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Innovation is a pivotal process in drug development to address unmet medical needs. Advanced therapy medicinal products (ATMPs) are amongst these innovations and the number of ATMPs entering clinical stages has been substantially increasing over the last few years. Bioanalysis and potency assessment has proven to be a challenge, as the mode of action for cellular therapeutics is often not fully characterized. Currently most companies apply a multi-assay approach, often consisting of cellular surface analysis, cytokine release and one form of functional cell-based assay. The challenge arises when these assays need to be validated. While walking you through some case studies, we will address these assay and regulatory challenges and how they can be handled.
A short introduction about T-cell independent ADA response and multimeric targets (TNFα and IL-17) followed by a case study based on COVA322, a dual TNFα and IL-17A inhibitor that went into a FIH SAD Psoriasis Study stopped due to the observed safety profile
Protein purity analysis by microfluidic separation offers distinct advantages over traditional capillary electrophoresis in sample consumption, ease of use, and speed of analysis. Using a smart microfluidic platform for high throughput quantitation and quality screening of proteins. The method utilizes a microchip which utilizes real-time electro-hydrodynamic optimization to eliminate variability in reported results, i.e. run-to-run and day-to-day variability, which may be caused by micro-scale fluctuations in the physical properties of chips, reagents, or instrument components.
The use of intrinsic fluorescence spectroscopy for the analysis of protein structure, stability, and aggregation has significant benefits in terms of sensitivity and minimal unwanted perturbation. However, most fluorescence-based assays use either single point measurements or two-dimensional spectra both of which have poor information content. The situation is further complicated by the presence of multiple-fluorophores in most therapeutic proteins (e.g. IgG > 100 fluorophores…) which makes spectral interpretation difficult because of extensive spectral overlap. To facilitate detailed structural analysis of multi-fluorophore proteins, Anisotropy Resolved Multi-Dimensional Emission Spectroscopy (ARMES) can be used to extract more information from a single fast measurement. ARMES is a 4D measurement (λex×λem×If×r) in which the anisotropy (r) measurement can provide information about both structural and physicochemical changes and aggregation of proteins. This novel method also involves the use and development of multivariate analysis tools to extract the most useful information from the 4D measurements which can be implemented using standard benchtop spectrometers. Here we present some of the latest ARMES developments for the characterisation of proteins with case studies involving Insulin, IgG, and albumin type proteins.
The self-assembly and aggregation of proteins can lead to the formation of protein assemblies with sizes ranging from nanometres to micrometres. For several biologic formulations, protein concentrations in excess of 150mg/ml are required. Analysis of size heterogeneity at high protein concentrations presents an analytical challenge. I will discuss methods we use for aggregation analysis at protein concentrations up to 200mg/ml.
The HexaBody® format is a novel platform for the potentiation of therapeutic antibodies by enhancement of antigen-dependent hexamer formation at the cell surface, which may drive subsequent target receptor activation or complement activation. The biophysical characteristics and stability of HexaBody-based model compounds in different formulations will be discussed, probed by a variety of analytical techniques.
As software tools become available for aiding in the design and formulation of biopharmaceuticals, it is necessary to evaluate their usefulness. Such an effort requires collaboration between groups, exchange of data, and ultimately the establishment of benchmark datasets. This presentation will discuss the available predictive tools, their molecular bases, and the scope for providing benchmark evaluations to the wider community of researchers.
A key obstacle to predicting aggregation is difficulties in isolating the partially folded states believed to be key intermediates in the aggregation pathways. One approach not yet fully explored is to study the self association behaviour of partially or unfolded states under chemically denaturing conditions when they occur at much larger populations. Typically, these measurements are carried out using static or dynamic light scattering in terms of an osmotic second virial coefficient or a diffusion interaction parameter kD. Here we show how to account for the high concentration of denaturants in the data analysis, which if unaccounted for, could lead to data mis-interpretation. We then correlate light scattering measurements of five monoclonal antibodies at high denaturant concentration with their shelf stability. The results indicate there is a window of denaturant concentration over which the self association behaviour provides an indicator for the aggregation propensity upon storage.