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. Therefore, understanding the behavior of flexible mAb molecules in acidic solution environments will benefit the development of stable mAb products. This study used small-angle X-ray scattering (SAXS) and complementary biophysical characterization techniques to investigate the conformational flexibility and protein-protein interactions (PPI) of a model mAb molecule under near-neutral and acidic conditions. The study also characterized the interactions between Fab and Fc fragments under the same buffer conditions to identify domain-domain interactions. The results suggest that solution pH significantly influences mAb flexibility and thus could help mAbs remain physically stable by maximizing local electrostatic repulsions when mAbs become crowded in solution. Under acidic buffer conditions, both Fab and Fc contribute to the repulsive PPI observed among the full mAb at a low ionic strength. However, as ionic strength increases, hydrophobic interactions lead to the self-association of Fc fragments and, subsequently, could affect the aggregation state of the mAb.

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. In this study, we used National Institute of Standards and Technology monoclonal antibody (NISTmAb) reference material as a model antibody protein to examine the physical stability and viscosity of concentrated formulations using a series of excipients, by varying pH, salt composition, and the presence of cosolutes including amino acids, sugars, and nonionic surfactants. Small angle X-ray scattering (SAXS) together with differential scanning calorimetry (DSC), dynamic light scattering (DLS), and viscosity measurements were used to obtain various experimental parameters to characterize excipient modulated PPI and bulk solution viscosities. In particular, a good correlation was found between SAXS and DLS/SLS results, suggesting that the use of DLS/SLS is valid for predicting the colloidal stability of NISTmAb in concentrated solutions. Moreover, further analysis of effective structure factor S(q)eff measured from SAXS enabled the dissection of net PPI into hydrodynamic forces due to excluded volume as well as any additional attractive or repulsive interactions with the presence of excipients. It was found that although no denaturation or aggregation of NISTmAb was observed and that the net PPI were repulsive, the use of ionic excipients such as pH and salts leads to increased short-range attraction, whereas the nonionic excipients including sugars, amino acids, and polysorbate surfactants lead to increased repulsive PPI with increasing protein concentration. Results obtained from viscosity measurements showed that the use of excipients can lead to increased solution viscosities at high protein concentrations. The use of S(q)eff, interaction parameter kD, and second virial coefficient B22 as predictors for solution viscosity was also evaluated by comparing the predicted results with the measured viscosities. Although B22 and S(q)eff appeared to be better predictors than kD, disagreement between the predicted and measured results suggests other factors apart from PPI contribute to the bulk rheological properties of concentrated protein solutions.

Characterization of the NISTmAb Reference Material using small-angle scattering and molecular simulation : Part II: Concentrated protein solutions.

Protein-protein interactions in monoclonal antibody solutions are important for the stability of a therapeutic drug and directly influence viscosity in concentrated protein solutions. This study describes the use of small-angle scattering to estimate protein-protein interactions at high concentrations of the IgG1 NISTmAb reference material and validate colloidal models for interacting molecules. In particular, we studied the colloidal stability of the NISTmAb at high protein concentrations and analyzed protein-protein interactions upon adding sodium chloride and its effect on viscosity. Isotropic colloidal models for interacting molecules were combined with an ensemble of atomistic structures from molecular simulation to account for the flexibility of the NISTmAb in solution. In histidine formulation buffer, net repulsive electrostatic interactions are important for the colloidal stability of the NISTmAb at high concentrations. Addition of sodium chloride increased the viscosity of the NISTmAb and decreased the colloidal stability due to charge screening of the repulsive interactions. The interactions at high concentrations (up to ~ 250 mg/mL) were consistent with those from light scattering at low concentrations (below ~ 20 mg/mL). However, in the presence of sodium chloride, the screening of charges was less pronounced with increasing protein concentration and the interactions approached those of the repulsive hard-sphere models. Additionally, we studied the NISTmAb under frozen conditions using in situ neutron scattering to analyze the crowded state as proteins are excluded from the water-rich phase. In the frozen samples, where protein concentration can reach hundreds of mg/mL in the protein-rich phase, sodium chloride did not affect the molecular spacing and crowding of the NISTmAb. Graphical Abstract Net repulsive interactions in concentrated NISTmAb solutions assessed by small-angle neutronscattering.

Advances in the measurement of protein mobility using laser Doppler electrophoresis - the diffusion barrier technique.

A new technique for the measurement of protein mobility using laser Doppler electrophoresis (LDE) is introduced and characterised. The diffusion barrier approach loads a tiny protein sample volume into a much larger volume of dispersant, which contains the electrodes; the LDE measurement is then recorded before the sample can diffuse to the electrodes. We demonstrate that sample volumes are reduced by up to two orders of magnitude to volumes typically associated with separation techniques (∼50 µL), no reduction in measurement sensitivity occurs, samples can be retrieved usefully intact, post-measurement and typical measurement times are of the order of minutes. Measurements of BSA mobility up to 75°C and 1 M buffer concentration and lysozyme at a concentration as low as 0.5 mg/mL are demonstrated using the technique with good agreement with literature values.

Evaluation of digital optical method to determine plume opacity during nighttime.

