These formulation conditions contain amino acids, surfactants, and sugars (Figure 3B), however, any of these additives do not yield unwanted signs in 15N-recognized CRINEPT spectrum

These formulation conditions contain amino acids, surfactants, and sugars (Figure 3B), however, any of these additives do not yield unwanted signs in 15N-recognized CRINEPT spectrum. due to the high molecular excess weight of the system. With this review, we will discuss how NMR techniques have been applied to HOS analysis of mAbs, along with the recent advances of the novel 15N direct detection NMR strategy that allows for obtaining the structural fingerprint Encequidar of mAbs at lower temperatures under multiple formulation conditions. The potential application of these NMR strategies will benefit next-generation mAbs, such as antibody-drug conjugates and bispecific antibodies. Keywords: NMR, high ordered structure, monoclonal antibodies, multi domain name, formulation condition, 15N-direct detection 1. Introduction Developments in biotechnology have expanded the pharmacological modalities beyond the conventional small molecules. The growth provides an opportunity to establish a novel strategy to target diseases, even for those which have been hard to remedy. Among such new modalities, biologics, especially therapeutic monoclonal antibodies (mAbs), have been utilized more extensively in clinics over the last decade. There is a nearly three-fold increase in the number of therapeutic mAbs on the market in 2019, as compared to 2010, and more than 600 mAbs are currently in commercial clinical pipelines [1]. Most therapeutic mAbs are IgG1, representing the most abundant and most extensively analyzed subclass of human IgG. IgG1 is usually a tetrameric protein consisting of two heavy chains with VH, CH1, CH2, and CH3 domains, and two light chains with VL and CL domains. Structurally, VH, CH1, VL, and CL domains together form a Fab portion (Physique 1). On the other hand, two CH2 and two CH3 domains form an Fc portion. Thus, IgG1 also has dynamic nature as a multidomain protein. Fab is responsible for specific high-affinity binding to antigens, whereas the Fc region mediates immune response and effector functions. Fab and Fc fragments are connected by a hinge region, which is composed of 23 a.a. residues (216-EPKSCDKTHTCPPCPAPELLGGP-238). The two heavy chains are connected by two disulfide bridges, Cys-226 and Cys-229, at the hinge. A disulfide bridge at Cys-220 connects the light chain and heavy chain. The former half of the hinge provides flexibility to Fab, allowing IgG1 to bind to multiple antigens simultaneously. Currently, improvements in protein engineering allow for building bispecific antibodies, which can target two different epitopes using different Fabs in a single IgG [2]. Taking advantage of the multivalent nature, bispecific antibodies have been extensively utilized in clinical applications. Open in a separate window Physique 1 Schematic representation of the structure of IgG. An IgG consists of two heavy chains (yellow) and two light chains (reddish). The VH and CH1 domains from a heavy chain interact with a light chain to form a Fab portion, while CH2 and CH3 domain name dimer forms Fc portion. Fab and Fc portions are connected by the hinge region, where multiple conserved disulfide bridges are present. CH2 domain has a conserved N-glycosylation site. The Fc portion, in contrast, is usually indispensable for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis Encequidar (ADCP), and complement-dependent cytotoxicity (CDC), through Fc-FcR and Fc-Complement 1q (C1q) interactions [3]. The Fc portion is basically composed of two identical CH2 and CH3 domains, which are related to each other by a two-fold axis. Thus, each Fc portion has been considered to bind up to two FcR molecules. Indeed, two X-ray crystal structures suggested the 1:2 stoichiometry interactions, albeit the proposed models of the IgG-FcR interactions are controversial [4,5]. Structural characterization by answer NMR, along with other biophysical methods, however, revealed that this stoichiometry of the conversation is usually 1:1 in reality. The binding of FcR onto one of the two symmetrically related sites around the Fc region induces a conformational switch in the other site to prevent the binding of another FcR [6]. This fact clearly represents the importance of characterizing the conversation and structure of mAbs in physiological solutions. High ordered structures (HOSs) of mAbs are crucial in clinical applications, as these Rabbit polyclonal to Smac are directly reflected in their bioactivity [7]. The HOS of the biologics can be compromised by partial denaturation, aggregation, and chemical alterations. Tertiary structures of the biologics can be affected by the Encequidar difference in expression, purification, formulation, and storage conditions, and the undesired HOS of mAbs often reduces their efficacy and causes unexpected adverse effects. Therefore, biophysical assays to analyze their HOS should be performed throughout the research and development phases to avoid such issues. In such a circumstance, assessing the HOS.