenium and antibody, we generated a formula based on the Beer-Lambert law that calculates both concentration and DOL of these ruthenium-labeled antibodies. Conclusion: The concentration and DOL values determined by our method were comparable to those determined from bicinchoninic acid and LC/MS for the same reagents. This method creates a faster and more accessible reagent characterization process that uses far less reagent than the more traditional alternatives.

Critical reagent inventory management system and web portal specifically optimized for supporting external clients.

High-quality critical reagents are essential for the establishment of robust ligand binding assays to support regulated bioanalysis. To ensure consistency in assay performance over the lifetime of a project, a well-defined set of processes is needed for critical reagent life cycle management. Moreover, contract research organizations must support reagent life cycle management for diverse global clients. To address these needs, the authors designed and implemented a customized inventory management system, known as LCM+. This software solution provides external clients with efficient, secure access via a web portal to their critical reagent information, pertinent documentation and inventory tracking. Hence, the authors believe that LCM+ can serve as a useful prototype to aid the design of future inventory management systems for optimal management of critical reagents.

Critical reagents in flow cytometry, instrumentation and application in drug discovery development.

Flow cytometer is a powerful cellular analysis tool consists of three main components; fluidics, optics and electronics. Flow cytometry methods have been used in all stages of drug development as like ligand binding assays (LBA). Both LBA and flow cytometry methods require specific interaction between the critical reagents and the analytes. Antibodies and their conjugates, viable dyes and permeabilizing buffer are the main critical reagents in flow cytometry methods. Similarly, antibodies, engineered proteins and their conjugates are the main critical reagents in LBA. The main difference between the two methods is the lack of true reference standards for flow cytometry cellular analysis.

The importance of quality critical reagents for the entire developmental lifecycle of a biopharmaceutical: a pharmacokinetic case study.

Background: High-quality critical reagents are essential to the successful support of biotherapeutic drug development regardless of the analytical platform used for support. The lack of such a reagent, early in the development lifecycle of a biotherapeutic can have detrimental impact on resource and translation of data across development phases. Results: Here, a pharmacokinetic assay case study is shared that illustrates what can occur when there is a lack of a reproducible and sustainable critical reagent early in the development lifecycle of a biotherapeutic. Various assay formats and critical reagents, as well as reagents generation programs, were initiated to find a reagent and assay format which was fit for purpose. Conclusions: Identification of appropriate critical reagents early in the development lifecycle of a biotherapeutic as advantageous.

Best practices for the development, analytical validation and clinical implementation of flow cytometric methods for chimeric antigen receptor T cell analyses.

Chimeric Antigen Receptor (CAR) T cells are recognized as efficacious therapies with demonstrated ability to produce durable responses in blood cancer patients. Regulatory approvals and acceptance of these unique therapies by patients and reimbursement agencies have led to a significant increase in the number of next generation CAR T clinical trials. Flow cytometry is a powerful tool for comprehensive profiling of individual CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. There are unique challenges with regard to the development and validation of flow cytometric methods for CAR T cells; moreover, the assay requirements for manufacturing and clinical monitoring differ. Based on the collective experience of the authors, this recommendation paper aims to review these challenges and present approaches to address them. The discussion focuses on describing key considerations for the design, optimization, validation and implementation of flow cytometric methods during the clinical development of CAR T cell therapies.

Critical reagent characterization and re-evaluation to ensure long-term stability: two case studies.

Characterization of critical reagents can mitigate adverse impact to ligand-binding assay performance. We investigated the conjugation conditions of a bispecific protein to SULFO-TAG NHS-Ester™ ruthenium to resolve a steady increase in ligand-binding assay background signal. Functional and biophysical attributes in stability samples revealed low pH (4.0) conjugation and formulation buffers were key to decrease aggregate formation. We also identified pH-specific (3.0) purification conditions to reduce aggregate levels from 37% to <5% of a mouse IgG3 reagent antibody. These case studies support the utility of biophysical and functional characterization of critical reagents as a proactive approach to maintain long-term stability and provide the basis for our recommendations a risk-based approach to establish re-evaluation intervals for traditional and novel reagents.

Lot-to-lot reproducibility, stability and life cycle management of antibody reagents for flow cytometry.

The increasing number of biopharmaceuticals, gene and cell therapies in development has seen a growing use of flow cytometry to measure biomarkers, generate pharmacokinetic data, assess immunogenicity and investigate target engagement. The importance of these data types and their inclusion in regulatory submissions mean that flow cytometry analyses are now expected to demonstrate robust performance and comply with both regulatory and scientific recommendations during their validation and subsequent use in sample analysis. The control of the 'critical reagents' commonly used in flow cytometry presents some specific challenges, particularly when an assay is required for use over a long period of time across different phases of a drug development program, or where it is deployed in complex, multisite clinical studies. This paper highlights some key challenges in flow cytometry reagent management with some of the strategies employed to control and monitor flow cytometry critical reagents.

Monitoring CAR-T cell kinetics in clinical trials by multiparametric flow cytometry: Benefits and challenges.