United States Environmental Protection Agency (USEPA) set opacity standards for visual emissions from industrial sources to protect ambient air quality. USEPA developed Method 9, which is a reference method to describe how plume opacity can be quantified by human observers during daytime conditions. However, it would be beneficial to determine plume opacity with digital still cameras (DSCs) to provide graphical records of the plume and its environment during visual emission evaluation and to be able to determine plume opacity with DSCs during nighttime conditions. Digital optical method (DOM) was developed to quantify plume opacity from photographs that were provided by a DSC during daytime. Past daytime field campaigns have demonstrated that DOM provided opacity readings that met Method 9 certification requirements. In this paper, the principles and methodology of DOM to quantify plume opacity during nighttime are described. Also, results are described from a nighttime field campaign that occurred at Springfield, IL. Opacity readings provided by DOM were compared with the opacity values obtained with the reference in-stack transmissometer of the smoke generator. The average opacity errors were 2.3-3.5% for contrast model of DOM for all levels of plume opacity. The average opacity errors were 2.0-7.6% for the transmission model of DOM for plumes with opacity 0-50%. These results are encouraging and indicate that DOM has the potential to quantify plume opacity during nighttime.

Field evaluation of digital optical method to quantify the visual opacity of plumes.

Visual Determination of the Opacity of Emissions from Stationary Sources (Method 9) is a reference method established by U.S. Environmental Protection Agency (EPA) to quantify plume opacity. However, Method 9 relies on observations from humans, which introduces subjectivity. In addition, it is expensive to teach and certify personnel to evaluate plume opacity on a semiannual basis. In this study, field tests were completed during a "smoke school" and a 4-month monitoring program of plumes emitted from stationary sources with a Method 9 qualified observer to evaluate the use of digital photography and two computer algorithms as an alternative to Method 9. This Digital Optical Method (DOM) improves objectivity, costs less to implement than Method 9, and provides archival photographic records of the plumes. Results from "smoke school" tests indicate that DOM passed six of eight tests when the sun was located in the 140 degrees sector behind one of the three cameras, with the individual opacity errors of 15% or less and average opacity errors of 7.5% or less. DOM also passed seven of the eight tests when the sun was located in the 216 degrees sector behind another camera. However, DOM passed only one of the eight tests when the sun was located in the 116 degrees sector in front of the third camera. Certification to read plume opacity by a "smoke reader" for 6 months requires that the "smoke reader" pass one of the smoke school tests during smoke school. The average opacity errors and percentage of observations with individual opacity errors above 15% for the results obtained with DOM were lower than those obtained by the smoke school trainees with the sun was located behind the camera, whereas they were higher than the smoke school trainee results with the sun located in front of the camera. In addition, the difference between plume opacity values obtained by DOM and a Method 9 qualified observer, as measured in the field for two industrial sources, were 2.2%. These encouraging results demonstrate that DOM is able to meet Method 9 requirements under a wide variety of field conditions and, therefore, has potential to be used as an alternative to Method 9.

Quantification of plume opacity by digital photography.

The United States Environmental Protection Agency (USEPA) developed Method 9 to describe how plume opacity can be quantified by humans. However, use of observations by humans introduces subjectivity, and is expensive due to semiannual certification requirements of the observers. The Digital Opacity Method (DOM) was developed to quantify plume opacity at lower cost, with improved objectivity, and to provide a digital record. Photographs of plumes were taken with a calibrated digital camera under specified conditions. Pixel values from those photographs were then interpreted to quantify the plume's opacity using a contrast model and a transmission model. The contrast model determines plume opacity based on pixel values that are related to the change in contrast between two backgrounds that are located behind and next to the plume. The transmission model determines the plume's opacity based on pixel values that are related to radiances from the plume and its background. DOM was field tested with a smoke generator. The individual and average opacity errors of DOM were within the USEPA Method 9 acceptable error limits for both field campaigns. Such results are encouraging and support the use of DOM as an alternative to Method 9.

Mesophase separation and probe dynamics in protein-polyelectrolyte coacervates.

Protein-polyelectrolyte coacervates are self-assembling macroscopically monophasic biomacromolecular fluids whose unique properties arise from transient heterogeneities. The structures of coacervates formed at different conditions of pH and ionic strength from poly(dimethyldiallylammonium chloride) and bovine serum albumin (BSA), were probed using fluorescence recovery after photobleaching. Measurements of self-diffusion in coacervates were carried out using fluorescein-tagged BSA, and similarly tagged Ficoll, a non-interacting branched polysaccharide with the same size as BSA. The results are best explained by temporal and spatial heterogeneities, also inferred from static light scattering and cryo-TEM, which indicate heterogeneous scattering centers of several hundred nm. Taken together with previous dynamic light scattering and rheology studies, the results are consistent with the presence of extensive dilute domains in which are embedded partially interconnected 50-700 nm dense domains. At short length scales, protein mobility is unobstructed by these clusters. At intermediate length scales, proteins are slowed down due to tortuosity effects within the blind alleys of the dense domains, and to adsorption at dense/dilute domain interfaces. Finally, at long length scales, obstructed diffusion is alleviated by the break-up of dense domains. These findings are discussed in terms of previously suggested models for protein-polyelectrolyte coacervates. Possible explanations for the origin of mesophase separation are offered.