From our LC-MS/MS data we determined that the unstressed, 0-week bevacizumab samples trended towards higher shuffled disulfide bond levels initially when compared with rituximab samples. Antibodies: A Laboratory Manual. Automated Disulfide Bond Mapping in Comparing Innovator and Biosimilar mAbs Using UNIFI Software | Waters. (2018). doi:10.1080/19420862.2018.1512328, Shatat, S. M., Al-Ghobashy, M. A., Fathalla, F. A., Abbas, S. S., and Eltanany, B. M. (2021). In addition to the major immunoglobulin classes, several Ig subclasses exist in all members of a particular animal species. TABLE 1. 2015 Oct 9;14:158. doi: 10.1186/s12934-015-0340-3. The designation of disulfide bond type (i.e. (2011). 22 - 24 Though there is a vast body of knowledge of in-vitro disulfide reoxidation for antibodies, the majority of these studies focused on the solution environment with limited investigational conditions. Similarly, both antibodies showed degradation over the course of the 4-week incubation, but the two varied in how they changed over time. Would you like email updates of new search results? (1976). Non-classical disulfide bond structure was first identified in IgG 4 and later in IgG 2 antibodies. Disulfide bond structures of IgG molecules - PMC - National Center for Alkaline environments can promote disulfide bond scrambling, and may result in the reconfiguration of interchain disulfide bonds in IgG antibodies, particularly in the IgG2 and IgG4 subclasses. Cambridge: Woodhead Publishing, 377595. (2021). These bonds are all normally involved intrachain binding. doi:10.1002/elps.201600425, Nie, S., Greer, T., Huang, X., Zheng, X., and Li, N. (2022). In addition to monitoring the relative contribution of the shuffled disulfide bonds, we also monitored the location of the shuffled bonds to see whether they would change over time (Figure 3B). An official website of the United States government. For the trisulfide bonds, we repeated the same process looking at the total XIC sum of all trisulfides bonds compared to the XIC sum of all detected disulfide bonds. Biotechnol. The x-axis lists the reduced unconjugated and conjugated species, where L and H are light and heavy chains, respectively, and 0, 1, 2, 3, 4, 5 refer to the number of attached DL. Inter-chain disulfide bond-reduced low molecular weight (LMW) is considered as one of quality attributes of IgG drug substance and is . Unauthorized use of these marks is strictly prohibited. Samples were incubated 0, 2 or 4weeks at 37C, shaking at 240RPM. Biochim Biophys Acta. doi:10.1080/19420862.2018.1553476, Zhang, E., Xie, L., Qin, P., Lu, L., Xu, Y., Gao, W., et al. Optimization and kinetic modeling of interchain disulfide bond Careers. doi:10.1080/19420862.2018.1440165/SUPPL_FILE/KMAB_A_1440165_SM5845.ZIP. 8600 Rockville Pike Homogeneous antibody-drug conjugates via site-selective disulfide bridging. Sites, D.P., et al. Agency. doi:10.1093/glycob/cwr027, Gu, S., Wen, D., Weinreb, P. H., Sun, Y., Zhang, L., Foley, S. F., et al. . Many published studies have characterized aggregation/degradation profiles of these and other IgG1 therapeutics, but few have focused on providing, improving and/or optimizing methods by which to measure disulfide bond shuffling. Gray circles indicate IgG2-A; black circles, IgG2-B; white circles, IgG2 starting material containing a mixture of A, A/B, and B isoforms. Author MF was employed by the company MS Bioworks. As mentioned in the introduction, interchain bonds are more susceptible to reduction, incomplete formation and, therefore, shuffling than intrachain bonds. Its most prominent shuffled bond location was at Cys194-Cys373 for all samples except bevacizumab originator at 4-weeks, whose most prominent location was Cys214-Cys206. Therefore, we compared both fed-batch and perfusion process at the intracellular-redox level to . This bolsters our hypothesis that as proteins unfold during degradation, exposing buried cysteine residues, they increase their likelihood to form shuffled disulfide bonds. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. To further confirm this finding, we would need to perform these studies on additional lots of each drug product. The gel was run under the following conditions: 150V, 50mA, 5W for 1h on a PowerEase500 electrophoresis system (Invitrogen). A trisulfide bond is described as the insertion of a third sulfur between the cysteines of a disulfide bond. Available at: https://www.waters.com/nextgen/en/library/application-notes/2016/automated-disulfide-bond-mapping-comparing-innovator-biosimilar-mabs.html (Accessed December 10, 2021). Colcher D, Pavlinkova G, Beresford G, Booth BJ, Choudhury A, Batra SK. The column was maintained at room temperature. Biotechnol., 14 March 2022, View all For the LC, a 30-minute reverse phase gradient was used. Aside from glycans, there are other noteworthy PTMs that influence protein activity and safety, including deamidation at asparagine and glutamine residues, oxidation at methionine and tryptophan residues, and disulfide bond shuffling (Strohl and Strohl, 2012). Recently, additional intra-domain disulfide bonds have been shown to stabilize Ig-fold domains and whole IgGs. Chem. Rapid, Automated Characterization of Disulfide Bond Scrambling and IgG2 Isoform Determination. Gray lines are IgG2-A and black lines are IgG2-B. Rituximab lots were aliquoted in 50l increments into 0.5ml Eppendorf tubes. Each sample was run in triplicate and results were reported as averages standard deviation. Then again, sometimes unconventional disulfide bond formation can be detrimental. Bioanal. Disulfide bonds play a crucial role in folding and structural stabilization of monoclonal antibodies (mAbs). Front. Each immunoglobulin molecule is made up of two heavy chains (green) and two light chains (yellow) joined by disulfide bonds so that each heavy chain is (more.) Disulfide bond structures of IgG molecules: structural variations, chemical modifications and possible impacts to stability and biological function. MAbs 10, 583595. (2019). 2023 Jan 3;28(1):425. doi: 10.3390/molecules28010425. Samples were run in triplicate. Free thiols, unexpected linkages and modifications such as trisulfides are embedded within this CQA (Alt et al., 2016). Quality by Design-Based Assessment for Analytical Similarity of Adalimumab Biosimilar HLX03 to Humira. Geddie ML, Kirpotin DB, Kohli N, Kornaga T, Boll B, Razlog M, Drummond DC, Lugovskoy AA. This correlates with the greater percent decrease in monomer for bevacizumab as detected by SEC across the 4-week incubation. *Correspondence: Anna A. Schwendeman, annaschw@med.umich.edu, These authors have contributed equally to this work and share first authorship, Characterization of Biotherapeutic Products, Front. Lu, Y., Seng Wong, C., Xing, J., and Zhan, Z. Drug Discov Today Technol. For the 4-week samples, Cys214-Cys206 (80.97 16.49) became the most prominent disulfide bond location for the originator while Cys194-Cys327(42.20 7.26) and Cys194-Cys373 (45.39 8.10) were nearly equal in their contribution for the biosimilar (Figure 3D). Med. Also on the robot were two other plates, one containing an AccuMAP low pH resistant rLys-C pre-digest and a second containing a digestion solution comprised of 10x low pH reaction buffer, AccuMAP low pH resistant rLys-C, Trypsin Platinum and water. 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. Because it contains this heterogeneous collection of antigen-binding immunoglobulins, an antibody purified from such a sample is called a polyclonal antibody. The pH for the digestion reaction was 5.4. Stressed samples were shaking at 240 RPM, incubating at 37C for 2 or 4weeks. Heavy and light chains are held together by a combination of non-covalent interactions and covalent interchain disulfide bonds, forming a bilaterally symmetric structure. 29, 923929. HHS Vulnerability Disclosure, Help Post translational modifications (PTMs) have been shown to negatively impact protein efficacy and safety by altering its native conformation, stability, target binding and/or pharmacokinetics. Data was reported as average % contribution of each peak type (monomer, aggregate and fragment) standard deviation. (2021). Antibodies are classified into subclasses based on minor differences in the heavy chain type of each Ig class. PMID:20178122 [Google Scholar] 13. MAbs. Alt, N., Zhang, T. Y., Motchnik, P., Taticek, R., Quarmby, V., Schlothauer, T., et al. Epub 2018 Oct 12. Full article: Detection and quantification of free sulfhydryls in A Multidimensional Analytical Comparison of Remicade and the Biosimilar Remsima. Disulfide Bond - an overview | ScienceDirect Topics How many disulfide bonds are in an antibody? - Studybuff - Knowing too Anal. Learn more: Antibody isotyping and characterization Learn more: Antibody purification methods. Heavy and light chains are held together by a combination of non-covalent interactions and covalent