AMBER HAYNES FRADKIN,1 JOHN F. CARPENTER,2 THEODORE W. RANDOLPH1
1Department of Chemical and Biological Engineering, University of Colorado, Boulder
2Department of Pharmaceutical Sciences, University of Colorado, Denver
Posted with permission from Amber Fradkin,
ABSTRACT: Parenteral administration of aggregates of therapeutic proteins has been shown to induce antibodies to the monomeric protein, which can jeopardize the safety and efficacy of therapeutic protein products. The risk that aggregates of therapeutic proteins pose is well-known; however, the characteristics that make an aggregate immunogenic and the mechanisms through which protein aggregates provoke immune responses are not fully understood. We used aggregates of recombinant murine growth hormone (mGH) to test the ability of homogeneous and heterogeneous aggregates of a self protein to break tolerance in mice. Homogeneous aggregates of mGH were produced by freeze-thawing and agitation stresses. Heterogeneous aggregates were formed by adsorption of mGH to Alhydrogel™ and glass microparticles. Aggregates were characterized by size exclusion chromatography (SE-HPLC), infrared spectroscopy (FTIR), front-face fluorescence and micro-flow imaging. Aggregate doses as low as 1.6 ng/dose broke tolerance in mice and induced immune responses to monomeric protein. Both homogeneous and heterogeneous aggregates of mGH produced T-cell dependent immune responses when administered subcutaneously. Heterogeneous aggregates contained protein molecules with more native-like secondary and tertiary structures than homogeneous aggregates and produced the highest antibody titers in mice. The use of high hydrostatic pressure to reduce aggregate levels to 0.02 ng/dose eliminated immunogenicity.
PRESENTED AT:
2010 AAPS National Biotechnology Conference, San Francisco CA, USA
2010 AAPS NBC UofC T-cell dependant responses