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. Int J Mol Sci. Disulfide Bond Formation and its Impact on the Biological Activity and Stability of Recombinant Therapeutic Proteins Produced by Escherichia coli Expression System. London, 1-9. Heavy and light chains are held together by a combination of non-covalent interactions and covalent interchain disulfide bonds, forming a bilaterally symmetric structure. In general, this SDS-PAGE data matches the SEC data. Homogeneous antibody-drug conjugates via site-selective disulfide bridging. Search (2018). Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach. FOIA Moreover, the Ig-fold domains are characterized by a buried intra-domain disulfide bond, which is important for its structural stability. The pH for the digestion reaction was 5.4. Principles and engineering of antibody folding and assembly. Spectrom. This gave us insights into antibody variability as antibodies can act differently, especially when exposed to undesirable conditions (Nowak et al., 2017; Xu et al., 2018; Halley et al., 2020). Epub 2018 Oct 12. 29, 923929. Therefore, increases in shuffled bond contributions at low levels can still support our hypothesisas a protein unfolds during degradation, buried cysteine residues are exposed and can participate in disulfide bond shuffling. (2017). doi:10.4161/MABS.3.6.17922, Keywords: disulfide bond shuffling, IgG1 therapeutics, biosimilars, LC-MS/MS, protein degradation, Citation: Coghlan J, Benet A, Kumaran P, Ford M, Veale L, Skilton SJ, Saveliev S and Schwendeman AA (2022) Streamlining the Characterization of Disulfide Bond Shuffling and Protein Degradation in IgG1 Biopharmaceuticals Under Native and Stressed Conditions. Creative Commons Attribution License (CC BY). Chromatograms are zoomed in to depict the increase in aggregates and/or fragments detected in each sample across each timepoint. Biotechnol. 8, 18, 19, 25, 26 . doi:10.1016/j.bbapap.2016.05.011. Turner KB, Hardy S, Liu JL, Zabetakis D, Lee PAB, Goldman ER, Anderson GP. doi:10.4161/MABS.4.1.18347, Liu-Shin, L., Fung, A., Malhotra, A., and Ratnaswamy, G. (2018). (2019). Would you like email updates of new search results? Garland Publishing, Inc., New York, NY. The next day samples were acidified with 20% TFA and prepared for lyophilization prior to reconstitution and MS injection. JC wrote the draft manuscript. Coupling of Trastuzumab Chromatographic Profiling with Machine Learning Tools: A Complementary Approach for Biosimilarity and Stability Assessment. 12 intra-chain (red) and 4 inter-chain (orange) bonds are typical for IgG1. J. Proteome Res. Sample plates for the digestion reaction were purchased through Agilent. Development Issues: Antibody Stability, Developability, Immunogenicity, and Comparability, in Therapeutic Antibody Engineering. The five primary classes of immunoglobulins are IgG, IgM, IgA, IgD, and IgE. For example, in humans, functioning IgM antibodies have five Y-shaped units (pentamer) containing a total of ten light chains, ten heavy chains, and ten antigen-binding. 2021 Jan-Dec;13(1):1963094. doi: 10.1080/19420862.2021.1963094. Originators are referred to as OR and biosimilars are referred to as BS. The two bevacizumabs (Avastin and Avegra) are referred to as Bev throughout the manuscript. However, a complete ranking of the susceptibility of disulfide bonds in IgG1 molecules is lacking. Liu JL, Goldman ER, Zabetakis D, Walper SA, Turner KB, Shriver-Lake LC, Anderson GP. Finally, we assessed the MSMS and mass error plots (Figures 2D,E) and ensured that we were seeing good fragmentation and ion coverage. Bethesda, MD 20894, Web Policies Antibodies have evolved to function in oxidative, extracellular environments. All samples were diluted to 1.5mg/ml to load 15g of antibody on the column. ADC; IgG2; antibody-drug conjugate; conjugation process; conjugation profile; disulfide isoform; disulfide scrambling; mAb; monoclonal antibody. However, while B lymphocytes can be isolated from suspensions of spleen or lymph node cells excised from immunized animals, they have a limited life span and cannot be cultured directly to produce