RSV neutralizing antibodies in dried blood.

BACKGROUND: The key correlate of protection of respiratory syncytial virus (RSV) vaccines and monoclonal antibodies (mAb) is virus neutralization, measured using sera obtained through venipuncture. Dried blood obtained with a finger prick can simplify acquisition, processing, storage, and transport in trials, and thereby reduce costs. In this study we validate an assay to measure RSV neutralization in dried capillary blood. METHODS: Functional antibodies were compared between matched serum and dried blood samples from a phase I trial with RSM01, an investigational anti-RSV Prefusion F mAb. Hep-2 cells were infected with a serial dilution of sample-virus mixture using RSV-A2-mKate to determine half-maximal inhibitory concentration. Stability of dried blood was evaluated over time and during temperature stress. RESULTS: Functional antibodies in dried blood were highly correlated with serum (R2 = 0.98, p < 0.0001). The precision of the assay for dried blood was similar to serum. The function of mAb remained stable for 9 months at room temperature and frozen dried blood samples. INTERPRETATION: We demonstrated the feasibility of measuring RSV neutralization using dried blood as a patient-centered solution that may replace serology testing in trials against RSV or other viruses, such as influenza and SARS-CoV-2.

Immunogenicity Risk Assessment for Multi-specific Therapeutics.

The objective of this manuscript is to provide the reader with a hypothetical case study to present an immunogenicity risk assessment for a multi-specific therapeutic as part of Investigational New Drug (IND) application. In order to provide context for the bioanalytical strategies used to support the multi-specific therapeutic presented herein, the introduction focuses on known immunogenicity risk factors. The subsequent hypothetical case study applies these principles to a specific example HC-12, based loosely on anti-TNFα and anti-IL-17A bispecific molecules previously in development, structured as an example immunogenicity risk assessment for submission to health authorities. The risk of higher incidence and safety impact of anti-drug antibodies (ADA) due to large protein complexes is explored in the context of multi-specificity and multi-valency of the therapeutic in combination with the oligomeric forms of the targets.

Strategies for method comparison when changes in the immunogenicity method are needed within a clinical program.

Aim: To present the reader with different approaches used to compare immunogenicity methods when changes are needed during a clinical program. Results: Five case studies are presented, in the first two case studies, the approach utilized a small sample size for the comparison. In the third case, all samples from a study were analyzed by both methods. In the fourth case, the intended use of noncomparable assays in an integrated summary drove design of experiments to establish the expected limits of pooling data. In the fifth case, a selectivity approach was used as an alternate to use of incurred samples. Conclusion: When data pooling across methods is needed, it is important to define the limits of comparability.

Immunogenicity Risk Assessment for PEGylated Therapeutics.

The objective of this manuscript is to provide the reader with two examples on how to present an immunogenicity risk assessment for a PEGylated therapeutic as part of Investigational New Drug (IND) application or during other stages of the drug development process. In order to provide context to the bioanalytical strategies used to support the PEGylated therapeutics presented here, a brief summary of information available for marketed PEGylated biologics is provided. Two case studies are presented, a PEGylated enzyme and a PEGylated growth factor. For the former, the risk assessment covers how to deal with a narrow therapeutic window and suggestions to utilize a PD marker as surrogate for neutralizing antibody assessments in Phase I. The latter has recommendations on additional analytes that should be monitored to mitigate risk of immunogenicity to endogenous counterparts.

Time-Varying Clearance and Impact of Disease State on the Pharmacokinetics of Avelumab in Merkel Cell Carcinoma and Urothelial Carcinoma.

Avelumab, a human anti-programmed death ligand 1 immunoglobulin G1 antibody, has shown efficacy and manageable safety in multiple tumors. A two-compartment population pharmacokinetic model for avelumab incorporating intrinsic and extrinsic covariates and time-varying clearance (CL) was identified based on data from 1,827 patients across three clinical studies. Of 14 tumor types, a decrease in CL over time was more notable in metastatic Merkel cell carcinoma and squamous cell carcinoma of the head and neck, which had maximum decreases of 32.1% and 24.7%, respectively. The magnitude of reduction in CL was higher in responders than in nonresponders. Significant covariate effects of baseline weight, baseline albumin, and sex were identified on both CL and central distribution volume. Significant covariate effects of black/African American race, C-reactive protein, and immunogenicity were found on CL. None of the covariate or time-dependent effects were clinically important or warranted dose adjustment.

Strategies to Determine Assay Format for the Assessment of Neutralizing Antibody Responses to Biotherapeutics.

