Behavior Of Fab Research Articles

Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

Mathematical Modelling of Protein Precipitation Based on the Phase Equilibrium for an Antibody Fragment from E. coli Lysis

Precipitation is an important operation in biopharmaceutical purification yet the mechanism of protein precipitation in multi-component solutions is not well understood. Existing models lack fundamental understanding of the process. In this paper, a new model describing how the protein solubility changes in the protein precipitation is proposed and is based on the phase equilibrium of the light liquid phase and dense solid phase. The model structure is generic and robust. It adequately reflects the non-linearity of protein precipitation kinetics and thus provides new fundamental insights into the protein precipitation in multi-component, complex protein solution. Two feed stocks of a pure fragment antigen-binding (Fab’) solution obtained by chromatographic purification and a clarified Fab’ homogenate solution from E. coli were used to examine the effect of ammonium sulphate concentrations and pH conditions on precipitation. It was found that the model can describe pure Fab’ precipitation well, and identify the non-ideal behavior of Fab’ precipitation in multi-component homogenates. Through statistical analysis, the model parameters have been further reduced from 8 to 4. The quality of the model is such that errors were within the acceptable statistical confidence limits, even when applied to multi-component impurity precipitation. The new model with fewer parameters is better than existing empirical models in reflecting the salting-in and salting-out effect of the protein precipitation. This demonstrated that the structure of the model is sound and over-fitting in the parameter estimation is avoided. The model can be applied directly to industrial processes for protein precipitation process design after appropriate calibration with the required operating conditions of pH and salt concentration.

Using affinity capillary electrophoresis to evaluate average binding constant of 18-mer diphosphotyrosine peptide to antiphosphotyrosine Fab.

We used affinity electrophoresis in capillaries to investigate the interaction between a monovalent antiphosphotyrosine antibody fragment, antigen-binding fragment (Fab), and a divalent antigen (dAg), an 18-mer diphosphopeptide phosphorylated on two-site tyrosine residues. The migration shift behavior of Fab in electrophoretic solution was observed and the quantitative expression was presented to estimate the arithmetical average value of the intrinsic affinities for two epitopes on the dAg with the Ag binding site on the Fab. In dAg excess, based on measurement of mobility changes of Fab analytes at different dAg concentrations, the experimental average dissociation constant (Kd = 27.7 microM) was calculated. It was also found that the structural variation of the two epitopes for binding specificity to the Ag-binding domain of Fab is not apparent. Moreover, the Kd values of Fab-dAg complexes were measured at higher electric fields and shown to be independent of changes in the electric field. Thus, under conditions where the total dAg concentration is in excess of the total Fab concentration, the method and quantitative expression which we developed is generally useful for the understanding of molecular interaction for an unlabeled monovalent receptor and its divalent ligand in free solution.

Velocity cross correlations in binary mixtures of simple fluids

The velocity cross correlation integrals $$D_{{\text{ab}}}^{\text{J}} = (N/3)\mathop \smallint \limits_{\text{o}}^\infty {\text{dt,}} {\text{a}} {\text{ = }} {\text{1,2;}} {\text{b}} {\text{ = }} {\text{1,2}}$$ can be estimated from the intradiffusion coefficients D1° and D2° at each mole fraction x1 of component 1 on the basis of the exact relations among the Onsager phenomenological coefficients together with an assumed equation relating the joint diffusion coefficients DabJ. The results from several such equations are compared with experimental data and with similar results derived by Hertz in a different way to represent the behavior of fab≡DabJxb in ideal reference systems. In some cases the agreement with experimental data for relatively ideal systems is even better than given by Hertz's results. For greater accuracy in predicting the DabJ from Dadg data one would need a prediction of the limiting value of DaaJ at xa=0 for a=1,2. Presently known theory does not give a basis for estimating this limit reliably.

Conformational studies on the tryptic digestion fragments of human immunoglobulin G

Conformational studies on Fab(t) and Fc(t) (normal and myeloma) have been carried out by circular dichroism, difference spectra, sedimentation, and viscosity measurements. Both Fab(t) and Fc(t) were largely unfolded in acid (pH 2.1) and alkali (pH 11.7), and the unfolding was greater in alkali than in acid for Fab(t). However, for Fc(t) the circular dichroism spectral change was slightly higher in acid (pH 2.1) than in alkali (pH 11.7). It was also noted that the disorganizations were not complete under these conditions. Similar types of disorganizations were observed in heat denatured Fab(t) and Fc(t). Difference spectral studies demonstrated the presence of a significant number of buried tyrosine and tryptophan residues in both fragments, and a large proportion of the residues became exposed on acid denaturation. The results led us to conclude that both Fab(t) and Fc(t) may be characterized by structures of different stabilities. There seem to be small stable structured regions containing some of the β structures while the other portions are more susceptible to denaturation. Also, there are regions in these proteins which are rich in hydrophobic interactions. Sodium dodecyl sulfate refolded both Fab(t) and Fc(t) into partial helical structures, although the accessibility and rotational freedom of the side chain aromatic chromophores were increased. In 6 m GuHCl containing 0.1 m 2-mercaptoethanol, complete disorganization of Fab(t) was demonstrated despite the fact that its circular dichroism spectra around 217 nm differed from the spectra of random coil polyamino acids. In spite of certain differences, the behavior of Fab(t) and Fc(t) towards different denaturing agents was similar pointing out homology in their basic structural features.