Noncompetitive immunoassay optimized for pharmacokinetic assessments of biologically active efruxifermin.

Efruxifermin (EFX) is a homodimeric human IgG1 Fc-FGF21 fusion protein undergoing investigation for treatment of liver fibrosis due to nonalcoholic steatohepatitis (NASH), a prevalent and serious metabolic disease for which there is no approved treatment. Biological activity of FGF21 requires its intact C-terminus, which enables binding to its obligate co-receptor ß-Klotho on the surface of target cells. This interaction is a prerequisite for FGF21 signal transduction through its canonical FGF receptors: FGFR1c, 2c, and 3c. Therefore, the C-terminus of each FGF21 polypeptide chain must be intact, with no proteolytic truncation, for EFX to exert its pharmacological activity in patients. A sensitive immunoassay for quantification of biologically active EFX in human serum was therefore needed to support pharmacokinetic assessments in patients with NASH. We present a validated noncompetitive electrochemiluminescent immunoassay (ECLIA) that employs a rat monoclonal antibody for specific capture of EFX via its intact C-terminus. Bound EFX is detected by a SULFO-TAG™-conjugated, affinity purified chicken anti-EFX antiserum. The ECLIA reported herein for quantification of EFX demonstrated suitable analytical performance, with a sensitivity (LLOQ) of 20.0 ng/mL, to support reliable pharmacokinetic assessments of EFX. The validated assay was used to quantify serum EFX concentrations in a phase 2a study of NASH patients (BALANCED) with either moderate-to-advanced fibrosis or compensated cirrhosis. The pharmacokinetic profile of EFX was dose-proportional and did not differ between patients with moderate-to-advanced fibrosis and those with compensated cirrhosis. This report presents the first example of a validated pharmacokinetic assay specific for a biologically active Fc-FGF21 fusion protein, as well as the first demonstration of use of a chicken antibody conjugate as a detection reagent specific for an FGF21 analog.

Immunogenicity of DaxibotulinumtoxinA for Injection in Glabellar Lines.

DaxibotulinumtoxinA-lanm for injection (DAXI), a novel botulinum toxin type A formulation, contains a purified 150-kD core neurotoxin (daxibotulinumtoxinA) and proprietary stabilizing peptide (RTP004), and is approved for glabellar line treatment. As with any biologic product, DAXI may potentially be immunogenic and elicit unwanted antibody formation, possibly resulting in partial or complete treatment failure. The immunogenicity of DAXI was assessed in 2 double-blind, placebo-controlled, single-dose studies and an open-label safety study of up to 3 repeat treatments. Of the 2737 evaluable patients, none developed neutralizing antibodies to daxibotulinumtoxinA and 0.8% developed treatment-related nonneutralizing anti-daxibotulinumtoxinA-binding antibodies. Of evaluable patients exposed to RTP004 with either DAXI or placebo, 1.3% developed treatment-related anti-RTP004-binding antibodies, which were mostly transient. No patient developed binding antibodies to both daxibotulinumtoxinA and RTP004. All patients with treatment-related binding antibodies to daxibotulinumtoxinA or RTP004 achieved a clinical response (none or mild glabellar line severity) at Week 4 following each DAXI treatment cycle. The duration of clinical response was not different between treatment cycles when antibodies were detected vs when they were absent. Although the analysis population was small compared to the number of patients likely to receive repeated treatment in clinical practice, these results suggest that DAXI administration at the approved glabellar lines dose has low immunogenic potential and that nonneutralizing antibodies to daxibotulinumtoxinA or RTP004 occur infrequently and often transiently, and have no impact on clinical efficacy, safety, or duration of action. Real-world data encompassing larger numbers of patients is needed to substantiate these results.

Clinical Immunogenicity of DaxibotulinumtoxinA for Injection in Glabellar Lines: Pooled Data from the SAKURA Phase 3 Trials.

