(291a) A Novel, High Throughput Assay for Evaluation of Protein Release Kinetics

The advent of recombinant technologies has led to a large number of protein drugs that are currently being evaluated by the FDA for human use. However, many current protein delivery systems have limitations associated with their ability to deliver stable proteins and their range of protein release rates. Polyanhydrides are a class of biodegradable and biocompatible polymers that have been shown to stabilize proteins and provide sustained protein release profiles. Much of the current work being carried out in this field is performed in a ?one-sample-at-a time? format, which can be time- and resource-intensive. With the limited supply of expensive therapeutic protein drugs, the large sample sizes required for the conventional format provide a non-viable option for studying in vitro release and stability. In addition, the conventional format requires repeated sampling, which introduces experimental error, and the use expensive protein assays with low sensitivity. We have developed a novel, fluorescence-based method for high throughput protein detection and quantification upon release from polyanhydride copolymers composed of sebacic acid (SA) and 1,6-bis(p-carboxyphenoxy)hexane (CPH). In this work we have employed a multiplexed format to simultaneously investigate the release of Texas Red bovine serum albumin (TRBSA) as a function of multiple parameters, including polymer chemistry, polymer device geometry (film or nanosphere), and release medium pH. The multiplexed format consists of modified 96-well plates such that neighboring wells are joined at the top. The protein-loaded nanospheres or films were deposited in the first well and buffer was added to both neighboring wells. With the protein-loaded nanospheres and films localized to the first well, the released protein was uniformly distributed across both wells. A fluorescent imager was used to rapidly detect and quantify released protein. TRBSA was encapsulated into five varying chemistries of CPH:SA films and nanospheres and released into three different pH environments. The data indicated that protein release was fastest with SA-rich chemistries, nanosphere device geometry, and neutral pH release medium when compared with their respective counterparts: CPH-rich chemistries, film device geometry, and acidic pH release medium. By employing this high throughput method a 10-fold faster evaluation of protein release kinetics was achieved over conventional techniques. These findings add to the large body of evidence supporting the use of polyanhydrides for use in controlled protein delivery applications.