Impact of short range hydrophobic interactions and long range electrostatic forces on the aggregation kinetics of a monoclonal antibody and a dual-variable domain immunoglobulin at low and high concentrations.

The purpose of this work was to determine the nature of long and short-range forces governing protein aggregation kinetics at low and high concentrations for a monoclonal antibody (IgG1) and a dual-variable-domain immunoglobulin (DVD-Ig). Protein-protein interactions (PPI) were studied under dilute conditions by utilizing the methods of static (B(22)) and dynamic light scattering (k(D)). PPI in solutions containing minimal ionic strengths were characterized to get detailed insights into the impact of ionic strength on aggregation. Microcalorimetry and susceptibility to denature at air-liquid interface were used to assess the tertiary structure and quiescent stability studies were conducted to study aggregation characteristics. Results for IgG1 showed that electrostatic interactions governed protein aggregation kinetics both under dilute and concentrated conditions (i.e., 5 mg/mL and 150 mg/mL). For DVD-Ig molecules, on the other hand, although electrostatic interactions governed protein aggregation under dilute conditions, hydrophobic forces clearly determined the kinetics at high concentrations. This manuscript shows for the first time that short-range hydrophobic interactions can outweigh electrostatic forces and play an important role in determining protein aggregation at high concentrations. Additionally, results show that although higher-order virial coefficients become significant under low ionic strength conditions, removal of added charges may be used to enhance the aggregation stability of dilute protein formulations.

The use of asymmetrical flow field-flow fractionation in pharmaceutics and biopharmaceutics.

Field-flow fractionation (FFF) is a family of flexible analytical fractionating techniques which have the advantage that the separation of analytes is achieved, solely through the interaction of the sample with an external, perpendicular physical field, rather than by the interaction with a stationary phase. The rapid progress in pharmaceutical biotechnology goes along with an increasing demand in potent, high-efficient analytical methods. Thus, FFF techniques are gaining increasing attention for their ability to separate and characterize populations of polymers, colloids and particles of up to about 100 microm in size. It is the intention of this review to provide an overview on common FFF techniques, to summarize inherent advantages and limitations and to introduce both established and challenging applications in the (bio)pharmaceutical field. Thereby, asymmetrical flow FFF is addressed predominantly, since it is the most versatile applicable FFF technique.

Asymmetrical flow field-flow fractionation and multiangle light scattering for analysis of gelatin nanoparticle drug carrier systems.

The physicochemical properties of nanosized colloidal drug carrier systems are of great influence on drug efficacy. Consequently, a broad spectrum of analytical techniques is applied for comprehensive drug carrier characterization. It is the primary objective of this paper to present asymmetrical flow field-flow fractionation (AF4), coupled online with multiangle light scattering detection, for the characterization of gelatin nanoparticles. Size and size distribution of drug-loaded and unloaded nanoparticles were determined, and data were correlated with results of state-of-the-art methods, such as scanning electron microscopy and photon correlation spectroscopy. Moreover, the AF4 fractionation of gelatin nanoparticulate carriers from a protein model drug is demonstrated for the first time, proposing a feasible way to assess the amount of loaded drug in situ without sample preparation. This hypothesis was set into practice by monitoring the drug loading of nanoparticles with oligonucleotide payloads. In this realm, various fractions of gelatin bulk material were analyzed via AF4 and size-exclusion high-pressure liquid chromatography. Mass distributions and high-molecular-weight fraction ratios of the gelatin samples varied, depending on the separation method applied. In general, the AF4 method demonstrated the ability to comprehensively characterize polymeric gelatin bulk material as well as drug-loaded and unloaded nanoparticles in terms of size, size distribution, molecular weight, and loading efficiency.