Most biotherapeutics can elicit immune responses in dosed recipients generating anti-drug antibodies (ADAs). Neutralizing antibodies (NAbs) are a subpopulation of ADAs that can potentially impact patient safety and directly mediate loss of drug efficacy by blocking the biological activity of a therapeutic product. Therefore, NAb detection is an important aspect of immunogenicity assessment, requiring sensitive and reliable methods reflective of the therapeutic mechanism of action (MoA). Both cell-based and non cell-based assays are viable options for NAb assessment. However, the scientific approach for the selection of a suitable assay format (cell-based or non cell-based) for NAb assessment is not currently well defined. In this manuscript, the authors summarize the design and utility of cell-based and non cell-based NAb assays and recommend a NAb assay format selection approach that relies on a combination of three factors. These include (i) the therapeutic MoA, (ii) the evidence of desirable assay performance characteristics, and (iii) risk of immunogenicity. The utility of correlating NAb response with pharmacodynamic data is also discussed. The aim of this paper is to provide a consistent strategy that will guide the selection of scientifically justified assay formats capable of detecting clinically relevant NAbs for biotherapeutics with varying MoAs and diverse complexity.

Incidence, characterization, and clinical impact analysis of peginterferon beta1a immunogenicity in patients with multiple sclerosis in the ADVANCE trial.

BACKGROUND: Efficacy of interferon beta in multiple sclerosis (MS) can be dampened in patients who develop neutralizing antidrug antibodies (NAbs). Peginterferon beta1a is an interferon conjugated with a polyethylene glycol (PEG) moiety. Pegylation increases a drug's half life and exposure, and may also reduce immunogenicity. OBJECTIVE: The objective of this study was to characterize the incidence and impact of immunogenicity to peginterferon beta1a over 2 years in patients with MS. METHODS: Patients with relapsing-remitting MS (N = 1512) were randomized to subcutaneous peginterferon beta1a 125 µg every 2 or 4 weeks, or placebo, for 1 year; patients in the placebo group were rerandomized to active treatment in year 2. The incidence and titers of binding antibodies (BAbs) and NAbs to interferon and antibodies to PEG (anti-PEG) were assessed in analytically validated assays. The clinical impact of immunogenicity on relapse and magnetic resonance imaging endpoints was evaluated. RESULTS: Over 2 years, 6%, less than 1%, and 7% of patients developed anti-interferon BAbs, NAbs, and anti-PEG antibodies, respectively. There was no discernible clinically meaningful effect of antibody status on the pharmacodynamic, efficacy, or safety parameters evaluated, although these analyses were limited by the low incidence of treatment-emergent antibodies. CONCLUSION: The treatment effect of peginterferon beta1a in patients with relapsing-remitting MS is not expected to be attenuated by immunogenicity.

Immunogenicity evaluation strategy for a second-generation therapeutic, PEG-IFN-ß-1a.

AIMS: Neutralizing antibodies can diminish clinical efficacy of IFN-ß in multiple sclerosis patients. Therefore, monitoring immunogenicity was considered critical during clinical development of a second-generation, pegylated IFN-ß product, PEG-IFN-ß-1a. MATERIALS & METHODS: Assays previously used to evaluate immunogenicity of IFN-ß-1a were used to assess PEG-IFN-ß-1a immunogenicity, with modifications to apply current best bioanalytical practices. A separate testing paradigm was used to monitor antibodies to polyethylene glycol. RESULTS & CONCLUSION: Final assay cut points and relevant titer levels were established in-study. Immunogenicity evaluation strategies for second-generation therapeutics should take into consideration current best bioanalytical practices while retaining consistency with legacy assays to facilitate data comparison and interpretation. This study illustrates challenges in assessing immunogenicity of second-generation therapeutics.

Incurred sample reproducibility and stability assessment in a cell-based drug concentration assay.

Joleen White is Principal Scientist in Translational Sciences at Biogen Idec. Throughout her career, she has applied her background in biophysical protein chemistry to pharmaceutical development in therapeutic indications with significant unmet medical need. In her current role, she supports method development and regulated bioanalysis of biomarkers, biopharmaceuticals, and immunogenicity in biological samples from nonclinical and clinical studies. Her experience with measuring macromolecules includes enzymes, monoclonal antibodies, Fc fusions, oligonucleotides, PEGylated proteins, and other novel protein constructs. She has supported studies from discovery through all phases of development including GLP nonclinical, clinical, and post-marketing commitments. Incurred samplereproducibility is one aspect of in-study validation, with white papers outlining expectations for chromatographic assays and immunoassays. This manuscript outlines an approach for performing incurred sample reproducibility for a bioequivalence study using a cell-based assay, with the complication of time elapsed between original and repeat assays. The incurred sample reproducibility passed the pre-established acceptance criteria of 45% for at least 2/3 of the samples: 174/216 samples (80.6%). Data trends between the two crossover arms were qualitatively similar. The passed incurred sample reproducibility and stability further supports the validity of the original study conclusion that the two manufacturing processes were bioequivalent. This illustrates one approach to extrapolating industry and regulatory recommendations for situations outside current guidance.

