importance of disulfide bonds in antibodiesimportance of disulfide bonds in antibodies

The dimerization of the CH3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. Therefore, the addition of GdmCl induces both the dissociation of the dimer and unfolding of the monomers. (A) Molecular mass determination of reduced (gray) and oxidized (black) human CH3 homodimer by dynamic light scattering. Since dimer dissociation is a second-order reaction that is protein-concentration dependent (Benson et al. Interestingly, the native monomer peak was not observed by this technique. 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. U How do you know if a protein is a disulfide bond? Huang B, Bai Z, Ye X, Zhou C, Xie X, Zhong Y, Lin K, Ma L. Comput Struct Biotechnol J. The nature and biological significance of the inter-epsilon chain disulfide-bond arrangement is . The excitation wavelength was set to 280 nm. In such a case, a native monomer (N) or monomeric intermediate (I) is unstable and does not accumulate significantly during the transition. Kinetic role of the intrachain disulfide bond. An excitation wavelength of 280 nm was used, and emission spectra were recorded between 290 and 400 nm. 2002) and the native (oxidized) CH3 domain of human IgG1 (pFc) (Isenman et al. Our finding is in good agreement with previous studies conducted on the CH3 domain of murine monoclonal antibody MAK33 (Thies et al. Thus, the disulfide bond stabilizes both murine and human CH3 domains. Six disulphide bond locations have been cited within the literature but the potential for the broad use of each has not been examined. 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. Both reduced and oxidized CH3 were eluted at identical elution times. A pair of cysteines in close proximity will oxidatively react to form a disulfide bond that fixes and stabilizes the tertiary structure of a protein. Harris, L.J., Larson, S.B., Hasel, K.W., Day, J., Greenwood, A., McPherson, A. 1979), corresponding to a free energy change upon dissociation of >13 kcal/mol. An official website of the United States government. It should be noted, however, that the thermal unfolding of reduced CH3 was not reversible. 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. Before 1992). In the near-UV spectra (Fig. Annual Review of Biochemistry, 48(1), 961-997. 1988). Bessa, J. , Boeckle, S. , Beck, H. , Buckel, T. , Schlicht, S. , Ebeling, M. , Kiialainen, A. , Koulov A. , Boll B. , Weiser T. , Singer T. , Rolink A.G. , Iglesias A. The enhanced intensity was more profound for the oxidized form when compared to the reduced form. 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. Enhanced stability of the oxidized form against pH denaturation was also reported for the constant fragment of the immunoglobulin light chain (Ashikari et al. Conformational stability and mechanism of folding of ribonuclease T1. 1) may be applied to human CH3 because the accumulation of native monomer was not detected by denatured SE-HPLC (Fig. Biotechnol Bioeng. Alvarez, M. , Tremintin, G. , Wang, J. , Eng, M. , Kao, Y.-H. , Jeong, J. , Ling, V.T. Our result also suggests that the net contribution of the disulfide bond to the dimerization and folding is 3 kcal/mol. Careers. m values for the reduced and oxidized forms were 0.7 and 1.9 M, respectively. The distance between Trp45 and the disulfide bond is 3.5 . 2001). {"type":"entrez-protein","attrs":{"text":"P01857","term_id":"2500440404","term_text":"P01857"}}. Three-dimensional structure of immunoglobulins. However, the reduced CH3 domain was significantly less stable than the oxidized form against denaturation by GdmCl, acidic pH, and heat. Crystal Structure of Human CD47 in Complex with Engineered SIRP.D1(N80A). The three-dimensional structures of the IgG1 CH3 domain between human and murine species are very similar (Huber et al. Which amino acids can make disulfide bonds? N I Our approach achieves a prediction accuracy of 0.8702 for inter-chain disulfide bond prediction using 128 features and 0.9219 for intra-chain disulfide bond prediction using 261 features. 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. Interestingly, the G values of unfolding coincide well with those G values of dissociation. m) of heat-induced unfolding of reduced and oxidized CH3 domain were 68.5 and 75.5C, respectively (Fig. Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. Purification of secreted recombinant proteins from. 