importance of disulfide bonds in antibodies

The AUC results are shown in Figure 5B. In an oxidative environment, disulfide bonds fix and stabilize the tertiary structures of proteins. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C (H)3 domain. The native disulfide linkage patterns of all four subclasses of IgG antibodies have been . 1988) and lysozyme (Matsumura et al. Yu J, Li S, Chen D, Liu D, Guo H, Yang C, Zhang W, Zhang L, Zhao G, Tu X, Peng L, Liu S, Bai X, Song Y, Jiang Z, Zhang R, Tian W. Molecules. Analytical Biochemistry, 419(1), 17-25. Proba, K., Honegger, A., Pluckthun, A. What is the difference between an interchain and an intrachain bond? Since the CH3 domain exists as a homodimer in the native state, denaturation using GdmCl should lead to both dissociation of the dimer and unfolding of the monomers. In recent years, with the development of high titer mammalian cell culture processes to meet the rapidly growing demand for antibody biopharmaceuticals, disulfide bond reduction has been observed more frequently. As discussed before, disulfide bonds are expected to be partly reduced in therapeutic antibody preparations. Would you like email updates of new search results? HHS Vulnerability Disclosure, Help Demeler, B. UltraScana comprehensive data analysis software package for analytical ultracentrifugation experiments. The class-specific effector sites are located in the epsilon chains; these form covalent dimers via two cystine residues (Cys241 and Cys328) linking opposite C epsilon 2 domains. A major point of discussion has been the suitable choice of v. The stability of both reduced and oxidized CH3 domain was analyzed by GdmCl-induced unfolding transitions at pH 5.0 via tryptophan fluorescence. The native dimer was stable up to 1.5 M GdmCl, although it appears that the peak contained a small portion of native monomer, as indicated by the decrease in molar mass of the later eluting species. 3B). Error bars are generated from three data sets. To further characterize the nature of dissociation and unfolding of the CH3 domain, denatured SE-HPLC was conducted at various GdmCl concentrations. However, human CH3 is considerably more resistant to GdmCl-induced denaturation than the murine counterpart. Reduction of structurally important interchain disulfide bonds to liberate the free cysteines may compromise the stability of the protein 40,44. Green, L.L. Different environments around these aromatic residues may contribute to the observed dissimilarity of near-CD spectra between the two species. Azuma, H., Hayashi, T., Dent, J.A., Ruggeri, Z.M., Ware, J. Disulfide bond requirements for assembly of the platelet glycoprotein Ib-binding domain of von Willebrand factor. Size-exclusion chromatograms of reduced (A) and oxidized (B) human CH3 with their corresponding molar masses. The dimer was maintained until the GdmCl concentration reached 0.5 M. As the GdmCl concentration increased beyond 0.5 M, the reduced CH3 dimer dissociated to monomer and completed the dissociation at 1.0 M GdmCl, yielding a molar mass of 13 kDa. In: Scott D.J., et al., editors. . Signals were normalized relative to unfolded and folded values. 2020 Jan-Dec;12(1):1829336. doi: 10.1080/19420862.2020.1829336. The hinge cysteines and the cysteines linking the light and heavy chains had particularly low free sulfhydryl levels, as might be expected given the structural importance of these disulfide bonds. One of the most important aspects of ADC development for cancer is the identification of the unique antigenic target of the mAb component. It is important to note that the IgG4s with the S228P mutation still . Connecting two -sheets is a buried, highly conserved disulfide bond between cysteine residues 31 and 89. Oxygen-induced maturation of SOD1: A key role for disulfide formation by the copper chaperone CCS. Furthermore, the molar mass values decreased as hydrodynamic radii increased. Data correspond to the molecular mass of CH3 at equilibrium. The elution time of the reduced CH3 dimer was identical to that of the oxidized form. Hence, these results may have implications for the stability of the intact antibody. m) for the murine protein were 0.3 M GdmCl for the reduced form and 1.0 M GdmCl for the oxidized form (Thies et al. This distance increased to 3.2 in the modeled reduced state. 