Pharmacokinetics and pharmacodynamics of peginterferon beta-1a in patients with relapsing-remitting multiple sclerosis in the randomized ADVANCE study.

AIMS: To evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) of subcutaneous peginterferon beta-1a in patients with relapsing-remitting multiple sclerosis (RRMS) in the phase 3 ADVANCE study (n = 1512). METHODS: During year 1, patients were randomized (1:1:1) to placebo or peginterferon beta-1a 125 µg every 2 or 4 weeks. After year 1, patients randomized to placebo were re-randomized to 125 µg peginterferon beta-1a administered every 2 weeks or every 4 weeks for year 2. Patients randomized to peginterferon beta-1a in year 1 remained on the same dosing regimen in year 2. Intensive blood samples for PK and PD (neopterin elevation; a biomarker of pharmacological activity induced by interferon beta-1a) measurements were collected from 44 patients pre-dosing and at intervals over 240 h post-dosing at weeks 4 and 24. Sparse samples were collected from all patients after each dosing at weeks 4, 12, 24, 56 and 84. RESULTS: The PK profile of peginterferon beta-1a did not change over time or between dosing regimens. No accumulation was observed. Peak serum concentrations were reached 1-1.5 days post-dosing, with a mono-phasic decline and a median half-life of approximately 2-3 days. Dosing every 2 weeks provided approximately two-fold greater monthly cumulative area under the curve than every 4 weeks. Neopterin elevation was sustained for 10-14 days following each dose, indicating doubled cumulative duration of pharmacological activity for dosing every 2 weeks vs. every 4 weeks. CONCLUSIONS: These PK/PD profiles potentially explain the enhanced efficacy of dosing every 2 weeks in patients with RRMS.

Stability: recommendation for best practices and harmonization from the Global Bioanalysis Consortium Harmonization Team.

This paper provides a comprehensive overview of stability-related aspects of quantitative bioanalysis and recommends science-based best practices, covering small and large molecules as well as chromatographic and ligand-binding assays. It addresses general aspects, such as the use of reference values, transferability and treatment of failing stability results, and also focuses on specific types of stability assessment: bench-top, freeze/thaw and long-term frozen stability, stock stability, extract stability, stability in whole blood, tissue and urine, and stability of endogenous analytes, in special matrix types and in incurred samples.

Free and total biotherapeutic evaluation in chromatographic assays: interference from targets and immunogenicity.

Measurement of drug concentrations is critical during drug development, supporting evaluation of safety and efficacy in the context of pharmacokinetics. Protein-based therapeutics have been historically measured by immunoassay methods. Technological advances provide new opportunities to measure these biotherapeutics using previously incompatible chromatographic techniques, such as MS. These advances are breaking down the barriers between 'large-molecule' and 'small-molecule' bioanalysis, and pushing scientists outside their comfort zones. One challenge in measuring biotherapeutic concentration is potential impact from other matrix components, such as therapeutic target or antidrug antibodies. Depending on the specific assay development objective, target interference could be either desired (favoring free measurement) or undesired (favoring total measurement). Orthogonal techniques provide additional tools to meet this challenge. The goal of this review is to introduce both small- and large-molecule bioanalytical scientists to the opportunities and challenges to consider while evaluating orthogonal methods for biotherapeutic bioanalysis.

Intrathecal recombinant human 4-sulfatase reduces accumulation of glycosaminoglycans in dura of mucopolysaccharidosis VI cats.

