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.

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.

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.