In connection with the use of prefilled syringes, studies focused so far on the possible incompatibility of biologics such as monoclonal antibodies with hydrophobic surfaces such as silicone layers. Silicone spiking studies or studies with over siliconised syringes should test the silicone compatibility in advance. However, recent studies in our company show that the formulation and certain components can also attack the silicone layer and that there are certain protein properties that can potentiate this effect. The consequence is the release of silicone-like droplets into the solution and a loss of syringe functionality over the storage period. The lecture will present the current studies on this topic.

The physical stability of therapeutic proteins during bioprocessing and formulation is a crucial property to guarantee their safety and efficacy. Air-water and solid-liquid interfaces are well known to potentially trigger protein instability and aggregation. Challenges in the investigation of interface-induced protein aggregation include the control of the amount and type of surfaces, as well as the presence of synergistic effects between interfaces and hydrodynamic flows. Here, we present a newly developed surface stress assay based on polymeric nanoparticles, which could complement developability studies in early-stage development.

Distinguishing aggregated API from other particle types is important for understanding the root cause of instability. Existing methods are unreliable, too cumbersome and difficult to use in many workflows. With Aura, you can now finally count, size, and characterize aggregates and identify them as proteins, non-proteins, or other molecules.

Residual HCPs can impact product quality in different ways. Besides effects on patient safety, product stability might be affected. Here, we present workflows based on LCMS to identify HCPs inducing particle formation. LCMS is the method of choice for identification of unknown HCPs as ELISA is usually not capable to identify individual HCPs. In addition to HCP identification, LCMS allows the relative and absolute identification of single HCPs.

We present key insights into protein structural factors controlling aggregation behaviour for scFv mutants. Aggregate growth rates are controlled by non-specific electrostatic interactions, while increasing the scFv lysine to arginine content lowers aggregation by preventing partially unfolded regions from associating. We show protein-protein interaction measurements made under chemically denaturing conditions reflect colloidal stability of scFv partially unfolded states, which play key roles in aggregation pathways.

A mutant of the interleukin-2 molecule without interaction with the alpha chain of the IL-2 receptor was designed at the CIM. This new mutant shows higher antitumor activity and less toxicity than human IL-2, that is why it is a better candidate for human cancer therapy. In order to improve the quality of the final protein some of the purification steps and conditions were modified. The proposed purification process allowed obtaining a consistent purified product, with similar physical-chemical and biological properties to the product, purified with the current process and higher quality.

I will discuss why proteins aggregate and how aggregation relates to protein stability. Next I will cover how nature selects against protein aggregation and discuss the implications of these findings for protein engineering and redesign.

Protein refoldability is an essential feature of therapeutic protein formulations with lower aggregation tendency during storage. I will introduce you to two approaches that allow the assessment of protein refoldability after unfolding caused by heat or chemical denaturants. Further, I will demonstrate how the presented methods can be useful for the selection of protein molecules with lower aggregation propensity and formulations that impede the formation of aggregates during storage.

In the process of developing biotherapeutics early developability and manufacturability assessment is crucial to select the best candidate for the CMC phase. Therefore we focus on miniaturized screening approaches which allow us to predict stability and rank lead candidates for further development. This talk will give an overview of our formulation screening cascade and show its applicability in a case study.

Quantifying concentration, checking aggregation and testing stability of proteins and AAV often consumes more sample than you want. Stunner delivers fast, accurate quantification and aggregation information on 2 µL of sample. Uncle’s full-spectrum fluorescence gives full visibility on protein stability in less than 10 µL.

Characterization of protein higher order structure (HOS) is technically challenging. A simple normalization technique will be discussed, with a few examples, to demonstrate enhanced precision of circular dichroism (CD) spectroscopy to better monitor and interpret HOS changes in proteins. Additionally, a case study with six different monoclonal antibodies will be presented where the consequences of Tryptophan (Trp) oxidation on product HOS and biological activity will be discussed.

Protein aggregation remains a challenge for the development of biopharmaceuticals, and currently there are no screening assays validated to predict aggregation upon long-term storage. Identifying predictive assays would be an important advancement to guide the design and selection of druggable candidates in screening campaigns. In this talk, I will show how we probe multiple biophysical properties of different antibody formats to calculate correlations with the aggregation propensity upon long-term storage.

Essentially all biopharmaceutical drug substances and drug product are exposed to freeze-thaw and/or freeze-drying/reconstitution. Recently, frozen and freeze-dried proteins with various excipients (surfactants and lyoprotectors) are studied using small-angle neutron scattering (SANS) and small- and wide-angle X-ray scattering (SAXS/WAXS). Both protein-protein interaction and various crystalline and liquid-crystalline phases of excipients are monitored. SANS and SAXS/WAXS represent valuable orthogonal tools to study mechanisms of protein (de)stabilization during freeze-thaw and freeze-drying.

BEPO® in situ implant forming (ISIF) technology is based on the association of amphiphilic block poly (lactic acid) (PLA) - poly (ethylene glycol) (PEG) copolymers and provides a unique combination for ease of administration, stability of fragile molecules and in vivo control of the drug release rate and duration of action after injection. This talk will provide insight into the many advantages of this new, highly versatile, long-acting injectable formulation technology and present the promising results obtained for the controlled delivery of a small, bispecific short half-life antibody for immunotherapy in prostate cancer. The antibody remains functional and active through formulation and release processes and demonstrates high anti-tumor activity in animal models, while improving subcutaneous bioavailability and half-life of the biomolecule.