Biologics such as monoclonal antibodies are much more complex than small-molecule drugs, which raises challenging questions for the development and regulatory evaluation of follow-on versions of such biopharmaceutical products (also known as biosimilars) and their clinical use once patent protection for the pioneering biologic has expired. With the recent introduction of regulatory pathways for follow-on versions of complex biologics, the role of analytical technologies in comparing biosimilars with the corresponding reference product is attracting substantial interest in establishing the development requirements for biosimilars. Here, we discuss the current state of the art in analytical technologies to assess three characteristics of protein biopharmaceuticals that regulatory authorities have identified as being important in development strategies for biosimilars: post-translational modifications, three-dimensional structures and protein aggregation.
Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin ␥1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the Fc␥RIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. Fc␥RIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications. Molecular & Cellular Proteomics 9:1716 -1728, 2010.
The function, efficacy, and safety of protein biopharmaceuticals are tied to their three-dimensional structure. The analysis and verification of this higher-order structure are critical in demonstrating manufacturing consistency and in establishing the absence of structural changes in response to changes in production. It is, therefore, essential to have reliable, high-resolution and high sensitivity biophysical tools capable of interrogating protein structure and conformation. Here, we demonstrate the use of hydrogen/deuterium exchange mass spectrometry (H/DX-MS) in biopharmaceutical comparability studies. H/DX-MS measurements can be conducted with good precision, consume only picomoles of protein, interrogate nearly the entire molecule with peptide level resolution, and can be completed in a few days. Structural comparability or lack of comparability was monitored for different preparations of interferon-β-1a. We present specific graphical formats for the display of H/DX-MS data that aid in rapidly making both the qualitative (visual) and quantitative assessment of comparability. H/DX-MS is capable of making significant contributions in biopharmaceutical characterization by providing more informative and confident comparability assessments of protein higher order structure than are currently available within the biopharmaceutical industry.
The phosphorylation of IkappaB by the IKK complex targets it for degradation and releases NF-kappaB for translocation into the nucleus to initiate the inflammatory response, cell proliferation, or cell differentiation. The IKK complex is composed of the catalytic IKKalpha/beta kinases and a regulatory protein, NF-kappaB essential modulator (NEMO; IKKgamma). NEMO associates with the unphosphorylated IKK kinase C termini and activates the IKK complex's catalytic activity. However, detailed structural information about the NEMO/IKK interaction is lacking. In this study, we have identified the minimal requirements for NEMO and IKK kinase association using a variety of biophysical techniques and have solved two crystal structures of the minimal NEMO/IKK kinase associating domains. We demonstrate that the NEMO core domain is a dimer that binds two IKK fragments and identify energetic hot spots that can be exploited to inhibit IKK complex formation with a therapeutic agent.
The aden late cyclase of Bordetella pertussis is stimulated 100-to 1000I old in a dose-dependent manner by calf brain calmodulin. The system has the following properties. (i) The activation is prevented by ethylene glycol bis(faminoethyl ether) NNN :Ft enzyme * calmodulin * Ca2+(active).The relatively few adenylate cyclases so far shown to be regulated by calmodulin are from neural tissues (7-14), adrenal medulla (15), and pancreatic islets (16). -It seemed reasonable, therefore, to investigate the effect of this regulator protein on B. pertussis adenylate cyclase. The present study shows activation of this enzyme up to 100-to 1000-fold and is an example of such an effect of calmodulin in a prokaryote. METHODSGrowth of the Organism. B. pertussis (strain 114) organisms were grown from a 5% inoculum in 250 ml of modified Stainer-Scholte medium (in 500-ml flasks) on an oscillating shaker at 35.50C with room air as the gas phase as described (17); incubation was for 20-24 hr. The flasks were then stored at 4°C and the suspension was used for several weeks unless otherwise indicated. Under these conditions, basal adenylate cyclase activity increased with time but the maximal stimulation attainable did not change.Supernatant culture medium which contains 0.1-0.2 of the total cyclase activity (18) was obtained by centrifugation of the suspension at 5000 X g for 10 min. Washed cells were prepared from the cell pellet by resuspending in 0.154 M NaCl that had been passed through a Chelex-100 column to remove divalent cations. This was repeated four times. ,Ml containing 60mM N-tris(hydroxymethyl)methylglycine-HCI (pH 7.8), 10 mM MgCl2, 1 mM [a-32P]ATP (1-1.5 X 106 cpm), and 10 Al of the bacterial suspension. GTP has no effect on this enzyme and was not included. The order of addition of the components of the assay mixture proved to be of importance. In some experiments, bacteria and activator were preincubated for 10 min and then the reaction was started by addition of the prewarmed substrate and was allowed to run for 10 min as described (1, 2). In most assays, the bacteria were added lastto start the reaction as described in the legends.Preparation of Calmodulin. Calmodulin was prepared from calf brains as described by Klee (20) with the following modifications: (i) the starting material was the first warm high-speed supernatant from microtubule preparations (see ref. 21 for details on procedures used for obtaining this supernatant), and (ii) the gel filtration step used a composite column (5 X 68 cm)
Protein function is dictated by protein conformation. For the protein biopharmaceutical industry, therefore, it is important to have analytical tools that can detect changes in protein conformation rapidly, accurately and with high sensitivity. In this paper we show that hydrogen/deuterium exchange mass spectrometry (H/DX-MS) can play an important role in fulfilling this need within the industry. H/DX-MS was used to assess both global and local conformational behavior of a recombinant monoclonal IgG1 antibody, a major class of biopharmaceuticals. Analysis of exchange into the intact, glycosylated IgG1 (and the Fab and Fc regions thereof) showed that the molecule was folded, highly stable and highly amenable to analysis by this method using less than a nanomole of material. With improved chromatographic methods, peptide identification algorithms and data-processing steps, the analysis of deuterium levels in peptic peptides produced after labeling was accomplished in 1–2 days. Based on peptic peptide data, exchange was localized to specific regions of the antibody. Changes to IgG1 conformation as a result of deglycosylation were determined by comparing exchange into the glycosylated and deglycosylated forms of the antibody. Two regions of the IgG1 (residues 236-253 and 292-308) were found to have altered exchange properties upon deglycosylation. These results are consistent with previous findings concerning the role of glycosylation in the interaction of IgG1 with Fc receptors. Moreover, the data clearly illustrate how H/DX-MS can provide important characterization information on the higher order structure of antibodies and conformational changes that these molecules may experience upon modification.
Electrophoresis of microtubule preparations purified from calf brain by repeated cycles of assembly and disassembly shows that they contain many proteins in addition to alpha- and beta-tubulin. These additional proteins constitute about 17% of the total material present after five cycles of assembly and disassembly. Both one-dimensional and two-dimensional (P.H. O'Farrell (1975), J. Biol. Chem. 250, 4007) electrophoretic techniques have been used to characterize them. They can be divided into two groups: one that contains proteins which remain in constant quantitative ratio to tubulin during the purification cycles, and one composed of proteins which are removed during purification, although inefficiently. Gel-filtration chromatography of cold-depolymerized microtubule preparations yields a polydisperse fraction of high molecular weight containing most of the non-tubulin proteins. This fraction contains flexible filaments about 100 A in diameter similar to those reported by R.A.B. Keats and R.H. Hall ((1975), Nature (London) 247, 418). It is suggested that these fibers are neurofilaments, and that they may be the major source of the group of inefficiently removed proteins.
Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.