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. Inclusion in an NLM database does not imply endorsement of, or agreement with, Aliquots of 1.0 mL sample were placed in a 1-cm cuvette with a stir bar. Mechanism of copper-catalyzed autoxidation of cysteine. Taniyama, Y., Ogasahara, K., Yutani, K., Kikuchi, M. Folding mechanism of mutant human lysozyme C77/95A with increased secretion efficiency in yeast. Optimization and kinetic modeling of interchain disulfide bond reoxidation of monoclonal antibodies in bioprocesses. This is most notable at 1.0 M GdmCl, where the reduced CH3 domain is monomeric yet the majority of the protein is folded. 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 . The estimated G value for the oxidized form is consistent with human CH3 dimers having a dissociation constant of <1010 M (Isenman et al. Pharmaceutical Research, 32(7), 2344-2359. u 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. 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. u was calculated using G Epub 2018 Jan 23. Le, H.V., Trotta, P.P. Size-exclusion chromatograms of reduced (A) and oxidized (B) human CH3 with their corresponding molar masses. 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. An official website of the United States government. 2002). 1. The protein was detected by UV absorbance at 214 and 280 nm, and monomerdimer content was simultaneously monitored by a multi-angle laser light scattering detector. How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system. However, human CH3 is considerably more resistant to GdmCl-induced denaturation than the murine counterpart. 16.1. In contrast, the CH2 domains do not interact with each other, as is evident from the antibody crystal structure (Huber et al. 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. (2002) studied the stability of recombinant murine IgG CH3 domain. The partial specific volume of the CH3 domain was calculated to be 0.7218 mL/g at 25C. Disclaimer. u and Y The pH values at the midpoint of the acid-induced unfolding transition of reduced and oxidized CH3 were 3.29 and 2.97, respectively. All measurements were performed at 20C with the sample in a 1.0 0.2 cm quartz cuvette. Structural analysis and binding sites of inhibitors targeting the CD47/SIRP interaction in anticancer therapy. The unfolding free energy is assumed to have a linear dependence on the denaturant concentration given by G A 1 cm path-length quartz cuvette was used for near-UV spectra measurements, whereas a 1 mm path-length quartz cuvette was used for far-UV spectra measurements. the contents by NLM or the National Institutes of Health. 1999) (note that the value for reduced murine CH3 is not available, as refolding of the protein was not entirely reversible) and other dimeric proteins (Neet and Timm 1994). A two-state model (N2 2U, where K 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. To eliminate the possibility of re-oxidation, 0.5 mM of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to all the reduced samples. (2005) reported that an oxidatively refolded Fc fragment was partially composed of reduced CH3 domain, but CH2 domain is fully oxidized. government site. Scientists across the industry have been examining the root causes and developing mitigation strategies to address the challenge. The viewpoint is parallel (A) and perpendicular (B) to the axis of symmetry with each monomer colored separately, and the Cys and Trp residues are in stick representation. What is the importance of disulfide bonds? Kato, M., Azuma, T., Isobe, T., Hamaguchi, K. Formation of interchain disulfide bonds in Bence Jones proteins and Fab(t) fragments of immunoglobulin G through thiol-disulfide interchange. Approximate C Oxygen-induced maturation of SOD1: A key role for disulfide formation by the copper chaperone CCS. The reduced CH3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. The light scattering result showed that both reduced and oxidized human CH3 formed a dimer with an approximate molar mass of 24 kDa. 1989). All measurements were performed from 25 to 91C with a 2C temperature increment and 1 min equilibration time. 1978; Tanaka et al. The baseline equations were used to determine the fraction of folded protein using the equation below: where Y is the data at each pH value, and Y 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. This suggests that kinetic stabilization mainly contributes to the thermal stability of the rabbit antibody. The stability of hydrogen-bonded peptide structures in aqueous solution. The measurements were carried out at 20C at a protein concentration of 230 g/mL (8.6 M dimer). 2002). 1992; Proba et al. 