A randomised, single-blind, single-dose, three-arm, parallel-group study in healthy subjects to demonstrate pharmacokinetic equivalence of ABP 501 and adalimumab.

OBJECTIVE: To demonstrate pharmacokinetic (PK) similarity of biosimilar candidate ABP 501 relative to adalimumab reference product from the USA and European Union (EU) and evaluate safety, tolerability and immunogenicity of ABP 501. METHODS: Randomised, single-blind, single-dose, three-arm, parallel-group study; healthy subjects were randomised to receive ABP 501 (n=67), adalimumab (USA) (n=69) or adalimumab (EU) (n=67) 40 mg subcutaneously. Primary end points were area under the serum concentration-time curve from time 0 extrapolated to infinity (AUCinf) and the maximum observed concentration (Cmax). Secondary end points included safety and immunogenicity. RESULTS: AUCinf and Cmax were similar across the three groups. Geometrical mean ratio (GMR) of AUCinf was 1.11 between ABP 501 and adalimumab (USA), and 1.04 between ABP 501 and adalimumab (EU). GMR of Cmax was 1.04 between ABP 501 and adalimumab (USA) and 0.96 between ABP 501 and adalimumab (EU). The 90% CIs for the GMRs of AUCinf and Cmax were within the prespecified standard PK equivalence criteria of 0.80 to 1.25. Treatment-related adverse events were mild to moderate and were reported for 35.8%, 24.6% and 41.8% of subjects in the ABP 501, adalimumab (USA) and adalimumab (EU) groups; incidence of antidrug antibodies (ADAbs) was similar among the study groups. CONCLUSIONS: Results of this study demonstrated PK similarity of ABP 501 with adalimumab (USA) and adalimumab (EU) after a single 40-mg subcutaneous injection. No new safety signals with ABP 501 were identified. The safety and tolerability of ABP 501 was similar to the reference products, and similar ADAb rates were observed across the three groups. TRIAL REGISTRATION NUMBER: EudraCT number 2012-000785-37; Results.

Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products.

The appropriate evaluation of the immunogenicity of biopharmaceuticals is of major importance for their successful development and licensure. Antibodies elicited by these products in many cases cause no detectable clinical effects in humans. However, antibodies to some therapeutic proteins have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In addition, antibodies can affect drug efficacy. In non-clinical studies, anti-drug antibodies (ADA) can complicate interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, it is important to develop testing strategies that provide valid assessments of antibody responses in both non-clinical and clinical studies. This document provides recommendations for antibody testing strategies stemming from the experience of contributing authors. The recommendations are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry. The strategies proposed are also expected to contribute to better understanding of antibody responses and to further advance immunogenicity evaluation.

Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products.

Most biological drug products elicit some level of anti-drug antibody (ADA) response. This antibody response can, in some cases, lead to potentially serious side effects and/or loss of efficacy. In humans, ADA often causes no detectable clinical effects, but in the instances of some therapeutic proteins these antibodies have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In nonclinical (preclinical) studies, ADA can affect drug exposure, complicating the interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, the immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess the immunogenic potential of biological drug molecules, and be able to correlate laboratory results with clinical events, it is important to develop reliable laboratory test methods that provide valid assessments of antibody responses in both nonclinical and clinical studies. For this, method validation is considered important, and is a necessary bioanalytical component of drug marketing authorization applications. Existing regulatory guidance documents dealing with the validation of methods address immunoassays in a limited manner, and in particular lack information on the validation of immunogenicity methods. Hence this article provides scientific recommendations for the validation of ADA immunoassays. Unique validation performance characteristics are addressed in addition to those provided in existing regulatory documents pertaining to bioanalyses. The authors recommend experimental and statistical approaches for the validation of immunoassay performance characteristics; these recommendations should be considered as examples of best practice and are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry.

Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics.

An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.

Cerebrospinal fluid ACTH and cortisol in opsoclonus-myoclonus: effect of therapy.

