High-Throughput Therapeutic Antibody Interference-Free High-Resolution Mass Spectrometry Assay for Monitoring M-Proteins in Multiple Myeloma.

Measurement of monoclonal antibodies (M-proteins) plays an important role in the diagnosis and treatment monitoring of multiple myeloma. Currently available M-protein assays have several limitations, particularly because of their lack of sensitivity and propensity to therapeutic antibody (t-mAb) interference. A previously described mass spectrometry method termed monoclonal immunoglobulin rapid accurate mass measurement (miRAMM) is more sensitive than current clinical tests and can provide a solution for resolving t-mAb interferences. However, the original miRAMM workflow is too complex for the throughput needed to analyze a large number of samples. Here, we describe a high-throughput liquid chromatography-high-resolution mass spectrometry (HT-LC-HRMS) approach that employs a fully automated immunocapture step, significantly improved immunoglobulin recovery, simplified chromatography, and high throughput (HT) data processing. In this HT-LC-HRMS approach, raw spectra of the peaks eluting from the LC column during the predefined time period are automatically deconvoluted without the need to identify and monitor the retention time of each patient-specific M-protein. The deconvoluted peak heights of M-protein and therapeutic antibody light chain are conveniently used for quantitation. With the total LC-HRMS measurement time being only 11.0 min, this method was able to differentiate between the M-protein and elotuzumab mass signatures in 91 out of 92 (98.9%) multiple myeloma serum samples tested. The single interference case was resolved using the mass signature of a heavy chain. In addition to resolving t-mAb interference, the developed assay has a 25-fold improvement in sensitivity over immunofixation electrophoresis and can potentially provide an objective tracking of M-proteins in patients with complete response.

Antipeptide Immunocapture with In-Sample Calibration Curve Strategy for Sensitive and Robust LC-MS/MS Bioanalysis of Clinical Protein Biomarkers in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Despite huge promises, bioanalysis of protein biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for clinical applications is still very challenging. Here, we describe a sensitive and robust LC-MS/MS assay to quantify clinical protein biomarkers in FFPE tumor sections using automated antipeptide antibody immunocapture followed by in-sample calibration curve (ISCC) strategy with multiple isotopologue reaction monitoring (MIRM) technique. ISCC approach with MIRM of stable isotopically labeled (SIL) peptides eliminated the need for authentic matrices for external calibration curves, overcame the matrix effects, and validated the quantification range in each individual sample. Specifically, after deparaffinization, rehydration, antigen retrieval, and homogenization, the protein analytes in FFPE tumor tissues were spiked with a known concentration of one SIL peptide for each analyte, followed by trypsin digestion and antipeptide immunocapture enrichment prior to MIRM-ISCC-based LC-MS/MS analysis. This approach has been successfully used for sensitive quantification of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) in 15 representative FFPE tumor samples from lung, colorectal, and head and neck cancer patients. Except for one sample, PD-L1 and PD-1 in all samples were quantifiable using this assay with concentrations of 27.85-798.43 (amol/µg protein) for PD-L1 and 16.96-129.89 (amol/µg protein) for PD-1. These results were generally in agreement with the immunohistochemistry (IHC) data but with some exceptions. This approach demonstrated the feasibility to quantify low abundant protein biomarkers in FFPE tissues with improved sensitivity, specificity, and robustness and showed great potential as an orthogonal analytical approach to IHC for clinical applications.

In-Sample Calibration Curve Using Multiple Isotopologue Reaction Monitoring of a Stable Isotopically Labeled Analyte for Instant LC-MS/MS Bioanalysis and Quantitative Proteomics.

