A survey of pharmacokinetic bioanalytical methods in biosimilar biological license applications for the assessment of target and antidrug antibody effects.

The presence of circulating targets and antidrug antibodies can influence the ability of a bioanalytical method to measure therapeutic protein (TP) concentration relevant to exposure-response evaluations. This project surveyed biosimilar submissions for their bioanalytical methods. Survey results revealed that 97% of pharmacokinetic methods designed to measure theoretically free or partial-free TPs with respect to target indeed measured free or partial-free TPs when considering experimental testing results for target effects. Antidrug antibody effect is less often evaluated. The observed trend of measuring biologically active forms of TP is consistent with the scientific understanding that pharmacokinetics of biologically active forms is more likely to be relevant to the clinical responses and evaluation of clinically meaningful differences to contribute to biosimilarity assessments.

Non-clinical similarity of biosimilar ABP 798 with rituximab reference product.

ABP 798 is a biosimilar to Rituxan® (rituximab reference product [RP]). Non-clinical assessments relevant to the primary and secondary mechanisms of action (MOA) contribute to the totality of the evidence (TOE) in supporting biosimilarity and are critical in providing scientific evidence for extrapolation of indications. Similarity of ABP 798 with rituximab RP was investigated across a range of biological activities which have potential impact on pharmacokinetics and clinical efficacy with non-clinical assessments relevant to MOA such as CD20 internalization, trogocytosis, binding to primary human natural killer (NK) cells as well as the ability to induce antibody-dependent cellular phagocytosis (ADCP) in peripheral blood mononuclear cells. Additionally, in vitro synergy of ABP 798 or RP with chemotherapeutic agents, in vivo xenograft studies in mice, and toxicological assessments in cynomolgus monkeys (including B cell depletion and toxicokinetics) were also conducted. Results from these non-clinical assessments contribute to the TOE supporting the biosimilarity between ABP 798 and rituximab RP across a range of primary and secondary MOAs and support justification for extrapolation to all indications of use for ABP 798 for which the RP is approved.

A Simulation Approach to Evaluate the Impact of Patterns of Bioanalytical Bias Difference on the Outcome of Pharmacokinetic Similarity Studies.

Pharmacokinetic (PK) similarity studies are vital to assess the biosimilarity of a biosimilar to a reference product. Systematic bias in a bioanalytical method that quantify products could be a potential source of error affecting the variability of the data and influencing the outcome of a PK similarity study. We investigated the impact of six varying patterns of bioanalytical bias difference (biasdiff ) between the similar products on the probability passing the PK similarity test. A population PK model was used to simulate concentration-time profiles for a biosimilar and a reference product and added biasdiff ranging from 030%. The probability of achieving the PK similarity criteria (90% confidence interval between 0.8 and 1.25) for the maximum serum concentration (Cmax ) and area under the curve (AUC) was assessed. The data indicate that an increase in absolute biasdiff between products of ≥ 10% would decrease the power to assess the similarity criteria for Cmax and AUC.

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.

Retrospective Analysis of Bioanalytical Method Validation Approaches in Biosimilar Biological Product Development.

Development and validation of a bioanalytical method for biosimilar biological product development (BPD) can be challenging. It requires the development of a bioanalytical method that reliably and accurately measures both proposed biosimilar and reference products in a biological matrix. This survey summarizes the current state of bioanalysis in BPD. Bioanalytical data from 28 biosimilar biologic license applications submitted to U.S. Food and Drug Administration (FDA) up to December 2018 were analyzed. The aim of the analysis was to provide (i) a summary of the bioanalytical landscape for BPD, (ii) a cumulative review of bioanalytical method validation approaches to aid in understanding how a specific method was selected, and (iii) a summary of data regarding bioanalytical bias differences between products. Results show diversity of the bioanalytical approaches used, as well as the observed differences in bioanalytical bias. Our findings highlight the need for understanding the critical aspects of BPD bioanalysis and clarifying BPD bioanalytical best practices, which could help ensure consistent method validation approaches in the BPD community.

Role of Modeling and Simulation in the Development of Novel and Biosimilar Therapeutic Proteins.

Modeling and simulation (M&S) is an important enabler of knowledge integration in novel biological product development programs. Given the volume of data generated from clinical trials and the complexity of pharmacokinetic (PK) and pharmacodynamic (PD) properties for reference products, extending the use of M&S to biosimilar development is logical. Assessing PK and PD similarity is normally a critical part of demonstrating biosimilarity to a reference product. Thoughtful considerations are necessary in study design to minimize the PK and PD variability, thereby increasing the sensitivity for detecting potential differences between products. In addition, the sensitivity of PD biomarkers depends partly on their relevance to the mechanism(s) of action and the dynamic range of PD response(s), including the impact of certain structural differences on PD in the relevant population. As such, opportunities exist for leveraging the available M&S knowledgebase to maximize the efficiency in the design and interpretation of PK and PD similarity studies. This article describes M&S applications which have contributed to and can continue to enhance biosimilar development programs.

Immune Suppression During Preclinical Drug Development Mitigates Immunogenicity-Mediated Impact on Therapeutic Exposure.

