Affinity adsorbents have been the cornerstone in protein purification.
The plethora of affinity adsorbents available in the market reflects the importance of affinity chromatography in the bioseparation industry. The main goal for the design of affinity ligands is to discover or improve functionality, such as increased stability or selectivity. However, the methodologies must adapt to the current needs, namely to the number and diversity of biologicals being developed, and the availability of new tools for big data analysis and artificial intelligence. In this presentation, we will show how rationally designed chemical combinatorial libraries support the development of robust peptidomimetics. We studied the potential of these scaffold affinity reagents to find binding partners against several biological targets.

Novel biological drugs, including monoclonal antibodies, are recurrently approved by the FDA. To address the demand for mild and specific purification, a protein purification ligand with calcium-dependent binding to IgG has been developed. Further improvement of this concept will be presented, including a novel protocol for antibody purification, as well as the development of new tailor-made ligands for purification of other biological therapeutics.

We describe the beneficial inclusion of the Emphaze™ AEX Hybrid Purifier from 3M, which was evaluated compared to a conventional clarification process, for removal of problematic HCPs during downstream bioprocessing of mAbs. Total host cell protein and host cell DNA were determined using the ProteinSEQ and ResDNASeq assay kits. Advanced LC-MS-based proteomic methods were used to track and identify known ‘problematic’ HCPs through a multi-cycle Protein A purification process.

The increasing number of projects in early R&D, combined with the need to characterize and evaluate potential new therapeutics, result in a continuously increasing number of requests to produce and QC proteins. Each production comes with its own challenges and requires different approaches to generate the requested amount of protein. The presentation will address some of the day-to-day issues in lab-scale protein production and suggest ways to overcome them.

We developed a new continuous precipitation process where the mass flow of the product is not interrupted. This process is robust, because fluctuations in the feed stream can be readily handled and easily realized as a disposable unit, because the necessary equipment, such as tubing, fittings, static mixers, and hollow fiber modules, are commercially available and do not require surge tanks. This process is truly continuous compared to other, quasi-/semi-continuous chromatography processes, which require cyclic operation.

The increased complexity of the new bio-therapeutics, as well as the growing pressure on cost reduction, have led to define new development and production strategies. The COMPAC2T™, an End-to-End Continuous Manufacturing process integrating perfusion bioreactor with continuous purification, is our solution to face these challenges. This approach allows significant reduction in the Cost of Good, while increasing the agility of the biotherapeutics manufacturing and supply models.

As the scientific community strives to make published research ever more transparent and reliable, the quality of recombinant proteins used comes into focus and there is a real concern about the problem of irreproducible results. In order to improve the reliability and reproducibility of data using purified proteins in life science research, we have drafted guidelines for improved quality control. These will be presented together with an evaluation of their impact on the success and reproducibility of downstream experiments.

In recent work (patent pending), we have discovered a peptide that binds bacterial lipopolysaccharides with nano- to picomolar affinity and no observable off-rate. We are currently exploring ways to optimize the binding even further, and to define the binding site on the LPS molecule better. Current data suggests binding to the invariant parts of LPS. We believe that this peptide could be used in detection applications to replace the current, non-sustainable endotoxin test procedures, and for improved endotoxin removal technologies.

MALS coupled with size exclusion chromatography (SEC-MALS) is a standard and common method for characterizing protein mass, shape, aggregation, oligomerization, interactions, and purity. The limited resolution of SEC interferes in some cases with the accurate analysis that can be achieved by MALS. These include mixtures of protein populations with identical or very similar masses, oligomers with poor separation and small peptides. We recently developed a new technology that combines MALS with protein separation according to charge, IEX (IEX-MALS). The high-resolution separation technique IEX (IEX-MALS) allows a precise analysis of samples that cannot be resolved (or have limited analysis) by SEC-MALS. The technology is very simple, and unlike SEC-MALS that needs prolonged column equilibration, here the equilibration is performed in a few minutes, and the injection volume is not restricted. We will discuss advantages and disadvantages of IEX-MALS vs. SEC-MALS, as well as its applicability at low scale during processing development, allowing a fast on-line identification of the target with the correct oligomeric conformation, from other conformations or other proteins. Its applicability in membrane proteins purification, characterization, and separation from free micelles, and many other possible applications will also be presented.

Membrane proteins are important drug targets (GPCRs, ion channels), yet are notoriously difficult to work with. We have developed a novel one-step approach for the incorporation of membrane proteins directly from crude cell membranes into lipid Salipro particles. This direct approach presents new opportunities for the analysis of novel drug targets. Furthermore, we present how the Salipro system enables the generation of antibodies against challenging drug targets.

Magnetic beads-based purification permits the incubation of the beads directly into cell culture (for secreted proteins) or crude lysates regardless of sample volume. This provides a simplified approach to direct target capture while eliminating much of the sample preparation steps and potentially improving the quality of the purified product.