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. Immunoglobulin G (IgG) includes several disulfide bonds, and the pattern On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format. This domain contains a single buried, highly conserved disulfide bond. The making of bispecific antibodies. More importantly, disulfide bond reduction could also impact drug safety and efficacy. 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. 1989; Denton and Scheraga 1991; Taniyama et al. 1997; Thies et al. N, where c is the total protein concentration (in monomer units) and f (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. 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. Optimization and applications of CDAP labeling for the assignment of cysteines. 2022 Feb;106(3):1057-1066. doi: 10.1007/s00253-022-11778-5. 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. Disulfide bonds can make a substantial contribution to protein stability. Unable to load your collection due to an error, Unable to load your delegates due to an error. On the other hand, the oxidized CH3 dimer was maintained until dissociation at 1.5 M GdmCl. 1984; Pace et al. Therefore, disulfide linkage mapping is an important component of mAB characterization for ensuring drug safety and efficacy. 280. 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. 1993; Furukawa et al. Huber, R., Deisenhofer, J., Colman, P.M., Matsushima, M., Palm, W. Crystallographic structure studies of an IgG molecule and an Fc fragment. the most important and common application of iodoacetyl-activated agarose is to immobilize cysteine-containing peptides for antibody purification after . The near-UV spectra were similar for both forms, though the magnitude of the signal was larger for the reduced form. As seen in Figure 2C, unfolding causes the fluorescence emission wavelength to red-shift from 330 to 357 nm, which is accompanied by an increase in the fluorescence intensity. With the recent advances in heterologous expression of antibodies, more than a dozen monoclonal antibody-based drugs have reached the market. Amzel, L. M. , & Poljak, R. J. 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. There are 328 unique antigens used in antibody-based . (A) Acid-induced denaturation of reduced () and oxidized () forms of human CH3 at pH 2.08.0. In many applications, however, thereceptor binding is sufficient to achieve the therapeuticeffect. 2002). The fluorescence maximum was plotted against pH. IgE antibodies are best known for their pathological role in allergy. (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. Hence, these results may have implications for the stability of the intact antibody. 2020 Jan-Dec;12(1):1829333. doi: 10.1080/19420862.2020.1829333. Hence, Equation 1 resembles a simple two-state model as shown in Equation 3. 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. The AUC results are shown in Figure 5B. U, where R is the gas constant and T is the absolute temperature. 3B). 2 is significantly larger than K 6B) underwent a dimermonomer transition between 2.0 and 2.75 M GdmCl, demonstrating that the oxidized dimer is more stable than the reduced dimer. 2021 Oct 1;19:5494-5503. doi: 10.1016/j.csbj.2021.09.036. 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. The pH titration data were analyzed by plotting wavelength against fluorescence emission (in counts per second) for each sample. Our results demonstrated that the disulfide bond not only stabilizes a conformation of monomeric CH3 but also enhances the dimer interactions. The midpoints of the transition (C U), where I This disulfide bond was not required for dimerization, since a recombinant human CH3 domain, even in the reduced state, existed as a dimer. 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 inset SE-HPLC chromatograms show the entire chromatograms monitored by A . Furthermore, we found that the disulfide bond could not be formed efficiently by unfolding and refolding the protein at pH 8.0 or 9.0 without copper or other redox reagents, such as a reduced/oxidized glutathione mixture. Ultracentrifugation was performed at 20C at a protein concentration of 50 g/mL (1.9 M dimer) in 10 mM sodium acetate, pH 5.0. The native disulfide linkage patterns of all four subclasses of IgG antibodies have been . Briefly, the human IgG1 CH3 domain was cloned into pET21a plasmid (Invitrogen) and expressed in E. coli strain BL21. Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies. How many disulfide bonds are in insulin? Please enable it to take advantage of the complete set of features! As a library, NLM provides access to scientific literature. Received 2007 Jul 20; Revised 2007 Oct 4; Accepted 2007 Oct 5. All measurements were performed at 20C. Second, in human CH3 the intensity of far-UV spectra at 220 nm changed very little upon the disulfide bond formation (approximately 1500 deg2 cm dmol1 for both forms). meas) at each denaturant concentration is related to f The final protein concentration during oxidation was adjusted to be 0.6 mg/mL to avoid aggregation. Laue, T.M., Shah, B.D., Ridgeway, T.M., Pelletier, S.L. These structural changes could potentially impede the formation of the disulfide bond within a reduced, folded CH3 domain. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized CH3 dimer were similar, but differences were observed. N I However, in recent years, single-domain antibodies have gained traction due to their smaller size and comparable binding affinity.Antibodies (Abs) and single-domain antibodies (sdAbs) differ in the structures of their binding sites: most significantly, single-domain antibodies lack a light chain and so have just three CDR loops. The excitation wavelength was set at 280 nm. For reduced CH3 samples, 1 mM TCEP was added to ensure the reduced state in the presence of GdmCl during the 3-d incubation. 8600 Rockville Pike 1976; Harris et al. The protein was refolded by dialyzing against 0.1 M Tris-HCl, pH 7.0 plus 0.45 M l-arginine for 24 h at 4C (Arakawa and Tsumoto 2003; Umetsu et al. HHS Vulnerability Disclosure, Help mAbs, 9(2), 182-212. 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. m) were 1.3 and 2.2 M GdmCl for the reduced and oxidized CH3 domain, respectively. 2002). 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. However, occasionally the protein was partially oxidized. U = 2cf Singh N, Arunkumar A, Chollangi S, Tan ZG, Borys M, Li ZJ. In the case of the equilibrium GdmCl denaturation curve measured by fluorescence, the data were fitted to an equilibrium two-state model corresponding to Equation 1 in the Discussion section. . At 1.5 M GdmCl, there is a split peak with two separate molar masses. The CD spectra measurements were carried out on an AVIV Model 202-01 spectropolarimeter (AVIV Biomedical). 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. Schellman, J.A. 0 denotes the unfolding free energy in the absence of denaturant and m is a measure of the cooperativity of the transition. Non-classical disulfide bond structure was first identified in IgG 4 and later in IgG 2 antibodies. While many antibody-based drugs are . 0/b)]}, using SigmaPlot software. Recently, Pipes et al. One of the most important aspects of ADC development for cancer is the identification of the unique antigenic target of the mAb component. u Glockshuber, R., Schmidt, T., Pluckthun, A. Hundreds more are at various stages of research and development for the treatment of a wide range of diseases, from cancer to inflammation. TCEP was purchased from Sigma Aldrich. A previous study has shown that the introduction of intermolecular disulfide bond increased the. For the reduced CH3 domain (Fig. For both forms of CH3, the dimer dissociation transition occurred at lower GdmCl concentrations than the monomer unfolding transition, although the two events were distributed over a narrower range of GdmCl concentrations for the oxidized form. The measurements were performed at 20C at a protein concentration of 30 g/mL (1.1 M dimer). Also, it was difficult to fully reduce the disulfide bond using DTT without partially unfolding the protein first with GdmCl. The fractions of unfolded and dimer were calculated from the data shown in Figure 5, A and B, respectively. u = G Both partial specific volume and densities of various GdmCl solutions were determined using SEDNTERP software (Laue et al. 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 fractions of unfolded protein and dimer in the figure were calculated using the original data shown in Figure 5, A and B, respectively. For convenience, only N is used to display the two equilibrium constants in Equation 2. High purity grade GdmCl was purchased from MP Biomedicals, Inc. 0/b)])), using SigmaPlot software. (1979). Although, c The emission spectra were measured for all samples, and the intensity at 357 nm was plotted. The antibody structure is complex, consisting of -sheet rich domains stabilized by multiple disulfide bridges. This implies that the disulfide bond shifts the monomerdimer equilibrium. 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. Samples used in measuring GdmCl-induced unfolding were prepared in 10 mM sodium acetate buffer at pH 5.0 in 06 M GdmCl at a protein concentration of 50 g/mL (1.9 M dimer). 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). Accessibility 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. 