importance of disulfide bonds in antibodies

Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomerdimer equilibrium of the human CH3 domain. 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. These observations may suggest that the disulfide formation in the hydrophobic core of CH3 domain may perturb its secondary structure for murine but not human molecule. Given this . A previous study has shown that the introduction of intermolecular disulfide bond increased the. 2020 Jan-Dec;12(1):1829333. doi: 10.1080/19420862.2020.1829333. Nonetheless, the human CH3 domain showed slightly different secondary and tertiary structural characteristics compared to the murine CH3 domain (Thies et al. The folding kinetics of the C. Kachur, A.V., Koch, C.J., Biaglow, J.E. The human IgG1 antibody CH3 domain was expressed in the cytosol of E. coli and purified to homogeneity by Ni-affinity chromatography. Tan Z, Ehamparanathan V, Ren T, Tang P, Hoffman L, Kuang J, Liu P, Huang C, Du C, Tao L, Chemmalil L, Lewandowski A, Ghose S, Li ZJ, Liu S. MAbs. Similarly, the C Furthermore, recently, it has been discovered that . Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies. Typically, immunoglobulin G (IgG) has 6 intra-domain disulfide bonds. The reduced form is shown under native conditions (0.0 M GdmCl; gray solid line) and denaturing conditions (4.8 M GdmCl; gray dotted line). Reprint requests to: Masazumi Matsumura, Department of Pharmaceutics, Amgen, Inc., One Amgen Center Drive, Mailstop 2-1-A, Thousand Oaks, CA 91320, USA; e-mail: moc.negma@imuzasam; fax: (805) 375-5794. In the near-UV spectra (Fig. Hundreds more are at various stages of research and development for the treatment of a wide range of diseases, from cancer to inflammation. The peak at 16.5 min had a molar mass of 13.9 kDa, whereas the peak at 17.2 min had a molar mass of 20.3 kDa. What is the difference between an interchain and an intrachain bond? 1988) and lysozyme (Matsumura et al. Camelidae recombinant antibodies in VHH format possess one intra-chain disulfide bond between framework residues and may have a further one to constrain the CDR3 loop. Folding-Assisted Peptide Disulfide Formation and Dimerization Since dimer dissociation is a second-order reaction that is protein-concentration dependent (Benson et al. In contrast, the far-UV spectra of reduced CL domains of both human and murine are comparable to those of oxidized forms (Goto and Hamaguchi 1986; Feige et al. Recent Mass Spectrometry-based Techniques and Considerations for For instance, at 1.0 M GdmCl the reduced CH3 domain was a dimer by SE-HPLC but monomer by AUC. However, human CH3 is considerably more resistant to GdmCl-induced denaturation than the murine counterpart. The HC and LC of the heterodimer are linked through disulfide bonds. Epub 2022 Jan 24. For antibodies, the effect of a disulfide bond on stability has been investigated using various . 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. 4B), indicating that oxidized CH3 is more thermally stable than the reduced form. 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). 2018 Apr;410(10):2467-2484. doi: 10.1007/s00216-017-0772-1. Equilibrium denaturation data are shown in Figure 5A. 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 Therefore, it is important to gain understanding of the effect of disulfide bond cleavage on different antibody functionalities. RP-HPLC chromatograms of reduced and oxidized CH3 showed that the oxidized and reduced forms eluted at 24.0 and 25.2 min, respectively (data not shown). 1992). Abstract Engineered introduction of interface interchain disulphide bonds is perceived to be a simple method to increase the stability of single chain Fv (scFv). Development issues: antibody stability, developability, immunogenicity Pace, C.N., Grimsley, G.R., Thomson, J.A., Barnett, B.J. 2018 Apr;10(3):500-510. doi: 10.1080/19420862.2018.1424609. Spectra were recorded on a PTI fluorometer at a controlled cell temperature of 20C with gentle mixing. Prior to chromatography, 200 g/mL (7.5 M dimer) of either reduced or oxidized CH3 protein was incubated in 10 mM sodium acetate buffer at pH 5.0 containing 06 M GdmCl for 3 d at 25C to reach equilibrium. However, these events were not coupled in the reduced CH3 dimer, as dissociation occurred at a different concentration of denaturant prior to unfolding. An official website of the United States government. 1999). Received 2007 Jul 20; Revised 2007 Oct 4; Accepted 2007 Oct 5. 