1992). 1, the dissociation dominates the overall transition. At 2.0 M GdmCl, the reduced CH3 domain eluted at 16.0 min as a homogenous peak representing the unfolded monomer with a molar mass of 13.1 kDa. (1999) showed that recombinant murine CH3 expressed in the E. coli periplasm contained the native disulfide bond. The antibody structure is complex, consisting of -sheet rich domains stabilized by multiple disulfide bridges. Thies, M.J., Mayer, J., Augustine, J.G., Frederick, C.A., Lilie, H., Buchner, J. Folding and association of the antibody domain C(H)3: Prolyl isomerization precedes dimerization. For example, the binding activity may be affected by open disulfide bonds in the CDR. FOIA Hence, Equation 1 resembles a simple two-state model as shown in Equation 3. U = 2cf 2004). 2022 Feb;106(3):1057-1066. doi: 10.1007/s00253-022-11778-5. Engineering disulfide bonds within an antibody Antibodies have evolved to function in oxidative, extracellular environments. The reversed-phase chromatography was performed with an Agilent 1100 high-performance liquid chromatography (RP-HPLC) system using Agilent ChemStation software. Intermolecular disulfide bond generally contribute to the conformational protein stability. Epub 2018 Jan 23. The far-UV spectrum (Fig. The estimated G value for the oxidized form is consistent with human CH3 dimers having a dissociation constant of <1010 M (Isenman et al. The light chain consists of a variable domain (VL) and a constant domain (CL), whereas the heavy chain is composed of a variable domain (VH) and three constant domains (CH1, CH2, and CH3). The fraction of folded CH3 was then calculated and plotted versus temperature, and the resulting curve was fit to a three-parameter sigmoid, (f = a/(1 + exp[(x x Non-classical disulfide bond structure was first identified in IgG4 and later in IgG2 antibodies. Epub 2022 Jan 24. The mobile-phase buffer used was 100 mM NaH2PO4, 150 mM NaCl at pH 5.0 in the presence of 06 M GdmCl. Goto, Y., Hamaguchi, K. Unfolding and refolding of the reduced constant fragment of the immunoglobulin light chain. How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system. Thies et al. government site. To prepare fully reduced CH3 domain, the protein was incubated in 3 M GdmCl, 0.1 M Tris-HCl, pH 7.0 with 10 mM dithiothreitol (DTT) for 1 h at room temperature. 2001) with a PDB ID of 1HZH. The effect of disulfide bonds on stability has been studied in many small globular proteins, such as ribonuclease (Pace et al. The fluorescence emission spectra (Fig. In combination, the total movement of the two cysteine side chains (26 away from each other) put these residues out of proper disulfide bond geometry. The fraction of folded CH3 was then plotted versus pH, and the resulting curve was fit to a three-parameter sigmoid, f = a/{1 + exp[(x x An official website of the United States government. The enhanced intensity was more profound for the oxidized form when compared to the reduced form. In spite of the sequence and structural similarities between the CH2 and CH3 domains and the fact that the thiol groups in the both domains were buried in the interior of molecules, the CH3 domain is more likely to exist in a partially oxidized state. The antibody structure is complex, consisting of -sheet rich domains stabilized by multiple disulfide bridges. These studies have shown that the intrachain disulfide bond has little effect on the tertiary structure of the respective domain, but its stability is significantly increased. Epub 2015 Sep 29. Likewise, Cys31, Trp45, Trp81, and Cys89 of the recombinant CH3 protein correspond to the residues at 250, 264, 300, and 308, respectively, of IGHG1_HUMAN. 