Biopharmaceuticals, such as monoclonal antibodies, are considered as life-saving drugs for autoimmune diseases, cancer and infectious diseases. However, biotherapeutics tend to undergo chemical degradation during various stages of manufacturing. The conditions of chemical degradation, along with the physical degradation pathways, have a direct influence on the overall stability, safety and efficacy of these therapeutics. While site-specific chemical changes have been well-explored and investigated using various analytical approaches, the resulting conformational and structural changes have not been much studied. Thus, we explored various biophysical techniques for assessing the influence of three representatives forced degradation conditions viz. oxidation, deamidation, and glycation, in a model therapeutic trastuzumab biosimilar. The site-specific modifications caused by these stress conditions were analysed using high resolution mass spectrometry. While their thermodynamic and conformational consequences were investigated by using differential scanning colorimetry (Nano-DSC), circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), and dynamic light scattering (DLS). The investigated stress conditions resulted in reduced thermodynamic stability of mAb, as confirmed using Nano-DSC. Secondary structure analysis performed with CD spectroscopy indicated detectable structural alterations in the beta sheets of stressed samples. DLS and SV-AUC studies demonstrated an enhanced level of aggregation and fragmentation in presence of all stress conditions. Thus, the biophysical analytical toolkits, when used simultaneously, could offer deeper insights into the subtle conformational changes that result from site-specific chemical modifications in mAbs. Hence, these analytical approaches may serve as significant additions to the battery of techniques used for forced degradation analysis of biopharmaceuticals.

Terbutaline sulphate (TS) is a selective short-acting β2 adrenoceptor agonist used for asthma treatment. The pharmacological activity of TS depends on its binding to the transmembrane protein, β2 adrenoceptor. Thus, the interactions of this drug with biological membranes are expected, affecting its pharmacological activity. Using in vitro models to study the interaction of TS with biological membranes can provide important information about the activity of the drug. Here, liposomes with different lipid compositions were used as biomimetic models of cell membranes to evaluate the effect of composition, complexity, and physical state of membranes on TS-membrane interactions. For that, liposomes containing dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and liposomes containing DMPC and cholesterol (CHOL) were prepared. For the study of TS-membrane interactions, the TS lipophilicity was evaluated in terms of i) partition coefficient; ii) the preferential location of the drug within the membrane; iii) and the effect of TS on the membrane fluidity. The obtained data suggest that TS has an affinity for the lipid membrane, partitioning from the aqueous to the lipid phase. The affinity was dependent on the liposomes\' compositions, showing a greater affinity for DMPC membranes than for DMPC:CHOL model. Dynamic light scattering (DLS) results revealed that this is due to the rigidizing effect caused by CHOL molecules. These findings provide valuable insights in the understanding of the complex interaction of TS with biomembrane models as well as the relevance of lipid compositions and membrane structure in such interactions, which may be related to its pharmacological activity and side effects.

Monoclonal antibodies (mAbs) are the modalities of choice for immunotherapy. This class of products are known to exhibit considerable heterogeneity with respect to size, aggregation states, and charge. This makes it challenging for biopharmaceutical manufacturers, in particular biosimilar producers, to maintain consistency in product quality. In order to fingerprint these biotherapeutic products, multiple, high-resolution analytical tools are used to characterize the numerous critical quality attributes. Recently, there has been growing interest in enhancing adaptability of 1D and 2D NMR platforms for characterization of higher order structure with emphasis on 1D 1H, 2D 1H-15N and 1H-13C NMR experiments at natural abundance. In this communication, we report the applicability of 2D-DOSY NMR for quantification of colloidal diffusivities, namely diffusion coefficient (and associated hydrodynamic radius) for monomeric IgG1 mAb formulations at physiological conditions. Similarity assessment has been performed for trastuzumab originator (multiple batches) and marketed biosimilars to showcase the applicability of this approach. While dynamic light scattering measurements are known to be sensitive to presence of larger particles with a concentration dependence for estimation of colloidal diffusivities, size estimated by NMR experiments was found to be more in agreement with the computational hydrodynamic size estimations derived from the published crystal structures of intact mAb at formulation concentration.

