Effect of Polysorbate 20 on Nucleation Rate of Interferon Beta-1b Aggregation

Introduction Interferon beta-1b (IFNβ1b) is a form of interferon beta which has shown biological activity in a variety of in vitro and in vivo systems. Interferon beta belongs to a class of proteins known as interferons (IFNs). Interferons were originally classified based on the cell type from which they were derived [1]. The non-glycosylated IFNβ1b (Betaseron®) was the first IFNβ product approved by (United State Food and Drug Administration) FDA in 1994. The non-glycosylated IFNβ1b has an apparent molecular weight of 18.5 kDa. It is produced in Escherichia coli cells, and Cys-17 is mutated to Ser-17 to reduce misfolding and/or aggregation during the refolding process [2]. Aggregates have been observed to form in therapeutic proteins during purification and storage, and the administration of proteins containing aggregates has been shown to stimulate immune responses, causing effects ranging from mild skin irritation to anaphylaxis [3]. Many studies have shown that aggregates in IFNβ1b products are a risk factor for immunogenicity [4]. In patients using formulated IFNβ protein, aggregates are a cause of Neutralizing Antibodies (NAbs). The therapeutic effect of IFNβ is influenced bythe formation of Binding Antibodies (BAbs) and NAbs with negative impact on its bioactivity. Therefore, the aggregation is of great concern affecting the biological activity of IFNβs [4]. Furthermore the low solubility of hydrophobic proteins becomes an issue, when the target concentration for the formulation cannot be achieved. Hydrophobic proteins often show limited solubility. For example, solubility of IFNβ1b is 0.05 mg/ml at physiological pH [5]. One of the procedures that now are used to increase the solubility of IFNβ1b is the adding Human Serum Albumin (HAS) to its formulation, but presence HAS in the formulation is a problem for development of significant analytical tools to characterize the protein and its degradation products, and furthermore might induce the risk of immunogenicity reactions in the patient. HSA itself exhibits a low risk for immunogenicity, however in presence of a second protein the formation of mixed aggregates can lead to immunogenicity reactions. So the investigation to use some excipients in the formulation can be an effective stage in the development of formulation of IFNβ1b without the HAS. In parenteral formulations of hydrophobic protein formulations (such as IFNβ1b), non-ionic surfactants, mostly Polysorbate 20 and 80, are frequently used [5]. Surfactants have been used not only to purify, isolate, or solubilize proteins, but also to maintain biological activity by binding to proteins through electrostatic and hydrophobic interactions [4]. Many nonionic surfactants have been tried or used in protein formulations. These nonionic surfactants have the hydrophobic tails, which can bind to hydrophobic patches on protein surfaces [6]. It has been extensively documented that surfactants suppress protein aggregation against various stresses, including heating and agitation [7]. A commonly used type of nonionic surfactant for this purpose is Polysorbate. Also, Pluronic F-127 was successfully used to decrease the aggregation of some proteins such as Alcohol dehydrogenase [8]. Pluronic F-127 is the block copolymers of polyethylene oxide (PEO) and polypropylene oxide (PPO) represent a class of thermo responsive polymer materials approved by FDA and (United State Environmental Protection Agency) EPA as food additives, pharmaceutical excipients and The aggregation of protein is the most prevalent and the most disturbing kind of instability and this challenge exists in almost every stage of the development of protein drug. The presence of insoluble aggregations in protein drugs will make the supply of the product a tough job. This study identifies the inhibition of the folded Interferon beta 1-b’s aggregation with the assistance of some excipients. It uses some thermal stress and mechanical methods to accelerate the aggregation, and also the spectroscopic method to identify the protein aggregation and its growth. Experimental data of the tests show compliance with the autocatalytic model. This model has been used to obtain the Kinetic constants of aggregation in different states and to make comparison with one another in the presence of some excipients. The kinetic constants were obtained by fitting the Autocatalytic model on data. Among these excipients, Polysorbate 20 of 0.01% (w/v) showed the best result in decreasing the aggregation. Using this excipient of 0.01% (w/v) in thermal stress causes dramatic reduction of nucleation constant from 8.3 ×10-3 (min-1) to 4.14 ×10-6 (min-1), which indicates the reduction of protein aggregation in the solution.