antibody in useful amounts. In an effort to gain more insights into the effects of shuffled disulfide bonds on protein therapeutics safety and efficacy, we designed and further optimized a semi-automated LC-MS/MS method for disulfide bond characterization on two IgG1 protein therapeuticsrituximab and bevacizumab. Datola A, Satwekar A, Barron N, DeRosa S, Tomascak B, Dawson J, Palmese A, Rossi M. Biotechnol Bioeng. Additionally, we compared originator and biosimilars versions of the drugs to determine their batch comparability and biosimilarity levels when exposed to various periods of stress. (2021). Shion, H., Du, M., and Yu, Y. Q. Abstract Therapeutic monoclonal antibodies (mAbs), primarily immunoglobin G1 (IgG1) and IgG4 with an engineered CPPC motif in its hinge region, are predominant biologics. Geddie ML, Kirpotin DB, Kohli N, Kornaga T, Boll B, Razlog M, Drummond DC, Lugovskoy AA. 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). Aggregates and fragments include summations of multiple peaks, where applicable. In IgG1s there are normally 16 disulfide bonds4 interchain and 12 intrachain (Figure 1). As the proteins are exposed to prolonged heat and shaking, a greater level of shuffling occurs. 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. Epub 2022 Nov 17. 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. As depicted in Figures 3A,C, we observed that over the course of 4 weeks under stressed conditions, both rituximab sample types had minor, possibly artificial increases in their average relative percent contribution of shuffled disulfide bonds: 0.24 0.21% to 0.51 0.11% for the originator and 0.27 0.07% to 0.35 0.08% for the biosimilar. Before In previous studies, disulfide bonds were removed by amino-acid substitution indicating that Val and/or Ala (i.e. (2020). % contribution values were based off of the entire area under the curve reported for each sample type. Please enable it to take advantage of the complete set of features! This level is low but is still worth mentioning because it was significantly greater than rituximab, which had no detectable trisulfides. Would you like email updates of new search results? The originator fragment contribution was 1.76 0.06% at 0weeks and 1.78 0.01% at 4weeks. Antibodies play a central role in the adaptive immune system, as they provide a highly specific response to foreign molecules. Based on our data, disulfide bond shuffling does occur in IgG1s, even when they are unstressed. Upon completion of the run, the gel was washed 3 times with water, shaking each time for 5min. 6, 62. doi:10.3390/bioengineering6030062, Goetze, A. M., Liu, Y. D., Zhang, Z., Shah, B., Lee, E., Bondarenko, P. V., et al. In May 2023, Frontiers adopted a new reporting platform to be Counter 5 compliant, in line with industry standards. doi:10.1080/19420862.2017.1368602, Ouellette, D., Alessandri, L., Chin, A., Grinnell, C., Tarcsa, E., Radziejewski, C., et al. official website and that any information you provide is encrypted Therefore, we compared both fed-batch and perfusion process at the intracellular-redox level to . MAbs 10, 583595. The V regions of H and L chains comprise the antigen-binding sites of the immunoglobulin (Ig) molecules. The Properties of Cysteine-Conjugated Antibody-Drug Conjugates Are Impacted by the IgG Subclass. Sapozhnikova KA, Misyurin VA, Ryazantsev DY, Kokin EA, Finashutina YP, Alexeeva AV, Ivanov IA, Kocharovskaya MV, Tikhonova NA, Popova GP, Alferova VA, Ustinov AV, Korshun VA, Brylev VA. Int J Mol Sci. The results from this study will help provide groundwork for comparative disulfide shuffling analysis by LC-MS/MS and standard analytical methodology implementation for the development and regulatory approval of biosimilars. The hinge region is the area of the H chains between the first and second C region domains and is held together by disulfide bonds. Zhang W, Wang H, Feng N, Li Y, Gu J, Wang Z. Antib Ther. We also measured more fragments than aggregates initially in rituximab, with fragment formation in the rituximab samples slightly increasing over time. Electrophoresis. HHS Vulnerability Disclosure, Help Generalized structure of an immunoglobulin (IgG). (2007). Then again, sometimes unconventional disulfide bond formation can be detrimental. J Exp . The robot added 35l of the pre-digest to the sample plate, incubated for 2.5h at 37C, then added 81l of digestion solution. MAbs. All antibodies secreted by a B cell clone are identical, providing a source of homogeneous antibody having a single defined specificity. Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules. In addition to disulfide bond shuffling, a rare modification called a trisulfide bond can occur in IgGs. Therefore, antibodies contain a number of disulfide bonds. doi:10.1007/S00216-017-0772-1, Lamanna, W. C., Mayer, R. E., Rupprechter, A., Fuchs, M., Higel, F., Fritsch, C., et al. Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies. Federal government websites often end in .gov or .mil. As mentioned in the introduction, interchain bonds are more susceptible to reduction, incomplete formation and, therefore, shuffling than intrachain bonds. By using the FASTA protein sequence and existing databases, the software was able to match the bonds detected from our samples with known, expected disulfide bonds. This means that the integrated XICs are representative of the monoisotopic, or most intense isotope, peak detected for a peptide. doi:10.1016/j.biologicals.2016.06.005, PubMed Abstract | CrossRef Full Text | Google Scholar, Cai, C. X., Schneck, N. A., Cozine, T., Ivleva, V. B., Ragheb, D., Gollapudi, D., et al. Antibodies: A Laboratory Manual. This unexpected, incorrect bonding of cysteines is referred to as disulfide bond shuffling or scrambling. The established SEC and SDS-PAGE methods were critical in demonstrating the variability in degradation pathways across IgG1 therapeutics. Anal. Colcher D, Pavlinkova G, Beresford G, Booth BJ, Choudhury A, Batra SK. Engineering disulfide bonds within an antibody Antibodies have evolved to function in oxidative, extracellular environments. The aggregate contributions also increased, yielding darker gel bands, in the stressed samples for both the bevacizumab originator and biosimilar. Characterization of Trisulfide Modification in Antibodies. Full size image It was found that the weak charge interaction of site 1 occurs among Asn26, Asn27, Ser85.1 of CL . When they are shuffled, they can have detrimental effects on the proteins stability and, therefore, potentially its safety and potency as well. Studies on structural units of the -Globulins. To assess the extent and location of disulfide bond shuffling in our monoclonal antibodies, we completed a non-reduced protein digestion using a modified version of the robots in-solution digestion protocol. By combining these methods, we were able paint a . 2018 Dec;30:11-20. doi: 10.1016/j.ddtec.2018.09.004. Stressed samples were shaking at 240 RPM, incubating at 37C for 2 or 4weeks. HHS Vulnerability Disclosure, Help This flexible hinge (found in IgG, IgA, and IgD, but not IgM or IgE) region allows the distance between the two antigen-binding sites to vary. Biotechnol Bioeng. With regards to regulatory agencies, our experimental workflow can become a standardized way to characterize expected and shuffled disulfide bonds within a protein therapeutic. Disulfide bond shuffling can negatively impact a therapeutic proteins safety and functionality by increasing its aggregation and degradation, modifying its folding, and/or reducing its target binding (Zhang et al., 2011; Sung et al., 2016; Weinfurtner, 2018). Biochem. The variant with a P14C-K64C disulfide bond shows a conformational shift in the loop conformations, . Immunoglobulin G (IgG) includes several disulfide bonds, and the pattern 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.) Assessment of Disulfide and Hinge Modifications in Monoclonal Antibodies. CHAPTER 1.4. 