Antibody Production and Testing Core (APTC)

PROJECT SUMMARY Extensive testing of our therapeutic candidate, MS-Hu6 in Projects 1 and 3 will require that we produce, purify and test 7.5 g of antibody over ~4 years. The primary function of the Antibody Production and Testing Core (APTC, Core B) thus remains to scale up the production and purification of MS-Hu6, and when required, the parent molecule, Hf2. Towards Specific Aim 1, we currently have stably–transfected CHO-K1 cells and Hf2 hybridomas, and are producing antibodies en masse in 3–liter spinner flasks, followed by FPLC purification using HiTrap Protein G columns. We have also developed a final formulation for MS-Hu6 that provides thermal and accelerated stability, prevents aggregation and degradation, maintains structural integrity, and enables retained FSH binding. Thus, in Specific Aim 2, we propose to evaluate the physicochemical properties of each batch of MS-Hu6 using a battery of tests––protein thermal shift assay, size exclusion chromatography, dynamic light scattering, differential scanning calorimetry, accelerated stability testing, tests for viscosity, turbidity and clarity, circular dichroism spectroscopy, Fourier–transform infrared spectroscopy, hydrophobic–, self–, and cross–interaction chromatography, and ELISAs for polyspecificity and FSH binding. This expansion allows us to determine if each batch of purified MS-Hu6 meets strict industry standards. Towards, Specific Aim 3, Core B will continue to confirm the binding affinity (KD) of each batch of MS-Hu6 to mouse and human FSH by surface plasmon resonance (SPR) and validate FSH–blocking activity using both osteoclast– and adipocyte–based in vitro assays, and when required, in vivo confirmation using the ThermoMouse. As we move MS-Hu6 into the clinic, Core B will also extend these capabilities to test small batches of clinical–grade MS-Hu6 once our master cell bank is ready for manufacturing and continue to support U01 AG073148 and R01 AG074092. Overall, we propose Core B as a mature, ongoing and expanded asset to the U19 renewal application.