In the clinical setting, anti-drug antibodies (ADA) against biotherapeutics can influence patient safety and interfere with product efficacy. High immunogenicity has been addressed in clinic by concomitant immune suppression, such as co-administration of methotrexate with enzyme replacement therapy (ERT) and combination tacrolimus/sirolimus treatment for prophylaxis against organ transplant rejection. This study investigates the use of such immune suppressants in mitigating ADA responses to a fully human monoclonal antibody (mAb1) in preclinical animal studies. Three groups of Sprague Dawley rats (n = 18) were treated with low (0.01 mg/kg), moderate (50 mg/kg), or high (300 mg/kg) doses of mAb1. Experimental groups also received either methotrexate or tacrolimus/sirolimus immune suppressive regimens. ELISA-based methods were utilized to measure and characterize ADA and mAb1 pharmacokinetics (PK). Results demonstrated a stepwise increase in immunogenicity with mAb1 dosage. Methotrexate significantly lowered incidence of anti-variable region antibodies at moderate mAb1 dose (P < 0.05), while tacrolimus/sirolimus did likewise at moderate and high doses (P < 0.01) of mAb1. Except for low-dose mAb1 + methotrexate, all immunosuppressed groups displayed more than a 70-fold decrease in ADA magnitude (P < 0.05). This abrogation in ADA response correlated with more mAb1 in circulation by week 4 for moderate- and high-dosed mAb1 groups. These data provide an approach to mitigate preclinical immunogenicity by the use of immunosuppressant regimens. Such preconditioning can support preclinical drug development of human therapeutics that are antigenic to animals. Similar approaches could be investigated for wider application to novel therapeutics.

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 3 - LBA, biomarkers and immunogenicity).

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 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. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

Utility of a Bayesian Mathematical Model to Predict the Impact of Immunogenicity on Pharmacokinetics of Therapeutic Proteins.

The impact of an anti-drug antibody (ADA) response on pharmacokinetic (PK) of a therapeutic protein (TP) requires an in-depth understanding of both PK parameters and ADA characteristics. The ADA and PK bioanalytical assays have technical limitations due to high circulating levels of TP and ADA, respectively, hence, significantly hindering the interpretation of this assessment. The goal of this study was to develop a population-based modeling and simulation approach that can identify a more relevant PK parameter associated with ADA-mediated clearance. The concentration-time data from a single dose PK study using five monoclonal antibodies were modeled using a non-compartmental analysis (NCA), one-compartmental, and two-compartmental Michaelis-Menten kinetic model (MMK). A novel PK parameter termed change in clearance time of the TP (α) derived from the MMK model could predict variations in α much earlier than the time points when ADA could be bioanalytically detectable. The model could also identify subjects that might have been potentially identified as false negative due to interference of TP with ADA detection. While NCA and one-compartment models can estimate loss of exposures, and changes in clearance, the two-compartment model provides this additional ability to predict that loss of exposure by means of α. Modeling data from this study showed that the two-compartment model along with the conventional modeling approaches can help predict the impact of ADA response in the absence of relevant ADA data.

Fundamentals of large-molecule protein therapeutic bioanalysis using ligand-binding assays.

This article provides an overview of ligand-binding assays, including the origin and evolution of the primary concepts, in addition to reviewing commonly used assay formats. The birth of ligand-binding assays began with a radioimmunoassay developed to measure insulin in 1960. Fundamental to ligand-binding assay design is the requirement of at least one protein that interacts with the analyte of interest. Enzyme immunoassay has largely supplanted radioimmunoassay as the ligand-binding assay of choice in today's laboratory environment. This article illustrates various assay formats such as competitive, sandwich and bridging, in addition to, describing critical reagents necessary for their design. The utility of ligand-binding assays in therapeutic protein development and comparison to alternative bioanalysis platforms is discussed.

2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 3--LBA, biomarkers and immunogenicity).

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was 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 and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 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 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.

Measurement of Free Versus Total Therapeutic Monoclonal Antibody in Pharmacokinetic Assessment is Modulated by Affinity, Incubation Time, and Bioanalytical Platform.

Decisions about efficacy and safety of therapeutic proteins (TP) designed to target soluble ligands are made in part by their ex vivo quantification. Ligand binding assays (LBAs) are critical tools in measuring serum TP levels in pharmacokinetic, toxicokinetic, and pharmacodynamic studies. This study evaluated the impact of reagent antibody affinities, assay incubation times, and analytical platform on free or total TP quantitation. An ELISA-based LBA that measures monoclonal anti-sclerostin antibody (TPx) was used as the model system. To determine whether the method measures free or total TPx, the effects of K on, K off, and K D were determined. An 8:1 molar ratio of sclerostin (Scl) to TPx compared to a 1:1 molar ratio produced by rabbit polyclonal antibodies to TPx was required to achieve IC50, a measure of TPx interference effectiveness, making it unclear whether the ELISA truly measured free TPx. Kinetic analysis revealed that Scl had a rapid dissociation rate (K off) from TPx and that capture and detection antibodies had significantly higher binding affinities (K D) to TPx. These kinetic limitations along with long ELISA incubation times lead to the higher molar ratios (8:1) required for achieving 50% inhibition of TPx. However, a microfluidic platform with the same reagent pairs required shorter incubations to achieve a lower Scl IC50 molar ratio (1:1). The findings from this study provide the bioanalytical community with a deeper understanding of how reagent and platform selection for LBAs can affect what a particular method measures, either free or total TP concentrations.

