Characterization of the sucrose/glycine/water system by differential scanning calorimetry and freeze-drying microscopy.

The objective of this study was to characterize the thermal properties of systems containing various ratios of amorphous and crystalline components using both differential scanning calorimetry (DSC) and freeze-drying microscopy. The glycine/sucrose system was used as a model system, since it is routinely used in protein formulations. DSC analysis revealed that the addition of glycine to sucrose solutions resulted in a decrease in the glass transition (T'g) of the system. The T'g of a pure sucrose solution (7% w/v) decreased from -32.3 to -51.5 degrees C for a mixture containing a sucrose/glycine ratio of 2:5. The glass transition of the sucrose/glycine mixture decreased linearly as more glycine was added to the system. This decrease in glass transition resulted in severe collapse during freeze-drying of these mixtures above T'g. However, collapse was not observed during freeze-drying if the DSC thermogram of the sucrose/glycine mixture exhibited a transition resulting from recrystallization of the amorphous glycine. Mixtures having a sucrose/glycine ratio of 3:4 and 2:5 had a glass transition of -48 degrees C and -51.5 degrees C, respectively. Despite their low glass transition temperatures, these samples freeze-dried readily at a product temperature > T'g using a fast freeze-drying cycle (primary drying at a shelf temperature of +20 degrees C and chamber pressure of 100 mTorr) without any sign of collapse. The crystallization of the amorphous glycine from the frozen mixture of sucrose and glycine provided support during freeze-drying which prevented the macroscopic collapse of the final product. Freeze-drying microscopy visually revealed the crystallization and allowed for prediction of cake quality upon lyophilization. Although the freeze-drying microscope is not as sensitive as the DSC in detecting all transitions (it cannot detect a glass transition), it clarifies the interpretation of DSC, and together they provide valuable information regarding the relevance of each of the transitions to the final freeze-dried product elegance.

Random and site-specific immobilization of catalytic antibodies.

The effects of immobilization on the immunologic and catalytic activity of a catalytic antibody were compared for randomly immobilized (via glutaraldehyde) whole antibody and site-specifically immobilized (via the reactive sulfhydryl group at the base of the fragment) Fab' fragments. Upon immobilization, the specific binding capacity (n) and the catalytic activity decreased significantly for both systems. Increases in the Michaelis constant (KM) were accompanied by corresponding decreases in the equilibrium binding constant determined through immunoassays. For the immobilized Fab', n decreased dramatically with increased protein loading, suggesting that, despite the site-specific attachment and favorable orientation, molecular crowding denatured the Fab' fragments. These results also show that there is an optimal surface coverage, not necessarily at the maximum loading, for both immunologic and catalytic activity. Finally, the combining/active site conformation was probed using electron paramagnetic resonance (EPR) spectroscopy. In all antibody samples, there was no spectral evidence of conformational changes in the antibody active site.

Surface-density and orientation effects on immobilized antibodies and antibody fragments.

The binding parameters of randomly immobilized protein 315 and Fv fragments, as well as site-specifically immobilized Fab' fragments, have been measured for a small hapten (MW = 341 Daltons) and a large synthetic antigen (MW = 50 kD). Immobilized Fv fragments had the highest binding capacities; hence, removing unnecessary protein domains can be beneficial for improving the total capacity of an immunosorbent. For all immunosorbents, high protein loadings led to relatively low specific activities (n values). This effect was reversible, however, as the loss of immobilized antibody upon prolonged storage partially restored the specific activity. At high loadings the specific activity of immobilized whole antibody was lower for the large antigen than for the small hapten, whereas no effect of hapten size on n was evident for either immobilized Fab' or Fv fragments. Although a fraction of immobilized antibody was inactive at the higher loadings, EPR spectroscopy revealed no significant changes in the conformation of active immobilized antibody.

Dissociation kinetics of antigen-antibody interactions: studies on a panel of anti-albumin monoclonal antibodies.

Kinetic parameters and equilibrium association constants (K) are reported for a panel of anti-bovine serum albumin (BSA) monoclonal antibodies (MAb) immobilized onto agarose particles. For 12 covalently immobilized MAb of moderate affinity (K = 0.25 x 10(8)-1.2 x 10(8) M-1) measured dissociation time constants varied two orders of magnitude, from 2.1 to 410 min. Directly measured association rate parameters agree with values calculated from measured equilibrium and dissociation rate parameters. Dissociation time constants and equilibrium association constants were also determined for eight MAb immobilized biospecifically (via their Fc regions). A significantly lower K was observed with those MAb which were covalently immobilized as opposed to biospecifically immobilized. These decreases in K appear to reflect decreased association rates rather than increased dissociation rates. The data suggest that, for the MAb described herein, dissociation rates do not correlate with equilibrium association constants.