DaxibotulinumtoxinA for Injection (DAXI) is a novel botulinum toxin type A product containing daxibotulinumtoxinA with a stabilizing excipient peptide (RTP004). DAXI immunogenicity was assessed in three phase 3 glabellar line studies (two placebo-controlled, single-dose studies and an open-label repeat-dose safety study). Binding antibodies to daxibotulinumtoxinA and RTP004 were detected by validated ELISAs. Samples positive for daxibotulinumtoxinA-binding antibodies were evaluated further for titer and neutralizing antibodies by mouse protection assay. Overall, 2786 subjects received DAXI and 2823 subjects were exposed to RTP004 as DAXI (n = 2786) or placebo (n = 37). Treatment-related anti-daxibotulinumtoxinA binding antibodies were detected in 21 of 2737 evaluable subjects (0.8%). No subject developed neutralizing antibodies. Treatment-related anti-RTP004 binding antibodies were detected in 35 (1.3%) of 2772 evaluable subjects. Binding antibodies were generally transient, of low titer (<1:200), and no subject had binding antibodies to both daxibotulinumtoxinA and RTP004. All subjects with treatment-induced binding antibodies to daxibotulinumtoxinA or RTP004 achieved none or mild glabellar line severity at Week 4 following each DAXI cycle, indicating no impact on DAXI efficacy. No subjects with binding antibodies to daxibotulinumtoxinA or RTP004 reported immune-related adverse events. This evaluation of anti-drug antibody formation with DAXI shows low rates of antibody formation to both daxibotulinumtoxinA and RTP004.

Critical reagent inventory management system and web portal specifically optimized for supporting external clients.

High-quality critical reagents are essential for the establishment of robust ligand binding assays to support regulated bioanalysis. To ensure consistency in assay performance over the lifetime of a project, a well-defined set of processes is needed for critical reagent life cycle management. Moreover, contract research organizations must support reagent life cycle management for diverse global clients. To address these needs, the authors designed and implemented a customized inventory management system, known as LCM+. This software solution provides external clients with efficient, secure access via a web portal to their critical reagent information, pertinent documentation and inventory tracking. Hence, the authors believe that LCM+ can serve as a useful prototype to aid the design of future inventory management systems for optimal management of critical reagents.

Anti-drug Antibody Validation Testing and Reporting Harmonization.

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting.

Sensitive assay design for detection of anti-drug antibodies to biotherapeutics that lack an immunoglobulin Fc domain.

Today the evaluation of unwanted immunogenicity is a key component in the clinical safety evaluation of new biotherapeutic drugs and macromolecular delivery strategies. However, the evolving structural complexity in contemporary biotherapeutics creates a need for on-going innovation in assay designs for reliable detection of anti-drug antibodies, especially for biotherapeutics that may not be well-suited for testing by a bridging assay. We, therefore, initiated systematic optimization of the direct binding assay to adapt it for routine use in regulatory-compliant assays of serum anti-drug antibodies. Accordingly, we first prepared a SULFO-TAG labeled conjugate of recombinant Protein-A/G to create a sensitive electrochemiluminescent secondary detection reagent with broad reactivity to antibodies across many species. Secondly, we evaluated candidate blocker-diluents to identify ones producing the highest signal-to-noise response ratios. Lastly, we introduced use of the ratio of signal responses in biotherapeutic-coated and uncoated wells as a data transformation strategy to identify biological outliers. This alternative data normalization approach improved normality, reduced skewness, and facilitated application of a parametric screening cut point. We believe the optimized direct binding assay design employing SULFO-TAG labeled Protein-A/G represents a useful analytical design for detecting serum ADA to biotherapeutics that lack an immunoglobulin Fc domain.

An Integrated Analysis of Dostarlimab Immunogenicity.

Monoclonal antibodies that block the interaction between programmed cell death 1 (PD-1) and its ligand (PD-L1) have revolutionized cancer immunotherapy. However, immunogenic responses to these new therapies-such as the development of antidrug antibodies (ADAs) and neutralizing antibodies (NAbs)-may represent a significant challenge to both efficacy and safety in some patients. Dostarlimab (TSR-042) is an approved, humanized, anti-PD-1 monoclonal antibody that has shown efficacy in multiple solid tumor types. Here, we report the results of an immunogenicity analysis of dostarlimab monotherapy in patients enrolled in the GARNET trial, a multicenter, open-label, single-arm phase 1 study. Overall, 477 of 478 patients (99.8%) were included in the analysis of dostarlimab antibody prevalence, and 349 out of 478 enrolled patients (73.0%) were evaluable for treatment-emergent antibodies to dostarlimab. The incidence of treatment-emergent ADAs was 2.5% at the recommended therapeutic dose (500 mg Q3W for the first 4 doses, 1000 mg Q6W until discontinuation), which is comparable to other anti-PD-(L)1 drugs. NAbs were detected in only 1.3% of patients. In the small percentage of patients who developed ADAs, there was no evidence of altered efficacy or safety of dostarlimab at the recommended dosing regimen. These findings demonstrated that treatment with dostarlimab was associated with a low risk of eliciting clinically meaningful ADAs over the course of this study, and dostarlimab is already approved by health authorities.