Integral membrane proteins are important drug targets that are critical in supporting many biological processes. Despite that, the study of their structure-function relationships remains a major goal in structural biology, yet progress has been hampered by inherent challenges in the production for stable and homogeneous protein samples. Dynamic light scattering provides a straightforward probe of protein quality in solution, particularly in relation to stability and aggregation. However, the necessity to use large amounts of sample and the low-throughput nature of the analysis remain as major bottlenecks of the technique.Here, we present a protocol for dynamic light scattering measurements that are executed in a fully automated fashion for low-volume samples, in situ. The protocol offers a generic pre-screening method for downstream structural studies of biomolecules using higher-resolution approaches such as X-ray crystallography, electron microscopy, small-angle X-ray scattering, and NMR .

The effect of Ca2+ and alginate on the stability of CeO2 nanoparticles (NPs) was investigated using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA); the two methods were then compared. DLS showed rapid aggregation of CeO2 NPs in 8 mM Ca2+ solution; however, NTA showed that some primary aggregates (200-400 nm) still remained-a result that was obscured in DLS measurements. Aggregation of alginate molecules was also studied using DLS and NTA, where NTA particle concentration and video provided additional information on the alginate aggregation progress. Finally, DLS showed that in the presence of alginate, the aggregation rate and size of CeO2 NPs increased. NTA intensity measurements provided insight into a heteroaggregation and bridging mechanism. NTA particle concentration and video also showed CeO2 NPs were linked by alginate gel in high Ca2+ concentration (>4 mM). the DLS and NTA had different advantages in measuring particle size. DLS could better study the initial aggregation stage and large aggregates, while NTA could better detect small aggregates. NTA also measured particle number concentration, individual intensity, and particle motion video, which provided additional insight. Combining these two methods could help us to better understand the behavior and fate of NPs in water.

The toxicity of carbon nanotubes (CNTs) is closely related to their physico-chemical characteristics as well as the physico-chemical parameters of the media where CNTs are dispersed. In a climate change scenario, changes in seawater salinity are becoming a topic of concern particularly in estuarine and coastal areas. Nevertheless, to our knowledge no information is available on how salinity shifts may alter the sensitivity (in terms of biochemical responses) of bivalves when exposed to different CNTs. For this reason, a laboratory experiment was performed exposing the Manila clam Ruditapes philippinarum, one of the most dominant bivalves of the estuarine and coastal lagoon environments, for 28 days to unfunctionalized multi-walled carbon nanotube MWCNTs (Nf-MWCNTs) and carboxylated MWCNTs (f-MWCNTs), maintained at control salinity (28) and low salinity 21. Concentration-dependent toxicity was demonstrated in individuals exposed to both MWCNT materials and under both salinities, generating alterations of energy reserves and metabolism, oxidative status and neurotoxicity compared to non-contaminated clams. Moreover, our results showed greater toxic impacts induced in clams exposed to f-MWCNTs compared to Nf-MWCNTs. In the present study it was also demonstrated how salinity shifts altered the toxicity of both MWCNT materials as well as the sensitivity of R. philippinarum exposed to these contaminates in terms of clam metabolism, oxidative status and neurotoxicity.

The major challenges in biophysical characterization of human protein-carbohydrate interactions are obtaining monodispersed preparations of human proteins that are often post-translationally modified and lack of detection of carbohydrates by traditional detection systems. Light scattering (dynamic and static) techniques offer detection of biomolecules and their complexes based on their size and shape, and do not rely on chromophore groups (such as aromatic amino acid sidechains). In this study, we utilized dynamic light scattering, analytical ultracentrifugation and small-angle X-ray scattering techniques to investigate the solution properties of a complex resulting from the interaction between a 15 kDa heparin preparation and miniagrin, a miniaturized version of agrin. Results from dynamic light scattering, sedimentation equilibrium, and sedimentation velocity experiments signify the formation of a monodisperse complex with 1:1 stoichiometry, and low-resolution structures derived from the small-angle X-ray scattering measurements implicate an extended conformation for a side-by-side miniagrin‒heparin complex.

Gaining insight into intermolecular interactions between multiple species is possible at an atomic level by looking at different parameters using different NMR techniques. In the specific case of the astringency sensation, in which at least three molecular species are involved, different NMR techniques combined with dynamic light scattering and molecular modeling contribute to decipher the role of each component in the interaction mode and to assess the thermodynamic parameters governing this complex interaction. The binding process between a saliva peptide, a polyphenol, and polysaccharides was monitored by following 1H chemical shift variations, changes in NMR peak areas, and size of the formed complex. These NMR experiments deliver a complete picture of the association pathway, assessed by dynamic light scattering and molecular dynamics simulations: all of the data collected converge toward a comprehensive mode of interaction in which sugars indirectly play a role in astringency by sequestering part of the polyphenols, reducing their effective concentration to bind saliva proteins.