2/f National Library of Medicine In addition, brief perspectives on the development of future mitigation strategies are also reviewed, including platform alignment, mitigation strategy application for the emerging new modalities such as bispecific and trispecific antibodies as well as using machine learning to identify molecule susceptibility of disulfide bond reduction. At the same wavelength, the magnitude at the minimum was lower for the reduced CH3 domain, showing an additional minimum at 218 nm. These observations suggest that the structure of folded CH3 is sufficiently stable to prevent action of the oxidative/reductive agents on the hydrophobic core of CH3 where the disulfide bond is located. American Association of Pharmaceutical Scientists, 5(2), 21. Size-exclusion chromatograms of reduced (A) and oxidized (B) human CH3 with their corresponding molar masses. u The excitation wavelength was set to 280 nm. Saphire, E.O., Parren, P.W., Pantophlet, R., Zwick, M.B., Morris, G.M., Rudd, P.M., Dwek, R.A., Stanfield, R.L., Burton, D.R., Wilson, I.A. Antibody disulfide bond reduction and recovery during - PubMed Keywords: Antibody stability: A key to performance - ScienceDirect Before A and B). It is the major class of the five classes of immunoglobulins in human beings, IgM, IgD, IgG, IgA, and IgE. Denton, M.E., Scheraga, H.A. What do disulfide bonds do in antibody? National Library of Medicine The purified, reduced CH3 protein was denatured and oxidized in 3 M GdmCl, 0.1 M Tris-HCl, pH 7.0, and 0.5 mM CuSO4 for 1 h at room temperature (Winterbourn and Carrell 1977). Our finding is in good agreement with previous studies conducted on the CH3 domain of murine monoclonal antibody MAK33 (Thies et al. Our results demonstrated that the disulfide bond not only stabilizes a conformation of monomeric CH3 but also enhances the dimer interactions. Bookshelf Crosslinking disulfide bonds govern solution structures of diabodies Analytical Biochemistry, 419(1), 17-25. The pH titration data were analyzed by plotting wavelength against fluorescence emission (in counts per second) for each sample. A 2-mm path-length quartz cuvette was used for far-UV spectra measurements at 218 nm. Engineering disulfide bonds within an antibody - ScienceDirect 1) may be applied to human CH3 because the accumulation of native monomer was not detected by denatured SE-HPLC (Fig. 2004) where the shift of the monomerdimer equilibrium is affected by the disulfide bond. Here, inter-domain disulfide bond would . Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules. 2B) the main minimum was observed at 273 nm in both reduced and oxidized forms, and a second minimum was observed at 293 nm. Therefore, antibodies contain a number of disulfide bonds. A two-state model (N2 2U, where K The emission is quenched in the folded state for the oxidized form because of the proximity of the disulfide bond to Trp45 (see Fig. 2001). 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. Careers, Unable to load your collection due to an error. 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. 2002). 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. The redox status of the disulfide bond of CH3 domain was investigated by RP-HPLC. official website and that any information you provide is encrypted unfold and aggregate), which leads to loss of functions and increase of adverse risks.Although engineering of an additional disulfide bond has been performed in Fc or Fc domains for optimization, the relationships between introduced disulfide bond and alteration of the . The purified protein was dialyzed against 10 mM sodium acetate, pH 5.0 at 4C. Mechanism of copper-catalyzed autoxidation of cysteine. Computer-aided interpretation of sedimentation data for proteins. m values for the human protein were determined to be 1.3 M GdmCl for the reduced form and 2.2 M GdmCl for the oxidized form. U = 2cf 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. The SARS-CoV-2 Omicron variant evades most currently approved neutralizing antibodies (nAbs) and caused drastic decrease of plasma neutralizing activity elicited by vaccination or prior infection . 1985) and endostatin (Zhou et al. Structural analysis and binding sites of inhibitors targeting the CD47/SIRP interaction in anticancer therapy. How many disulfide bonds are in IgM? A comprehensive analysis of novel disulfide bond introduction - Nature Influence of an extrinsic cross-link on the folding pathway of ribonuclease A. Conformational and thermodynamic analysis of cross-linked (lysine7-lysine41)-ribonuclease A. Matsumura, M., Becktel, W.J., Levitt, M., Matthews, B.W. It has been known that inter chain disulfide bonds are . The previous work by Thies et al. Please enable it to take advantage of the complete set of features! The lack of separation between unfolded monomer and folded dimer may be attributed by a more compact structure of the oxidized form when compared to the reduced CH3 domain in the unfolded state, which makes it more difficult to distinguish the two forms by SE-HPLC. Thisformat has proved extremely useful in the development ofanti-cancer biotherapeutics due to the associated cell killingactivities viaeffector functions located in the Fc portions ofthese antibodies. 1978). The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in. (1999) showed that recombinant murine CH3 expressed in the E. coli periplasm contained the native disulfide bond. (B) SE-HPLC elution times of reduced and oxidized CH3 () were plotted against their molecular masses with the following molecular mass standards: human antibody (IgG, 150 kDa, open squares), bovine serum albumin (BSA, 68 kDa, open triangles), and granulocyte-colony stimulating factor (G-CSF, 14 kDa, open inverted triangles). 