Opsoclonus-myoclonus syndrome is one of a few corticotropin (ACTH)-responsive central nervous system disorders of childhood. We measured cerebrospinal fluid ACTH and cortisol in 69 children with opsoclonus-myoclonus and 25 age- and sex-matched control subjects to determine endogenous levels and look for hypothesized differential hormonal effects of ACTH and corticosteroid treatment. Cerebrospinal fluid cortisol was 10-fold higher with ACTH treatment (n = 26), but was unchanged with oral steroid treatment (n = 18) or no treatment (n = 25). It was significantly higher in children receiving daily high-dose ACTH than alternate day ACTH. In ACTH-treated children, cerebrospinal fluid and serum cortisol were highly correlated (r = 0.96, P = 0.0001), with a mean ratio of cerebrospinal fluid to serum cortisol of approximately 1:10. Cerebrospinal fluid ACTH concentration did not differ significantly between untreated opsoclonus-myoclonus and control subjects but was lower with ACTH (-29%) or steroid treatment (-36%), suggesting feedback inhibition of ACTH release. These data delineate differences in the central effects of ACTH and corticosteroid therapy, as well as between high and low ACTH doses, and support the integrity of the brain-adrenal axis in pediatric opsoclonus-myoclonus.

Comparison of pharmacokinetics and safety of pegfilgrastim administered by two delivery methods: on-body injector and manual injection with a prefilled syringe.

PURPOSE: For patients with clinically significant risk of febrile neutropenia, pegfilgrastim administration should occur the day after myelosuppressive chemotherapy; however, a variety of factors may preclude patients from returning to the clinic the next day for pegfilgrastim administration, necessitating other strategies. This study compared the pharmacokinetics and safety of pegfilgrastim administered via an on-body injector applied to the subject's skin versus manual injection using a prefilled syringe. METHODS: Healthy subjects aged 18-50 years were randomized 1:1 to receive a single 6-mg subcutaneous pegfilgrastim dose from an on-body injector or a prefilled syringe. Blood for pharmacokinetic measurements was collected at baseline and prespecified time points after pegfilgrastim administration; safety was assessed throughout the 6-week study. Primary endpoints were maximum concentration (C max) and area under the concentration curve from time 0 to infinity (AUC0-inf). Secondary endpoints included safety, tolerability, and immunogenicity. RESULTS: Pegfilgrastim mean AUC0-inf values for the on-body injector (n = 125) and manual injection (n = 128) were 10,900 and 11,100 h ng/mL, respectively; mean C max values were 248 and 262 ng/mL, respectively. The least squares geometric mean ratios were 0.97 for C max and 1.00 for AUC0-inf; the corresponding 90 % CIs were within the prespecified range (0.80-1.25), indicating comparable pegfilgrastim pharmacokinetics between delivery methods. Treatment-emergent adverse events (AEs) were similar between groups (injector, 86 %; manual, 85 %). Injector- or syringe-related AEs were more prevalent with the injector (13 %; manual, 4 %); none were serious. No pegfilgrastim-neutralizing antibodies were detected. CONCLUSIONS: Pegfilgrastim pharmacokinetics and safety were comparable between the on-body injector and manual injection groups.

Strategic characterization of anti-drug antibody responses for the assessment of clinical relevance and impact.

All therapeutic proteins have the potential to induce anti-drug antibodies (ADA). Clinically relevant ADA can impact efficacy and/or safety of a biological therapeutic. Immunogenicity assessment strategy evaluates binding and neutralizing ADA, and the need for additional characterization (e.g., epitope, titer and so on) is determined using a risk-based approach. The choice of characterization assays depends on the type, application and immunogenicity of the therapeutic. ADA characterization can impact the interpretation of the risk profile of a given therapeutic, and offers insight into opportunities for risk mitigation and management. This article describes common ADA characterization methods. Strategic assessment and characterization of clinically relevant ADA are discussed, in order to support clinical options for safe and effective patient care and disease management.

Epitope characterization of pre-existing and developing antibodies to an aglycosylated monoclonal antibody therapeutic of G1m17,1 allotype.

Allotypes of IgG1 molecules can influence the immunogenicity of therapeutic monoclonal antibodies and may account for the presence of some pre-existing antibodies. An electrochemiluminescent (ECL) bridging immunoassay was used to characterize the binding epitopes of anti-therapeutic antibodies (ATAs) in a Phase 1 single ascending dose clinical trial of a therapeutic aglycosylated IgG1monoclonal antibody (mAb). There was no evidence for ATAs specific for a possible neo-epitope created due to the lack of glycosylation. ATAs that developed post-treatment were specific for the F(ab')2, whereas, pre-existing ATAs were specific to the Fc region. Further characterization of the pre-existing ATAs identified the specific epitope to be the G1m1 allotype determinant in the Fc of the therapeutic. A novel competitive bridging assay was developed to verify that serum IgG1 from subjects with pre-existing anti-G1m1 antibodies was homozygous for the antithetical allotype (G1m3). The endogenous G1m allotype of all subjects was assessed and correlation to ATA incidence and adverse events was evaluated. Interestingly, the pre-existing anti-allotype antibody in subjects persisted but was not augmented after dosing, indicating the lack of a secondary immune response to this epitope. These studies indicate the relationship of the therapeutic allotype and the corresponding allotype of subjects is an important component to further understand the impact of immunogenicity on the safety and efficacy of therapeutic antibodies.