Similar immunogenicity profiles between the proposed biosimilar MYL-1501D and reference insulin glargine in patients with diabetes mellitus: the phase 3 INSTRIDE 1 and INSTRIDE 2 studies.

BACKGROUND: MYL-1501D is a proposed biosimilar to insulin glargine. The noninferiority of MYL-1501D was demonstrated in patients with type 1 diabetes and type 2 diabetes in 2 phase 3 trials. Immunogenicity of MYL-1501D and reference insulin glargine was examined in both studies. METHODS: INSTRIDE 1 and INSTRIDE 2 were multicenter, open-label, randomized, parallel-group studies. In INSTRIDE 1, patients with type 1 diabetes received MYL-1501D or insulin glargine over a 52-week period. In INSTRIDE 2, patients with type 2 diabetes treated with oral antidiabetic drugs, insulin naive or not, received MYL-1501D or reference insulin glargine over a 24-week period. Incidence rates and change from baseline in relative levels of antidrug antibodies (ADA) and anti-host cell protein (anti-HCP) antibodies in both treatment groups were determined by a radioimmunoprecipitation assay and a bridging immunoassay, respectively. Results were analyzed using a mixed-effects model (INSTRIDE 1) or a nonparametric Wilcoxon rank sum test (INSTRIDE 2). RESULTS: Total enrollment was 558 patients in INSTRIDE 1 and 560 patients in INSTRIDE 2. The incidence of total and cross-reactive ADA was comparable between treatment groups in INSTRIDE 1 and INSTRIDE 2 (P > 0.05 for both). A similar proportion of patients had anti-HCP antibodies in both treatment groups in INSTRIDE 1 at week 52 (MYL-1501D, 93.9 %; reference insulin glargine, 89.6 %; P = 0.213) and in INSTRIDE 2 at week 24 (MYL-1501D, 87.3 %; reference insulin glargine, 86.9 %; P > 0.999). CONCLUSIONS: In INSTRIDE 1 and INSTRIDE 2, similar immunogenicity profiles were observed for MYL-1501D and reference insulin glargine in patients with type 1 diabetes and type 2 diabetes, respectively. TRIAL REGISTRATION: ClinicalTrials.gov, INSTRIDE 1 ( NCT02227862 ; date of registration, August 28, 2014); INSTRIDE 2 ( NCT02227875 ; date of registration, August 28, 2014).

FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein.

A phage library displaying 1010 variants of the fibronectin type III (FN3) domain was affinity selected with the biotinylated form of the receptor binding domain (RBD, residues 319-541) of the SARS-CoV-2 virus spike protein. Nine binding FN3 variants (i.e. monobodies) were recovered, representing four different primary structures. Soluble forms of the monobodies bound to several different preparations of the RBD and the S1 spike subunit, with affinities ranging from 3 to 14 nM as measured by bio-layer interferometry. Three of the four monobodies bound selectively to the RBD of SARS-CoV-2, with the fourth monobody showing slight cross-reactivity to the RBD of SARS-CoV-1 virus. Examination of binding to the spike fragments and its trimeric form revealed that the monobodies recognise at least three overlapping epitopes on the RBD of SARS-CoV-2. While pairwise tests failed to identify a monobody pair that could bind simultaneously to the RBD, one monobody could simultaneously bind to the RBD with the ectodomain of the cellular receptor angiotensin converting enzyme 2 (ACE2). All four monobodies successfully bound the RBD after overexpression in Chinese hamster ovary (CHO) cells as fusions to the Fc domain of human IgG1.

Purification, characterization and identification of rat brain cytosolic tyrosine transaminase as glutamine Transaminase-K.

