Characterizing the Pharmacokinetics and Biodistribution of Therapeutic Proteins: An Industry White Paper.

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.

Absorption, Distribution, Metabolism, and Excretion of Therapeutic Proteins: Current Industry Practices and Future Perspectives.

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.

The first-in-human study of CNTO 7160, an anti-interleukin-33 receptor monoclonal antibody, in healthy subjects and patients with asthma or atopic dermatitis.

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.

First-in-human study to assess guselkumab (anti-IL-23 mAb) pharmacokinetics/safety in healthy subjects and patients with moderate-to-severe psoriasis.

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.

Clinical Pharmacology Perspective on Development of Adeno-Associated Virus Vector-Based Retina Gene Therapy.

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.

An IQ Consortium Perspective on Best Practices for Bioanalytical and Immunogenicity Assessment Aspects of CAR-T and TCR-T Cellular Therapies Development.

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.

Preexisting antibody assays for gene therapy: Considerations on patient selection cutoffs and companion diagnostic requirements.

Recombinant adeno-associated virus (AAV) vectors are the leading delivery vehicle used for in vivo gene therapies. Anti-AAV antibodies (AAV Abs) can interact with the viral capsid component of an AAV-based gene therapy (GT). Therefore, patients with preexisting AAV Abs (seropositive patients) are often excluded from GT trials to prevent treatment of patients who are unlikely to benefit1 or may have a higher risk for adverse events outweighing treatment benefits. On the contrary, unnecessary exclusion of patients with high unmet medical need should be avoided. Instead, a risk-benefit assessment that weighs the potential risks due to seropositivity vs. severity of disease and available treatment options, should drive the decision if patient selection is required. Assays for patient selection must be validated according to their intended use following national regulations/standards for diagnostic assays in appropriate laboratories. In this review, we summarize the current process of patient selection, including assay cutoff criteria and related assay validation approaches. We further provide considerations on regulatory requirements for the development of in vitro diagnostic tests supporting market authorization of a corresponding GT.

Optimization of Peripheral Blood Mononuclear Cell Processing for Improved Clinical ELISpot Assay Performance.

Cell and gene therapies have demonstrated impressive therapeutic efficacy in various human diseases. Nevertheless, cellular immune response directed against these therapeutic agents is an obstacle for achieving long-lasting clinical efficacy. Therefore, it is crucial to develop robust assays to accurately monitor cellular immunogenicity towards these therapies. Enzyme-linked immunospot (ELISpot) assay is one of the primarily used methods for measuring cellular immune response in clinical programs, which requires isolation of the peripheral blood mononuclear cells (PBMCs). The quality of this clinical material is one of the most critical factors that impact the robust assessment of cellular immune responses. The optimal blood sample processing conditions, however, remain poorly understood. In this study, we examined the impact of blood sample processing time on the performance characteristics of ELISpot to measure antigen-specific cellular responses. Blood samples that were processed after overnight delay resulted in a loss of ELISpot signals. We subsequently optimized several parameters of sample processing, and successfully recovered ELISpot signals for the blood samples that are processed within 32 h. Furthermore, several mitigation strategies were employed that would potentially address the impact of granulocyte contamination on detection of antigen-specific cellular responses. Our investigation provides an extension of sample processing window for clinical studies and is significant for resolving the logistical challenge of whole blood sample shipment for timely PBMC preparation in cell/gene therapy clinical studies.

Best Practices and Considerations for Clinical Pharmacology and Pharmacometric Aspects for Optimal Development of CAR-T and TCR-T Cell Therapies: An Industry Perspective.

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.

A Phase 1 First-in-Human Pharmacokinetic and Pharmacodynamic Study of JNJ-64264681, a Covalent Inhibitor of Bruton's Tyrosine Kinase.

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.

PK/PD and Bioanalytical Considerations of AAV-Based Gene Therapies: an IQ Consortium Industry Position Paper.

Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in understanding of the pharmacokinetics/pharmacodynamics (PK/PD) and disposition of this modality exist. This position paper comes from the Novel Modalities Working Group (WG), part of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). The pan-industry WG effort focuses on the nonclinical PK and clinical pharmacology aspects of AAV gene therapy and related bioanalytical considerations.Traditional PK concepts are generally not applicable to AAV-based therapies due to the inherent complexity of a transgene-carrying viral vector, and the multiple steps and analytes involved in cell transduction and transgene-derived protein expression. Therefore, we explain PK concepts of biodistribution of AAV-based therapies and place key terminologies related to drug exposure and PD in the proper context. Factors affecting biodistribution are presented in detail, and guidelines are provided to design nonclinical studies to enable a stage-gated progression to Phase 1 testing. The nonclinical and clinical utility of transgene DNA, mRNA, and protein analytes are discussed with bioanalytical strategies to measure these analytes. The pros and cons of qPCR vs. ddPCR technologies for DNA/RNA measurement and qualitative vs. quantitative methods for transgene-derived protein are also presented. Last, best practices and recommendations for use of clinical and nonclinical data to project human dose and response are discussed. Together, the manuscript provides a holistic framework to discuss evolving concepts of PK/PD modeling, bioanalytical technologies, and clinical dose selection in gene therapy.