interchain disulfide bonds, forming a bilaterally symmetric structure. Abstract. The XIC is then integrated to determine area under the curve, and this integrated value is compared with other peptides to report the relative abundances of the peptide. These bonds are then able to participate in forming the connection between the antibody and the drug (Liu-Shin et al., 2018). Bethesda, MD 20894, Web Policies Lim, S. (2018). National Library of Medicine Cys193-Cys325 had a higher abundance for the unstressed sample. sharing sensitive information, make sure youre on a federal While there are five different types of heavy chains, there are only two main types of light chains: kappa () and lambda (). JC, AB, and AS proposed the research topic and conceptualized the research studies. We also measured more fragments than aggregates initially in rituximab, with fragment formation in the rituximab samples slightly increasing over time. The addition of 17l of this solution into the protein plate, followed by a 30-minute incubation at 37C, yielded denatured mAbs with blocked free cysteines. LV, SJS, AS, and AB edited the manuscript. The https:// ensures that you are connecting to the Toward Drug-Like Multispecific Antibodies by Design. Conversely, we observed that the bevacizumab samples had more degradation in the form of aggregates. doi:10.1007/S00216-019-02298-9, Kshirsagar, R., McElearney, K., Gilbert, A., Sinacore, M., and Ryll, T. (2012). Because an individual B lymphocyte produces and secretes only one specific antibody molecule, clones of B lymphocytes produce monoclonal antibodies. Generalized structure of an immunoglobulin (IgG). We also saw fragments present at 115 and 85kDa in both proteins. Chem. Turner KB, Hardy S, Liu JL, Zabetakis D, Lee PAB, Goldman ER, Anderson GP. The originator fragment contribution increased from 6.76 0.24% (0weeks) to 7.61 0.24% (4weeks) and the biosimilar fragment contribution increased from 7.09 0.05% (0weeks) to 8.02 0.38% (4weeks). Soc. Assessment of disulfide and hinge modifications in monoclonal antibodies 17, 104118. Thomann, M., Reckermann, K., Reusch, D., Prasser, J., and Tejada, M. L. (2016). (2018). 407, 7994. 10, 12001213. Post-translational Structural Modifications of Immunoglobulin G and Their Effect on Biological Activity. 412 (412), 763775. Influence of disulfide bond isoforms on drug conjugation sites in cysteine-linked IgG2 antibody-drug conjugates. Bevacizumab had a general upward trend in shuffled disulfide bond levels over the course of a 4-week incubation (0.58 0.08% to 1.46 1.10% for originator) whereas rituximab maintained similar levels throughout the incubation (0.24 0.21% to 0.51 0.11% for originator). 2021 Nov 27;22(23):12845. doi: 10.3390/ijms222312845. To address this lack of knowledge, we have designed a semi-automated, streamlined method for characterizing disulfide bonds on two IgG1s, rituximab and bevacizumab, using an Agilent AssayMAP Bravo liquid handling platform and LC-MS/MS. 15l of the ladder (HiMarkTM pre-stained protein standard, Invitrogen) were added into well 1. Acta (Bba) - Proteins Proteomics. No use, distribution or reproduction is permitted which does not comply with these terms. 32, 18371840. Silver Springs, MD: FDA, Cordoba, A. J., Shyong, B.-J., Breen, D., and Harris, R. J. Dong et al. Tubes were placed on an orbital shaker at 240 RPM, incubating at 37C for up to 4weeks. Khetan R, Curtis R, Deane CM, Hadsund JT, Kar U, Krawczyk K, Kuroda D, Robinson SA, Sormanni P, Tsumoto K, Warwicker J, Martin ACR. Bioeng. Eur. Delicate balance among thermal stability, binding affinity, and conformational space explored by single-domain V. Untargeted proteomics reveals upregulation of stress response pathways during CHO-based monoclonal antibody manufacturing process leading to disulfide bond reduction. Evaluation of Factors Influencing Antibody Reduction for Development of As a post-translational modification, a disulfide bond is formed by reoxidizing two neighboring free cysteine residues. MAbs. AB and AS designed the experiments. Alberts, B., et al. The solid lines are the mAbs incubated at pH 5.3 and dashed lines are the mAbs incubated at pH 8.9. nrRP-UHPLC chromatograms of partially-reduced IgG2-A (gray) and IgG2-B (black) mAbs generated by reactions at pH 8.9 (left column) and pH 5.3 (right column). Disulfide bonds are made in nearly one-third (7000) of the proteins in the eukaryotic proteome,11 many of which are destined for contact