The current study was undertaken to investigate the spectrum of tyrosine transaminases enzymes in a cytosolic fraction of rat brain and to specifically purify and characterize a previously identified cytosolic brain enzyme possessing tyrosine/glyoxylate transaminase activity. Based upon extensive biochemical and immunochemical characterization of purified brain tyrosine/glyoxylate transaminase, we concluded the purified enzyme is glutamine transaminase-K (EC 2.6.1.64). This conclusion was based on: 1.) a concurrent enrichment in the tyrosine/glyoxylate and glutamine/phenylpyruvate transaminase activities during purification, 2.) demonstration of a co-substrate specificity for amino acids and α-keto acids that was highly consistent with published information for glutamine transaminase-K, 3.) results from detailed kinetic analysis, 4.) glutamine was a potent inhibitor of in vitro tyrosine/glyoxylate transamination, 5.) biochemical characterization, including pH optimum of 8.5 and spectrophotometric analysis and 6.) immunoanalytical analysis using a specific antiserum to rat renal glutamine transaminase-k. In addition, immunochemical characterization of a crude soluble extract of whole brain suggests that the in vitro tyrosine transaminase activity for several different α-keto acid co-substrates likely reflect the activity of glutamine transaminase-K. In conclusion, this investigation confirmed the presence of multiple tyrosine transaminase enzymes in a cytosolic extract of rat brain. Moreover, we concluded glutamine transaminase-K represents a predominant cytosolic enzyme in rat brain that's capable of catalyzing in vitro transamination of p-tyrosine and other aromatic amino acids, including the neurotransmitter precursors L-dopa and 5-hydroxytryptophan. The purified transaminase possesses a broad co-substrate specificity with preferential reactivity with α-keto acids derived from neutral aliphatic and aromatic amino acids. Lastly, we identified a heterogeneous regional distribution of tyrosine/glyoxylate transaminase (glutamine transaminase-K) in rat brain with a significantly higher level of in vitro activity in cerebellum.

Development and validation of a noncompetitive electrochemiluminescence-based immunoassay (ECLIA) for specific determination of insulin lispro (Humalog®) in human serum to support pharmacokinetic assessments.

Despite the importance of insulin and insulin analogs as therapeutic agents for the treatment of type I and II diabetes mellitus (DM), bioanalysis to support regulatory submissions of analogs remains a challenging endeavor. In particular, quantitation of insulin lispro by immunoanalytical methods has largely been limited to assays that display a high degree of cross-reactivity to native insulin because this analog shares extensive primary sequence homology with endogenous insulin and its efficacious circulating concentrations are low. We report herein development of the first noncompetitive electrochemiluminescence-based immunoassay (ECLIA) for specific determination of insulin lispro in serum or plasma. The new sandwich ECLIA permits accurate assessment of insulin lispro pharmacokinetics without interference from endogenous insulin. Integral to the development of this specific immunoassay was establishment of a proprietary process for affinity production of an oligoclonal monospecific guinea pig antiserum to the unique subtle structural modification in insulin lispro. We specifically optimized the ECLIA to provide reliable performance for supporting pharmacokinetic assessments in the pharmacologically relevant concentration range from 50.0 to 5,000 pM with robust performance up to 100,000 pM upon dilution. We concluded the new noncompetitive ECLIA represents a useful and convenient immunoassay for accurate quantitation of insulin lispro during pharmacokinetic assessments.

Recommendations for Systematic Statistical Computation of Immunogenicity Cut Points.

Today, the assessment of immunogenicity is integral in nonclinical and clinical testing of new biotherapeutics and biosimilars. A key component in the risk-based evaluation of immunogenicity involves the detection and characterization of anti-drug antibodies (ADA). Over the past couple of decades, much progress has been made in standardizing the generalized approach for ADA testing with a three-tiered testing paradigm involving screening, confirmation, and quasi-quantitative titer assessment representing the typical harmonized scheme. Depending on a biotherapeutic's structural attributes, more characterization and testing may be appropriate. Unlike bioanalytical assays used to support the evaluation of pharmacokinetics or toxicokinetics, an important component in immunogenicity testing is the calculation of cut points for the identification (screening), confirmation (specificity), and titer assessment responses in animals and humans. Several key publications have laid an excellent foundation for statistical design and data analysis to determine immunogenicity cut points. Yet, the process for statistical determination of cut points remains a topic of active discussion by investigators who conduct immunogenicity assessments to support biotherapeutic drug development. In recent years, we have refined our statistical approach to address the challenges that have arisen due to the evolution in biotherapeutics and the analytical technologies used for quasi-quantitative detection. Based on this collective experience, we offer a simplified statistical analysis process and flow-scheme for cut point evaluations that should work in a large majority of projects to provide reliable estimates for the screening, confirmatory, and titering cut points.

Recommendations for adaptation and validation of commercial kits for biomarker quantification in drug development.

