Monoclonal antibody fragment removal mediated by mixed mode resins.

Efforts to increase monoclonal antibody expression in cell culture can result in the presence of fragmented species requiring removal in downstream processing. Capto adhere, HEA Hypercel, and PPA Hypercel anion exchange/hydrophobic interaction mixed mode resins were evaluated for their fragment removal capabilities and found to separate large hinge IgG1 antibody fragment (LHF) from monomer. Removal of greater than 75% of LHF population occurred at pH 8 and low conductivity. The mechanism of fragment removal was investigated in two series of experiments. The first experimental series consisted of comparison to chromatographic behavior on corresponding single mode resins. Both single mode anion exchange and hydrophobic interaction resins failed to separate LHF. The second experimental series studied the impact of phase modifiers, ethylene glycol, urea, and arginine on the mixed mode mediated removal. The addition of ethylene glycol decreased LHF removal by half. Further decreases in LHF separation were seen upon incubation with urea and arginine. Therefore, it was discovered that the purification is the result of a mixed mode phenomena dominated by hydrophobic interaction and hydrogen bonding effects. The site of interaction between the LHF and mixed mode resin was determined by chemical labeling of lysine residues with sulfo-NHS acetate. The labeling identified the antibody hinge and light chain regions as mediating the fragment separation. Sequence analysis showed that under separation conditions, a hydrophobic proline patch and hydrogen bonding serine and threonine residues mediate the hinge interaction with the Capto adhere ligand. Additionally, a case study is presented detailing the optimization of fragment removal using Capto adhere resin to achieve purity and yield targets in a manufacturing facility. This study demonstrated that mixed mode resins can be readily integrated into commercial antibody platform processes when additional chromatographic abilities are required.

Receiver operating characteristic curve analysis of beach water quality indicator variables.

Receiver operating characteristic (ROC) curve analysis is a simple and effective means to compare the accuracies of indicator variables of bacterial beach water quality. The indicator variables examined in this study were previous day's Enterococcus density and antecedent rainfall at 24, 48, and 96 h. Daily Enterococcus densities and 15-min rainfall values were collected during a 5-year (1996 to 2000) study of four Boston Harbor beaches. The indicator variables were assessed for their ability to correctly classify water as suitable or unsuitable for swimming at a maximum threshold Enterococcus density of 104 CFU/100 ml. Sensitivity and specificity values were determined for each unique previous day's Enterococcus density and antecedent rainfall volume and used to construct ROC curves. The area under the ROC curve was used to compare the accuracies of the indicator variables. Twenty-four-hour antecedent rainfall classified elevated Enterococcus densities more accurately than previous day's Enterococcus density (P = 0.079). An empirically derived threshold for 48-h antecedent rainfall, corresponding to a sensitivity of 0.75, was determined from the 1996 to 2000 data and evaluated to ascertain if the threshold would produce a 0.75 sensitivity with independent water quality data collected in 2001 from the same beaches.