Journal of Chromatography B, 1022 (2016) 206–212 Contents lists available at ScienceDirect Journal of Chromatography B jou rn al hom epage: www.elsevier.com/locate/chromb Pre-study and in-study validation of a size-exclusion chromatography method with different detection modes for the analysis of monoclonal antibody aggregates Alexis Oliva ∗ , Jose B. Fari ˜ na, Matías Llabrés Departamento de Ingeniería Química y Tecnología Farmacéutica, Facultad de Ciencias de la Salud-Sección Farmacia, Universidad de La Laguna, 38200, Tenerife, Spain a r t i c l e i n f o Article history: Received 7 January 2016 Received in revised form 5 April 2016 Accepted 9 April 2016 Available online 13 April 2016 Keywords: Bevacizumab Light-scattering Validation Aggregation kinetics Stability a b s t r a c t Size exclusion chromatography (SEC) with different detection modes was assessed as a means to char- acterize the type of bevacizumab aggregate that forms under thermal stress, quantitatively monitoring the aggregation kinetics. The combination of SEC with light-scattering (SEC/LS) detection was validated using in-study validation process. This was performed by applying a strategy based on a control chart to monitor the process parameters and by inserting quality control samples in routine runs. The SEC coupled with a differential refractive-index detector (SEC/RI) was validated using a pre-study validation process in accordance with the ICH-Q2 (R1) guidelines and in-study monitoring in accordance with the Analytical Target Profile (ATP) criteria. The total error and -expectation tolerance interval rules were used to assess method suitability and control the risk of incorrectly accepting unsuitable analytical methods. The aggregation kinetics data were interpreted using a modified Lumry-Eyring model. The true order of the reaction was determined using the initial-rate approach. All the kinetic data show a linear Arrhenius dependence within the studied temperature range. The Arrhenius approach over-predicted the aggre- gation rate for 5 ◦ C, but provides an idea of the aggregation process and amount of aggregate formed. In any case, real-time stability data are necessary to establish the product shelf-life. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Antibody therapeutics is undergoing rapid growth rates, the major areas of application being cancer and various immunological disorders. Bevacizumab (Avastin R , Genetech, San Francisco, USA) is a recombinant humanized monoclonal IgG1 antibody that prevents or reduces the formation of blood vessels (angiogenesis), thereby preventing or reducing metastatic disease progression [1]. It has been also shown to be effective as an adjunct treatment for neovas- cularization of the iris and neovascular glaucoma with or without vitreous hemorrhage [2]. The successful application of this impor- tant class of drugs requires avoiding any form of degradation. For this, the main product characteristics to be monitored are aggregate and fragment content, glycosylation pattern and charged isoforms. To define the identity or purity of biopharmaceutical drugs requires methodologies that differ from classical pharmaceu- ∗ Corresponding author. E-mail address: amoliva@ull.es (A. Oliva). tical practices [3]. However, due to the complexity of protein drugs, different analytical methods must be applied in protein character- ization and development of stability-indicating assays. The effect of heat, shear, surface phenomena, and solvent additions on the native-state protein should also be studied [4]. All the techniques are expected to detect, quantify and distinguish different forms of the active ingredients and their degradation products. The standard method used in biopharmaceutical quality control (QC) for monoclonal antibody (mAb) aggregate and fragment anal- ysis is size exclusion chromatography (SEC). Its main advantage is the mild elution conditions that allow protein characteriza- tion with minimal impact on conformational structure and local environment [5]. The potential and applications of this analytic technique in the characterization of biopharmaceutical drugs was recently reported by Fakete et al. [5]. SEC in combination with light- scattering (SEC/LS) detection offers an easy, accurate and reliable alternative technique to assess the association of macromolecules in solution [6,7]. In some instances it can provide information regarding the conformation of a protein (folded or unfolded), if the http://dx.doi.org/10.1016/j.jchromb.2016.04.022 1570-0232/© 2016 Elsevier B.V. All rights reserved.