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A rotating disk shear device was used to study the effect of interfacial shear on the structural integrity of human monoclonal antibodies of IgG4 isotype. Factors associated with the solution conditions (pH, ionic strength, surfactant concentration, temperature) and the interface (surface roughness) were studied for their effect on the rate of IgG4 monomer loss under high shear conditions. The structural integrity of the IgG4 was probed after exposure to interfacial shear effects by SDS-PAGE, IEF, dynamic light scattering, and peptide mapping by LC-MS. This analysis revealed that the main denaturation pathway of IgG4 exposed to these effects was the formation of large insoluble aggregates. Soluble aggregation, breakdown in primary structure, and chemical modifications were not detected. The dominant factors found to affect the rate of IgG4 monomer loss under interfacial shear conditions were found to be pH and the nanometer-scale surface roughness associated with the solid-liquid interface. Interestingly, temperature was not found to be a significant factor in the range tested (15-45 degrees C). The addition of surfactant was found to have a significant stabilizing effect at concentrations up to 0.02% (w/v). Implications of these findings for the bioprocessing of this class of therapeutic protein are briefly discussed., (2009 American Institute of Chemical Engineers Biotechnol.)

The purpose of this study was to assess the stability of protein formulations using a device designed to generate defined, quantifiable levels of shear in the presence of a solid-liquid interface. The device, based on a rotating disk, produced shear strain rates of up to 3.4 x 10(4) s(-1) (at 250 rps) and was designed to exclude air-liquid interfaces and enable temperature to be controlled. Computational fluid dynamics (CFD) was used to study the fluid flow patterns within the device and to determine the shear strain rate (s(-1)) at a range of disk speeds. The device was then used to study the effect on a monoclonal IgG4 of high levels of shear at the solid-liquid interface. Monomeric antibody concentration and aggregation of the protein in solution were monitored by gel permeation HPLC and turbidity at 350 nm. High shear strain rates were found to cause significant levels of protein aggregation and precipitation with reduction of protein monomer following first-order kinetics. Monomer reduction rate was determined for a range of disk speeds and found to have a nonlinear relationship with shear strain rate, indicating the importance of identifying and minimizing such environments during processing.

Domain fragments of human serum albumin corresponding to domains 1 and 2 (D12) and domains 2 and 3 (D23) were expressed in yeast. The kinetics of warfarin binding to these fragments were investigated using stopped-flow fluorescence spectroscopy. Binding can be characterized by a two-step process, a rapid diffusion-controlled step and a slower rate-limiting step in which a stable drug-protein complex is formed. The equilibrium constant for step 1 is greater for both D12 and D23 than for albumin, probably as a result of reduced steric hindrance offered by the domain fragments. Binding step 2, thought to be the result of a conformational change as warfarin is accommodated by the protein, is faster for D12 and D23. Albumin and the domain fragments show an increased preference for the R enantiomer, but the preference is particularly enhanced for domain fragment D12. These preferences can largely be explained by the domains having different rates for step 2 of the binding process.

To determine an effective nutritional regimen for management of growth failure in infants with congenital heart disease and congestive heart failure, the authors studied 19 infants with cardiac anomalies who were not candidates for early corrective surgery. Patients were randomly assigned to one of three feeding groups: group 1 (n = 7) received continuous, 24-hour nasogastric alimentation; group 2 (n = 5) received overnight, 12-hour nasogastric infusions plus daytime oral feedings as tolerated; and group 3 (n = 7) received oral feedings alone. For all patients, commercial infant formula (cow's milk or soy protein) was supplemented to a calorie density of approximately 1 kcal/mL. During a 5.25 +/- 0.45 month study period, only group 1 infants achieved intakes greater than 140 kcal/kg per day (mean = 147 kcal). Serial anthropometric measurements demonstrated that only 24-hour infusions (group 1) were associated with significantly improved nutritional status, when assessed by z scores for weight (P less than .01) and length (P less than .05). Group 1 infants also showed marked increases in midarm muscle circumference and triceps and subscapular skinfold thicknesses (P less than .01, compared with groups 2 and 3). These data suggest that infants with congenital cardiac defects complicated by malnutrition manifest increased nutrient requirements for growth and weight gain. Continuous, 24-hour, nasogastric alimentation is a safe and effective method for achieving both increased nutrient intake and improved overall nutritional status in these infants.