Summary of confirmation cut point discussions.

A subgroup of AAPS NBC Immunogenicity Workshop attendees met to discuss the current recommendations in white papers and guidance documents, to describe and discuss current practices, and to resolve concerns as to the biologically and statistically appropriate approaches to determining a confirmatory cut point for immunogenicity assays. This is a summary of our discussions and recommendations.

Bioanalytical considerations in the comparability assessment of biotherapeutics.

Comparison of biotherapeutic products before and after manufacturing changes is required to show that the products are highly similar. Besides in vitro assessment on the critical quality attributes and potency, biocomparability studies are sometimes required to demonstrate similarities in pharmacokinetic and pharmacodynamic characteristics. The complex and diverse nature of biotherapeutics requires multifaceted considerations in the biocomparability study design, bioanalytical measurements of drug concentrations and/or pharmacodynamic responses, immunogenicity analysis, data interpretation and decision making. A major perspective is to understand the structure and biological functions of the biotherapeutics in relation to the indication. Issues of a common standard and the importance of the use of ligand-binding assays that are sensitive to structural changes are discussed. It would not be possible to use the same process and one-size-fit-all criteria for biocomparability studies of all biologics. Previous examples from industry and our experience of the bioanalytical considerations for fit-for-purpose pharmacokinetic support and immunogenicity assessments are presented.

Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics.

Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs. When using a sensitive electrochemiluminescent (ECL) bridging immunoassay, samples from subjects with rheumatoid arthritis demonstrated much higher baseline reactivity than healthy subjects. Interference was found to be dependent on the aggregation state of the therapeutic antibody that had been conjugated with the detection reagent (ruthenium). Size exclusion high performance liquid chromatography (SE-HPLC) demonstrated that of the total integrated peaks, as little as 0.55% high molecular weight aggregates (>600kDa) were sufficient to cause increased reactivity. Stability studies of the ruthenium and biotin conjugated therapeutic antibody indicated that storage time, temperature and buffer formulation were critical in maintaining the integrity of the reagents. Through careful SE-HPLC monitoring we were able to choose appropriate storage and buffer conditions which led to a reduction in the false reactivity rate in therapeutic-naïve serum from a rheumatoid arthritis population.

Comparing exponentially weighted moving average and run rules in process control of semiquantitative immunogenicity immunoassays.

The immunogenicity immunoassay validation process ensures development of a robust, reproducible method. However, no matter how well developed, validated, and maintained a method is, in the course of running a large number of samples over time, it is not uncommon to see bad reagents, poorly calibrated equipment, personnel errors, or other unknown and unpredictable factors that have an impact in the performance of the method and quality of the sample results. The immunogenicity immunoassay thus needs to be closely monitored with an internal statistical quality control process overtime to ensure a consistent and reliable output. The statistical process control has been widely applied to monitor manufacturing processes and in clinical laboratories. Its application to immunogenicity immunoassays is relatively novel. Limited guidance is available to implement the process to monitor semiquantitative immunogenicity immunoassay performance. Here, we have performed a suitability evaluation for process control charts with actual laboratory data from three immunogenicity immunoassay methods each utilizing a different technology platform. Additionally, a panel of prepared samples designed to assess long-term method performance were periodically evaluated for over a year. Finally, we make recommendations for an internal quality control process based on the results of these evaluations.

A step-wise approach for transfer of immunogenicity assays during clinical drug development.

We designed a three-step statistical approach to transfer bioanalytical assays (ELISA and Biacore) which evaluates the (1) average equivalence between the two labs (2) concordance in individual sample results between the two labs, and (3) long-term stability of assay performance. Each experimental design evaluated the contribution of four critical variables to the overall variability. Two lots of each variable were examined in a controlled experiment. The variables tested for ELISA were analyst, plate washer, biotinylated-therapeutic protein, and streptavidin-horseradish peroxidase; and for Biacore were analyst, instrument, chip lot, and conjugation chemistry reagent lots. Equivalence in the mean signal to noise (S/N) or mean relative units (RU) between the two labs was established through statistical evaluation of the assay performance characteristics across multiple assay variables. Concordance between the two labs in the individual sample results was subsequently verified both quantitatively and qualitatively. The long-term maintenance of assay stability was monitored by performance testing of a predefined set of samples which were prepared in sufficient quantities to last several years. The process of method validation for biomarker testing in clinical trials is to analyze the variability of the assay performance. However, different factors contribute to this variability and need to be evaluated when the method is transferred to another site/lab. Lack of understanding the critical variables can potentially result in unexpected problems and delays. The three-step statistical approach of assay transfer provides a robust process for transferring complex biological assays.

