Developability assessment is a well-established concept at pharmaceutical industry to mitigate early on possible risks for drug candidates, which in absence of identification might endanger whole programs. But are these concepts addressing all the critical parameters? And how can the higher candidate assessment demand be optimised to not overburden the resources of the organisation? Examples of new modules in the developability assessment concept for biologics will be shown.

I will present the latest advances in end-to-end automation, digitalisation and data management of the protein analytics workflows in the pharma research and early development (pRED) unit of Roche, focusing on mass spectrometry analysis of complex antibody-based drug candidates. It is routinely applied to a variety of sample types and throughput, from early binder screening to clone selection, and bioprocess development.

SARS-CoV-2 spike protein has emerged as a prime target for vaccine development  and serological assays. The heavily glycosylated structure with host-derived glycans two O-glycans sites not only aids the virus in evading the host immune response but further makes it challenging to characterise its physicochemical attributes such as its stability and fragmentation. This was overcome by performing buffer exchange and deglycosylation, thereby optimising inherent migration behaviour in capillary electrophoresis.

Due to the chemical properties of mRNA, including its size, charge, polydispersity, and susceptibility to degradation, the quality control of mRNA therapeutics is complex. Approaches for control strategies and the quality attributes for release and stability testing of mRNA lipid nanoparticle drug products will be outlined. The main impurities and degradation pathways to consider will be described. In addition, key methods for mRNA LNP analysis will be showcased.

Characterisation of antibody biopharmaceuticals largely relies on mass-spectrometric approaches which involves various sample preparation steps including protein digestion. These steps are time-consuming and can increase the risk of inducing artificial modifications. Using multidimensional LC-MS we have developed a platform that enables automated sample preparation (including digestion with different enzymes) and MS analysis. The results were comparable to offline approaches, while drastically reducing analysis and hands-on time.

Disulfide bond formation plays an important role in protein folding, stability, and biological functions, and therefore should be well-characterised during biotherapeutics development to ensure correct cysteine pairing and protein-structural integrity. A mass spectrometry-based disulfide mapping method was developed to provide accurate relative quantitation of free thiols, and high recovery of disulfide-linked peptides. This platform has been successfully applied to a diverse variety of therapeutic proteins.

The talk underscores the pivotal role of polyA tailed mRNA in mRNA-based therapeutics and presents a unique approach to assay development. By crafting a generic method, we efficiently characterized both tailed and tailless mRNA molecules. Leveraging this initial data, we optimised the method into a robust analytical assay, now serving as a release assay and enabling high-throughput screening of multiple mRNA lots and plasmid batches. This innovative assay not only ensures product consistency, but also expedites the development and manufacturing processes, highlighting its paramount significance in the rapidly evolving landscape of mRNA-based therapeutics.

Recent progress in MS characterisation platform will be presented. Topics include automated fraction collection from any type of LC separation (including non-MS compatible LC methods) combined with a broad range of MS characterisation workflows (including intact LC-MS, peptide mapping by LC-MS/MS, and direct-infusion MS) of the individual LC fractions. The characterisation platform provides highly flexible, robust, ultra-sensitive, and in-depth characterisation of proteoforms. Case studies and perspective will be presented.

Product development spans many years and includes transitions in personnel, facilities, and overall strategy. By necessity, we prioritise the items required to reach the next milestone. Yet, the studies that are critical to identifying gaps are often performed late in development. Changes in technology and regulations may invalidate historical work. This presentation provides advice on gap mitigation as we approach our BLA/marketing applications.

The ability of proteins to aggregate is a challenge upon the development of therapeutic products. By fluorescence microscopy, X-ray scattering, spectroscopy and molecular dynamics, we identify the interactions responsible for protein phase separation, conformational changes, and colloidal instability, and how those aspects are linked to the variability in the morphologies. Our findings provide a scenario in which heterogeneous structures can be generated as a result of interconnected aggregation pathways.

• How we can proceed with the High throughput analytical methods (size, charge, fragments, and glycan) to support the NGM process?
• Is there opportunity for characterization methods to be scaled up for High throughput analysis?
• What are the risk and challenges?​

Defining limits for analytical method outputs is a central element of a control strategy. Traditional approaches usually focus on ensuring process consistency, while, in more advanced approaches, the understanding of structural-function relationships is utilised. This enables definition of limits that may extend outside those determined by clinical experience. Where indicated, this is required to incorporate more worst-case assessments to predict future manufacturing variability as the basis to mitigate risks to patients and supply.

Analytical characterisation of cell and gene therapy manufacturing processes is essential for process understanding and driving quality-by-design development. Incorporation of higher-content analytical techniques that result in meaningful outputs which can be used to drive higher-quality process development and integrate into a manufacturing setting is a challenge and one that the Cell and Gene Therapy Catapult is looking to address for allogeneic cell therapy manufacturing processes.