INTRODUCTION: Mucopolysaccharidosis VI (MPS-VI) is caused by a deficiency in N-acetylgalactosamine-4-sulfatase activity, resulting in lysosomal accumulation of partially degraded glycosaminoglycans (GAGs). Compressive myelopathy in early-onset MPS-VI patients has been partly attributed to thickening of the dura mater following engorgement with GAG. In this study, we therefore tested whether the dural abnormalities could be prevented in a feline model of the disorder. RESULTS: All intrathecal injections (IT-INJs) were well tolerated. MPS-VI cats treated with IT-INJ of recombinant human N-acetylgalactosamine-4-sulfatase (rhASB) exhibited reduced vacuolation in the dural fibroblasts, diminished levels of sulfated-N-acetylhexosamine (HNAc(+S)) in the cerebrospinal fluid (CSF) and no hind-limb paresis. Serum anti-rhASB antibodies remained low in MPS-VI cats treated with intravenous enzyme replacement therapy (IV-ERT) and increased slightly in normal cats treated with IT-INJ of rhASB alone. Anti-rhASB antibodies in CSF remained undetectable. DISCUSSION: These data indicate that repeated IT-INJ of rhASB can safely prevent GAG storage in MPS-VI dura. METHODS: Cats were assigned to three groups: (i) receiving weekly IV-ERT of rhASB from birth plus six monthly IT-INJs of rhASB from age 2 months; (ii) receiving six monthly IT-INJs of vehicle; or (iii) untreated. Additional normal cats received five fortnightly IT-INJs of rhASB or vehicle alone.

Biodistribution and pharmacodynamics of recombinant human alpha-L-iduronidase (rhIDU) in mucopolysaccharidosis type I-affected cats following multiple intrathecal administrations.

The storage disorder mucopolysaccharidosis type I (MPS I) is caused by a deficiency in lysosomal α-L-iduronidase activity. The inability to degrade glycosaminoglycans (GAG) results in lysosomal accumulation and widespread tissue lesions. Many symptoms of MPS I are amenable to treatment with recombinant human α-L-iduronidase (rhIDU), however, peripherally administered rhIDU does not cross the blood-brain barrier and has no beneficial effects in the central nervous system (CNS). A feline model of MPS I was used to evaluate the CNS effects of rhIDU following repeated intrathecal (IT) administration. Twelve animals were randomized into four groups based on the time of euthanasia and tissue evaluation following three repeat IT administrations of 0.1 mg/kg rhIDU or placebo on Study Days 1, 4 or 5, and 9. Two days after the final IT injection, the mean tissue α-L-iduronidase (IDU) activity in the brains of the two treated animals were approximately 3-times higher (50.1 and 54.9 U/mg protein) than the activity found in normal cat brains (mean of 18.3 U/mg), and remained higher than untreated MPSI brain at 1 month (2.4 and 4.1 U/mg protein) before returning to near-baseline levels after 2 months. This activity corresponded with decreased brain GAG concentrations after 2 days (1.4 and 2.0 µg/mg) and 1 month (0.9 and 1.1 µg/mg) which approached levels observed in normal animals (0.7 µg/mg). Attenuation of GAG, gangliosides GM2 and GM3, and cholesterol reaccumulation was identified at both two days and one month following final IT injection. No adverse effects attributable to IT rhIDU administration were observed. IT rhIDU may be an effective means for providing enzyme replacement therapy for the central manifestations of MPS I.

2010 white paper on recent issues in regulated bioanalysis & global harmonization of bioanalytical guidance.

The 4th Calibration and Validation Group Workshop on Recent Issues in Regulated Bioanalysis, a 2-day full immersion workshop, was organized by the Calibration and Validation Group. Contract research organizations, pharmaceutical companies and regulatory agencies came together to discuss several 'hot' topics concerning bioanalytical issues and regulatory challenges and to reach a consensus among panelists and attendees on many points regarding method validation of small and large molecules.

Repeated intrathecal injections of recombinant human 4-sulphatase remove dural storage in mature mucopolysaccharidosis VI cats primed with a short-course tolerisation regimen.

All MPS-VI cats treated thus far with weekly intravenous enzyme replacement therapy (IV ERT) with recombinant human N-acetylgalactosamine-4-sulphatase (rhASB) from 3 months of age onwards developed circulating anti-rhASB antibodies. In view of this, the possibility of inducing immune tolerance by using a short-course tolerisation regimen was tested. Starting at 4 months of age, MPS-VI (n=5) and unaffected cats (n=2) received cyclosporine and azathioprine over a 22-day period plus weekly IV ERT with 0.1mg/kg rhASB. After a 4-week resting period, these cats were administered weekly IV ERT with 1mg/kg rhASB until 11 or 17 months of age. Four unaffected cats (n=4) received weekly IV ERT only. Health, growth and seroconversion were regularly monitored. Four out of five MPS-VI cats tolerated rhASB well, as indicated by negligible or low antibody titres and absence of hypersensitivity reactions. One MPS-VI cat exhibited elevated antibody titres and hypersensitivity reactions during some IV treatments. The two unaffected cats that received the tolerisation regimen remained seronegative, however, only half of the unaffected cats not submitted to this regimen seroconverted. Only minor side-effects were attributed to the short-course of cyclosporine and azathioprine. Two MPS-VI cats also well-tolerated four weekly intrathecal injections of rhASB and consequently exhibited less oligosaccharide fragments in cerebrospinal fluid and less vacuolation within their dura mater. These data indicate that a relatively high rate of immunotolerance towards rhASB can be achieved in MPS-VI cats with a short-course tolerisation regimen ultimately permitting removal of lysosomal storage within the dura mater with the use of intrathecal therapy.