A novel methodology of in-sample calibration curves (ISCC) using multiple isotopologue reaction monitoring (MIRM) of multiple naturally occurring isotopologue transitions of a stable isotopically labeled (SIL) analyte for instant liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis of biomarkers, biotherapeutics, and small-molecule compounds is proposed and demonstrated for the first time. The theoretical isotopic abundances of the SIL analyte in its MIRM channels can be accurately calculated based on the isotopic distributions of its daughter ion and neutral loss. The isotopic abundances in these MIRM channels can also be accurately measured with a triple quadrupole mass spectrometer. By spiking a known amount of a SIL analyte into each study sample, an ISCC can be established based on the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) in the selected MIRM channels of the SIL analyte and the measured MS/MS peak areas in the corresponding MIRM channels in each individual study sample. The analyte concentration of each study sample can then be calculated individually with the ISCC instantly without using an external calibration curve. The MIRM-ISCC-LC-MS/MS methodology was evaluated and demonstrated in this work with the examples of quantitation of a protein biomarker in human and monkey serum processed with immunocapture and trypsin digestion; three surrogate peptides in trypsin-digested human colon tissue homogenates; and a small-molecule drug in human and rat plasma extracted with liquid-liquid extraction. The potential applications of the MIRM-ISCC-LC-MS/MS methodology in quantitative proteomics, clinical laboratories, and other areas are also discussed in this paper. Without the need for using external calibration curves, this novel MIRM-ISCC-LC-MS/MS methodology can provide accurate and reliable bioanalysis in many potential applications, especially for cases where authentic matrices for external calibration curves are not available.

Eliminating Preparation of Multisample External Calibration Curves and Dilution of Study Samples Using the Multiple Isotopologue Reaction Monitoring (MIRM) Technique in Quantitative LC-MS/MS Bioanalysis.

Preparation of multisample external calibration curves and dilution of study samples are critical steps in bioanalytical sample processing for quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) based bioanalysis of small-molecule compounds, biotherapeutics, and biomarkers, but they can be time-consuming and prone to error. It is highly desired to simplify or eliminate these two steps in order to improve the assay throughput and robustness. While multisample external calibration curve preparation using authentic matrices can be eliminated with a previously reported in-sample calibration curve (ISCC) approach using multiple isotopologue reaction monitoring (MIRM) of a stable isotopically labeled (SIL) analyte, dilution of study samples is still inevitable due to limited LC-MS/MS assay ranges. In this work, a one-sample multipoint external calibration curve and isotope sample dilution, both using MIRM of an analyte, for quantitative LC-MS/MS based bioanalysis are proposed and demonstrated. By spiking a known amount of an analyte into one blank authentic matrix sample, a one-sample multipoint external calibration curve in an authentic matrix can be established on the basis of the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) and the MS/MS responses in the corresponding MIRM channels. This one-sample multipoint external calibration curve can be used in the same way as the traditional multisample external calibration curve for quantitative LC-MS/MS-based bioanalysis. As isotopic abundance in each MIRM channel can be calculated and measured accurately, isotope sample dilution can be achieved by simply monitoring one or a few of the MIRM channels of the analyte in addition to the most abundant MIRM channel for study samples. While the most abundant MIRM channel (isotopic abundance of 100%) is used for the quantitation of samples having concentrations within the assay calibration curve range, less abundant MIRM channels (isotopic abundance of IA%) can be used for the quantitation of samples having concentrations beyond the assay upper limit of quantitation (ULOQ), resulting in isotope dilution factors (IDF) of 100%/IA%. The approaches of one-sample multipoint external calibration curve and isotope sample dilution were evaluated and demonstrated in this work with an example of the quantitation of daclatasvir in human plasma extracted with liquid-liquid extraction. Using these approaches together with the MIRM-ISCC methodology, accurate and reliable LC-MS/MS bioanalysis can be achieved without the need of preparation of multisample external calibration curve and dilution of study samples.

Determination of Real Time in Vivo Drug Receptor Occupancy for a Covalent Binding Drug as a Clinical Pharmacodynamic Biomarker by Immunocapture-LC-MS/MS.

