AUC measurements of diffusion coefficients of monoclonal antibodies in the presence of human serum proteins.

The goal of this work is to develop a preclinical method for quantitative hydrodynamic and thermodynamic analysis of therapeutic proteins in crowded environments like human serum. The method utilizes tracer amounts of fluorescently labeled monoclonal antibodies and the Aviv AU-FDS optical system. We have performed sedimentation velocity experiments as a function of mAb, human serum albumin and human IgG concentration to extract self- and cross-term hydrodynamic nonideality effects. SV measurements are consistently complicated by weak mAb-mAb and mAb-IgG interactions (Wright et al. in Anal Biochem 550:72-83, 2018). In an attempt to explore different approaches we have investigated measurements of diffusion coefficients by traditional synthetic boundary experiments. Here we present a new technique incorporated into SEDANAL that can globally analyze the full time course of synthetic boundary experiments. This approach also utilizes F-mAb against a high concentration of unlabeled carrier protein (HSA or IgG). In principle both diffusion and sedimentation coefficient information can be extracted including hydrodynamic and thermodynamic nonideality. The method can be performed at a traditional low speed (5-7K rpm) or at high speeds. The high speed method can also be used to measure D and s for small molecules like fluorescein (often contaminants of F-HSA and F-mAb). The advantage of synthetic boundary over the standard sedimentation velocity method is that it allows for higher precision determination of diffusion coefficients. The concentration dependence of D can be corrected for hydrodynamic nonideality effects by plotting D * (1 + kijcj) vs total carrier concentration. The slope of the fitted data allows an alternate approach to determine self- and cross-term thermodynamic nonideality. This method can also explore cross-term diffusion coefficient effects. These results are compared to dynamic light scattering approaches which are limited to kD determinations for solutions of pure protein.

Characterization of therapeutic antibodies in the presence of human serum proteins by AU-FDS analytical ultracentrifugation.

The preclinical characterization of biopharmaceuticals seeks to determine the stability, state of aggregation, and interaction of the antibody/drug with other macromolecules in serum. Analytical ultracentrifugation is the best experimental method to understand these factors. Sedimentation velocity experiments using the AU-FDS system were performed in order to quantitatively characterize the nonideality of fluorescently labeled therapeutic antibodies in high concentrations of human serum proteins. The two most ubiquitous serum proteins are human serum albumin, HSA, and γ-globulins, predominantly IgG. Tracer experiments were done pairwise as a function of HSA, IgG, and therapeutic antibody concentration. The sedimentation coefficient for each fluorescently labeled component as a function of the concentration of the unlabeled component yields the hydrodynamic nonideality (ks). This generates a 3x3 matrix of ks values that describe the nonideality of each pairwise interaction. The ks matrix is validated by fitting both 2:1 mixtures of HSA (1-40 mg/ml) and IgG (0.5-20 mg/ml) as serum mimics, and human serum dilutions (10-100%). The data are well described by SEDANAL global fitting with the ks nonideality matrix. The ks values for antibodies are smaller than expected and appear to be masked by weak association. Global fitting to a ks and K2 model significantly improves the fits.

Folding and hydrodynamics of a DNA i-motif from the c-MYC promoter determined by fluorescent cytidine analogs.

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C(+) basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C(+) hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.