1997; Thies et al. 4A). Purification of secreted recombinant proteins from. The figures were generated using DS ViewerPro (Accelrys). Brinkmann, U. , & Kontermann, R. E. (2017). The inset SE-HPLC chromatograms show the entire chromatograms monitored by A Approximate C 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). Tanaka, Y., Azuma, T., Hamaguchi, K. Ionization and reactivities of the thiol groups which participate in the formation of interchain disulfide bonds of Bence Jones proteins and an Fab(t) fragment. Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Becker, G.W., Hsiung, H.M. Thus, the disulfide bond stabilizes both murine and human CH3 domains. How many disulfide bonds are in insulin? In addition, neither of the tryptophan residues is located near the dimer interface, suggesting that the tryptophan fluorescence signal would not be affected by dissociation of the dimer (Isenman et al. The https:// ensures that you are connecting to the Optimization and kinetic modeling of interchain disulfide bond reoxidation of monoclonal antibodies in bioprocesses. Epub 2015 Sep 29. meas) at each denaturant concentration is related to f 1992). 20 . Reduced and oxidized CH3 were prepared to a concentration of 150 g/mL (5.7 M dimer) in 10 mM sodium acetate buffer at pH 5.0. 2007), as in the case for human CH3 in this study. Introduction MAbs and FcFPs are complex, large molecule biologics produced in cell cultures, purified from those cultures, and formulated for use in some form of stabilization buffer. This apparent disagreement may be explained by the higher protein concentration used in this experiment (7.5 M for SE-HPLC vs. 1.9 M for AUC) in order to achieve an adequate signal-to-noise ratio for the light-scattering detector. As described below, the purified CH3 protein was mostly in the reduced form (SH) and existed as homodimer in solution. The estimated G value for the oxidized form is consistent with human CH3 dimers having a dissociation constant of <1010 M (Isenman et al. For the murine CH3 domain, the reduced protein started to unfold at 0.05 M GdmCl, while the oxidized form was stable up to 0.4 M GdmCl. In addition, some of these aromatic residues are located at different positions. It is possible that the apparent lack of native monomer peak may be due to the fast association of the native monomer to native dimer. PMC The three-dimensional structure of an intact monoclonal antibody for canine lymphoma. X-, left Y-, and right Y-axes represent time, molar mass, and UV absorbance at 280 nm (A Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. (A) Reduced form; (B) oxidized form. Isenman, D.E., Lancet, D., Pecht, I. Folding pathways of immunoglobulin domains. The site is secure. At 1.5 M GdmCl, there is a split peak with two separate molar masses. 2002), as judged by their respective CD spectra. Reduced and oxidized CH3 were diluted to a concentration of 70 g/mL (2.6 M dimer) in a buffer solution containing 50 mM Tris-HCl, 20 mM sodium acetate, 20 mM MES at pH 2.010.0. TCEP was purchased from Sigma Aldrich. Antibodies are the largest class of biotherapeutics. monoclonal antibody and our results show that no such alternative disulfide bonds exist. 1979). This scenario (K HHS Vulnerability Disclosure, Help Furthermore, disulfide bonds have been shown to influence the folding pathways and kinetics in murine antibody CL and CH3 domains (Thies et al. The emission maximum occurs in the region of 357 nm in the unfolded state and is shifted to a lower wavelength (330 nm) in the folded state. 1995), it is reasonable to expect a shift in equilibrium. Approximately 200 g/mL (7.5 M dimer) of CH3 in 10 mM sodium acetate at pH 5.0 was equilibrated at the corresponding GdmCl concentration prior to chromatography. 5, cf. MeSH (A) Molecular mass determination of reduced (gray) and oxidized (black) human CH3 homodimer by dynamic light scattering. 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. antibody; disulfide bond; process development; reduction/oxidation. The CH3 domain of human IgG1 was cloned, expressed, and purified by Abgent, Inc. These structural changes could potentially impede the formation of the disulfide bond within a reduced, folded CH3 domain. Six disulphide bond locations have been cited within the literature but the potential for the broad use of each has not been examined. MAbs. It should be noted, however, that the thermal unfolding of reduced CH3 was not reversible. 