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. Received: 25 January 2022; Accepted: 16 February 2022;Published: 14 March 2022. Cys194, Cys373 and Cys206 are typically involved in intrachain bonds but Cys214 is typically involved in an interchain bond. MF performed LC-MS/MS analysis, generated raw data and provided instrumental method information. Pairing Alpaca and Llama-Derived Single Domain Antibodies to Enhance Immunoassays for Ricin. However, the intra-domain disulfide bond can be replaced without crucial effects on the structure and function, if the domain structure is intrinsically stable or has been stabilized by protein engineering. Authors LV and SJS were employed by the company Protein Metrics. Typically, immunoglobulin G (IgG) has 6 intra-domain disulfide bonds. 22, 6914. The concentration of TCEP used to achieve each average DAR is indicated. (Editor) (2018). (2020). The total amount of shuffled bonds and changes in shuffled bond levels over time differed between rituximab and bevacizumab. Influence of Disulfide Bond Isoforms on Drug Conjugation Sites in Cysteine-Linked IgG2 Antibody-Drug Conjugates. Representative samples from each antibody at each timepoint (0, 2, and 4weeks) were run on an Invitrogen NuPAGE 3%8% Tris-Acetate Gel. A similar story played out for bevacizumab. Cys193-Cys325 had a higher abundance for the unstressed sample. Red represents the position of native disulfide bond and the mutated amino acids. Data was processed using the Byos disulfide bond workflow (Protein Metrics, Inc.), accounting for trypsin and Lys-C cleavage. Front. A 2-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) was conducted to compare the statistical significance of BS and OR results for the same protein at the same timepoint. TABLE 1. Overview of the Regulatory Framework and FDAs Guidance for the Development and Approval of Biosimilar Products in the US. 8600 Rockville Pike Tubes were placed on an orbital shaker at 240 RPM, incubating at 37C for up to 4weeks. This bolsters our hypothesis that as proteins unfold during degradation, exposing buried cysteine residues, they increase their likelihood to form shuffled disulfide bonds. 15l of the ladder (HiMarkTM pre-stained protein standard, Invitrogen) were added into well 1. MAbs. As depicted in Figures 5, 6 and Table 1, bevacizumab was more likely to aggregate than fragment when exposed to stress while the opposite was true for rituximab. 2022 Jan-Dec;14(1):2083466. doi: 10.1080/19420862.2022.2083466. FIGURE 1. The disulfide bond structures established decades ago for immunoglobulins have been challenged by findings from extensive characterization of recombinant and human monoclonal IgG antibodies. Total shuffled bond contribution relative to the XIC sum of all identified disulfide bonds for (A) rituximab originator and biosimilar and (C) bevacizumab originator and biosimilar. 73, 6975. 2010; 106:452-61. Monoclonal antibodies are especially useful as primary antibodies in applications that require single epitope specificity and an unchanging supply over many years of use. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. By combining these methods, we were able paint a full picture on the stability of IgG1 therapeutics exposed to normal and stressed conditions. The gel was imaged using a FluorChem M Imaging System (Protein Simple). Sci. (B) Highlighted bevacizumab shuffled disulfide bond used for representation of LC-MS/MS data plots. 1 Once bound, the ADC-antigen complex is usually internalised into the malignant cell and trafficked through the endosomes and lysosomes. 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. We noticed that there was a greater decrease in the percent monomer for bevacizumab compared to rituximab (Table 1). IgG2-A and IgG2-B antibodies generated under basic conditions yielded ADCs with comparable average drug-to-antibody ratios and conjugate distributions. J Biol Chem. Data was reported as average % contribution of each peak type (monomer, aggregate and fragment) standard deviation. Inter-chain disulfide bond-reduced low molecular weight (LMW) is considered as one of quality attributes of IgG drug substance and is . 9, 12171230. 407, 7994. doi:10.1016/j.ab.2009.09.027, Pereira, N. A., Chan, K. F., Lin, P. C., and Song, Z. 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).
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disulfide bonds in antibodies
