Fast and accurate methods for functionally evaluating and characterizing the stability of bispecific antibodies (bsAb) are necessary during both discovery and development stages. Here, we describe high throughput no wash homogenous immunoassays using two different technologies to evaluate the binding activity and to show if both sites on the bsAb can bind the target proteins. We also developed a homogeneous multiplex assay to measure the two separate binding events simultaneously.

CAR T drug products generally use commercial formulation media with complex, undisclosed stoichiometry of ingredients. The proprietary nature of the commercial formulation limits complete understanding of the impact of formulation excipients on product quality, safety and efficacy. In this talk, we present our formulation efforts to develop defined formulations for T and NK cells and also discuss the ability of  formulation excipients to increase the product quality and recovery administration.

Recombinant adeno-associated virus (rAAV) for gene therapy has a size distribution due to the presence of empty and intermediate rAAV particles and aggregates. Though the influence of such particles on the efficacy and safety is under investigation, reliable and proper assessments of rAAV size distribution are highly preferable. In this talk, I'll introduce currently available methods for rAAV size distribution analysis and advantageous point of each method including analytical ultracentrifugation.

A new technology called LassoGraft Technology® was developed by combining a successful macrocyclic discovery platform system (RaPID system) and a structure-based protein design effort. We found that the pharmacophore sequences of a RaPID-derived peptides can be implanted to a surface-exposed loop of recombinant proteins without losing the function of both the grafted peptide and the scaffold protein. By this method, any chosen proteins could be endowed with binding capability toward various receptors, allowing us to quickly formulate bi-, tri-, and even tetra-specific binder molecules.

Monoclonal antibodies and therapeutic proteins for vaccines require extensive stability characterization during their development phase to improve their probability of successful treatment. We will discuss several case studies that demonstrate how the robust intrinsic fluorescence (nanoDSF) and dynamic light scattering (DLS) data obtained from NanoTemper’s Prometheus instruments helped many groups find better formulations and storage buffers to ultimately expedite the lab to clinic pipeline.

The use of kD and B22 in predicting the PPI in concentrated mAb formulations was evaluated through comparison with S(q)eff extracted from SAXS/SANS measurements. The disagreements between PPI determined from dilute (kD/B22) and concentrated solutions (S(q)eff) highlight the necessities of performing measurements directly from concentrated mAb solutions. Also, the correlation between the measured and predicted viscosity results suggests a better understanding of the relationship between PPI and solution viscosity is needed so that more reliable predictions can be made from PPI information.

We implemented a powerful workflow for the determination of multiple PQAs in a single analysis in combination with NPD capability which enables simultaneous determination of purity and identity. Use of high-resolution mass detection is essential for unambiguous analysis of PQAs when using the retention time plus accurate mass-targeted approach. We investigate the implementation of high-resolution MAM for PQA determination at various stages of the biopharmaceutical manufacturing process.

The structural integrity of polysorbate (PS) is important for its function as a surfactant to protect proteins from aggregation. We have explored the role of light-induced, protein-derived radicals on the cis/trans isomerization of unsaturated fatty acids in PS80. A mechanistic study performed with a combination of N-acetyltryptophan amide and glutathione disulfide suggested the involvement of thiyl radicals, generated by photoinduced electron transfer from Trp to the disulfide, in cis/trans isomerization.

Adeno associated viruses (AAV) are gene-delivery vectors carrying a genetic payload inside a protein capsid comprised of three viral proteins (VP1/2/3). The viral coat proteins interact with various structural elements and target cell types within the body to which the gene of interest will be transported. Aspects of VP primary structure including post-translational modifications (PTMs) potentially impact AAV activity and the efficacy of gene delivery. 

Polysorbate 20 and 80 are essential excipients to stabilize biopharmaceutical formulations. However, polysorbate is prone to degradation induced by (enzymatic) hydrolysis and/or oxidation. An overview on degradation pathways and analytical tools for polysorbate (focus on LC-CAD and LC-MS) will be given. For LC-MS analysis, novel universal markers for oxidation will be presented, as well as a novel hypothesis on the role of micelles for polysorbate oxidation.

Polysorbate 80 is a commonly used excipient for multiple Sanofi biotherapeutics, including enzyme replacement therapies (ERTs), antibodies, antibody drug conjugates (ADCs), and gene therapies, to ensure their stability. A minimal concentration of PS80 is required to maintain its effectiveness for preventing aggregation, and unwanted degradation leads to a decrease of PS80 concentration and particle formation in drug substance and drug product.  Here we present several hyphenated chromatographic methods recently developed in analytical development for characterization of PS80, including HPLC-CAD, 2D HPLC-CAD, and UPLC-QDa. We demonstrate that these methods can be used to quantitatively and qualitatively determine the PS80 content and investigate the degradation pathways, to support product understanding and formulation development.