Exceptional clinical responses produced by the first chimeric antigen receptor T [CAR-T] cell therapies, and their entry into commercial markets prompted a logarithmic increase in the number of next generation CAR-T clinical trials. As a result, there is a growing interest in understanding the analytical approaches utilized for reliable monitoring of these "living" drugs, and the challenges encountered during their clinical development. Multiparametric flow cytometry (MFC) assays have played a crucial role in understanding the phenotype and function of first approved CAR-T therapies. Herein, three main areas for monitoring CAR-T therapies in clinical trials are discussed: (1) analytical considerations critical for development of MFC assays for the reliable enumeration of CAR-T levels, (2) operational challenges associated with clinical trial sampling and transportation, and (3) differential cellular kinetics observed by MFC and qPCR analyses and their relationship with efficacy (measurable residual disease levels). Initial experiences described here may enable design of fit-for-purpose tools and help to more rapidly advance the development of next generation CAR-T therapies.

Magnetic bead extraction with acid dissociation immunoassay for the determination of serum CD80-Fc fusion protein.

Background: To detect concentrations of subtherapeutic doses of the CD80-Fc fusion protein FPT155 in serum in Phase I studies, a highly sensitive assay was developed. Materials & methods: FPT155 was purified from human serum using magnetic beads coupled to cytotoxic T-lymphocyte-associated antigen-4. After washing away the serum components, FTP155 was released by acid dissociation and neutralization. The eluted drug was quantified in an ELISA using cytotoxic T-lymphocyte-associated antigen-4 as a capture reagent and biotinylated anti-human Fc for detection. The assay was validated with a calibration range of 5-40 ng/ml and a dilutional integrity of up to 100,000 ng/ml. Conclusion: A highly sensitive assay to determine serum concentrations of FPT155 using readily available reagents was developed. The results were in conformity with theoretical calculations.

Critical reagents issues & solutions for anti-drug antibody assays.

Background: Anti-Drug Antibody assays (ADA) are developed and constructed with biological and chemical reagents. Capture and detector reagents as well as ADA standard are considered critical for the performance's characteristics of a bridging assay. Current literature well describes theoretical considerations to manage critical reagents (CR) life cycle management. Nevertheless, those recommendations must be completed by a pragmatic approach which have to be exemplified. Methodology: This article intends to present and describe two study cases of bioanalytical challenge coming from the practical experience of dealing with ADA CR and offers a concrete explanation of how to solve issues. Conclusion: An appropriate management of ADA CR goes through availability anticipation, characterization and by a scientific understanding process of assay and reagents inconsistency.

EBF recommendation on practical management of critical reagents for antidrug antibody ligand-binding assays.

Immunogenicity assays are required to measure antidrug antibodies that are generated against biotherapeutic modalities. As for any ligand-binding assays, critical reagents (CR) play a crucial role in immunogenicity assays, as the robustness and reliability of an assay are defined by the quality and long-term availability of these reagents. The current regulatory guidelines do not provide clear directions on how to implement and verify lot-to-lot changes of CR during an assay life cycle, or the acceptance criteria that should be used when implementing new lots of CR. These aspects were extensively discussed within the European Bioanalysis Forum community. In this paper, CR for immunogenicity assays are identified and the minimum requirements for introducing new lots of CR in immunogenicity assays are described.

New insights on critical reagent optimization for antidrug antibody assays.

Aim: Conjugated critical reagents are a pillar of ligand binding analysis. Although good practices for characterization have already been discussed, little is known about how assays are affected by specific conjugation parameters. Results: Here we developed, characterized and screened a toolset of bioconjugates that provided new insights about the optimization of conjugated critical reagent attributes. Biotinylated and sulfo-tagged trastuzumab were utilized as capture and detection antibodies, respectively, in an antidrug antibody (ADA) assay. The optimal conjugation window was identified by functional assessment. Excess of unlabeled biotin, but not sulfo-tag, affected the assay performance. An increase in the assay baseline was observed when sulfo-tagged trastuzumab underwent increasing freeze-thaw cycles. Conclusion: Upfront systematic screening and characterization of conjugated critical reagent attributes benefit assay robustness.

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.

Critical reagent screening and characterization: benefits and approaches for protein biomarker assays by hybrid LC-MS.

In recent years, hybrid ligand-binding assays (LBAs)/LC-MS assays have been increasingly used for quantitation of protein biomarkers in biological matrices. However, unlike in LBAs where the importance of critical reagent screening and characterization is well understood and widely reported, benefits of well-characterized hybrid LC-MS assay reagents are frequently underestimated. Two groups of analyte-specific reagents, binding reagents and assay calibrators, are considered the critical reagents for biomarker assays. In this article, we summarize the similarities and differences of critical reagents used in LBAs and hybrid LC-MS assays, overview the benefits and approaches of critical reagent screening, characterization, antibody conjugation and discuss bioanalytical considerations in hybrid LC-MS assay development for robust measurements of protein biomarkers in biological matrices.

EBF recommendation on practical management of critical reagents for PK ligand-binding assays.

Critical reagents play a crucial role in ligand-binding assays; the robustness and reliability of an assay is defined by the quality and long-term availability of these reagents. However, neither regulatory guidelines nor relevant scientific papers provide clear directions for set-up, life cycle management and, more importantly, the acceptance criteria required for the testing of the critical reagents for pharmacokinetic, biomarker and immunogenicity assays. The ambiguity from current guidelines can be a challenge for the bioanalytical community. Members of the European Bioanalysis Forum community undertook a more pragmatic approach on how to assess the impact of critical reagents. In this paper, a review and corresponding gap analysis of the current guidelines and relevant papers will be provided as well as decision trees proposed for lot-to-lot changes of critical reagents for pharmacokinetic assays.