Comparison of neutralizing antibody assays for receptor binding and enzyme activity of the enzyme replacement therapeutic Naglazyme (galsulfase).

Most patients receiving Naglazyme (galsulfase, rhASB) enzyme replacement therapy for mucopolysaccharidosis type VI develop an antibody response. To evaluate the impact of this response, two in vitro neutralizing antibody (NAb) assays were developed based on the two steps of the mechanism of action. Neutralization of enzyme activity was detected by inhibition of rhASB cleavage of a fluorogenic substrate. Neutralization of receptor binding was detected by decreased binding of labeled rhASB to immobilized soluble receptor. For the enzyme activity NAb assay, serum pretreatment was required to isolate antibodies from interfering phosphate ions, with sensitivity of < or =5 microg/mL. The receptor binding NAb assay used a five-fold dilution, with sensitivity of < or =40 microg/mL. Cutpoints for percent inhibition were based on 95% confidence intervals from naïve sera. Clinical samples were similarly likely to be positive in both assays than positive for neutralization of only one step in the mechanism of action. The two NAb assays yielded complementary information about potential neutralization of rhASB. Relative estimated sensitivity between neutralization assays did not correlate with the number of positive clinical samples or patients. In vitro NAb assays based on a well-understood mechanism of action provide specific information about the NAb mechanism.

Development, validation, and clinical implementation of an assay to measure total antibody response to naglazyme (galsulfase).

Naglazyme (galsulfase, rhASB) was developed as enzyme replacement therapy for mucopolysaccharidosis type VI. Naglazyme generated an IgG antibody response in most patients. To better characterize Naglazyme immunogenicity, a solution phase bridged immunoassay was developed to measure total antibody response regardless of isotype. Overnight incubation of serum dilutions with rhASB labeled with biotin and ruthenium-based tags allowed antibody-antigen complexes to form prior to capture on a streptavidin plate. Neat serum was tolerated in the assay, with a 1:10 screening dilution implemented for testing. At this dilution, the assay was sensitive to 75 ng/ml anti-rhASB. Titers were reported as the highest dilution factor with signal above a 95% confidence interval from naïve individual sera. Precise measurement of titers, within two consecutive dilution factors, was observed across analysts and days. Clinical samples showed similar positive/negative results between the IgG ELISA and the total antibody ECLA, although with an imperfect correlation. Improvements in assay performance and implementation strategy altered some positive clinical samples to negative and vice versa. Comparison of the titer readout for clinical samples with the screening signal illustrates a range of relationships for signal versus sample dilution factor, confirming that signal from a screening dilution cannot directly predict the reported titer.

R104H may suppress transthyretin amyloidogenesis by thermodynamic stabilization, but not by the kinetic mechanism characterizing T119 interallelic trans-suppression.

The tetrameric protein transthyretin (TTR) forms amyloid fibrils upon dissociation and subsequent monomer misfolding, enabling misassembly. Remarkably, the aggregation of one of over 100 destabilized TTR variants leads to familial amyloid disease. It is known that trans-suppression mediated by the incorporation of T119M subunits into tetramers otherwise composed of the most common familial variant V30M, ameliorates disease by substantially slowing the rate of tetramer dissociation, a mechanism referred to as kinetic stabilization of the native state. R104H TTR has been reported to be non-pathogenic, and recently, this variant has been invoked as a trans-suppressor of amyloid fibril formation. Here, we demonstrate that the trans-suppression mechanism of R104H does not involve kinetic stabilization of the tetrameric structure, instead its modest trans-suppression most likely results from the thermodynamic stabilization of the tetrameric TTR structure. Thermodynamic stabilization increases the fraction of tetramer at the expense of the misfolding competent monomer decreasing the ability of TTR to aggregate into amyloid fibrils. As a consequence of this stabilization mechanism, R104H may be capable of protecting patients with modestly destabilizing mutations against amyloidosis by slightly lowering the overall population of monomeric protein that can misfold and form amyloid.