The most common tool used for purification of antibodies is protein A affinity chromatography, a method that offers high productivity and pure protein product. However, elution of captured antibodies requires low pH and that might be deleterious. We have addressed this issue by developing a protein domain displaying calcium-dependent binding to IgG. To evaluate the domain in affinity chromatography, a matrix based on a tetrameric version of this domain was produced. From this column, elution in physiological pH was possible and IgG recovery was shown to be comparable to commercial matrices.

This presentation will focus on the development of high-affinity, highly-specific diagnostic and therapeutic binding molecules based on the creation of two novel phage displayed libraries of a uniquely stable and hydrophilic protein scaffold. This novel scaffold has molecular properties designed to achieve significant impact as a new generation of powerful tools in areas of clinical need, both as diagnostic and therapeutic entities. Processing of specific candidates will be described.

Protein A affinity chromatography is the workhorse for antibody purification. Novel media exhibit some amazing features, like extreme pH transitions, which can be triggered by specific buffer selection leading to process conditions that can benefit the respective antibody to be purified. Furthermore, protein A ligand properties can change significantly upon alkaline treatment and eventually lead to enhanced mass transfer.

The last few decades have witnessed a huge development in the design of new virus-based biopharmaceuticals. They have been used for cancer treatment, neurodegenerative and genetic diseases, as well as in immunotherapies. However, their purification still presents challenges related to the high dosages and high purity required. New insights on how the additive manufacturing and new affinity matrices improve the global recoveries are presented and discussed.

Establishing a virus filtration step in biopharmaceutical development and manufacturing process may require a large number of resources. These can be minimized with swift process development, smooth scale-up, and worry-free operations. In this presentation, you will learn through different case studies the filtration performances of PlanovaTM BioEX with regards to robustness, scalability, flexibility, and consistency, even in challenging conditions. Contribution to rapid cost-effective process development during different clinical phases will be also explained.

Continuous antibody precipitation combined with tangential flow filtration allows a constant mass flow process. Fine-tuning the conditions is imperative for achieving acceptable recovery and purity rates. Using a cross-linking agent as the main precipitant, we describe the critical factors and its effects on the precipitation of different recombinant monoclonal antibodies.

Integrated continuous biomanufacturing requires big hold tanks for buffer and media preparation. The current trend to reconstitute from stock solutions is especially problematic for fermentation media. We show a proof-of-concept for a continuous reconstitution of media and buffers from solids in lab scale. This opens a new playground for development of strategies in upstream and downstream and several applications of this concept will be demonstrated.

Development of next-generation medicines to treat or cure some of the most serious of human diseases is on the near horizon. Majority of treatments rely on recombinant proteins, however this landscape is now changing to include oligonucleotides and viral delivery technologies. This presentation will focus on the critical considerations around the development of a scalable manufacturing process to produce plasmid DNA as final product or intermediate to gene therapies.

Enveloped virus-like particles (eVLPs) are a complementary strategy for the manufacturing of vaccines against enveloped viruses. The baculovirus expression vector system (BEVS) in insect cells is an attractive and widely used platform for the expression of eVLPs. Inherent to this system is the co-expression of baculoviruses and other extracellular vesicles, which we could nicely demonstrate by high-resolution electron microscopy on one of our expression supernatants (Figure 1). Such heterogenous sample mixtures pose a major bottleneck for effective downstream processing of insect cell-expressed, enveloped virus-like particles. eVLPs, baculoviruses, and extracellular vesicles are similar in size and share a lot of common surface properties which make chromatographic separation difficult. In addition, immune responses against baculoviruses have been reported in humans, therefore efficient removal is mandatory. We produced influenza HA-Gag virus-like particles by baculovirus infection of our Nodavirus-free Trichoplusi ni-derived Tnms42 insect cell line (Figure 2) in 10-liter bioreactors using suspension cell technology. A fast and simple purification method for these VLPs with simple processing of the feed and using one chromatography step has been developed.

A gram-scale prototype employing membrane assisted crystallisation has been developed for the single step purification of monoclonal antibodies. Learnings and results from purifying a model monoclonal antibody using this prototype are guiding the development of scale-up and transition to continuous purification using the same technology, progressing towards the goal of improving efficiencies in the downstream processing of therapeutic proteins.

The number of biotechnology-based pharmaceuticals in the late-stage pipeline has been increasing more than ever; in particular monoclonal antibodies (mAbs) represent a quarter of all biopharmaceuticals in clinical trials. As a result, there is an enhanced demand for more efficient and cost-effective processes. Here, the potential of miniaturization as a high-throughput screening tool to speed up process optimization is explored, considering optimization of antibody extraction conditions with ATPS and chromatographic conditions optimization using a multimodal ligand.

The most commonly applied approach for intermediate acceptance range definition, a 3-sigma range, leads to higher out-of-specification probabilities and a restrained manufacturing flexibility. Using a digital twin – with the same amount of data – we derive specification-driven acceptance criteria that ensure a predefined out-of-specification probability. These specification-driven ranges enable us to set up a control strategy that prevents failed batches while maintaining the highest possible manufacturing flexibility.