1997; Thies et al. The peak at 17.2 min was mostly composed of native dimer. Biotechnol Bioeng. Goto, Y., Hamaguchi, K. Conformation and stability of the constant fragment of the immunoglobulin light chain containing an intramolecular mercury bridge. It is the major class of the five classes of immunoglobulins in human beings, IgM, IgD, IgG, IgA, and IgE. Clarification technologies for monoclonal antibody manufacturing processes: Current state and future perspectives. The free energy of dissociation (dimer to monomer change monitored by AUC; Fig. This dramatic quenching of tryptophan fluorescence signal in the folded, oxidized CH3 is likely due to the proximity of Trp45 to the disulfide bond (Fig. 2022 Aug 30;27(17):5574. doi: 10.3390/molecules27175574. Data analysis was performed using both local and global data sets using the Kdalton proprietary software developed by John Philo and Amgen, Inc. An Agilent 1100 HPLC system equipped with a diode array detector, a multi-angle laser light-scattering detector, and a refractive index detector (Wyatt Technology) was used to conduct SE-HPLC with a TSK G2000SWXL (7.8 60 mm) column. The recombinant production of therapeutic antibodies requires each chain within the multidomain complex to be accurately synthesized, correctly folded, and self-assembled. In contrast, dissociation and unfolding of oxidized CH3 domain occurred simultaneously within the same GdmCl concentration window between 1.5 and 2.5 M and therefore looks coupled. sharing sensitive information, make sure youre on a federal Approximate C However, the far-UV CD spectra of reduced murine CH3 showed a significant increase in the intensity of the signal at 220 nm (from 2500 to 7000 deg2 cm dmol1) compared to the oxidized domain (Thies et al. The cloned sequence contains four (MGSS) and six (HHHHHH) extra amino acid residues at the N- and C-termini, respectively, for expression, cloning, and purification. Hundreds more are at various stages of research and development for the treatment of a wide range of diseases, from cancer to inflammation. The https:// ensures that you are connecting to the The midpoints of the transition (C At concentrations >2.5 M, the oxidized CH3 was identified as a single species with a molar mass of 13 kDa. Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The contribution of disulfide bonds to protein stability is presumed to be mainly due to loss of conformational entropy in the unfolded state (Schellman 1955), assuming the change in enthalpy (H) is negligible. monoclonal antibody and our results show that no such alternative disulfide bonds exist. 20 . Clipboard, Search History, and several other advanced features are temporarily unavailable. Hennecke, J., Sillen, A., Huber-Wunderlich, M., Engelborghs, Y., Glockshuber, R. Quenching of tryptophan fluorescence by the active-site disulfide bridge in the DsbA protein from. Mechanism and suggested role of the thiol group of residue -93. 2/f Ashikari, Y., Arata, Y., Hamaguchi, K. pH-induced unfolding of the constant fragment of the immunoglobulin light chain: Effect of reduction of the intrachain disulfide bond. The unfolding free energy is assumed to have a linear dependence on the denaturant concentration given by G Only the soluble fraction of the lysate was purified using Ni-NTA resin (Qiagen GmbH) according to the manufacturer's protocol. Cysteines that are canonically disulfide bonded to each other tended to have similar free sulfhydryl abundances. The carboxyl-terminal regions (CH2 CH3) of two heavy chains associate with each other through two disulfide bridges in the hinge region between the CH1 and CH2 domains and by strong noncovalent interactions between the two CH3 domains (Huber et al. Abstract Engineered introduction of interface interchain disulphide bonds is perceived to be a simple method to increase the stability of single chain Fv (scFv). and transmitted securely. In an oxidative environment, disulfide bonds fix and stabilize the tertiary structures of proteins. Recombinant human monoclonal antibodies or Fc fragments have been observed to contain some amounts of reduced disulfide bond in the buried regions, especially in the CH3 domain (Zhang and Czupryn 2002; Pipes et al. It should be noted, however, that the thermal unfolding of reduced CH3 was not reversible. MeSH terms Animals Antibodies, Monoclonal / chemistry* Binding Sites Chromatography, High Pressure Liquid This disulfide bond was not required for dimerization, since a recombinant human CH3 domain, even in the reduced state, existed as a dimer. 