Considerations in the Immunogenicity Assessment Strategy for Oligonucleotide Therapeutics (ONTs).

Oligonucleotide therapeutics (ONTs) are a diverse group of short synthetic nucleic acid-based molecules that exploit innovative intracellular molecular strategies to create novel treatments for a variety of medical conditions. ONT molecules (~7-15 kDa) reside between traditional large and small molecules, and there has been debate regarding their immunogenicity risk. To date, 13 ON drugs have been approved, and as the field is relatively new, there are currently no specific regulatory guidelines to indicate how to develop, validate, and interpret the immunogenicity assays of ONTs. Some investigators do not test for immune responses to ONs while others test for antibodies (Abs) to components within the formulation, which may or may not include aspects of characterization such as domain mapping of ONT conjugates. Similar to other biopharmaceuticals, the immunogenic properties of ONTs could be influenced by sequence, route, dosage, target population, co-medications, etc. The current anti-drug antibody (ADA) data for different approved ONTs suggest that their administration poses a low immunogenicity risk without any significant impact on pharmacokinetics (PK), pharmacodynamics (PD), and safety; nevertheless, until the field matures with data from many more ON drugs, it remains prudent to assess immunogenicity. The emphasis of this article is to highlight how current ADA methodologies might be applied to the development of ONTs, discuss factors that may pose immunogenicity risks, and provide the authors' current position on immunogenicity assessment strategies for ONTs. We also discuss assay parameters that may be appropriate for the detection and characterization of ADAs, including the evaluation of neutralizing ADAs, ADA isotyping, Abs to dsDNA, and pre-existing ADA. Immunogenicity risk assessments (IRAs) and early interactions with regulators will inform how to proceed in late stage/pivotal studies.

Immunogenicity assessment of AAV-based gene therapies: An IQ consortium industry white paper.

Immunogenicity has imposed a challenge to efficacy and safety evaluation of adeno-associated virus (AAV) vector-based gene therapies. Mild to severe adverse events observed in clinical development have been implicated with host immune responses against AAV gene therapies, resulting in comprehensive evaluation of immunogenicity during nonclinical and clinical studies mandated by health authorities. Immunogenicity of AAV gene therapies is complex due to the number of risk factors associated with product components and pre-existing immunity in human subjects. Different clinical mitigation strategies have been employed to alleviate treatment-induced or -boosted immunogenicity in order to achieve desired efficacy, reduce toxicity, or treat more patients who are seropositive to AAV vectors. In this review, the immunogenicity risk assessment, manifestation of immunogenicity and its impact in nonclinical and clinical studies, and various clinical mitigation strategies are summarized. Last, we present bioanalytical strategies, methodologies, and assay validation applied to appropriately monitor immunogenicity in AAV gene therapy-treated subjects.

Translational Modeling Predicts Efficacious Therapeutic Dosing Range of Teclistamab for Multiple Myeloma.

BACKGROUND: Teclistamab (JNJ-64007957), a B-cell maturation antigen × CD3 bispecific antibody, displayed potent T-cell-mediated cytotoxicity of multiple myeloma cells in preclinical studies. OBJECTIVE: A first-in-human, Phase I, dose escalation study (MajesTEC-1) is evaluating teclistamab in patients with relapsed/refractory multiple myeloma. PATIENTS AND METHODS: To estimate the efficacious therapeutic dosing range of teclistamab, pharmacokinetic (PK) data following the first cycle doses in the low-dose cohorts in the Phase I study were modeled using a 2-compartment model and simulated to predict the doses that would have average and trough serum teclistamab concentrations in the expected therapeutic range (between EC50 and EC90 values from an ex vivo cytotoxicity assay). RESULTS: The doses predicted to have average serum concentrations between the EC50 and EC90 range were validated. In addition, simulations showed that weekly intravenous and subcutaneous doses of 0.70 mg/kg and 0.72 mg/kg, respectively, resulted in mean trough levels comparable to the maximum EC90. The most active doses in the Phase I study were weekly intravenous doses of 0.27 and 0.72 mg/kg and weekly subcutaneous doses of 0.72 and 1.5 mg/kg, with the weekly 1.5 mg/kg subcutaneous doses selected as the recommended Phase II dose (RP2D). With active doses, exposure was maintained above the mean EC90. All patients who responded to the RP2D of teclistamab had exposure above the maximum EC90 in both serum and bone marrow on cycle 3, Day 1 of treatment. CONCLUSIONS: Our findings show that PK simulations of early clinical data together with ex vivo cytotoxicity estimates can inform the identification of a bispecific antibody's therapeutic range. CLINICAL TRIAL REGISTRATION: NCT03145181, date of registration: May 9, 2017.

Considerations in the development and validation of real-time quantitative polymerase chain reaction and its application in regulated bioanalysis to characterize the cellular kinetics of CAR-T products in clinical studies.

