The cross-linking of novel Fab’ fragments is one of the latest efforts to improve the use of monoclonal antibodies as radiolabeled diagnostic markers. A relationship is expected to exist between how the antigen binding sites are spaced at the extremes of the Fab’ fragments in solution and their immunological performance in vivo and in vitro.
Gaining an appreciation of the size, solution conformation and self-association behavior (or preferably lack of) of these new antibody fragments is critical to properly understanding their function in vivo. The only single technique that can provide this breadth of information is appropriate application of the ProteomeLab XL-A analytical ultracentrifuge.
The flexibility conferred upon F(ab’)2 by its linker makes it impossible to use protein X-ray crystallography to determine its conformation. However, combining the sedimentation coefficients of both the F(ab’)2 and Fab’ fragments with the radius of gyration data obtained from small angle X-ray scattering experiments provides a useful gauge of solution conformation.
Here, sedimentation velocity and sedimentation equilibrium experiments strongly indicate the monodispersity and absence of self-association phenomena of Fab’ and F(ab’)2 solutions.
The weight average molecular weights measured are in complete agreement with the molecular weights as calculated from amino acid sequences. Sedimentation coefficients have been measured and it is shown how these can be used to access conformation when combined with other solution measurements.
Celltech supplied the Fab’ and (Fab’)2 fragments. A standard phosphate chloride buffer (I = 0.1, pH = 6.8) was used to dissolve these fragments.
Sedimentation velocity and sedimentation equilibrium experiments were performed using the Beckman ProteomeLab XL-A analytical ultracentrifuge. Solute distributions at 20°C were recorded via their absorption at 278nm.
The “autoscan” facility was used to record consecutive scans at regular intervals. Sedimentation coefficient (s20,w) values were measured in the standard way and plotted against concentration, corrected for radial dilution. Extrapolation to infinite dilution yielded s020,w.
This study used the low-speed sedimentation equilibrium method, which is more accurate because it provides more data points over the whole radial path length than the high-speed sedimentation equilibrium method.
Equilibrium was considered to have been established when two consecutive scans, recorded several hours apart, seemed to be identical. Simultaneous measurement of multiple samples was enabled through use of multichannel (Yphantis type (3)) centerpieces.
The IBM 3084Q Phoenix mainframe at the University of Cambridge and the FORTRAN MSTARA program were used to capture and analyze the final solute distribution ASCII data.
The limiting value at the cell base of the M* (point average molecular weight) function was used to extract the whole-cell weight average molecular weights (M0w). An independent estimate was not required for the initial loading concentration. Partial specific volumes were calculated from the amino acid sequence for the fragments.
Results and Discussion
Confirmation of Monodispersity and Absence of Self-Association Phenomena
The monodispersity and absence of self-association phenomena were confirmed by the following:
- Only single boundaries were observed from sedimentation velocity (Figures 1a and 2a).
- There was no evidence of an increase in sedimentation coefficients with increase in concentration (Figures 1b and 2b)
- Linear plots of log (absorbance) versus distance squared from sedimentation equilibrium (Figures 3b and 4b)
Figure 1a. Sedimentation velocity profiles for Fab’. Loading concentration = 2.0 mg/mL; rotor speed = 49,000 rpm; scan interval = 27 min. The direction of sedimentation is from left to right.
Figure 1b. Plot of sedimentation coefficient versus concentration for Fab’. Run conditions were as above.
Absolute Molecular Weights
The absolute molecular weights (i.e. assumptions concerning calibration standards are not needed), M0w, calculated from the limiting form of the function M* to the cell base were 47,000 ± 2000 and 94,000 ± 2000 for Fab’ (Figure 3c) and F(ab’)2 (Figure 4c), respectively.
These values agree almost exactly with the polypeptide sequence molecular weights of 47,499 and 94,996, respectively, demonstrating the homogeneity of the antibody preparations as well as the accuracy of the ProteomeLab XL-A.
Figure 2a. Sedimentation velocity profiles for F(ab’)2. Loading concentration = 6.6 mg/mL; rotor speed = 49,000 rpm; scan interval = 18 min. The direction of sedimentation is from left to right.
Figure 2b. Plot of sedimentation coefficient versus concentration for F(ab’)2. Run conditions were as above.
For the Fab’ and F(ab’)2 fragments, the s020,w values were found to be 3.6 ± 0.2 S and 5.1 ± 0.2 S, respectively (Figure1b and 2b). The relationship between these values and the molecular weight is in accordance with the sedimentation behavior of other well characterized globular proteins.
Figure 3a. Sedimentation equilibrium of Fab’Solute distribution recorded at 9,000 rpm. Loading concentration = 0.5 mg/mL.
Figure 3b. Plot of log (absorbance) versus ξ for Fab’.
Figure 3c. Plot of M* function versus ξ (the normalized radial displacement parameter) for Fab’. ξ = (r2 - a2)/(b2 - a2) where r is the radial displacement and a and b are the corresponding values at the cell meniscus and base, respectively.
Figure 4a. Sedimentation equilibrium of F(ab’)2. Solute distribution recorded at 9,000rpm. Loading concentration = 0.5mg/mL.
Figure 4b. Plot of log (absorbance) versus ξ for F(ab’)2
Figure 4c. Plot of M* function versus ξ (the normalized radial displacement parameter) for F(ab’)2.
Solution Conformation Modeling
The s020,w values determined can serve as a useful handle on the conformation of these antibody fragments in solution. Using simple formulae, a combination of the s020,w values and molecular weight data yields estimates for the frictional ratios (f/f0) of 1.31 ± 0.11 and 1.47 ± 0.08, respectively.
It is possible to interpret these values in terms of simple ellipsoid models or, more usefully, this data can be used to model conformation in terms of sophisticated hydrodynamic bead models using the FORTRAN program TRV.
However, additional information from other solution measurements such as X-ray scattering is required for this increased sophistication. For instance, the hydrodynamic bead model for Fab’ shown in figure 5 was developed on the basis of an initial estimate for the solution conformation based on static X-ray crystallographic coordinates for the monoclonal antibody R19.9’s Fab’ fragment and then modified to reproduce f/f0 (or equivalently s020,w) and a radius of gyration of 26 ± 3Å obtained from small angle X-ray scattering experiments. This information is currently being extended by us to model the conformation of F(ab’)2, especially with regard to antigen binding site separation. The ultimate objective is that the solution conformation of the intact parent antibody is adequately represented.
Figure 5. Hydrodynamic bead model for Fab’ of chimeric B72.3 (Celltech Ltd), based on sedimentation coefficient and radius of gyration data. The sphere coordinates and radii were generally such that their maximum diameter is 10Å. The overall dimensions of this model are approximately 50 × 80 × 50Å, inclusive of hydration.
Produced from content authored by Peter J. Morgan, Olwyn D. Byron, Stephen E. Harding, Department of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington, U. K.
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