Stability of murine, chimeric and humanized antibodies against pre-S2 surface antigen of hepatitis B virus.

We have constructed a humanized antibody with specificity for the pre-S2 surface antigen of hepatitis B virus (HBV) by grafting the complementarity determining regions (CDRs) of parental murine monoclonal antibody (mAb) into human anti-Sm antibody framework regions. The humanized antibody has a substitution at position 94 in a framework region of the heavy chain variable region, and exhibits the same antigen binding affinity as the parental murine monoclonal and chimeric antibodies. In order to assess the stability of these antibodies, thermal inactivation of the parental, chimeric and humanized antibodies was analyzed. Fifty percent inactivation of the chimeric and humanized antibodies was observed at 63.7 degrees C and 68.7 degrees C, respectively, compared to 55.0 degrees C for murine antibody. The humanized antibody also exhibited increased stability against denaturant. Guanidine-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that midpoints of the transition of the chimeric and humanized antibodies were 2.47 M and 2.56 M, respectively, whereas that of the murine antibody was 1.36 M.

van't Hoff and calorimetric enthalpies II: effects of linked equilibria.

The complexity of binding reactions, including the linkage with other equilibria, is becoming increasingly apparent in biological processes such as signal transduction. Understanding these interactions requires obtaining thermodynamic profiles for each of the equilibria that occur in a binding event. Concern has been raised as to whether linked equilibria contribute differently to thermodynamics, such as DeltaH degrees and DeltaC(p), obtained from calorimetric and van't Hoff methods. We have previously shown that linked equilibria do not contribute differently to the van't Hoff and calorimetrically determined DeltaH degrees for processes such as linked folding or hydration. Here, examples of proton and ion linkage are examined. We show that there is no reason to expect the calorimetric and van't Hoff DeltaH degrees to be different, even without prior knowledge of the presence or absence of linked equilibria, as long as the system is permitted to equilibrate. However, it is possible to create experimental scenarios that result in and discrepancies. Furthermore, it is found that the presence of linked equilibria in all cases can result in "nonconventional" DeltaH degrees and DeltaC(p) profiles, making data analysis nontrivial.

Determination of the volumetric properties of proteins and other solutes using pressure perturbation calorimetry.

Pressure perturbation calorimetry is a new technique that measures the heat change in a solution that results when the pressure above the solution is changed. When used in a differential calorimeter containing a dilute solution of solute in the sample cell and the corresponding buffer in the reference cell, the measured differential heat can be used to calculate the thermal coefficient of expansion of the partial volume of the solute, alpha. For proteins in dilute aqueous solution, alpha is dominated by a temperature-dependent contribution arising from the interaction of protein groups with water at the protein-solvent interface. This arises due to the effect of the protein groups on the hydrogen-bonded structure of water, and thereby clearly differentiates between structure-making hydrophobic groups and structure-breaking hydrophilic groups. This solvation contribution to alpha can be accentuated in solvents having more structure (deuterium oxide) than water and attenuated in solvents having less structure (2.8 M guanidinium sulfate). Six different proteins (chymotrypsinogen, pepsinogen, lysozyme, bovine pancreatic trypsin inhibitor, ribonuclease A, and T4 lysozyme) were examined carefully by this technique, allowing estimates of various volumetric parameters including the volume change resulting from thermal unfolding of each protein. For ribonuclease A, results obtained in both water and deuterium oxide led to an estimate of the accessible surface area of the native protein of approximately 45% relative to the fully reduced unfolded protein. Also, it was also found that ligand binding to ribonuclease A led to changes in alpha, suggesting a burial of some surface area in the ligand-protein complex.

An autosampling differential scanning calorimeter instrument for studying molecular interactions.

A new ultrasensitive differential scanning calorimeter (DSC) instrument is described, which utilizes autosampling for continuous operation. High scanning rates to 250 deg/h with rapid cooling and equilibration between scans facilitates higher sample throughput up to 50 samples during each 24 h of unattended operation. The instrument is suited for those pharmaceutical applications where higher throughput is important, such as screening drug candidates for binding constant or screening solution conditions for stability of liquid protein formulations. Results are presented on the binding of five different anionic inhibitors to ribonuclease A, which included cytidine 2'-monophosphate (2'CMP), 3'CMP, uridine 3'-monophosphate, pyrophosphate, and phosphate. Binding constants K(B) (or dissociation constants K(d)) are obtained from the shift in the transition temperature T(M) for ribonuclease thermal unfolding in the presence of ligand relative to the transition temperature in the absence of ligand. Measured binding constants ranged from 155 M(-1) (K(d) = 6.45 mM) for the weak-binding phosphate anion to 13100 M(-1) (K(d) = 76.3 microM) for the strongest binding ligand, 2'CMP. The DSC method for measuring binding constants can also be extended to ultratight interactions involving either ligand-protein or protein-protein binding.