We report a novel immunocapture (IC)-LC-MS/MS methodology to directly measure real time in vivo receptor occupancy (RO) for a covalent binding drug in blood lysate. A small molecule quencher was added immediately after sample collection to convert the free receptor to a quencher-bound receptor (QB-R) which was measured with the drug-bound receptor (DB-R) simultaneously by LC-MS/MS after immunocapture enrichment, followed by trypsin digestion. Addition of the quencher is necessary to prevent the free receptor from ex vivo binding with the drug. The real time RO was calculated based on the concentrations of DB-R and the free receptor (which is now QB-R) that were obtained from each sample. This strategy has been successfully applied to the measurement of the RO for Bruton's tyrosine kinase (BTK) in the blood lysate of monkeys after dosing with branebrutinib (BMS-986195), a covalent BTK inhibitor being evaluated to treat rheumatoid arthritis. A custom-made quencher, which is more reactive to BTK than branebrutinib, was added in excess amount to bind with all available free BTK to form quencher-bound BTK (QB-BTK) during blood sample collection. To measure a wide range of % BTK RO, including those of <5% or >95%, the required LLOQ at 0.125 nM for QB-BTK and 0.250 nM for drug-bound BTK (DB-BTK) in blood lysate were successfully achieved by using this IC-LC-MS/MS strategy. This proof-of-concept assay demonstrated its suitability with high throughput for real time in vivo BTK RO measurement as a pharmacodynamic (PD) biomarker for clinical drug development.

Slow Off-Rate Modified Aptamer (SOMAmer) as a Novel Reagent in Immunoassay Development for Accurate Soluble Glypican-3 Quantification in Clinical Samples.

Accurate quantification of soluble glypican-3 in clinical samples using immunoassays is challenging, because of the lack of appropriate antibody reagents to provide a full spectrum measurement of all potential soluble glypican-3 fragments in vivo. Glypican-3 SOMAmer (slow off-rate modified aptamer) is a novel reagent that binds, with high affinity, to a far distinct epitope of glypican-3, when compared to all available antibody reagents generated in-house. This paper describes an integrated analytical approach to rational selection of key reagents based on molecular characterization by epitope mapping, with the focus on our work using a SOMAmer as a new reagent to address development challenges with traditional antibody reagents for the soluble glypican-3 immunoassay. A qualified SOMAmer-based assay was developed and used for soluble glypican-3 quantification in hepatocellular carcinoma (HCC) patient samples. The assay demonstrated good sensitivity, accuracy, and precision. Data correlated with those obtained using the traditional antibody-based assay were used to confirm the clinically relevant soluble glypican-3 forms in vivo. This result was reinforced by a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay quantifying signature peptides generated from trypsin digestion. The work presented here offers an integrated strategy for qualifying aptamers as an alternative affinity platform for immunoassay reagents that can enable speedy assay development, especially when traditional antibody reagents cannot meet assay requirements.

Prevalence of Pre-Existing Neutralizing Antibodies Against Adeno-Associated Virus Serotypes 1, 2, 5, 6, 8, and 9 in Sera of Different Pig Strains.

Pre-existing neutralizing antibodies (NAb) to adeno-associated virus (AAV) may diminish the efficacy of AAV-based therapies depending on the titer. To support gene therapy studies in pigs, the seroprevalence of NAb to AAV1, 2, 5, 6, 8, and 9 serotypes were assessed in the sera of 3 different strains of pigs consisting of 60 Norsvin Topigs-20 strain, 22 Gottingen minipigs, and 40 Yucatan minipigs. Cell-based NAb assays were developed for various AAV serotypes. The sera were tested for NAb in a Lec-2 cell line for AAV9 vector and in a COS-7 cell line for the other AAV serotypes. In the 60 Topigs-20 strain 2 to 4 years of age, 100% were positive for AAV2 NAb, 45% positive for AAV6 NAb, and ∼20% positive for each of AAV1, 5, 8, and 9 NAb. These data showed that ∼80% of Norsvin Topigs-20 pigs evaluated were seronegative for pre-existing NAb to the AAV1, 5, 8, and 9 serotypes, respectively. In 22 Gottingen minipigs at 5-6 months of age, serum AAV serotype-specific NAb coexisted with that of various other AAV serotypes at 32% to 46% between two serotypes. These results suggested that coexisting NAb resulted either from multiple AAV serotype coinfection or from one (or more) serotypes that can crossreact with other AAV serotypes in some minipigs. Among the 40 Yucatan minipigs, 20 of the minipigs were <3 months old and were all negative for NAb against AAV5, 8, and 9, and only one of these 20 pigs was positive to AAV1 and 6. We further determined the titers in those positive pigs and found most Gottingen minipigs had low titer at 1:20, whereas some of Topigs-20 pigs had titers between 1:80 and 1:320, and some of Yucatan pigs had titers between 1:160 and 1:640. These results suggested that the majority of the pigs in the three strains would be amenable to gene therapy study using AAV1, AAV5, AAV8, and AAV9 and that prescreening on circulating AAV antibodies could be helpful before inclusion of pigs into studies.