In Silico Evaluation of the Potential Impact of Bioanalytical Bias Difference between Two Therapeutic Protein Formulations for Pharmacokinetic Assessment in a Biocomparability Study.

Formulation changes at later stages of biotherapeutics development require biocomparability (BC) assessment. Using simulation, this study aims to determine the potential effect of bias difference observed between the two formulations after spiking into serum in passing or failing of a critical BC study. An ELISA method with 20% total error was used to assess any bias differences between a reference (RF) and test formulations (TF) in serum. During bioanalytical comparison of these formulations, a 9% difference in bias was observed between the two formulations in sera. To determine acceptable level of bias difference between the RF and TF bioanalytically, two in silico simulations were performed. The in silico analysis showed that the likelihood of the study meeting the BC criteria was >90% when the bias difference between RF and TF in serum was 9% and the number of subjects was ≥20 per treatment arm. An additional simulation showed that when the bias difference was increased to 13% and the number of subjects was <40, the likelihood of meeting the BC criteria decreased to 80%. The result from in silico analysis allowed the bioanalytical laboratory to proceed with sample analysis using a single calibrator and quality controls made from the reference formulation. This modeling approach can be applied to other BC studies with similar situations.

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.

Validation of a microfluidic platform to measure total therapeutic antibodies and incurred sample reanalysis performance.

BACKGROUND: A microfluidic platform-based assay was validated to measure a humanized or fully human IgG in rat serum samples. MATERIALS & METHODS: The cumulative assessment for accuracy and precision was performed with three accuracy and precision runs. RESULTS: The inter-assay accuracy (mean %bias) ranged from -4.3 to 3.8%, and inter-batch %CV ranged from 5.0 to 9.2%. The method acceptance criterion was determined as 15% total error. The assay dynamic range was 50 to 10000 ng/ml. Incurred sample reanalysis passed with 95% of samples meeting incurred sample reanalysis acceptance criteria. Potential carryover effect was not observed. CONCLUSION: This study demonstrated the need for evaluating additional platform-specific processes when new technologies are employed to ensure the reproducibility of a successfully validated microfluidic platform method.

Evaluating the impact of matrix effects on biomarker assay sensitivity.

BACKGROUND: The accuracy of highly sensitive biomarker methods is often confounded by the presence of various circulating endogenous factors in samples causing matrix effects. METHOD: This article outlines two different biomarker methods: hepcidin enzyme-linked immunosorbent assay (ELISA) for which an orthogonal assessment of ELISA to liquid chromatography-tandem mass spectrometry was performed to examine the potential matrix effect, and sclerostin ELISA to evaluate the matrix effect. RESULTS: Although the potential interfering effects of the endogenous hepcidin variants (prohepcidin and clipped) showed that these proteins had >30% immunoreactivity in ELISA, the hepcidin ELISA preferentially measures full-length hepcidin when the molar ratios of full-length to variants remain >1. The correlation of ELISA to liquid chromatography-tandem mass spectrometry results showed full-length hepcidin as the major form in diseased populations. CONCLUSION: A fit-for-for-purpose assessment of matrix effect/selectivity was also performed for each method. This article demonstrates the utility of a fit-for-purpose approach to assess the validity of biomarker methods in evaluating the interconnected parameters of matrix effects, sensitivity and selectivity.

Impact of anti-drug antibodies in preclinical pharmacokinetic assessment.

The administration of human biotherapeutics is often associated with a higher incidence of immunogenicity in preclinical species. The presence of anti-drug antibodies (ADAs) in the test samples can affect the accurate measurement of therapeutic protein (TP) in bioanalytical methods designed to support pharmacokinetic (PK) and toxicokinetic (TK) assessments. The impact can vary depending on the bioanalytical method platform and study dosing design. The goal of this study is to evaluate the impact of ADA response on the bioanalytical methods in support of PK/TK and the associated study data interpretation. Sprague Dawley rats were administered with four weekly doses of 50 mg/kg TP, a humanized monoclonal antibody. The TP in serum samples was measured using three bioanalytical methods that quantified bound and/or unbound TP to ADA. The ADA response in the animals was classified into negative, low, medium, and high based on the magnitude of the response. The presence of ADA in samples led to discrepant TP measurements between the methods, especially at time points where the TP concentrations were low. This could be due to ADA interference to the accurate measurement of ADA-bound TP concentrations. The TP concentration at last time point (C last) was reduced by 82.8%, 98.6%, and 99.8%, respectively, for samples containing low, medium, and high levels of ADA. The interfering effects of the ADA on bioanalytical methods and exposure were evident as early as 2 weeks post-dosing. This modeling approach can provide the better understanding of ADA impact on PK exposure in multiple doses.