Increasingly, commercial immunoassay kits are used to support drug discovery and development. Longitudinally consistent kit performance is crucial, but the degree to which kits and reagents are characterized by manufacturers is not standardized, nor are the approaches by users to adapt them and evaluate their performance through validation prior to use. These factors can negatively impact data quality. This paper offers a systematic approach to assessment, method adaptation and validation of commercial immunoassay kits for quantification of biomarkers in drug development, expanding upon previous publications and guidance. These recommendations aim to standardize and harmonize user practices, contributing to reliable biomarker data from commercial immunoassays, thus, enabling properly informed decisions during drug development.

Using DTA and DTAARRAY variables and programming in WinNonlin ASCII models to streamline user-defined calculation and data analysis.

In pharmacokinetic (PK) analysis, there are many occasions where user-defined calculations need to be performed before or after the primary PK modeling/analysis. Conventionally, these calculations are often executed outside of the primary PK analysis by pre- or post-processing data from multiple sources, manually entering formulas and multiple additional set-ups. Such analysis approaches increase the risk of generating data defects and can employ software that is not fully compliant. We propose a method of leveraging DTA and DTAARRAY variables plus simple programming techniques in an ASCII model to automate these user-defined calculations in WinNonlin and eliminate the need for manual handling of data outside of the primary analysis. We demonstrated the application of this strategy through three case study examples. In case 1 (post-processing data), DTA variables were used to calculate three user-defined parameters in the primary PK model. In case 2 (pre-processing data), a baseline correction decision tree was programmed into the PK model to account for both the endogenous baseline level as well as the presence of residual drug. In case 3, DTAARRAY variables were used to perform a looping operation to calculate the difference factor (F1) and the similarity factor (F2) in support of in vitro bioequivalence evaluations.

Systematic verification of bioanalytical similarity between a biosimilar and a reference biotherapeutic: committee recommendations for the development and validation of a single ligand-binding assay to support pharmacokinetic assessments.

For biosimilar drug development, it is critical to demonstrate similar physiochemical characteristics, efficacy, and safety of the biosimilar product compared to the reference product. Therefore, pharmacokinetic (PK) and immunogenicity (antidrug antibody, ADA) assays that allow for the demonstration of biosimilarity are critical. Under the auspices of the American Association of Pharmaceutical Scientists (AAPS) Ligand-Binding Assay Bioanalytical Focus Group (LBABFG), a Biosimilars Action Program Committee (APC) was formed in 2011. The goals of this Biosimilars APC were to provide a forum for in-depth discussions on issues surrounding the development and validation of PK and immunogenicity assays in support of biosimilar drug development and to make recommendations thereof. The Biosimilars APC's recommendations for the development and validation of ligand-binding assays (LBAs) to support the PK assessments for biosimilar drug development are presented here. Analytical recommendations for the development and validation of LBAs to support immunogenicity assessments will be the subject of a separate white paper.

Development of a novel radioimmunoassay to detect autoantibodies to amyloid beta peptides in the presence of a cross-reactive therapeutic antibody.

An increasing need in the development of biotherapeutic agents is the ability to monitor a potential autoimmune response to the therapeutic target of interest. Unfortunately, the presence of high concentrations of therapeutic antibody can hinder such detection, because there is competition for binding in cases where epitopes are not structurally distinct. This situation was encountered in the development of LY2062430, a therapeutic mid-domain monoclonal anti-amyloid beta peptide (Aß) antibody undergoing clinical trials for the treatment of Alzheimer's disease. This communication reports the development and validation of a novel radioimmunoassay used to measure potential patient immune responses to Aß in the presence of LY2062430. This assay employs a radioiodinated analog of the human amyloid beta 1-40 peptide (Aß1-40) in which a single amino acid substitution of alanine for phenylalanine at position 19 (F19A) effectively eliminates binding by LY2062430. In contrast, F19A binding by monoclonal antibodies specific for the N- and C-termini of the human Aß1-40 peptide was shown to be unaltered. Additional experiments involving a polyclonal rabbit antibody raised against the midregion of Aß1-40 (residues 15-30) resulted in only a slight reduction in binding to the F19A tracer, suggesting that the modification does not affect distal epitopes in Aß1-40 and supporting the notion that this conservative substitution produces only subtle change in the overall peptide structure. The assay is therefore believed to detect most, if not all, patient antibodies to native Aß peptides. The assay was validated for use in clinical trials allowing detection of antibodies to Aß in human serum in the presence of therapeutic concentrations of LY2062430.