Understanding the Impacts of Molecular and Macromolecular Crowding Agents on Protein-Polymer Complex Coacervates.

Complex coacervation refers to the liquid-liquid phase separation (LLPS) process occurring between charged macromolecules. The study of complex coacervation is of great interest due to its implications in the formation of membraneless organelles (MLOs) in living cells. However, the impacts of the crowded intracellular environment on the behavior and interactions of biomolecules involved in MLO formation are not fully understood.

Role of Domain-Domain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt.

Monoclonal antibodies (mAbs) make up a major class of biotherapeutics with a wide range of clinical applications. Their physical stability can be affected by various environmental factors. For instance, an acidic pH can be encountered during different stages of the mAb manufacturing process, including purification and storage.

The Assembly Mechanism and Mesoscale Architecture of Protein-Polysaccharide Complexes Formed at the Solid-liquid Interface.

Protein-polysaccharide composite materials have generated much interest due to their potential use in medical science and biotechnology. A comprehensive understanding of the assembly mechanism and the mesoscale architecture is needed for fabricating protein-polysaccharide composite materials with desired properties. In this study, complex assemblies were built on silica surfaces through a layer-by-layer (LbL) approach using bovine beta-lactoglobulin variant A (βLgA) and pectin as model protein and polysaccharide, respectively.

Effects of Monovalent Salt on Protein-Protein Interactions of Dilute and Concentrated Monoclonal Antibody Formulations.

In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl () and monoclonal antibody (mAb) concentration () were assessed using the diffusion interaction parameter and second virial coefficient measured from solutions with low to moderate . The effective structure factor measured from concentrated mAb solutions using small-angle X-ray and neutron scattering (SAXS/SANS) was also used to characterize the PPI.

Studying Excipient Modulated Physical Stability and Viscosity of Monoclonal Antibody Formulations Using Small-Angle Scattering.

Excipients are substances that are added to therapeutic products to improve stability, bioavailability, and manufacturability. Undesirable protein-protein interactions (PPI) can lead to self-association and/or high solution viscosity in concentrated protein formulations that are typically greater than 50 mg/mL. Therefore, understanding the effects of excipients on nonspecific PPI is important for more efficient formulation development.

The so-called critical condition for polyelectrolyte-colloid complex formation.

Complexes formed between oppositely charged polyelectrolytes (PE's) and either biological or abiotic colloid particles play a central role in such remarkably diverse areas as enzyme immobilization, protein purification, growth factor delivery, personal care products, food formulations and as precursors to coacervates and multilayers. Unlike PE adsorption on oppositely charged planar surfaces-also driven by electrostatics-PE-colloid complexes are often equilibrium states exhibiting reversible formation at a well-defined "critical" colloid surface charge density. We consider how the experimentally observed breadth of this transition, for three polyelectrolyte-colloid systems, is broadened-compared to theoretical expectations-due to (1) colloid (protein) charge anisotropy, (2) colloid (micelle) polydispersity, and (3) colloid (micelle) instability.

Dilution induced coacervation in polyelectrolyte-micelle and polyelectrolyte-protein systems.

"Self-suppression", the instability of complex coacervates at high concentration, is well-known for polycation-polyanion systems, but the transient nature of those complexes impedes development of a convincing model. The stable polyelectrolyte-micelle complexes of the polycation poly(diallyldimethylammonium chloride) (PDADMAC) with mixed micelles of sodium dodecyl sulfate (SDS)/Triton X-100 (TX100); and the stable complexes of PDADMAC with bovine serum albumin (BSA) can be characterized and identified as coacervate precursors. We observe liquid-liquid phase separation upon isoionic dilution, a common facet of self-suppression.

Precipitate-Coacervate Transformation in Polyelectrolyte-Mixed Micelle Systems.

The polycation/anionic-nonionic mixed micelle, poly(diallyldimethylammonium chloride)-sodium dodecyl sulfate/Triton X-100 (PDADMAC-SDS/TX100), is a model polyelectrolyte-colloid system in that the micellar mole fraction of SDS (Y) controls the micelle surface charge density, thus modulating the polyelectrolyte-colloid interaction. The exquisite temperature dependence of this system provides an important additional variable, controlling both liquid-liquid (L-L) and liquid-solid (L-S) phase separation, both of which are driven by the entropy of small ion release. In order to elucidate these transitions, we applied high-precision turbidimetry (±0.

Sugar-coated proteins: the importance of degree of polymerisation of oligo-galacturonic acid on protein binding and aggregation.

We have simplified the structural heterogeneity of protein-polysaccharide binding by investigating protein binding to oligosaccharides. The interactions between bovine beta-lactoglobulin A (βLgA) and oligo-galacturonic acids (OGAs) with various numbers of sugar residues have been investigated with a range of biophysical techniques. We show that the βLgA-OGA interaction is critically dependent on the length of the oligosaccharide.

Structural mechanism of complex assemblies: characterisation of beta-lactoglobulin and pectin interactions.

Knowledge of how proteins and polysaccharides interact is the key to understanding encapsulation and emulsification in these composite systems and ultimately to understanding the structures of many biological network systems. As a model system we have studied β-lactoglobulin A (βLgA) interacting with pectins of various amounts and distribution patterns of charge. The studies were conducted at pH 4 at minimal ionic strength, where the βLgA and the pectins are oppositely charged, resulting in an electrostatic attraction to each other.