Research paper Induction and analysis of aggregates in a liquid IgG1-antibody formulation Hanns-Christian Mahler a, * , Robert Mu ¨ller a , Wolfgang Frieb b , Aurelie Delille a , Susanne Matheus a a Merck KGaA, Global Pharmaceutical Development, Darmstadt, Germany b Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University Munich, Munich, Germany Received 1 May 2004; accepted in revised form 13 December 2004 Available online 19 January 2005 Abstract The objective of this study was to compare different agitation stress methods (stirring in Reacti Vials TM versus horizontal shaking) in their effect on protein destabilization, to assess several analytical techniques (light obscuration, turbidimetric and light scattering analysis) for detection of aggregates of various sizes and to evaluate the protecting effect of polysorbate 80 on protein aggregation. A monoclonal IgG1 antibody was used as model protein. Both mechanical stress methods can provoke aggregate formation. The method of stirring induces particles in the range of 10–25 mm comparable to shaking stress. However, stirred samples show a much higher absorbance and reveal a second particle species in DLS analysis, suggesting that stirring stress induces a higher amount of smaller protein aggregates. Addition of polysorbate 80 protects the antibody against aggregation. Only in stirred samples a slight increase in sub-visible particles and turbidity was noted. However, a greater extent of aggregation products was detected by DLS as compared to surfactant-free formulations. Thus, polysorbate 80 appears to stabilise small aggregates and prevents further proceeding of the aggregation process. The induction of aggregates by stirring stress in Reacti Vials TM analysed by absorbance measurement seems to be a good combination for high-throughput formulation studies. q 2005 Elsevier B.V. All rights reserved. Keywords: Protein stability; Mechanical stress; Aggregation; Association; Light obscuration; Turbidity; Dynamic light scattering; Antibody 1. Introduction Advances in biotechnology in the last years have made it possible to produce a number of proteins for pharmaceutical use. The size and complex structure of these molecules cause chemical and/or physical instabilities, which make stable aqueous pharmaceutical protein formulations a challenging task. Exogenous factors affecting protein stability during production, storage, shipping and handling are for example temperature (e.g. heat, freeze-thaw cycles), solution conditions (e.g. pH, addition of co-solvents, surfactants or stabilizers) as well as surface interactions (e.g. at hydro- phobic surfaces or container surfaces) [1–4]. Physical instability reactions include protein denatura- tion, aggregation, precipitation and adsorption. Denatured protein (D) shows a failure of the protein tertiary and secondary structure and is often equated with protein instability. The transition from the native state (N) to a denatured state can either be a direct unfolding process or may pass through a series of partially or more extensively unfolded intermediate states leading to exposure of hydrophobic residues to the aqueous environment [2,4,5] (Fig. 1). Aggregate (Ag) formation, as the prevalent physical instability reaction in liquid protein formulations, is initiated by the intermolecular interaction of hydrophobic regions of at least two unfolded or partially folded, denatured protein molecules. Hydrophobic interaction is affected by temperature, ionic strength or shaking [2–4,6]. Aggregation of the denatured state is in competition refolding to the native structure. Proceeding of the aggregation process leads to clusters of aggregated proteins 0939-6411/$ - see front matter q 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejpb.2004.12.004 European Journal of Pharmaceutics and Biopharmaceutics 59 (2005) 407–417 www.elsevier.com/locate/ejpb * Corresponding author. Merck KGaA, Global Pharmaceutical Develop- ment, Frankfurter Strabe 250, 64293 Darmstadt, Germany. Tel.: C49 6151 72 4454; fax: C49 6151 72 7864. E-mail address: hanns-christian.mahler@merck.de (H.-C. Mahler).