Application of in-sample calibration curve methodology for regulated bioanalysis: Critical considerations in method development, validation and sample analysis.

Traditionally, for a liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalytical assay, an external calibration curve is required to achieve accurate quantitation of an analyte. Recently, a novel in-sample calibration curves (ISCC) methodology that can achieve quick and accurate LC-MS/MS bioanalysis without the use of an external calibration curve was reported. The ISCC methodology utilizes the presence of multiple naturally occurring isotopologues of a stable isotopically labeled analyte to construct an in-sample calibration curve for the quantification. This methodology has great potential in many applications, for example biomarker measurement, quantitative proteomics and clinical diagnosis. Here, we assessed the feasibility of applying this ISCC-LC-MS/MS methodology in regulated bioanalysis using BMS-984478, a drug candidate, as the model compound. We also proposed method validation procedures/processes for this new approach for industry peers' consideration and feedback. A LC-MS/MS method using the ISCC strategy was successfully developed and validated for the quantitative analysis of BMS-984478 in human plasma over the range of 1.33-993.42 ng/mL. The validated ISCC-LC-MS/MS method was compared with a previously validated method using the conventional external calibration curve approach, and the two methods showed equivalent performance. Critical considerations and practical approaches in method development, validation and sample analysis were also discussed. Our work demonstrated that the ISCC-LC-MS/MS methodology is a promising approach for regulated LC-MS/MS bioanalysis. ISCC-LC-MS/MS methodology has its unique advantages and has great potential to be widely applied for various quantitative applications, and may even change the landscape of quantitative analysis.

Recommendations for singlet-based approach in ligand binding assays: an IQ Consortium perspective.

Historically, ligand-binding assays for pharmacokinetic samples employed duplicate rather than singlet-based analysis. Herein, the Translational and absorption, distribution, metabolism and excretion (ADME) Sciences Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) presents a study aiming to determine the value of duplicate versus singlet-based testing. Based on analysis of data collected from eight organizations for 20 drug candidates representing seven molecular types and four analytical platforms, statistical comparisons of validation and in-study quality controls and study unknown samples demonstrated good agreement across duplicate sets. Simulation models were also used to assess the impact of sample duplicate characteristics on bioequivalence outcomes. Results show that testing in singlet is acceptable for assays with %CV ≤15% between duplicates. Singlet-based approach is proposed as the default for ligand-binding assays while a duplicate-based approach is needed where imprecision and/or inaccuracy impede the validation of the assay.

A Fit-for-Purpose Method for the Detection of Human Antibodies to Surface-Exposed Components of BMS-986263, a Lipid Nanoparticle-Based Drug Product Containing a siRNA Drug Substance.

