Contents lists available at ScienceDirect Thermochimica Acta journal homepage: www.elsevier.com/locate/tca Buffer and additive thermofluor screening of wild type human interferon gamma and mutant proteins M. Tileva a , E. Krachmarova a , S.G. Taneva b , S. Todinova b , K. Maskos c , I. Ivanov a , G. Nacheva a, ⁎ a Institute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria b Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria c Proteros Biostructures, D–82152 Martinsried, Germany ARTICLE INFO Keywords: Human interferon gamma Protein stabilization Buffer optimization Thermofluor assay Differential scanning calorimetry ABSTRACT Human interferon gamma (hIFNγ) plays a key role in the immune system and therefore this cytokine has many current and future therapeutic applications. hIFNγ is well known with its aggregation propensity and despite its clinical use, there is not much data about the stabilization of hIFNγ preparations. Nowadays, substantial evidence indicates that the pathogenesis of many autoimmune diseases is related to overproduction of hIFNγ. In this regard we have developed inactive hIFNγ analogues to act as receptor antagonists of the endogenous hIFNγ. Since they show even higher tendency for aggregation than the wild type protein, we designed two-step thermofluor screen of 61 buffer conditions to identify the best storage solution for all investigated proteins to be used in the form of research samples or biopharmaceuticals. Tris buffer pH 8.0 supplemented with NaCl and trehalose/betaine/glycerol as additives showed to be the most appropriate choice ensuring high solubility and thermal stability of both hIFNγ and its mutants. 1. Introduction Nowadays protein-based pharmaceuticals have increased dramati- cally in number and frequency of use [1]. Unlike small molecules, the protein drugs suffer from aggregation and other chemical and physico- chemical alterations affecting the active substance and thus leading to decrease or complete loss of therapeutic activity and/or immunogeni- city [2]. Therefore, the issue with protein instability in development of protein drugs is one of the major obstacles in the way of their application and commercialization. To protect recombinant proteins from such alterations stabilizers are added to the solutions used for their isolation and storage [3–5]. In general, the additives that increase the thermal stability of a protein have protective effect against aggregation and improve the protein product quality. Accordingly, the formulation is crucially important and the buffer and/or additive screening require to be performed even before the assessment of safety and toxicity of the drug [6]. Human interferon gamma (hIFNγ) is a cytokine which is endowed with multiple biological activities, such as antiviral, antiproliferative, antitumor, etc. [7]. Due to its immunomodulatory role hIFNγ is applied for treatment of chronic granulomatous disease and osteopetrosis, and has much more therapeutic potential [8,9]. hIFNγ displays propensity for quick aggregation even under mild stress conditions [10,11]. Although hIFNγ is often used as a model for studying protein aggregation mechanisms in vitro, there are insufficient data about the efficiency of additives that positively affect its stability [12,13]. In spite of the essential role of hIFNγ in immune system regulation, its overproduction is associated with the aetiology of a number of autoimmune diseases [14,15]. We have been exploring the idea to suppress the activity of the endogenous hIFNγ using interferon mutants bearing preserved affinity to the hIFNγ receptor but deprived of the capacity to trigger the signal transduction pathway. Pursuing this idea, we have created a library of mutants carrying mutations in the putative upstream NLS sequence and identified several of them as candidates for future research and development as autoimmune therapeutics [16]. These mutants show even higher tendency for aggregation than the wild type protein. Taking into consideration that there is no common way for selection of an optimal stabilizer for recombinant proteins and particularly for hIFNγ and/or its derivatives, we chose three mutants: a single point mutant (with Q substitution for K in position 88), a double mutant (with additional substitution D for K in position 86) and a triple mutant (with D, L and L substitutions for K, K and K in positions 86, 87 and 88, respectively) for evaluating the efficiency of 61 different buffer conditions using the protein melting temperature (T m ) as a criterion. The list of buffer conditions included a great variety of salts, amino http://dx.doi.org/10.1016/j.tca.2017.05.003 Received 13 January 2017; Received in revised form 11 April 2017; Accepted 7 May 2017 ⁎ Corresponding author. E-mail addresses: genoveva@bio21.bas.bg, genoveva_nacheva@hotmail.com (G. Nacheva). Thermochimica Acta 654 (2017) 1–7 Available online 10 May 2017 0040-6031/ © 2017 Elsevier B.V. All rights reserved. MARK