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. First, this paper intends to highlight different aspects of the root cause for disulfide reduction. Goto, Y., Hamaguchi, K. Unfolding and refolding of the reduced constant fragment of the immunoglobulin light chain. This may be due to the E. coli cytoplasm having a more reductive environment than the periplasm, where disulfide bond formation is promoted (Becker and Hsiung 1986; Le and Trotta 1991). . The recombinant protein was expressed in E. coli BL21 in the presence of 1 mM isopropyl--d-thiogalactopyranoside at room temperature for 4 h. Cells were lysed with 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 1% Triton-X100, pH 8.0 buffer with protease inhibitors (Roche Applied Science). Thies et al. This implies that the disulfide bond shifts the monomerdimer equilibrium. Disulfide Bonds in Protein Folding and Stability government site. Crystal structure of a neutralizing human IGG against HIV-1: A template for vaccine design. Hence, these results may have implications for the stability of the intact antibody. N and I 1976; Deisenhofer 1981), and in fact these isolated domains exist as monomers in solution (Feige et al. 3A). The disulfide linkage of Cys31 and Cys89 in the recombinant CH3 protein corresponds to that of Cys250 and Cys308 in the human immunoglobulin -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). Background: The presence of cystines connecting antigen-binding loops in single domain antibodies is puzzling.Results: Cysteines forming such cystine are substituted, and the performance of functional antibody fragments is determined.Conclusion: An interloop disulfide bond stabilizes the domain and rigidifies the long third antigen-binding loop, leading to stronger antigen interaction . At 2.0 M GdmCl, the observed molar mass of the peak was 21.8 kDa, which was slightly smaller than that of the native dimer. 2016 Apr;113(4):698-716. doi: 10.1002/bit.25810. 1978; Tanaka et al. Subsequently the protein was refolded in 0.45 M l-arginine buffer described above, dialyzed against 10 mM sodium acetate, pH 5.0, and stored at 4C. At 1.0 M GdmCl, the protein was solely native dimer. Bethesda, MD 20894, Web Policies Measurements were obtained at 20C at a protein concentration of 50 g/mL (1.9 M dimer) in 10 mM sodium acetate, pH 5.0. Goto, Y., Hamaguchi, K. Conformation and stability of the constant fragment of the immunoglobulin light chain containing an intramolecular mercury bridge. Disulfide bonds can make a substantial contribution to protein stability. The reduced sample was measured in 2 mM sodium acetate, pH 5.0, and the oxidized sample was in 5 mM sodium acetate, pH 5.0. u Mechanisms of aggregate formation and carbohydrate excipient stabilization of lyophilized humanized monoclonal antibody formulations. 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. Accessibility Biotechnol Bioeng. The human CH3 domain contains two tryptophans, five tyrosines, and four phenylalanines, while the murine has three tryptophans, four tyrosines, and four phenylalanines. 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. Given the disulfide reduction challenges that biotech industry is facing, in this review, we provide a comprehensive scientific summary of the root cause analysis of disulfide reduction during process development of antibody therapeutics, mitigation strategies and its potential remediated recovery based on published papers. However, occasionally the protein was partially oxidized. Both human and murine CH3 domains encode the same numbers of amino acid residues (107 in total), and the amino acid sequences are 59% identical and 94% homologous. Both reduced and oxidized samples were equilibrated for a period of 24 h at room temperature and for 4 d at 4C prior to measurement. Conformational stability of dimeric proteins: Quantitative studies by equilibrium denaturation. The unfolding free energy G Optimization and applications of CDAP labeling for the assignment of cysteines. Furthermore, the equilibrium unfolding and dissociation studies using analytical ultracentrifugation (AUC) and size-exclusion HPLC (SE-HPLC) coupled with dynamic light scattering (DLS) revealed that the oxidized CH3 dimer unfolded and dissociated to monomer simultaneously. 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). The amino acid sequence of the human IgG1 CH3 domain used in this study is as follows: MGSSGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH. The nature and biological significance of the inter-epsilon chain disulfide-bond arrangement is . Here we review antibody stability under five headings: (i) measurement techniques; (ii) stability issues in expression and production (expression, proteolysis, aggregation); (iii) effects of antibody format and engineering on stability and (iv) formulation, drying and storage conditions. Also these antibodies and have been successfully produced in the cytoplasm of trxB - , gor - bacteria [ 183 ]. (1979). What structure is disulfide bonds? A three-state model for dissociation and unfolding of the dimeric protein can be expressed by the following equation (Neet and Timm 1994): Comparison between the CH3 unfolding monitored by fluorescence (closed symbols; left Y-axis) and the dimer to monomer dissociation monitored by AUC (open symbols; right Y-axis).

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