ND-L02-s0201/BMS-986263 is a lipid nanoparticle (LNP) drug product containing a heat shock protein 47 (HSP47)-specific small interfering ribonucleic acid (siRNA) and being developed for the treatment of liver and idiopathic pulmonary fibrosis. To address immunogenicity-related issues, we developed a robust, fit-for-purpose (FFP) three-tier electrochemiluminescent (ECL) anti-drug antibody (ADA) assay for the detection of antibodies (Abs) generated to surface-exposed components of BMS-986263. The drug was coated directly on plates, and several Abs specific for polyethylene glycol (PEG) and other surface components were tested for use as positive quality controls (QCs). Following selection of a rabbit monoclonal anti-PEG Ab, the assay was optimized, and various method development challenges specific to the modality and pseudo surrogate rabbit control were addressed. Screening, confirmatory, and titer cut points were validated following a statistical evaluation of 41 individual K2EDTA human plasma samples at a minimum required dilution (MRD) of 100. Assay precision, sensitivity, selectivity, drug tolerance, and hook effect were determined for the rabbit Ab prepared in human K2EDTA plasma matrix. The assay was used to interrogate anti-drug Ab (ADA) responses in normal human subjects who were administered 90 mg of the drug intravenously (IV) once every week for 3 weeks in phase I clinical trials. All pre- and post-dose samples were found to be negative for ADA. Based on these results, we concluded that BMS-986263 is not immunogenic. To the best of our knowledge, this work represents the first ADA method developed and reported for an LNP-based drug product.

Perspectives on exploring hybrid LBA/LC-MS approach for clinical immunogenicity testing.

Biological drug products may elicit an antidrug antibody (ADA) response. The current widely used bridging ligand binding assay (LBA) is the gold standard for ADA assessments in drug development, which is a qualitative assay followed by a quasi-quantitative titer analysis but can be prone to interferences from biological matrices, drug targets and circulating drugs. We present our perspectives and findings in exploring a hybrid LBA/LC-MS as an orthogonal bioanalytical tool for clinical immunogenicity assessments. The hybrid LBA/LC-MS is a semiquantitative assay with acceptable specificity, drug tolerance and the capability of multiplexed detection of ADA isotypes. The assay results suggest this technology to be a promising and complementary bioanalytical tool that can provide informative immunogenicity data in drug development.

A practical workflow for capillary microsampling in nonclinical studies.

Aim: The result of investigation on the procedure of sample handling and bioanalysis of small volume of plasma sample for nonclinical studies stored in 0.5-ml micronic tubes was reported. Results/methodology: Sample integrity of the small volume (25 µl) during long-term storage and the feasibility and data reliability of performing multiple re-assays on the small volume sample using 5 µl aliquot per analysis was evaluated. Conclusion: Integrity was maintained in samples (25 µl) stored for up to 1 month in 0.5-ml micronic tubes at -20°C. A 25 µl sample is sufficient for four-times of re-assays. This evaluation demonstrated the feasibility of this workflow of handling and bioanalysis on small volume plasma sample for GLP studies under the US FDA guidance.

A convenient strategy to overcome interference in LC-MS/MS analysis: Application in a microdose absolute bioavailability study.

