Application Notes

4 However, there are certain buffer and pH conditions e.g. TRIS buffer at pH 7.5 (Figure 1D) and Na 2 HPO 4 ·2H 2 O buffer at pH 6.5 that cause a splitting of the last peak. This could either be induced by C H 3 or by the presence of partly denatured intermediates during the transition of the F ab fragment, which has been described in the literature. 6,7 In order to determine a T m -value of the F ab fragment in those systems the average was used. Figure 2 Buffer and pH screening of nine formulations (A) Summary of T m values of different domains of an IgG1- Antibody in nine formulations (B) Summary of C m values of different domains of an IgG1-Antibody in nine formulations Figure 2A reveals that the His/Gly buffer at pH 6.5 is the most stable formulation for the protein because of high T m -values, particularly of the F ab fragment (70.2 °C for C H 2 and 81.8 °C for F ab ). It has already been described in literature that the temperature of the F ab transition point may be a better indication of protein stability than the T m of the first transition point. 8 Measurements of the chemical stability can serve as a supporting element: here the His/Gly buffer system at pH 6.5 produces high C m -values for both domains as well (Figure 2B). Having a look at the other buffer types, Na 2 HPO 4 ·2H 2 O at pH 7.5 and TRIS at pH 7.5 are the best options. Nevertheless, one should note that the differences in C m - and T m - values between the nine formulations are quite small. Comparison of Figure 2A and 2B shows that the transition points of the domains have different sensitivities to pH changes: Lowering pH leads both to smaller C m - and T m -values of the C H 2 domain, whereas the F ab fragment does not seem to be sensitive to pH changes. This trend is in good agreement with some other studies on denaturation of immunoglobulins. 3,9 Results of the stability experiment upon six weeks storage are presented in Figure 3 using His/Gly- and TRIS buffer as examples. The T m -values of C H 2 did not change significantly; therefore these values are not shown in the figure. Despite of a short storage period of 42 days T m -values of the F ab fragment decreased continuously in particular systems, being His/Gly pH 7.5, TRIS pH 7.5 and TRIS pH 8.5. These systems seem to be sensitive to thermal stress, on the contrary His/Gly at pH 5.5 and 6.5 as well as TRIS at pH 6.5 appear to be more robust. Figure 3A reveals that the His/Gly buffer at pH 6.5 provides the most stable environment for the protein. This is in good agreement with the data in Figure 2A, from which this system was selected as the best option out of the nine tested systems. Figure 3B demonstrates that the antibody tested favors pH 6.5 as the best TRIS buffer option, whereas the data depicted in Figure 2 do not show a clear difference between the three TRIS systems. Consequently, a single T m measurement as indication of protein stability might not be sufficient. Instead, it could be helpful to monitor thermal stability over a period of storage at increased temperature. Such an experiment can serve as an additional tool to identify the most stable formulation, especially if T m 2 values at T 0 are very similar, as can be observed in Figure 3B.

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