Note that and bear opposite signs in NaI and NaSCN solutions. Our results demonstrate that and and that both and have the same sign (both Rucaparib (Camsylate) +or ?and values with opposite signs. diffusion interaction parameters and are determined from the first-order, concentration dependence of the Rucaparib (Camsylate) diffusion (=?and and and and have the same sign (either both +or Cand are often of Rucaparib (Camsylate) comparable magnitudes and can bear opposite signs. Therefore, concentration dependences of and need to be determined independently via orthogonal techniques. We demonstrate this with measurements provide an alternative high-throughput tool for predicting the colloidal stability of proteins and for screening studies to identify solution conditions that minimize protein aggregation. In support of our thesis, we present heat- and agitation-induced aggregation data for an IgG2 monoclonal antibody (mAb1) in different solutions along with the corresponding measurements as predictors of mAb1 aggregation. Materials and Methods Materials Hen-egg white lysozyme (HEWL; Cat. No. L7651) was obtained from Sigma (St. Louis, MO). Bulk drug lot of an IgG2 monoclonal antibody (mAb1) was received from the Amgen Rabbit Polyclonal to FZD6 Process Development group at a concentration of 70 mg/mL in 10 mM acetate with 9% (w/v) sucrose at pH 5.2 (A52Su). The formula molecular mass for mAb1 is 148 kDa and the pI is 8.8. All other chemicals used in the preparation of buffers were of analytical grade or better. Methods Sample preparation Second virial coefficient measurements for HEWL were conducted in solutions containing 10 mM acetate at pH 4.6 (A46) and 100C400 mM sodium chloride. First, a HEWL stock solution was prepared by mixing the lyophilized protein powder in a given acetate-NaCl solution to achieve a concentration of 60 mg/mL. The HEWL stock solution was subjected to further buffer exchange by passing it through an Illustra NAP-5 (GE Healthcare, Piscataway NJ) gel filtration column equilibrated with a given acetate-NaCl solution. The eluate was mixed with an appropriate volume of the acetate-NaCl solution to generate a series of HEWL samples ranging from 3 to 22 mg/mL. For mAb1, and and values for HEWL were measured by DLS and SV respectively, in 10 mM acetate, pH 4.6 (A46) buffer with varying concentrations ((Fig.?1 (Fig.?1 intercepts range from 123 to 125 and were calculated from the slopes and the intercepts. The results (Fig.?1 and were of comparable magnitudes. Both coefficients were positive at 100 mM NaCl and decreased with increasing salt concentration to negative values at 400 mM NaCl; decreased from 6.2 to??0.4 mL/gm and from 4.2 to ?5.7 mL/gm. At each NaCl concentration, values, and a sequence molecular mass of HEWL of 14.3 kDa (Fig.?1 and as a function of NaCl concentration. Note that and are of comparable magnitude and bear opposite signs at intermediate NaCl concentrations. ((Fig.?2 (Fig.?2 appeared to become independent of protein concentration whereas the plot still exhibited a positive slope. The and values (Fig.?2?and values were determined in the presence of Hofmeister sodium salts. Both parameters decreased with increasing chaotropic character of the anion in the order of CH3COO?(=?14.7 mL/gm,? =?8.7 mL/gm)? ? Cl?(13.7,? 3.0)? ?I?(9.9,? ???0.2)? ?SCN?(8.8,? ???2.1) (see Fig.?2 and in the presence of 0 mM and 5 mM NaCl, exceeds and in the presence of 50?mM Hofmeister, Na-anion salts. The control represents A5 buffer. Note that and bear opposite signs in NaI and NaSCN solutions. Our results demonstrate that and and that both and have the same sign (both +or ?and values with opposite signs. These solution conditions corresponded to assumption also failed for mAb1 under low-salt conditions. With 0, 5, and 10 mM NaCl, (36.9, 27.2, 22.8 mL/gm) was less than or comparable to (52.9, 28.1, 20.3 mL/gm) (Fig.?2 is essential. The need for independent measurement will especially hold for solution conditions wherein a relatively small change in the nature and extent of protein-protein interaction, and thus and Fig.?3 and with increasing turbidity or particulation propensity (Fig.?4, and and value (Fig.?5 and as a function of solution turbidity. Turbidity at the corresponding NaCl concentration calculated by interpolation of?the data represented in Fig.?3and plotted versus the rate of protein aggregation. Open in a separate window.