Stable isotope labeled (SIL) compounds have been commonly used as internal standards (IS) to ensure the accuracy and quality of liquid chromatography-mass spectrometry (LC-MS) bioanalytical assays. Recently, the application of SIL drugs and LC-MS assays to microdose absolute bioavailability (BA) studies has gained increasing attention. This approach can provide significant cost and time saving, and higher data quality compared to the accelerator mass spectrometry (AMS)-based method, since it avoids the use of radioactive drug, high-cost AMS instrumentation and complex measurement processes. It also eliminates potential metabolite interference with AMS-based assay. However, one major challenge in the application of this approach is the potential interference between the unlabeled drug, the microdose SIL drug, and the SIL-IS during LC-MS analysis. Here we report a convenient and cost-effective strategy to overcome the interference by monitoring the isotopic ion (instead of the commonly used monoisotopic ion) of the interfered compound in MS analysis. For the BMS-986205 absolute BA case study presented, significant interference was observed from the microdose IV drug [13C7,15N]-BMS-986205 to its SIL-IS, [13C7,15N, D3]-BMS-986205, since the difference of nominal molecular mass between the two compounds is only 3 mu, and there is a Cl atom in the molecules. By applying this strategy (monitoring the 37Cl ion for the analysis of the IS), a 90-fold reduction of interference was achieved, which allowed the use of a synthetically accessible SIL compound and enabled the fast progress of the absolute BA study. This strategy minimizes the number of stable isotope labels used for avoiding interference, which greatly reduces the difficulty in synthesizing the SIL compounds and generates significant time and cost savings. In addition, this strategy can also be used to reduce the MS response of the analyte, therefore, avoiding the detector saturation issue of LC-MS/MS assay for high concentration BMS-986205. A LC-MS/MS assay utilizing this strategy was successfully developed for the simultaneous analysis of BMS-986205 and [13C7, 15N]-BMS-986205 in dog plasma using [13C7,15N, D3]-BMS-986205 as the IS. The assay was successfully applied to a microdose absolute BA study of BMS-986205 in dogs. The assay was also validated in human plasma and used to support a human absolute BA study. The same strategy can also be applied to other compounds, including those not containing Cl or other elements with abundant isotopes, or other applications (e.g. selection of internal standard), and the applications were presented.

Evaluating a Multiscale Mechanistic Model of the Immune System to Predict Human Immunogenicity for a Biotherapeutic in Phase 1.

A mechanistic model of the immune response was evaluated for its ability to predict anti-drug antibody (ADA) and their impact on pharmacokinetics (PK) and pharmacodynamics (PD) for a biotherapeutic in a phase 1 clinical trial. Observed ADA incidence ranged from 33 to 67% after single doses and 27-50% after multiple doses. The model captured the single dose incidence well; however, there was overprediction after multiple dosing. The model was updated to include a T-regulatory (Treg) cell mediated tolerance, which reduced the overprediction (relative decrease in predicted incidence rate of 21.5-59.3% across multidose panels) without compromising the single dose predictions (relative decrease in predicted incidence rate of 0.6-13%). The Treg-adjusted model predicted no ADA impact on PK or PD, consistent with the observed data. A prospective phase 2 trial was simulated, including co-medication effects in the form of corticosteroid-induced immunosuppression. Predicted ADA incidences were 0-10%, depending on co-medication dosage. This work demonstrates the utility in applying an integrated, iterative modeling approach to predict ADA during different stages of clinical development.

2019 White Paper on Recent Issues in Bioanalysis: Chromatographic Assays (Part 1 - Innovation in Small Molecules and Oligonucleotides & Mass Spectrometric Method Development Strategies for Large Molecule Bioanalysis).

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 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 1) covers the recommendations on Innovation in Small Molecules and Oligonucleotides & Mass Spec Method Development Strategies for Large Molecules Bioanalysis. Part 2 (2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) and Part 3 (New Insights in Biomarkers Assays Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in drug discovery & development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and The Gene Therapy Bioanalytical Challenges) are published in volume 11 of Bioanalysis, issues 23 and 24 (2019), respectively.

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.

Development and validation of a functional cell-based neutralizing antibody assay for ipilimumab.

Ipilimumab is the first US FDA-approved immune checkpoint-blocking antibody drug to harness the patient's own immune cells. One of the postmarketing requirements is to develop a cell-based neutralizing antibody assay. Here, we share some of the most challenging aspects encountered during the assay development: new cell line construction; an unexpected inhibition of T-cell activation by low concentrations of ipilimumab; and two issues caused by sample pretreatment with acid dissociation to overcome drug interference: instability of neutralizing antibody positive control at low pH, and incompatibility of commonly used acid dissociation buffers in the cell assay. After troubleshooting and optimization, we successfully validated the assay and used the assay to test clinical samples to date.

Development of a Chemiluminescent ELISA Method for the Detection of Total Anti-Adeno Associated Virus Serotype 9 (AAV9) Antibodies.