2B) the main minimum was observed at 273 nm in both reduced and oxidized forms, and a second minimum was observed at 293 nm. Epub 2017 Dec 18. MAbs. Green, L.L. The .gov means its official. 1975; Lamhasni et al. Therefore, it is important to gain understanding of the effect of disulfide bond cleavage on different antibody functionalities. Andya, J. D. , Hsu, C. C. , & Shire, S. J. The https:// ensures that you are connecting to the For example, tyrosine at position 37 and phenylalanine at position 68 in human are replaced by phenylalanine and tyrosine in murine, respectively. Interestingly, comparison of top features between . Thus, it is necessary to continue evolving the disulfide reduction mitigation strategies and developing novel approaches to maintain high product quality. 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. The solid lines were fit to the data using Equation 3. Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules. The result was confirmed by size-exclusion chromatography using various molecular weight standards (Fig. It is the major class of the five classes of immunoglobulins in human beings, IgM, IgD, IgG, IgA, and IgE. 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. 18, 19 In 2023, over 120 antibody-based drugs were approved by the FDA. The thermal unfolding of the reduced and oxidized CH3 domain was monitored by CD spectroscopy for changes in secondary structure. Brinkmann, U. , & Kontermann, R. E. (2017). The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). For oxidized CH3, chromatograms and molar mass are shown at 1.0 M (purple), 1.5 M (blue), 2.0 M (green), and 2.75 M (red) GdmCl. Analytical Biochemistry, 419(1), 17-25. 2002), unfolding of both forms of human CH3 was reversible under our denaturing conditions (data not shown). 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. Crystal structure of a neutralizing human IGG against HIV-1: A template for vaccine design. 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). 7B) forms. The unfolding free energy G (2015). National Library of Medicine 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. 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. 2002). The measured fluorescence intensity (I antibody; disulfide bond; process development; reduction/oxidation. The result show that the melting temperatures (T (2015). To investigate the effect of the disulfide bond on the conformation of human CH3 domain, near- and far-UV CD spectra as well as the fluorescence emission spectra were obtained for both the reduced and oxidized CH3 domains (Fig. Cysteines that are canonically disulfide bonded to each other tended to have similar free sulfhydryl abundances. This may have possible stability implications for the drug products containing intact antibody. The dimermonomer transitions observed in the SE-HPLC experiment shifted to higher GdmCl concentrations compared to the dimer dissociation monitored by AUC, especially for the reduced form. As stated previously, the CH3 domain is dimeric in the native state. 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. The protein concentration was 230 g/mL (8.6 M dimer) in all cases, and the buffer used was 2 mM sodium acetate (for reduced CH3) and 5 mM sodium acetate (for oxidized CH3) at pH 5.0. More importantly, disulfide bond reduction could also impact drug safety and efficacy. Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme. Lamhasni, S., Larsen, A.K., Barray, M., Monnot, M., Delain, E., Fermandjian, S. Changes of self-association, secondary structure, and biological activity properties of topoisomerase II under varying salt conditions. The side chains of Cys31 and Cys89 were rotated 4 and 22, respectively, away from their original locations within the oxidized form. The excitation and emission slit widths were 2 nm and 5 nm, respectively. We investigated the importance of a major component of chlorotoxin's chemical structure - four disulfide bonds - to its tertiary structure and biological function. 2001) with a PDB ID of 1HZH. Antibody disulfide bond reduction has been a challenging issue in monoclonal antibody manufacturing. This data is well supported by other reports (Azuma et al. 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. (A) Far-UV CD spectra and (B) near-UV CD spectra of reduced (gray) and oxidized (black) forms of human CH3. Since the equilibrium denaturation curves are expected to be dependent on protein concentration because of the dimeric nature of the native state, the fluorescence measurements were performed at the same protein concentration (1.9 M dimer) at pH 5.0 for both the reduced and the oxidized forms.