How relevant are the matrix and ligands when developing adsorbents with improved functionality? In this talk, we will show how robust peptidomimetics can be easily adapted to several targets and to chromatographic and non-chromatographic matrices and purification processes.

Due to the costly and time-consuming production of biologicals, selective affinity purification methods are desirable. For efficient purification, several criteria are to be met. Strategies to address the harsh conditions often required for efficient affinity chromatography purification will be presented. Novel domains with calcium dependent affinity, developed through a semi-rational design combined with directed evolution, will be described. Further, insight on the molecular mechanism underlying the calcium switch, investigated by analyzing protein structure and dynamics with NMR spectroscopy will be shared.

This study investigates the utilisation of agarose gel electrophoresis (AGE) to identify byproducts in plasmid DNA (pDNA). Despite its perceived obsolescence, AGE successfully confirmed pDNA identity whilst uncovering additional bands for further analysis. Through meticulous examination using size-specific gel extraction and next-generation sequencing (NGS), the study identified these bands as deletion variants of the original pDNA, with low significance for subsequent processing steps. By reducing cell stress and lowering incubation temperature during seed culture fermentation, the occurrence of deletion variants in upstream processing (USP) was significantly minimised, eliminating the need for rigorous cleaning procedures and enhancing overall process efficiency.

This study introduces a novel proof of concept for antibody purification through potential-controlled membrane chromatography, addressing cost and efficiency in large-scale production. A 2.5 V cell potential was able to disrupt Protein A-antibody interactions and achieved ~90% elution rates. Membrane chromatography itself offers faster cycle times and eliminates mass transfer limitations. Additionally, it reduces reliance on buffer exchange, potentially enhancing sustainability. Its efficiency and sustainability render it a promising alternative for antibody production. Furthermore, the lack of low pH elution buffers offers promising new adaptations for difficult and pH-sensitive antibody species.

The DuoBody platform is a versatile and dependable technology for generating bispecific antibodies. Unlike other bispecific antibody platforms, the process is based on controlled Fab-arm exchange, which is performed post-production using purified monospecific antibodies. The process yields bispecific antibodies that retain the molecular structure and quality attributes of therapeutic IgGs. Case studies are presented detailing downstream processing approaches in the development of a range of bispecific antibody therapeutics.

With the goal to streamline and accelerate protein production, automated ÄKTA-based purification protocols have been developed consisting of autosampler systems (Teledyne, ALIAS) to facilitate injections during overnight and idling times, two- and three-column schemes to automate purifications, and novel affinity chromatography membrane technology to reduce cyclization time. Those protocols enable the cost-effective screening and purification of hundreds of single-chain variable fragments (scFvs) and multispecific antibody-based therapeutics per week with high monomeric purity.

Development of novel protein scaffolds through protein engineering strategies requires the complementary development of downstream processing methods for the non-natural proteins. Further steps taken in one expression host may need to be altered or optimised in another. We describe here the development of novel 10kDa scaffolds, SXkmers, and the development of techniques to purify and polish for in vitro and in vivo studies with monomers, dimers, and Fc fusion variants.

A lot of choices of membrane absorbers and fiber materials for virus purification, compared to the past. What is the selection criterion for a membrane adsorber and fiber material? The ideal separation process for a viral vaccine.

• Expectations
• What has changed
• Impact on bioprocessing

This presentation unveils an adaptive Model Predictive Control (MPC) framework designed to mitigate the challenges of column aging in process chromatography. Through a nonlinear MPC, extended Kalman filter, and a soft sensor mechanism, the framework dynamically adapts to resin capacity reduction, optimising bioprocesses. Experiments demonstrate a 50% reduction in processing time, improved resin and buffer utilisation, marking a stride towards sustainable production aligned with BioPharma 4.0.

Understanding how biomolecules interact with chromatography resin is a pivotal challenge in chromatographic procedures, requiring a deep grasp of fundamental adsorption mechanisms. Thermodynamic studies, using batch equilibrium experiments and calorimetric measurements, have illuminated liquid chromatography biomolecule adsorption processes. However, we continue to face limitations in resolving thermodynamic variances related to protein loading and temperature. Flow microcalorimetry (FMC) ability to measure heat signals during chromatographic events, both in linear and overloaded conditions, has proven invaluable, enhancing chromatography forces comprehension. It clarifies biomolecule adsorption mechanisms, shedding light on interactions with diverse supports (ion-exchange, silica-based matrices), aiding in exploring surface heterogeneous adsorption and biomolecule rearrangement.