Recombinant adeno associated viruses (rAAV) have become an important tool for the delivery of gene therapeutics due to long-standing safety and success in clinical trials. Since humans often become exposed to AAVs and develop anti-AAV antibodies (Abs), a potential impediment to the success of gene therapeutics is neutralization of the viral particle before it has had a chance to bind and enter target cells to release the transgene. Identification of subjects with preexisting Abs having neutralizing potential, and exclusion of such subjects from clinical studies is expected to enhance drug efficacy. In vitro cell-based reporter assays are most often employed to determine the level of neutralizing antibodies in a given population. Such assays measure the ability of the Abs to prevent viral binding and entry into cells by engaging epitopes on the viral capsid involved in host cell receptor binding. In general, cell-based assays are low throughput and labor intensive and may suffer from high variability and low sensitivity issues. In contrast, enzyme-linked immunosorbent assays (ELISAs) are simpler, less variable, and have higher throughput. Demonstrating a correlation between neutralizing Abs assessed by a cell-based assay and total binding Abs measured in an ELISA will enable the use and substitution of the latter for screening and exclusion of subjects. In this work, we describe the development of a highly sensitive, specific, robust, and reproducible chemiluminescent ELISA method for the detection of total anti-AAV9 Abs. Using this method, we analyzed the prevalence of preexisting anti-AAV9 Abs in 100 serum samples from heart disease patients. Analysis of neutralizing Abs in the same samples using an in vitro cell-based assay showed a strong correlation between total anti-AAV9 Abs and neutralizing Abs, indicating the feasibility of using the total Ab ELISA in the future for patient screening and exclusion.

Bead-extraction and heat-dissociation (BEHD): A novel way to overcome drug and matrix interference in immunogenicity testing.

Biological therapeutics are foreign antigens and can potentially induce immune response resulting in the formation of anti-drug antibodies (ADA), which in turn may lead to a wide range of side effects. Neutralizing Ab (NAb) is a subset of ADA that can bind to the pharmacological activity regions of therapeutic to inhibit or complete neutralize its clinical efficacy. A cell-based functional NAb assay is preferred to characterize its neutralization activity. However, cell-based NAb assays are often vulnerable to drug interference, as well as interference from numerous serum factors, including but not limited to growth factors and disease-related cytokines. Bead Extraction with Acid Dissociation (BEAD) has been successfully applied to remove circulating drug and/or other interfering factors from human serum samples, thereby enriching for ADA/NAb. However, the harsh acid used in the extraction procedure can cause irreversible denaturing of NAb and lead to underestimated NAb measurement. Herein we describe a new approach when acid-dissociation is not optimal for a PEGylated domain antibody (Ab). We further demonstrate that heating at 62 °C can not only dissociate drug/ADA/NAb immune complex but also selectively and irreversibly denature domain Ab drug due to much lower thermal stability of the domain Ab, when compared to that of full antibodies. The irreversible denaturing of the drug favors the formation of an immune complex between ADA/NAb and the added biotinylated drug thus increasing the recovery of ADA/NAb from samples. We call this new procedure Bead Extraction with Heat Dissociation (BEHD), which can potentially be applied to other NAb assays that have poor compatibility with acid dissociation.

Concerted application of LC-MS and ligand binding assays to better understand exposure of a large molecule drug.

AIM: A ligand-binding assay (LBA) was used to measure exposure of PRM-151, the recombinant form of human pentraxin-2 (PTX-2), a complex pentamer with multiple binding partners. However, the assay showed a lack of dose-dependent exposure in select preclinical species and it could not differentiate the infused PRM-151 from the endogenous PTX-2 in nonhuman primates. MATERIALS & METHODS: Instead of assessing interference from its multiple binding partners, which could be time consuming and laborious, a LC-MS assay avoid of these interference was implemented to measure 'total' drug without the use of immunoaffinity capture reagents. RESULTS & CONCLUSION: The resultant LC-MS data confirmed the original data and the lack of dose-dependent exposure is now understood to be due to the multiple and diverse targets and functions and resultant complex biodistribution rather than an assay artifact.