The impact of drug loading and distribution on higher order structure and physical stability of an interchain cysteine-based antibody drug conjugate (ADC) has been studied. An IgG1 mAb was conjugated with a cytotoxic auristatin payload following the reduction of interchain disulfides. The 2-D LC-MS analysis shows that there is a preference for certain isomers within the various drug to antibody ratios (DARs). The physical stability of the unconjugated monoclonal antibody, the ADC, and isolated conjugated species with specific DAR, were compared using calorimetric, thermal, chemical denaturation and molecular modeling techniques, as well as techniques to assess hydrophobicity. The DAR was determined to have a significant impact on the biophysical properties and stability of the ADC. The CH2 domain was significantly perturbed in the DAR6 species, which was attributable to quaternary structural changes as assessed by molecular modeling. At accelerated storage temperatures, the DAR6 rapidly forms higher molecular mass species, whereas the DAR2 and the unconjugated mAb were largely stable. Chemical denaturation study indicates that DAR6 may form multimers while DAR2 and DAR4 primarily exist in monomeric forms in solution at ambient conditions. The physical state differences were correlated with a dramatic increase in the hydrophobicity and a reduction in the surface tension of the DAR6 compared to lower DAR species. Molecular modeling of the various DAR species and their conformers demonstrates that the auristatin-based linker payload directly contributes to the hydrophobicity of the ADC molecule. Higher order structural characterization provides insight into the impact of conjugation on the conformational and colloidal factors that determine the physical stability of cysteine-based ADCs, with implications for process and formulation development.

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Abstract-The bioavailability of hydrophobic organic chemicals decreases with aging in soil because of sequestration. However, assessments of the risk of exposure to contaminated soils are usually dependent on either chemical concentrations, which are measured using vigorous extraction methods, or models that assume an equilibrium without considering the actual conditions. The objective of this research was to determine the availability and desorption kinetics of freshly added and aged naphthalene from a peat and a mineral soil; naphthalene was desorbed into solutions with pH levels that approximate those found in different gastric regions. Soil and peat samples were spiked with radiolabeled and unlabeled naphthalene at 2 and 20 g/g and were aged from 0 to 135 d. Desorption kinetics were determined using a simulated stomach solution (0.1 M NaCl, 0.1 M HCl, 0.01 M NH 4 Ac, pH ϭ 1.0) and a neutral solution (0.2 M NaCl, pH ϭ 6.7) that represented the pH of intestinal conditions and most soils. Peat sorbed much more naphthalene than did soil, and it allowed little desorption. Though both acidic and neutral extracting solutions could desorb naphthalene, little apparent effect of aging was observed in peat, whereas desorption from soil declined markedly with aging. In addition, the percentage of naphthalene that desorbed from soil was greater for the higher incubation concentration. The desorption of naphthalene from the peat and soil was higher into the neutral solution than into the gastric solution. These results suggest that aging, exposure conditions, concentration effect, and organic matter content should be taken into account in predictive models and risk assessments.

The bioavailability of hydrophobic organic chemicals decreases with aging in soil because of sequestration. However, assessments of the risk of exposure to contaminated soils are usually dependent on either chemical concentrations, which are measured using vigorous extraction methods, or models that assume an equilibrium without considering the actual conditions. The objective of this research was to determine the availability and desorption kinetics of freshly added and aged naphthalene from a peat and a mineral soil; naphthalene was desorbed into solutions with pH levels that approximate those found in different gastric regions. Soil and peat samples were spiked with radiolabeled and unlabeled naphthalene at 2 and 20 μg/g and were aged from 0 to 135 d. Desorption kinetics were determined using a simulated stomach solution (0.1 M NaCl, 0.1 M HCl, 0.01 M NH4Ac, pH = 1.0) and a neutral solution (0.2 M NaCl, pH = 6.7) that represented the pH of intestinal conditions and most soils. Peat sorbed much more naphthalene than did soil, and it allowed little desorption. Though both acidic and neutral extracting solutions could desorb naphthalene, little apparent effect of aging was observed in peat, whereas desorption from soil declined markedly with aging. In addition, the percentage of naphthalene that desorbed from soil was greater for the higher incubation concentration. The desorption of naphthalene from the peat and soil was higher into the neutral solution than into the gastric solution. These results suggest that aging, exposure conditions, concentration effect, and organic matter content should be taken into account in predictive models and risk assessments.

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