1976; Kato et al. The fraction of folded CH3 was calculated from the red shift of the maximum wavelength of emission spectra at each pH (see details in Materials and Methods). The glycine (G) at position 5 corresponds to the amino acid position 224 of the human immunoglobulin gamma-1 chain C region (Swiss-Prot entry name: IGHG1_HUMAN, primary accession number, {"type":"entrez-protein","attrs":{"text":"P01857","term_id":"2500440404","term_text":"P01857"}}P01857). mAbs, 9(2), 182-212. In contrast, another tryptophan (Trp81) is far from the disulfide bond, and hence it is unlikely that its fluorescence is quenched by the disulfide in the folded state. Typically, immunoglobulin G (IgG) has 6 intra-domain disulfide bonds. 280. The distance between Trp45 and the disulfide bond is 3.5 . The .gov means its official. Le, H.V., Trotta, P.P. Schellman, J.A. The fluorescence measurements were performed at 20C at a protein concentration of 70 g/mL (2.6 M dimer). The Immunogenicity of antibody aggregates in a novel transgenic mouse model. This allowed direct comparison of the stability of the two forms. m values for the reduced and oxidized forms were 0.7 and 1.9 M, respectively. TCEP was purchased from Sigma Aldrich. {"type":"entrez-protein","attrs":{"text":"P01857","term_id":"2500440404","term_text":"P01857"}}. 0/b)])), using SigmaPlot software. The portion of the curve 20 nm from the wavelength maximum was fitted to a second-order polynomial equation. Arakawa, T., Tsumoto, K. The effects of arginine on refolding of aggregated proteins: Not facilitate refolding, but suppress aggregation. 5, cf. , Borisov O.V. The measurements were carried out at 20C at a protein concentration of 230 g/mL (8.6 M dimer). (1988), the entropic loss by a disulfide bond of an unfolded protein can be estimated using the following equation: where n is the number of residues in the loop forming the disulfide bond. It is also important to understand how this disulfide bond contributes to affinity. All antibodies rely on interchain disulfide bonds to maintain the correct structure for function and activity. The solid lines were fit to a three-parameter sigmoidal using SigmaPlot software. Optimal sedimentation equilibrium speeds of 34,000, 44,000, and 54,000 rpm were determined using the equilibrium speed calculation module in the Ultrascan Software version 7.3 developed by Demeler (2005). Disclaimer. This led us to hypothesize that the lack of spontaneous disulfide bond formation in the human CH3 domain may be due to an unfavorable disulfide geometry or distance between the two sulfurs of Cys31 and Cys89. When the same two-state model was applied for the unfolding event, the G values of unfolding were calculated to be 12.5 kcal/mol for reduced and 15.2 kcal/mol for oxidized form. (2011). Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. Federal government websites often end in .gov or .mil. Therefore, antibodies contain a number of disulfide bonds. 4B), indicating that oxidized CH3 is more thermally stable than the reduced form. A previous study has shown that the introduction of intermolecular disulfide bond increased the. The pH titration data were analyzed by plotting wavelength against fluorescence emission (in counts per second) for each sample. 8600 Rockville Pike The results showed that reduced CH3 domain has intact secondary and tertiary structures and that both the reduced and oxidized forms exhibited similarities, suggesting that their secondary and tertiary structures may not be significantly different. The figure depicts both dimer dissociation and monomer unfolding at various concentrations of GdmCl for reduced (Fig. For human CH3, n is 57 (residues), and hence S at 20C (TS) is estimated to be 4.1 kcal/mol. 1979). u = RT ln K The fluorescence spectra were measured using an AVIV Model ATF 105 spectrofluorometer. Modern analytical ultracentrifugation: Techniques and methods. When the purified CH3 domain from E. coli lysate was analyzed by the same method, the protein eluted at the same time as the fully reduced form, indicating that purified CH3 domain was in the reduced state. High purity grade GdmCl was purchased from MP Biomedicals, Inc. 2001). Both the reduced and oxidized CH3 domains unfolded cooperatively as the pH decreased. Disulfide bonds can make a substantial contribution to protein stability. 1989; Denton and Scheraga 1991; Taniyama et al. Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins. To eliminate the possibility of re-oxidation, 0.5 mM of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to all the reduced samples. 