Application Notes

Chemical and thermal stability screening of an IgG1-antibody

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1 Chemical and Thermal Unfolding Application Note NT-PR-009 Chemical and Thermal Stability Screening of an IgG1- Antibody Barbara Julia Spix, Marieke Veurink Bayer Pharma AG, Wuppertal, Germany Abstract The main purpose in formulation development of biologicals, such as monoclonal antibodies, is to establish optimal conditions for long-term stability of the protein. Here we used the Prometheus NT.48 instrument by NanoTemper Technologies to perform a buffer screening for an IgG1-Antibody. This instrument uses nanoDSF technology to measure thermal and chemical stability as indicators to predict the best buffer conditions for the protein. The results show that thermal and chemical unfolding, together with short-term stability studies, provides a complementary screening tool for finding optimal buffer conditions. Introduction Biopharmaceuticals, especially monoclonal antibodies, have reached an increasingly important position on the drug market over the last years. To screen for a formulation with a long shelf-life, analyses focusing on the unfolding of the protein are often used as optimization tool, since unfolding may lead to aggregation and therefore needs to be avoided. There are two common procedures used to induce protein unfolding, i.e. by constantly increasing temperature (thermal unfolding) and by titrating a denaturing agent such as guanidine hydrochloride into the protein sample (chemical unfolding). The Prometheus NT.48 instrument can be used for both experimental approaches. It measures changes in intrinsic fluorescence of the protein. Once the unfolding process begins, induced either by thermal or chemical stress, the tryptophan and tyrosine groups expose themselves to the solvent. This causes a shift in fluorescence emission especially of the tryptophan groups, which is plotted as the ratio between 350 and 330 nm. The inflection point of the resulting sigmoidal curve provides the temperature (T m ) or the concentration of the denaturing agent (C m ), at which half of the protein amount is unfolded. 1 The Prometheus NT.48 is well suited for buffer screening because of its possibility to measure up to 48 samples simultaneously as well as its low consumption of sample (10 ┬ÁL per capillary). The needlessness of an additional fluorescence dye and the easy handling are further advantages. Figure 1: Structure of an Antibody: F ab , C H 2, C H 3 domains highlighted. The enlargement of the variable domain shows a ribbon representation of the Beta-sheet framework and CDR loops.

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