International Journal of Pharmaceutics 304 (2005) 124–134 Effect of lyophilization on the structure and phase changes of PEGylated-bovine serum albumin Virgilio Tattini Jr. a , Duclerc F. Parra b , Bronislaw Polakiewicz a , Ronaldo N.M. Pitombo a,∗ a Department of Biochemical and Pharmaceutical Technology, Pharmaceutical Sciences School, University of S˜ ao Paulo, Av. Prof. Lineu Prestes, 580, Bloco 16, CEP 05508-900 S˜ ao Paulo, SP, Brazil b Nuclear and Energetic Research Institute/University of S˜ ao Paulo, S˜ ao Paulo, Brazil Received 25 February 2005; received in revised form 30 June 2005; accepted 10 August 2005 Available online 26 September 2005 Abstract Poly (ethylene glycol) (PEG) conjugation masks the protein’s surface and increases the molecular size of the polypeptide, thus preventing the approach of antibodies or antigen processing cells and reducing the degradation by proteolytic enzymes. Proteins are readily denatured by numerous stresses arising in solution (e.g., heating, agitation, freezing and pH changes) or by chemical reactions (e.g., hydrolysis and deamidation), many of which are mediated by water. Lyophilization is most commonly used to prepare dehydrated proteins, which, theoretically, should have the desired long-term stability at ambient temperatures. Through Raman spectroscopy, differential scanning calorimetry (DSC) associated with the determination of water content by Karl Fisher titration, it was observed that after the modification of BSA–PEG in a ratio of 1:0.25 showed lower degree of structural alterations and consequently lower variation on the physical–chemical characteristics when it was compared to BSA–PEG (1:0.5). Moreover, the BSA–PEG (1:0.25) optimizes the conditions during the lyophilization process and storage of the protein. © 2005 Elsevier B.V. All rights reserved. Keywords: Freeze–drying; Lyophilization; PEGylation; Bovine serum albumin; Glass transition; Raman spectroscopy 1. Introduction PEGylation is of interest in applied biotechnology because upon modification it masks the protein’s sur- ∗ Corresponding author. Tel.: +55 11 3091 3665; fax: +55 11 3815 6386. E-mail address: pitombo@usp.br (R.N.M. Pitombo). face, increases the molecular size of the polypeptide, conveys to molecules its physical–chemical properties and therefore also modifies biodistribution and solu- bility of peptide and non-peptide drugs. This property provides new techniques in biocatalysis and in phar- maceutical technology where many insoluble drugs are solubilized by poly (ethylene glycol) (PEG) conjuga- tion and thus more easily administered (Herman et al., 1995). 0378-5173/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2005.08.006