1988) and lysozyme (Matsumura et al. MAbs. Unable to load your collection due to an error, Unable to load your delegates due to an error. (2002) suggested that the increased far-UV signal may indicate a more compact secondary structure due to the removal of the disulfide spacer between the two -sheets. Unauthorized use of these marks is strictly prohibited. Equilibrium denaturation data are shown in Figure 5A. Du C, Huang Y, Borwankar A, Tan Z, Cura A, Yee JC, Singh N, Ludwig R, Borys M, Ghose S, Mussa N, Li ZJ. A natural antibody missing a cysteine in VH: Consequences for thermodynamic stability and folding. 1976; Harris et al. The emission is quenched in the folded state for the oxidized form because of the proximity of the disulfide bond to Trp45 (see Fig. To understand the contribution of the CH3 disulfide bond on conformation and stability, we have prepared and conducted experiments on fully reduced and oxidized forms of recombinant human CH3 domain. During manufacturing processes, all antibodies are susceptible to reduction of the interchain disulfide bonds that hold the heavy and light chains together. The human IgG1 antibody CH3 domain was expressed in the cytosol of E. coli and purified to homogeneity by Ni-affinity chromatography. government site. u = G 1998). The effect of acid-induced unfolding of the reduced and oxidized CH3 domain was monitored by fluorescence spectroscopy (Fig. Secondly, to provide a broader understanding of the disulfide bond reduction in downstream process, this paper discusses disulfide bond reduction impact on product stability, associated analytical methods for disulfide bond reduction detection and characterization, process control strategies as well as their manufacturing implementation. U are the fluorescence intensities of the native and unfolded molecules, respectively. This domain contains a single buried, highly conserved disulfide bond. Andya, J. D. , Hsu, C. C. , & Shire, S. J. Hagihara and coauthors added a novel disulfide bond by using cysteines to replace the native alanine and isoleucine at positions 49 and 70 of a sdAb . For example, tyrosine at position 37 and phenylalanine at position 68 in human are replaced by phenylalanine and tyrosine in murine, respectively. Scientists across the industry have been examining the root causes and developing mitigation strategies to address the challenge. 1988). In contrast, the oxidized form showed excellent reversibility during thermal unfolding and refolding cycles. Pipes, G.D., Kosky, A.A., Abel, J., Zhang, Y., Treuheit, M.J., Kleemann, G.R. A and B). Thus, correct disulfide bond formation in human CH3 domain is critical for both stability and dimerization. IgG1 (hereafter referred to as antibody) is a complex molecule, composed of two identical light (L) and heavy (H) chains. U = [U]2/[N2]) was used to fit the data of unfolding (Fig. The origin of the major stabilizing effect of a disulfide bond is thought to be the decrease in conformational entropy in the unfolded state of a protein (Lin et al. It's important to get hold of the idea that they do not occur in the cytoplasm, but are only formed in proteins which pass through the secretory pathway: they are formed in the endoplasmic reticulum (ER), and there are resident ER proteins (protein disulphide isomerases) whose function is to assist in their formation. (2015). The result showed that the oxidized form was more resistant to acid-induced denaturation than the reduced form by 0.3 pH unit. Furthermore, disulfide bonds have been shown to influence the folding pathways and kinetics in murine antibody CL and CH3 domains (Thies et al. Data correspond to emission intensities measured at 357 nm at an excitation wavelength of 280 nm. One light chain (VLCL) associates with the amino-terminal region (VH CH1) of one heavy chain to form an antigen-binding site (VLVH) through noncovalent interactions and the interchain disulfide bond. Lin, S.H., Konishi, Y., Denton, M.E., Scheraga, H.A. What is the importance of disulfide bonds? The latter showed a mass difference of 2 Da between oxidized and reduced forms (data not shown).

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