Evolving biologics complexity and regulatory demands dictate using advanced bioanalytics for product and bioprocesses characterisation. We developed an MS-based Multiple Attribute Method (MAM) strategy for assessing PTMs of CHO-derived mAbs and established an HCP profiling workflow by SWATH-MS. Key PTMs like glycosylation, oxidation, and deamidation were profiled, and targeted high-risk HCPs were quantified. These results defined the best design space, maximising product quality and purification performance of our polishing platform.

The recombinant dual-tagged-E6 protein (His6-MBP-E6) was expressed from Escherichia coli cultures and successfully extracted by sonication/ice cycles. The isolation/capture step was obtained by affinity chromatography using MBPtrap column. The purification/polishing step was explored by applying anionic exchange (QSepharose), size exclusion (Superdex), or immobilised-metal affinity chromatography (HisTrap). The combination of MBPtrap with HisTrap obtained 94±3% of highly pure His6-MBP-E6, preserving its secondary structure and allowing its application for biointeraction studies.

The diagnosis potential of single domain antibodies (sdAb) has prompted their use in several research and biotechnological applications. This work describes the production in Pichia pastoris of two gluten-specific sdAbs fused to Lucia luciferase and two red fluorescent proteins (DsRed-Express2 and mCherry). The results evidenced the high applicability of this expression system for this goal, with mCherry fusions exhibiting the highest production yields due to their effective release into the supernatant. In contrast, DsRed-Express2 fusions showed the lowest production yields since they were not efficiently secreted into the yeast supernatant.

Vaults (protein nanoparticles naturally found in eukaryotic cells, but absent in prokaryotic ones) show an enormous potential as drug-delivery systems (DDS). Recombinant vaults are routinely produced in insect cells and purified by ultracentrifugation, both tedious and time-consuming procedures. Here, we propose a cost-efficient and faster protocol to produce vault-like nanoparticles in Escherichia coli cells, still one of the most widely used prokaryotic cell factories for recombinant protein production. This allows the spontaneous formation of vault-like nanoparticles and their loading with engineered cargo proteins. This approach paves the way to faster and easier engineering and production of vault-based DDSs.

To facilitate clinical grade lentiviral vector production, we are presenting a novel state of the art platform for LV production using HEK293T-based stable packaging cell lines in both adherent and suspension modalities. We developed an optimised perfusion process resulting in high Lentivirus (LV) titer and ensuring that high viability of cells during LV production is ensured. We also designed an efficient tailor-made DSP process to purify and sterile filter LVs.

The reduction of cell-specific productivity in transient gene expression (TGE) at high cell density (HCD) is known as the cell density effect (CDE). This study investigates the CDE through the production of HIV-1 Gag virus-like particles (VLPs) via transient transfection in HEK293 cells. Combining EV depletion and UGCG overexpression improved transfection efficiency by ~45% at 12 × 106 cells/mL also enhancing VLP budding and improving production by 60%.

Biochemical research often necessitates the optimisation of conditions to maximise product yield. However, Design of Experiments (DoE) approaches for rapid process optimisation present a challenge due to statistical complexities and high software costs. To address this, we introduce "Spreadsheet DoE," a user-friendly tool for non-statisticians. In conjunction with high-throughput protein purification methods developed in our lab, Spreadsheet DoE has optimised various biochemical processes along the protein production and characterisation pipeline.

For novel bispecific antibodies, methods exist for low-throughput large-scale production but combinatorial screens often still represent the bottleneck for the identification of the best possible bispecific antibody. This presentation will share insights into two robust and miniaturised heterodimerisation workflows based on inteins or controlled Fab-arm exchange (cFAE), discuss advantages and pitfalls, and show its compatibility with high-throughput functional screens of biparatopic antibodies and ADCs.

We report the development of a semi-automated platform to express panels of antibodies in high-throughput at mid-scale for developability and functional assays whilst minimising hands-on lab work. Our platform enables parallel production of 30 mg of material for 96 clones, keeping the discovery funnel wide for longer. We have created digital workflows for sample and data tracking enabling data reuse and driving refinement of AI/ML models.