Development of a maturing T-cell-mediated immune response in patients with idiopathic Parkinson's disease receiving r-metHuGDNF via continuous intraputaminal infusion.

The development of a maturing T-cell-mediated immune response was characterized in Parkinson's disease subjects receiving recombinant human glial-derived neurotrophic factor (r-metHuGDNF) via continuous bilateral intraputaminal infusion. Eighteen of 34 subjects tested positive for anti-r-metHuGDNF-binding antibodies. Four subjects developed neutralizing activity, three of which demonstrated classic immunoglobulin class switching from IgM to IgG. An increase of anti-r-metHuGDNF IgG-binding antibodies correlated with the development of neutralizing activity. All serum samples from two subjects with neutralizing activity were characterized for IgG subclasses. These data revealed an initial anti-r-metHuGDNF IgG population where IgG1 >> IgG2 >> IgG4, and IgG3 concentrations were negligible. However, continued antigenic stimulation resulted in concentration changes where IgG4 > IgG1> IgG2, indicating a mature immune response. In addition, using in silico techniques, two immunodominant MHC class II T-cell epitopes were predicted for the native GDNF sequence. These data demonstrate development of a mature T-cell-mediated immune response in these subjects.

Six-month continuous intraputamenal infusion toxicity study of recombinant methionyl human glial cell line-derived neurotrophic factor (r-metHuGDNF) in rhesus monkeys.

Recombinant human glial cell line-derived neurotrophic factor (r-metHuGDNF) is a potent neuronal growth and survival factor that has been considered for clinical use in the treatment of Parkinson's disease (PD). Here we present results of a 6-month toxicology study in rhesus monkeys conducted to support clinical evaluation of chronic intraputamenal infusion of r-metHuGDNF for PD. Monkeys (6-9/sex/group) were treated with 0 (vehicle), 15, 30, or 100 micro g/day r-metHuGDNF by continuous unilateral intraputamenal infusion (150 micro l/day flow rate) for 6 months; a subset of animals (2-3/sex/group) underwent a subsequent 3-month treatment-free recovery period. Notable observations included reduced food consumption and body weight at 100 micro g/day and meningeal thickening underlying the medulla oblongata and/or overlying various spinal cord segments at 30 and 100 micro g/day. In addition, multifocal cerebellar Purkinje cell loss (with associated atrophy of the molecular layer and, in some cases, granule cell loss) was observed in 4 monkeys in the 100-micro g/day group. This cerebellar finding has not been observed in previous nonclinical studies evaluating r-metHuGDNF. The small number of affected animals precludes definitive conclusions regarding the pathogenesis of the cerebellar lesion, but the data support an association with r-metHuGDNF treatment.

Six-month continuous intraputamenal infusion toxicity study of recombinant methionyl human glial cell line-derived neurotrophic factor (r-metHuGDNF in rhesus monkeys.

Recombinant human glial cell line-derived neurotrophic factor (r-metHuGDNF) is a potent neuronal growth and survival factor that has been considered for clinical use in the treatment of Parkinson's disease (PD). Here we present results of a 6-month toxicology study in rhesus monkeys conducted to support clinical evaluation of chronic intraputamenal infusion of r-metHuGDNF for PD. Monkeys (6-9/sex/group) were treated with 0 (vehicle), 15, 30, or 100 microg/day r-metHuGDNF by continuous unilateral intraputamenal infusion (150 microl/day flow rate) for 6 months; a subset of animals (2-3/sex/group) underwent a subsequent 3-month treatment-free recovery period. Notable observations included reduced food consumption and body weight at 100 microg/day and meningeal thickening underlying the medulla oblongata and/or overlying various spinal cord segments at 30 and 100 microg/day. In addition, multifocal cerebellar Purkinje cell loss (with associated atrophy of the molecular layer and, in some cases, granule cell loss) was observed in 4 monkeys in the 100-microg/day group. This cerebellar finding has not been observed in previous nonclinical studies evaluating r-metHuGDNF. The small number of affected animals precludes definitive conclusions regarding the pathogenesis of the cerebellar lesion, but the data support an association with r-metHuGDNF treatment.