Our laboratory offers a range of services from simple characterization to complete preformulation studies of potential protein pharmaceuticals, vaccines, viruses, bacterial and plasmid-based gene therapy delivering complexes, including both physical and chemical characterization studies. In some cases, complete formulation development is available. It is our belief that a systematic approach to the characterization process yields better results than more empirical approaches to stabilization. To this end, we approach stability studies with the goal of understanding the degradation pathways at the molecular level. With this information in hand, we identify stabilizers that block or slow the individual degradation pathways. Our physical stability studies are guided by the generation of empirical phase diagrams (EPD) that are generated either from high resolution 2nd derivative UV spectroscopy studies or employing multiple techniques such as CD, fluorescence (intrinsic and extrinsic) and dynamic light scattering. This approach, pioneered in our laboratory, utilizes a multidimensional vector space approach that permits the visualization of multidimensional data in an intuitive graphic format. The output of this approach is a multicolored plot as a function of pH and temperature or other variables such as ionic strength, macromolecule concentration, redox potential, freeze thaw stress or agitation. Color changes correspond to regions of changing stability (pseudo-phase boundaries). This approach is described in detail in J Pharm Sci 92:1793-1804 (2003).
The information obtained from the phase diagrams is used to design high throughput screening assays for the identification of potential stabilizers for the protein pharmaceuticals/vaccine antigens/gene therapy agent. The conditions for the screening assays are determined by the apparent phase boundaries of the empirical phase diagrams as defined by the conditions of abrupt color change. The type of assays (light scattering for aggregation, fluorescence for tertiary structural changes, etc.) is determined by the nature of the physical changes of the proteins and their potential adaptation to a microtiter plate format.
Once the assays are established, a GRAS (Generally Regarded as Safe) library is screened and potential stabilizers determined. In collaboration with the KU high throughput screening laboratory, we also offer screening of larger libraries including the ChenBridge, ChemDiv and Prestwick libraries. To insure the highest degree of stability for the macromolecular species, the number and concentration of stabilizers is optimized. For example, since it is possible that a stabilizer may prevent aggregation but not provide conformational stability, individual techniques (usually fluorescence and CD) are repeated in the presence and absence of potential excipients to determine thermal melting temperatures (Tms). Our goal is to obtain at least a 10 to 15 degree increase in the thermal melting temperature in the presence of excipients with complete elimination of aggregation under accelerated testing conditions.
Work can be performed under a service agreement which surrenders all intellectual and publication rights to clients and is performed on a fixed fee basis or as a research agreement which requires negotiation with the University Research Office (KUCR).
For more details on our work, see the link to our publications.
