This project is a follow on project to: "Practical Applications of circular dichroism spectroscopy for biopharmaceuticals." Reference number 102611 which finished in October 2017. Higher Order Structure (HOS) of a biopharmaceutical is a key quality attribute required to be controlled throughout the development and manufacture of a biologic drug or vaccine with changes in the HOS potentially impacting stability, function and safety of the drug. This necessity to control the HOS structure of biopharmaceuticals extends throughout the whole product life cycle of a pharmaceutical, including biosimilar and generics manufactures having to show comparable HOS to the original manufacturer's product. FDA guidance states a requirement for many orthogonal measurements of HOS using multiple different techniques. Orthogonality, the correlation of non connected measurements from different analytical instruments, to confirm a safety critical parameter, is an integral concept in the confirmatory data required to assert a biotherapeutic's performance and its consistency in manufacture. A number of approaches are used to analyse and understand the HOS structure of biopharmacueticals. From high resolution techniques like NMR and mass spectrometry, as well as optical spectroscopy techniques like circular dichroism (CD) spectroscopy, Infrared spectroscopy, Raman, and UV-fluorescence emission. This project will develop a comprehensive solution for HOS analysis of biopharmaceuticals incorporating multiple orthogonal measurements of HOS with a data analysis software system using a simplified and unified workflow on one platform. We will offer this as a Software as a Service (SaaS) model on an open platform basis. Therefore the software system would not only analyse data generated on our HOS system but be extensible allowing other probes of HOS from other instrumentation and analytical systems to be analysed in a consistent way.

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High throughput protein stability analysis systems for biopharma

"To ensure product safety and efficacy, protein therapeutic critical quality attributes must remained within tolerances for the duration of the shelf life of the product, as well as during transportation, storage and use. Consequently development of and testing for long term stability of the protein drug product is a major focus of biotherapeutic development and formulation activities. Thermodynamic stability of the higher order structure of the protein is used as a proxy for longer term stability of the drug product, allowing a large number of potential formulation conditions or variants on the drug product to be screened in a period of time significantly shorter than the potential shelf life, greatly increasing the speed the drug can be developed and commercialised. The tools currently deployed are Differential Scanning Calorimetry (DSC), that looks at the change in heat capacity for the protein as it is heated, and the optical spectroscopic methods that look at changes in the higher order structure (HOS) of the protein with temperature. These approaches have traditionally been restricted by low throughputs (1 sample per hour) and high sample consumption. Another approach repurposes qPCR machines by adding fluorescent dyes, that change their fluorescence properties on binding to the exposed hydrophobic core of the protein as it unfolds during heating. This approach allows screening of many conditions at once, in 384 well disposable microplates at relatively little cost per sample. But the utilisation of hydrophobic dye can produce false results due to other binding modes of the intact protein and interference with the dye binding from other buffer components. This project is a feasibility study to determine the technical and commercial viability of a new high throughput technology for the screening of protein stability that requires very little sample, is able to be extensively automated and does not require addition of an indicating dye. The target market will be biopharmaceutical development and formulation, allowing much higher numbers of drug candidate variants or formulation conditions to be screened earlier in the development cycle."

Circular dichroism (CD) spectroscopy is an analytical technique routinely used in the biopharmaceutical industry to study the effects of manufacturing, formulation, and storage conditions on protein conformation and stability. The difficulty in data interpretation of CD is limited to demonstrating conformational comparability after a manufacturing/formulation change using qualitative assessments of overlaid spectra, which is fundamentally subjective and prone to error. This project will standardise data collection and analysis and build model based approaches for quantitative analysis of Higher Order Structure using CD. The data collected will allow for the generation of platform models and data sets to widen the application of CD into bioprocess development.

Knowledge of a biopharmaceutical product’s higher order structure (HOS) and conformational dynamics, and how these relate to it’s mode of action and/or degradation, are central to enable effective and streamlined biopharmaceutical development through QbD-based approaches. Hydrogen deuterium exchange mass spectrometry (HDX MS) is a technique increasingly used to characterise the HOS of peptide and protein therapeutics. HDX MS can determine structural perturbations in a protein that elicit changes in conformational dynamics or solvent accessibility. Commercial implementation of automated HDX MS analysis, has greatly increased the utility of HDX MS in the biopharmaceutical industry for HOS analysis. But there are limitations with these systems where unstructured or highly dynamic, solvent exposed protein regions are not able to be differentiated or analysed. This team propose to develop a system that will allow routine analyse of these proteins and peptides with HDX MS.

Eight of the predicted top-ten drugs by revenue in 2016 will be biotherapeutics and as early as 2014, 50% of the top-100 pharmaceutical products will be accounted for by vaccines and biotherapeutics. Understandably, pharmaceutical companies are competing to establish new biotherapeutic drug pipelines, both as innovator and generic manufacturers. The societal benefit through improved treatment of any number of conditions by these new drugs cannot be over-stated. The FDA and the EMA are demanding that companies provide evidence of equivalence of higher order structure (HOS), not only for biosimilars when compared with innovator products but also for any licensed biotherapeutic where the process is changed, e.g. on scaleup or where some manufacturing efficiency improvement is sought. There are very few tools that provide HOS information in a manner that is amenable to rapid and routine analysis: the recent introduction by APL of a highly innovative, fully automated circular dichroism (CD) spectrometer has opened the door for CD to take on that role. Automated CD has been used for early-stage characterisation and formulation studies but a wider, unmet need is for routine, quantitative and robust comparison of HOS in innovator and generic products. Two key barriers stand in the way of using CD in this role: 1. An absence of primary certified reference materials for CD and hence a lack of traceability of the CD measurement, which lowers confidence in comparing data measured in different places on different instruments, or even the same instrument at different times. 2. An absence of defined analytical methods, particularly in the analysis and presentation of data in a simple, meaningful manner that is accepted industry-wide The key aims of this proposal are to deliver novel methods for the calibration and validation of CD instruments and to develop statistical analytical methods to provide a solution to the comparison of protein HOS.