== (A) Viscosity-concentration profiles of three monoclonal antibodies of the IgG1 isotype in a buffered solution at pH 5.5 and 200 mM arginine-HCl. to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 Nortadalafil isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities Rabbit Polyclonal to Smad1 increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high unfavorable net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery. Treatment of certain chronic ailments, e.g., rheumatoid arthritis, using mAb-based therapies require delivery via the s.c. route for patients at-home use, self-administration, and compliance (1). To deliver several hundred milligrams of the active drug in a small volume (1 mL) into the s.c. space, a liquid formulation containing high concentrations of mAb is required (1,2). Therefore it is essential that the lead clinical mAb candidate meets the following criteria: (i) an injectable solution of low viscosity (higher viscosity solutions are difficult to manufacture and administer, and could be painful to inject) (2); (ii) minimal chemical/physical degradation in solution such that the efficacy and safety is maintained; and (iii) a normal in vivo clearance profile to avoid multiple injections and/or more frequent dosing (3,4). Effort must be spent early on to screen for lead mAb candidates, which exhibit desirable physicochemical and biological attributes. Inclusion of computational in silico tools along with experimental approaches will enable rapid screening Nortadalafil of a larger number of mAb candidates (57). In this report, we describe in silico screening approaches that aid in selection of mAb candidates with respect to viscosity, in vivo clearance in Cynomolgus monkeys (a relevant preclinical model for human clearance), Trp oxidation, and Asp isomerization. We show that using the optimal parameters extracted from sequence and/or structure including molecular dynamic simulations, the desirable attributes of mAbs can be predicted to enable lead candidate selection. == Viscosity == mAbs, differing largely in the complementarity determining region (CDR) sequence or in the Fv domain, exhibit a variety of viscosity-concentration profiles under similar conditions of shear rate (Fig. 1A). Recognizing that for mAbs of similar Nortadalafil isotype (IgG1 in the present case), the variable domain Fv (and the CDRs within) presumably plays a critical role in defining intermolecular interactions leading to differences in viscosity (8,9), we set out to determine what parameters can be extracted to capture the contributing hydrophobic and electrostatic elements (10,11). It has been postulated recently that such interactions may produce molecular entanglements, leading to increased viscosity (12). We focused on sequence only because this provided the simplest means of data generation and analysis. However, we note that any of the parameters discussed below and calculated from the sequence can be readily calculated from structure as well (Figs. S1andS2). As shown (Figs. S1andS2), the sequence-based calculations correlate with structure-based calculations. == Fig. 1. == (A) Viscosity-concentration profiles of three monoclonal antibodies of the IgG1 isotype in a buffered solution at pH 5.5 and 200 mM arginine-HCl. The points represent the experimental data. The lines are used as a guide to the eye and were generated using the equation of the exponential formy=a+becx, whereyis viscosity,xis protein concentration, anda, b, andcare fitting parameters. Correlation of log viscosity with the calculated sequence-based parameters of (B) charge at pH 5.5, (C) Fv charge symmetry parameter (FvCSP) at pH 5.5, and (D) Fv hydrophobicity index (HI). The viscosity values were obtained in buffered solution at pH 5.5 and 200 mM arginine-HCl. (E) Principal component regression analysis plot showing the predicted viscosity values against the experimental viscosity values for 180 mg/mL mAb concentration. The observed viscosity values are the experimental values obtained in buffered solution at pH 5.5 and 200 mM arginine-HCl. The predicted viscosity values are the output values from PCR analysis and are described byEq.1..