with the relatively nonreducing extracellular environment as secretory or cell surface proteins. mAbs. 12 intra-chain (red) and 4 inter-chain (orange) bonds are typical for IgG1. The bevacizumab biosimilar samples saw a marginal increase, potentially due to analytical variability, between the 2-week (1.10 0.50%) and 4-week samples (1.25 0.20%). Site-selective modification strategies in antibody-drug - www.rsc.org Therefore, the hinge region and disulfide bonds require specific consideration during quality assessment of mAbs. 2014 Nov;1844(11):2024-2031. doi: 10.1016/j.bbapap.2014.06.004. This means that the integrated XICs are representative of the monoisotopic, or most intense isotope, peak detected for a peptide. and transmitted securely. Mol. 11, 239264. Both protein types also have a variety of other less abundant fragments below the main monomer band at 150kDa. AAPS J. Biotechnol. (2005). Hybridoma clones may be grown in cell culture for collection of antibodies from ascites fluid. The percent monomer for the bevacizumab originator changed from 95.34 0.33% to 91.14 0.38% and the biosimilar changed from 94.78 0.02% to 87.65 0.53% over the course of 4weeks. Trisulfide bonds detected for bevacizumab samples by LC-MS/MS. Open Access Published: 21 January 2020 Heavy chain dimers stabilized by disulfide bonds are required to promote in vitro assembly of trastuzumab Merc Farrs, Ramn Romn, Marc Camps, Joan Miret, scar Martnez, Xavier Pujol, Antoni Casablancas & Jordi Joan Cair BMC Molecular and Cell Biology 21, Article number: 2 ( 2020 ) Cite this article 10:862456. doi: 10.3389/fbioe.2022.862456. Antibody (or immunoglobulin) molecules are glycoproteins composed of one or more units, each containing four polypeptide chains: two identical heavy chains (H) and two identical light chains (L). Representative SEC chromatograms at 214nm for 15g of antibody. By identifying shuffled disulfide bonds upfront, companies can save themselves the inevitable headache that will occur if a product fails to meet its designated specifications. Upon completion of the run, the gel was washed 3 times with water, shaking each time for 5min. The site is secure. Fc region is shown in black and Fv region is shown in red. The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation. The Impact of Glycosylation on Monoclonal Antibody Conformation and Stability. Targeting Cancer with Antibody-Drug Conjugates: Promises and Challenges. official website and that any information you provide is encrypted Full article: Rapid, automated characterization of disulfide bond Disulfide Bonds in Protein Folding and Stability They also exemplify the uniqueness and complexity of protein therapeutics, as well as documents how not all IgG1s can be expected to act similarly. It was shown that the arrangement of disulfide bonds in this antibody subtype supports dimerization and disulfide bond shuffling: (i) IgG2 MAbs frequently exhibit low affinity to their antigens and were found to occur as bindingactive dimers . Generated data depicted as a (C) MS1 plot; (D) MSMS plot; (E) Mass error plot; and (F) XIC intensity plot. The established SEC and SDS-PAGE methods were critical in demonstrating the variability in degradation pathways across IgG1 therapeutics. Keywords: Spectrom. Being able to identify, locate and quantify protein modifications is paramount when developing new biologics and biosimilars. B. (2007). Although we recognize that degradation and disulfide shuffling are not directly proportional, completing these studies helps justify future research and innovation in this space. Thermo Fisher Scientific. Gray solid lines are the A isoforms and black dashed lines are the B isoforms. Polyclonal antibodies, which are generally purified directly from serum, are especially useful as labeled secondary antibodies in immunoassays. Characterization of Disulfide Bonds in Bevacizumab Biosimilar Using A Q-TOF Mass Spectrometer. Please enable it to take advantage of the complete set of features! From our resulting data, we noticed differences in how the two proteins degraded. One PTM in particular, shuffled disulfide bonds, has been linked to decreased potency and increased immunogenicity of protein therapeutics. 2010; 106:452-61. Given the prevalence of disulfide bonds in many natural products, considerable effort has been invested in site-selective disulfide bond formation approaches to control the folding of chemically synthesized peptides and proteins.