International Journal of Biological Macromolecules 66 (2014) 151–157 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l h o mepa ge: www.elsevier.com/locate/ijbiomac Formation of protein sub-visible particles during vacuum degassing of etanercept solutions Haibin Wang a,b , Hong-Jian Zheng b , Zhao Wang b , Hua Bai b , John F. Carpenter c , Shuqing Chen a,∗ , Wei-Jie Fang b,∗∗ a College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Xihu District, Hangzhou 310058, China b Division of Biopharmaceuticals, Zhejiang Hisun Pharmaceutical Inc, 46 Waisha Road, Jiaojiang District, 317000 Taizhou, China c Department of Pharmaceutical Sciences, University of Colorado Denver, Aurora, CO 80045, United States a r t i c l e i n f o Article history: Received 1 December 2013 Received in revised form 30 January 2014 Accepted 1 February 2014 Available online 7 February 2014 Keywords: Degassing Freeze-drying Protein particle Micro-flow imaging a b s t r a c t The main purpose of this manuscript is to describe a phenomenon in which vacuum degassing a reconstituted freeze-dried fusion protein etanercept formulation caused a significant amount of pro- tein sub-visible particles (SbVP). Physical stability of etanercept was monitored by micro-flow imaging (MFI), dynamic light scattering (DLS), size-exclusion high pressure liquid chromatography (SE-HPLC) and far- and near-ultraviolet circular dichroism (far- and near-UV CD). One potential explanation of this phenomenon is that bubble collapses when the vacuum is applied, leads to substantial heat formation, and ultimately free radical formation. Subsequently, the effect of a free-radical scavenger (ascorbic acid, AA) on SbVP formation was also evaluated. Degassing of etanercept solution by applying vacuum caused substantial increase of SbVP, as detected by MFI and DLS. However, traditional techniques such as SE- HPLC could not detect any change. The addition of free-radical scavenger had minimal effect on SbVP formation, therefore the formation of free radicals was probably not the main cause for this effect. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Biopharmaceuticals, or protein-based medicines, are a rapidly growing therapeutic modality in the treatment of human diseases, especially in the areas of infection, cancer and autoimmune dis- eases [1]. However, due to their complex and fragile structures, the development of protein therapeutics is a complicated process and presents unique challenges to manufactures. Unlike traditional small-molecule drugs, essentially all biopharmaceuticals are prone to induce immune response due to their large size and complexity in structure [2,3]. This in some cases will either abrogate all of the biological effects of the therapeutic proteins or change their phar- macokinetic profile. It has now been widely recognized that the presence of protein aggregates is a potential risk factor for causing immunogenicity and impacting the rate and strength of immune Abbreviations: AA, ascorbic acid; CD, circular dichroism; DLS, dynamic light scattering; ECD, equivalent circular diameter; HPF, 2-[6-(4-hydroxy)phenoxy-3H- xanthen-3-on-9-yl]benzoic acid; MFI, micro-flow imaging; ROS, reactive oxygen species; SbVP, sub-visible particle; SE-HPLC, size exclusion high-performance liquid chromatography; UV, ultraviolet. ∗ Corresponding author. Tel.: +86 571 88208411; fax: +86 571 88208410. ∗∗ Corresponding author. Tel.: +86 571 63281551; fax: +86 576 88828299. E-mail addresses: chenshuqing@zju.edu.cn (S. Chen), wjfang@hisunpharm.com (W.-J. Fang). response [2,4–6]. The insoluble high-molecular-weight, sub-visible particles (SbVP) presents the highest risk, with a relatively low level of aggregates on a percentage basis (i.e. less than 1% total mass) potentially inducing a robust immune response. This small amount of mass may not be accurately detected by conventional chromato- graphic methods such as size exclusion high-performance liquid chromatography (SE-HPLC) and minor secondary or tertiary struc- tural changes may not be detected by spectroscopic methods such as circular dichroism (CD). Light obscuration, the most widely applied technology to measure sub-visible particles in pharmaceutical products, is based on the ability of particles to block light intensity [7]. However, this technology was originally designed to count non- proteinaceous particles, and therefore may not be suitable to characterize proteinaceous particles. Currently more and more particle-characterization technologies with higher sensitivity and resolution are applied in the fields of biopharmaceuticals, such as dynamic light scattering (DLS), micro-flow imaging (MFI), coulter counter and nanoparticle tracking analysis (nanosight). Each has advantages and disadvantages in counting and characterizing par- ticles [7–9]. For example, DLS can analyze samples containing very broad distributions of species and can detect very small amount of the higher mass species (i.e. <0.01% mass in some cases) [10]. On the other hand, MFI is sensitive to measure highly transparent particles and can provide particle images and morphology [11–13]. 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.02.001