Small-angle neutron scattering (SANS) can be used to probe the solution structure of two protein therapeutics (monoclonal antibodies 1 and 2 (MAb1 and MAb2)) and their protein-protein interaction (PPI) at high concentrations. answer viscosity. Introduction Therapeutic monoclonal antibodies (MAbs) have been found to be highly effective brokers in the treatment of immunological and allergic disorders, as well as malignant growth (1C5), with a high level of success due to PSACH their structural specificity and low toxicity in contrast to many traditional small-molecule drug options (6,7). During the last several decades, more than 20 MAbs have been ENMD-2076 approved by the FDA for clinical use (7), and several hundred are currently in development (8). Because of their success and effectiveness, MAbs are one of the fastest growing therapeutic agents on the market (6). Currently, many therapeutic MAb products are often administered in high doses, typically in the hundreds of milligrams (9,10), by an intravenous route at dilute conditions. The pharmaceutical industry is now proposing the use of subcutaneous (SC) injection delivery methods for some MAbs due to the convenience (10) and reduced number/frequency of administrations (9). However, SC delivery imposes a constraint on the volume of MAb answer that can be injected (1.5?mL) (10). Thus, the high concentration of MAbs (>50?mg/mL) often required to attain efficacious dosages sometimes prospects to nonideal answer behavior, such as a large answer viscosity (11,12), which limits the use of SC delivery (13). Recent experimental results suggest that the increased viscosity (14C16) of concentrated MAb protein solutions is related to the reversible or dissociable aggregates/clusters that are dictated by the protein-protein connections (PPIs) (13,14,16C18). Understanding the type of the PPIs being a function of proteins focus and formulation method is thus essential and could result in ENMD-2076 more logical primary-structure design strategies and/or collection of effective excipient circumstances for the reduced amount of high-concentration MAb alternative viscosities. Many biophysical techniques, such as for example powerful and static light scattering (12,19), molecular modeling (20), zeta potential (10,12), and rheological strategies, have been utilized to extract information regarding PPIs between MAbs in alternative (10,12,14,18,21C28). Right here, we concentrate on two MAbs (MAb1 and MAb2) which have been broadly investigated, as both of these MAbs in alternative show significantly different viscosity reactions like a function of concentration despite the small difference in their main structure (10,13,14,17C19,22,23,27C30). In particular, solutions of MAb1 show a very large viscosity increase with increasing protein concentration compared to solutions of MAb2. Based on sedimentation equilibrium analysis of different concentrations of protein solutions, Liu et?al. proposed the electrostatic charge connection between MAb1 molecules may be responsible for the large increase in viscosity like a function of concentration (14). Kanai et?al. analyzed ENMD-2076 Fab and Fc fragments inside a MAb protein and observed that Fab-Fab relationships, in contrast to the Fab-Fc or Fc-Fc fragment relationships, resulted in an increase in viscosity (18). Using numerous bioanalytical techniques, it was confirmed the addition of salt decreases the viscosity in MAb1 as a result of the screening effects (14). A recent calculation of the electrostatic surface potential of MAb1 and MAb2 suggests that the nonuniform charge distribution may impact the PPI significantly (30). Therefore, ENMD-2076 direct measurement of the PPI becomes important for understanding the PPI at large protein concentrations. It is noted the similar ionic strength dependence of viscosity has been observed in additional MAbs as well (12). Therefore, despite variations in main structure between some MAbs, ENMD-2076 a general understanding of one MAb system will become helpful for understanding additional MAb systems. Scattering methods have been shown to be successful in probing PPIs in solutions of globular proteins (31C35). Here we use small-angle neutron scattering (SANS) to study MAb PPIs in concentrated solutions relevant for SC delivery. SANS probes size scales commensurate with the proteins size and standard interparticle range and, as such, provides a detailed measurement of the conformation and spatial set up of the macromolecules in answer. As a result, SANS measurements provide information about the conformation of individual proteins in answer, as well as the PPIs of concentrated samples. Most studies on MAb solutions using small-angle scattering have focused on the study of the conformation of individual proteins at dilute concentrations (36,37). Here, we directly probe PPIs at large protein concentrations at numerous conditions and explore the possible relation between.