Glucagon-like peptide-1 (GLP-1) analogs (GLPA) exist primarily as micelle-like associations when free in aqueous solution. The results here indicate that anisotropic electrostatic interactions play a central role in the instability and aggregation, which appear to arise predominantly from multipole, orientation-dependent electrostatics: net dipole moment and charge in GLPA affect attraction and repulsion, features not captured by mean-field, spherically symmetric approaches. Whereas hydrophobicity drives the micelle formation, electrolyte-dependent aggregation appears to be governed by these electrostatic interactions. Increasing ionic strength screens the Coulomb repulsion between micelles, reducing the electrostatic stabilization barrier and allowing orientation-dependent multipole attractions to promote aggregation. This behavior contrasts with globular protein aggregation, typically dominated by the classical hydrophobic effect. Spectroscopically monitoring forward and reverse dialysis with a custom device, stability of liraglutide and semaglutide samples was mapped vs electrolyte (NaCl) and denaturant concentrations (guanidinium chloride, Gdn). Gdn+ cation binding to negatively charged amino acids reduces net charge and dramatically destabilizes GLPA. In contrast, simple cations, such as Na+, merely screen electrostatically, and no binding term is required to explain the data. Aggregation caused by both NaCl and Gdn+ was semi-irreversible. An electrostatic model, based on attractive, screened monopole-dipole, dipole–dipole, and repulsive monopole-monopole interactions was developed to interpret results. This model may be applicable to other peptides and biologics with asymmetric and patchy charge distributions, and dipole moments. The work establishes a stability-testing paradigm that may accelerate development of these biologics, as well as other therapeutic peptides.

The second virial coefficient A2 (B22) and diffusion interaction parameter kD are used to assess colloidal stability and the risk of undesirable high viscosity behavior of monoclonal antibodies and other biologics at high concentration. These processes have conventionally been conducted through discrete serial dilutions or multiple measurements at various concentrations in a well-plate. Here, a single trajectory experiment is performed via an automatic cuvette-based method to produce continuous dilution of the starting concentration of the material, that is, automatic continuous dilution (ACD), while gathering static light scattering and/or dynamic light scattering data during the process. The sample volume in the cuvette is kept constant during the process, and the measurements are made without disturbance or motion of the spatial orientation of the incident laser beam and cuvette. This single-sweep approach yields weight average molar mass Mw, A2 (B22), apparent hydrodynamic diameter DH,app, kD, and, where measurable, A3. The method reduces sample consumption and eliminates artifacts due to plate motion, thereby increasing reliability and reproducibility. The sample can be recovered after the measurements, as the final withdrawal reservoir volume is only about 5× the cuvette volume. Examples include two globular proteins, where A2 is positive and kD small, which are expected for globular proteins resembling spheres. For natural polyelectrolytes, A2 and an “effective kD” are both strongly positive for sodium hyaluronate and A2 is strongly positive. In contrast, effective kD is strongly negative for alginate. This reflects the effects of segment overlap and interactions for random coil polymers, which makes the effective kD dramatically different than for kD of spheres. This concise method may prove useful and economical for assessing formulation stability and providing an indication of high viscosity behavior. ACD can also be used directly with any instrument accepting 1 cm cuvettes to determine the impact of dilution on association states and other concentration-dependent phenomena.

A recently introduced device and method for spectroscopically monitoring the behavior of macromolecules during chemical dialysis in a cuvette were applied to several synthetic and biomacromolecules, including biologic drugs. Via a dialysis membrane, the device partitions the cuvette contents into Fluid 1, containing the macromolecules, from Fluid 2 containing the dialysate, which is circulated through an external reservoir. When dialysis is carried out against electrolyte solutions, including ionic excipients, it is difficult to precisely measure the time-changing concentration of the electrolyte in the macromolecular solution in the cuvette, because conventional conductivity probes are too large to fit into a cuvette, together with the inserted dialysis device. Because of this, measurements of electrolyte concentration in Fluid 1 until now have been made by measuring the conductivity in the external Fluid 2 reservoir and inferring the conductivity in Fluid 1 via the ratio of volumes of Fluid 2 to Fluid 1 (typically 40x to 400x). This method is imprecise, since conductivity variations in Fluid 2 are small. Now, a recently available miniature (∼1 mm) ion selective field effect transistor (ISFET) for both conductivity and pH has been adapted to the dialysis system and fits into the cuvette. This gives an immense improvement in data precision and opens the door for the method being able to scan the effect on macromolecules over a wide range of electrolyte concentrations, which can aid in optimizing biologic formulations. A single dialysis experiment of this type may be functionally equivalent to high throughput testing of multiple discrete samples. Results are presented here for an integrated cuvette device containing (i) dialysis membrane, (ii) non-contact stirring impeller,(iii) dialysate circulation channels, and (iv) the miniature ISFET probe. It can be used in any instrument accepting 1 cm square cuvettes, without any modifications to the instrument or its native data gathering software.

The seemingly unrelated massive aggregation of free mRNA under certain solution conditions and the well-known autohydrolysis of mRNA are actually both closely linked through its secondary and possibly tertiary structure (s/t). This hypothesis posits that s/t partially stabilizes mRNA against both autohydrolysis and massive aggregation. Destabilization of s/t via denaturant guanidine-HCl (Gd), or temperature, has profound effects on both aggregation rates and final degree of autohydrolysis. These denaturant effects occurred for a variety of mRNA, ranging from 700 to 3000 nucleotides but showed very different quantitative behavior among themselves, suggesting some of the methods presented here might help characterize mRNA stability and robustness. Light scattering monitoring during dialysis of mRNA against Gd revealed an “aggregation window”, over 0.5–3 M Gd, whereas dialyzing against a nondenaturing electrolyte (NaCl) showed semireversible monotonic increase of aggregation up to 4 M. Massive aggregation of mRNA in solutions with monovalent ions and in denaturing solutions has not been previously reported. A phenomenological model involving intermolecular electrostatic repulsion and attractions due chiefly to π–π stacking helps interpret the various phenomena.

Monitoring membrane-mediated dialysis in real time with static and dynamic light scattering revealed distinctive differences, including reversibility/irreversibility, in the effects of ionic strength (NaCl) and the denaturant guanidine-HCl (Gd) on a synthetic polyelectrolyte and several types of biomacromolecules: protein, polysaccharide, and polyampholyte. Dialysis cycles against aqueous NaCl and Gd, and reverse back to the original aqueous solution, were monitored. The behavior of Na-polystyrenesulfonate was reversible and yielded a detailed polymer physics description. The biomacromolecules additionally showed hydrogen-bonding/hydrophobic (HP) interactions. An interpretive model was developed that considers the interplay among polyelectrolyte, polyampholyte, and HP potential energies in determining the different associative, aggregative, and dissociative behaviors. NaCl isolated purely electrostatic effects, whereas Gd combined electrostatic and HP effects. Some macromolecules showed partially reversible behavior, and others were completely irreversible. The dialysis monitoring method should prove useful for investigating fundamental macromolecular and colloid properties and for drug formulation and stability optimization.