Real-time quantitative polymerase chain reaction (qPCR) has become the standard method for monitoring cellular kinetics of CAR-T therapies with measurement of the CAR transgene copy numbers in peripheral blood mononuclear cells isolated from patients receiving the treatment. Unlike other biophysical and immunological methodologies for bioanalytical characterization of conventional small molecule drugs or protein biologics, there is no relevant regulatory guidance to date on the method development and validation for quantitative qPCR assays employed during clinical development of CAR-T products. This paper will provide an overview and considerations in the development and validation of a qPCR assay from sample extraction to assay parameters and its implementation in regulated bioanalysis for CAR-T or other types of cell therapies.

Bifunctional molecules targeting SARS-CoV-2 spike and the polymeric Ig receptor display neutralization activity and mucosal enrichment.

The global health crisis and economic tolls of COVID-19 necessitate a panoply of strategies to treat SARS-CoV-2 infection. To date, few treatment options exist, although neutralizing antibodies against the spike glycoprotein have proven to be effective. Because infection is initiated at the mucosa and propagates mainly at this site throughout the course of the disease, blocking the virus at the mucosal milieu should be effective. However, administration of biologics to the mucosa presents a substantial challenge. Here, we describe bifunctional molecules combining single-domain variable regions that bind to the polymeric Ig receptor (pIgR) and to the SARS-CoV-2 spike protein via addition of the ACE2 extracellular domain (ECD). The hypothesis behind this design is that pIgR will transport the molecule from the circulation to the mucosal surface where the ACE ECD would act as a decoy receptor for the nCoV2. The bifunctional molecules bind SARS-Cov-2 spike glycoprotein in vitro and efficiently transcytose across the lung epithelium in human tissue-based analyses. Designs featuring ACE2 tethered to the C-terminus of the Fc do not induce antibody-dependent cytotoxicity against pIgR-expressing cells. These molecules thus represent a potential therapeutic modality for systemic administration of neutralizing anti-SARS-CoV-2 molecules to the mucosa.

2020 White Paper on Recent Issues in Bioanalysis: Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation (Part 3 - Recommendations on Immunogenicity Assay Strategies, NAb Assays, Biosimilars and FDA/EMA Immunogenicity Guidance/Guideline, Gene & Cell Therapy and Vaccine Assays).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global 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 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.

Application of multiplexed pharmacokinetic immunoassay to quantify in vivo drug forms and coadministered biologics.

Aim: Meso Scale Discovery U-PLEX® provides an opportunity to develop multiplexed pharmacokinetic (PK) immunoassays. Two case studies demonstrate the utility of multiplexed PK methods. Materials & methods: Development of PK ligand-binding assays quantify of nonclinical plasma concentrations of a biotherapeutic that has degraded due to in vivo biotransformation, and clinical serum concentrations from two biotherapeutics spiked into a single sample. Results: Data from multiplexed U-PLEX PK methods are comparable to results from single-readout streptavidin Meso Scale Discovery gold PK methods. Multiplex measurement of a nonclinical study showed acceptable performance for accuracy, precision and dilutional linearity while a clinical study additionally passed selectivity, specificity and stability. Conclusion: Regulated, validation-ready multiplex PK methods for both nonclinical and clinical studies allow opportunities for high-throughput bioanalysis.

Approaches to improve drug tolerance and target tolerance in the assessment of neutralizing anti-drug antibodies.

Aim: Neutralizing anti-drug antibody (NAb) assays are inherently prone to the interference from drug and its soluble target, potentially resulting in erroneous results. An effective approach to improve drug tolerance of an NAb assay is pretreatment of samples with acid to dissociate immune complexes of NAb and drug, followed by separating NAbs from circulating drug before testing them in the assay. Methods and Results: The acid pretreatment conditions were optimized to improve drug tolerance of cell-based and non-cell-based NAb assays. NAbs were further separated from circulating drug either through direct drug removal or purification of NAb from the sample. In addition, an integrated experimental strategy was implemented to simultaneously improve drug and its soluble target tolerance for reliable NAb assessment. Conclusion: The approaches described herein would enable the development of reliable NAb assays that overcome drug and its target interference for more precise and sensitive NAb assessment.

Analysis of regulatory guidance on antidrug antibody testing for therapeutic protein products.

Therapeutic proteins have the potential to induce unwanted immune responses. The potential impact of immunogenicity on pharmacokinetics, pharmacodynamics, safety and efficacy are well established. Here, we analyze key aspects of current US FDA and EMA guidelines on the development and validation of antidrug antibody assays. Although FDA and EMA guidance documents are in harmony on most points, EMA allows greater leeway for scientific judgement, while FDA recommends specific approaches that may not be appropriate in some situations. Many white papers suggest approaches different from the guidance documents, however, these can conflict with each other and are themselves only scientifically valid in certain situations. Here, we indicate when alternatives to guidance may be needed and what those approaches might be.