To sustain the rapid expansion of biopharmaceuticals whilst preserving their quality, the Quality-by-Design (QbD) initiative was introduced, which has process intensification as a main pillar. Process integration and continuous operations are valuable strategies towards consistent product quality and high throughput. However, digitalisation is the keystone to express their full potential as it allows a model-based process control, a reduction in time-to-market and costs, and fulfillment of the QbD guidelines.

Biopharmaceutical industry traditionally relies on pharmaceutical manufacturing practices to monitor processes and release products. The use of Process Analytical Technologies (PAT) can improve the process monitoring and control, and increase the process understanding. PAT also enable real-time control when integrated into a digital twin. This talk is concerned with the implementation of PAT and development of digital twins in GSK for purification processes.

More than 25% of all biopharmaceuticals are produced in E. coli. However, a common consequence of recombinant protein production in E. coli is the formation of insoluble product aggregates, Inclusion Bodies (IBs). To obtain correctly folded protein, IBs are solubilised with strong chaotropic denaturants. I will present strategies for mild solubilisation, preserving secondary protein structures, thus alleviating IB processes.

Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) was demonstrated as a valuable process to improve the efficiency associated with the chromatographic purification of oligonucleotides through the automated internal recycling of impure side-fractions. A dynamic process controller AutoPeak, adjusting in real time the characteristic times for internal recycling and product collection based on the recorded UV signal, is presented as a key technology to improve process robustness, rejecting disturbances during manufacturing.

It is recommended by ICH guidelines to use residence time distribution (RTD) for characterizing material flow. RTD can be measured experimentally by injecting an inert tracer into the inlet and tracing it in the outlet. For antibody production in continuous mode, periodic counter-current chromatography (PCC) is widely used. Fluorescent-labeled antibody was used as an inert tracer. It was injected into the inlet, then it was traced during the loading phase and elution phase. The experimental findings were corroborated by a process model. The RTD of the PCC is rather wide due to both cyclic inlet flow and the constant exchange.

The Centre for Process Innovation (CPI) is part of the UK High Value Manufacturing Catapult (HVMC). CPI has been, and is currently, involved in several continuous biologics manufacturing and process analytical technology (PAT) projects. In this talk, I will discuss previous and current projects involving continuous manufacturing and the use of novel process analytical technologies (PATs).

Here, we present a first scale-up of perfusion cultivations of Rhodovulum sulfidophilum for the production of extracellular oligonucleotides. Starting from high-throughput micro-scale models, we assessed the role of different process variables which highlighted a maximum cell-specific perfusion rate. We then scaled-up the process to a 2L benchtop perfusion bioreactor. Here, we emphasized the impact of the perfusion rate on cellular growth, nutrient consumption, and oligonucleotides expression.

New modalities are a challenging task for downstream processing. Alternative purification strategies that can improve the purification yield, such as affinity chromatography or the use of new adsorbent materials are regarded nowadays as enabling technologies to overcome the capacity bottleneck in biomanufacturing. The current talk will focus on the development of new engineering ligands and new matrices aimed at the purification of viral vectors; some case studies will be discussed.

Process intensification and integration are often viewed as tools to improve bioprocess efficiency. This talk will explore the use of perfusion bioreaction and continuous chromatography to seamlessly integrate and connect upstream and downstream stages. AAV expression, harvesting, and clarification processes are integrated using tangential flow depth filtration. The cases of continuous AAV affinity capture and polishing will be presented, with an emphasis on the challenges and opportunities for future developments.

Bionanoparticles, like virus-like particles, are promising candidates for future vaccines and gene therapies. For the production of safe pharmaceuticals, a major focus lies on the downstream processing. Within our research, we are investigating the fundamental principles of bionanoparticle purification with the goal of moving from resin-based chromatography to more environmentally friendly, biodegradable, membrane-based methods. This shift enhances process efficiency and aligns with our commitment to sustainable production.

In this presentation, we show how advanced machine learning and hybrid modeling approaches can be exploited to significantly improve process understanding, performance, and automated operation as digital twins. All presentations will be centered on industrial implementation examples for mAb, cell & gene therapy, and mRNA processes with numerous big pharma and CDMO partners allowing to quantify efficiency gains in and improved understanding in process development.

The lytic nature of IC-BEVS limits its use for continuous bioprocessing. Here, a continuous multi-stage bioreactor process was established to produce influenza hemagglutinin-displaying virus-like particles (HA-VLPs) using IC-BEVS. Different process designs (varying cell line and rBAC construct) and residence times (RT=18, 36, and 54 hours) were tested. The best-performing design allowed consistent rBAC (108-109 pfu/mL) and HA-VLPs (34±14 HA titer/mL) titers; RT=54 hours outperformed other RTs. HA-VLPs were efficiently identified by electron microscopy after 20 days of continuous operation. This work showcases successful continuous HA-VLPs production using IC-BEVS, paving the way for establishing continuous, integrated setups using this expression system.

We present a novel purification method for recombinant multimeric virus-like particles (VLPs) using metal-ion affinity precipitation. The study focused on VLPs made of turnip yellow mosaic virus coat protein, produced in Escherichia coli. Remarkably, a mere 15 µM (or lower) of transition-metal salt, such as nickel chloride, proved sufficient to induce precipitation, leading to an impressive recovery rate of up to 70% with a purity exceeding 0.90.