Biomaterials 27 (2006) 3312–3320 Protein stability in the presence of polymer degradation products: Consequences for controlled release formulations Amy S. Determan a , Jennifer H. Wilson b , Matt J. Kipper a , Michael J. Wannemuehler b , Balaji Narasimhan a,Ã a Department of Chemical and Biological Engineering, Iowa State University, 2035 Sweeney Hall, Ames, IA 50011, USA b Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011, USA Received 10 November 2005; accepted 24 January 2006 Abstract When encapsulating proteins in polymer microspheres for sustained drug delivery there are three stages during which the stability of the protein must be maintained: (1) the fabrication of the microspheres, (2) the storage of the microspheres, and (3) the release of the encapsulated protein. This study focuses on the effects of polymer degradation products on the primary, secondary, and tertiary structure of tetanus toxoid, ovalbumin (Ova), and lysozyme after incubation for 0 or 20 days in the presence of ester (lactic acid and glycolic acid) and anhydride (sebacic acid and 1,6-bis(p-carboxyphenoxy)hexane) monomers. The structure and antigenicity or enzymatic activity of each protein in the presence of each monomer was quantified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism, and fluorescence spectroscopy were used to assess/evaluate the primary, secondary, and tertiary structures of the proteins, respectively. Enzyme-linked immunosorbent assay was used to measure changes in the antigenicity of tetanus toxoid and Ova and a fluorescence-based assay was used to determine the enzymatic activity of lysozyme. Tetanus toxoid was found to be the most stable in the presence of anhydride monomers, while Ova was most stable in the presence of sebacic acid, and lysozyme was stable when incubated with all of the monomers studied. r 2006 Elsevier Ltd. All rights reserved. Keywords: Tetanus toxoid; Ovalbumin; Lysozyme; Polyester; Polyanhydride 1. Introduction As the number of therapeutic proteins being marketed in the US each year continues to increase, new challenges are arising [1]. Proteins have shorter half-lives and are more prone to chemical and physical mechanisms of deactivation in vivo than small molecular weight drugs [2–7]. Thus, an injectable biodegradable controlled delivery device that is capable of encapsulating and providing a sustained release of biologically active proteins is desirable. Encapsulating proteins into polymer microspheres is the most common method of fabricating an injectable con- trolled delivery device. Numerous biodegradable polymers have received attention as protein carriers, including polyesters and polyanhydrides [8–11]. Polyesters are bulk-eroding materials prone to elevated moisture levels within the degrading device, resulting in aggregation of some proteins [12,13]. In contrast, polyanhydrides are surface-eroding materials that significantly reduce the moisture level within the degrading device [12,14]. Another difference between polyanhydrides and polyesters is the strength of acidity generated by the resultant degradation products. As polyesters erode, their degradation products are more soluble in water than anhydride monomers and create a microenvironment with a pH that is lower than that created by eroding polyanhydrides [15–17]. The acidic microenvironment produced by either degrading polymer could alter protein structure, and that induced by polyesters is likely to be more destructive to protein integrity [18]. Because proteins can be denatured by various mechan- isms [2–7,19], care must be taken to select a polymer formulation that can stabilize the protein of interest. The ARTICLE IN PRESS www.elsevier.com/locate/biomaterials 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.01.054 Ã Corresponding author. Tel.: +1 515 294 8019; fax: +1 515 294 2689. E-mail address: nbalaji@iastate.edu (B. Narasimhan).