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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.

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)effmeasured 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 B22as predictors for solution viscosity was also evaluated by comparing the predicted results with the measured viscosities. Although B22and S(q)effappeared 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.

AbstractVisual 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° 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° sector behind another camera. However, DOM passed only one of the eight tests when the sun was located in the 116° 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.

Negatively charged polyelectrolytes such as carboxymethylcellulose, pectin, and alginate are commonly present in food products. These polyelectrolytes serve a variety of functions such as controlling viscosity and stabilizing emulsions. Proteins are also present in many food formulations. Because of their high charge density, polyelectrolytes can be expected to interact with these proteins. Hence, an understanding of the parameters controlling protein‐polyelectrolyte interactions is useful.

Complexation of alcohol dehydrogenase (ADH) and trypsin with poly(diallyldimethyl-ammonium chloride) (PDADMAC) in dilute electrolyte solution was studied by turbidimetric titration, quasi-elastic light scattering (QELS), and electrophoretic light scattering (ELS). Both QELS and turbidimetric titration show that PDADMAC forms complexes with ADH and trypsin in 0.01M NaCl solution at pH ≥ 6.8 and pH ≥ 9.2, respectively. These complexes take the form of stable coacervates in 0.01M, pH 11.0, phosphate buffer solution. QELS shows sizes of 400 and 315 nm for the coacervates of ADH-PDMDAAC and trypsin-PDMDAAC, respectively, while ELS reveals that these coacervates carry a net positive charge. Activity measurements show that both ADH and trypsin are enzymatically active in their coacervated states. Complexation of trypsin and PDADMAC was also studied by fluorescence in 0.01M, pH 11.0, phosphate buffer, and the protein emission was found to be quenched by complexation. The fluorescence quenching data show that trypsin retains its three-dimensional structure in the complex. These and other results are consistent with the quenching of the two tryptophans on the protein surface, but not the interior ones.© 1997 John Wiley & Sons, Inc.