disulfide bonds in antibodies

Chem. Copyright 2022 Coghlan, Benet, Kumaran, Ford, Veale, Skilton, Saveliev and Schwendeman. 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. We were also curious about the number of trisulfide bonds present in the samples. Antibodies were detected at dual wavelengths of 214 and 280nm. 9, 12171230. The column was maintained at room temperature. All samples were diluted to 1.5mg/ml to load 15g of antibody on the column. MAbs 10, 583595. A. Teasdale, D. Elder, and R. W. Nims. Please enable it to take advantage of the complete set of features! Non-classical disulfide bond structure was first identified in IgG 4 and later in IgG 2 antibodies. The site is secure. 21, 949959. sharing sensitive information, make sure youre on a federal Protein samples were diluted from 10mg/ml down to 0.33mg/ml with water. To further confirm this finding, we would need to perform these studies on additional lots of each drug product. doi:10.1021/AC100575N/SUPPL_FILE/AC100575N_SI_002.PDF, Liu, H., and May, K. (2012). Biotechnol. Clipboard, Search History, and several other advanced features are temporarily unavailable. Influence of disulfide bond isoforms on drug conjugation sites in cysteine-linked IgG2 antibody-drug conjugates. Trisulfide bonds detected for bevacizumab samples by LC-MS/MS. This held true for both the originator and biosimilar samples. Med. The disulfide bond structures established decades ago for immunoglobulins have been challenged by findings from extensive characterization of recombinant and human monoclonal IgG antibodies. SS designed digestion parameters using Promega materials and acted as a reference for questions regarding non-reduced digestion. The relatively low dissociation energy of disulfide bonds and the high flexibility of the hinge region frequently lead to modifications and cleavages. Liu, H., Chumsae, C., Gaza-Bulseco, G., Hurkmans, K., and Radziejewski, C. H. (2010). In contrast, the antibody disulfide configuration affected the distribution of ADCs generated under acidic conditions. With regards to regulatory agencies, our experimental workflow can become a standardized way to characterize expected and shuffled disulfide bonds within a protein therapeutic. 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. Would you like email updates of new search results? Biochim Biophys Acta. 44, 291305. 2023 Jan 3;28(1):425. doi: 10.3390/molecules28010425. For shuffled disulfide bonds, we reported the disulfide bond data as the XIC sum contribution of all shuffled disulfide bonds relative to the total XIC sum of all (shuffled and expected) detected disulfide bonds. It should be noted, though, that to further bolster our findings and ensure that intra- and inter-batch variability are not dictating our results and theorized trends, we need to perform these same studies on more than one lot per originator and biosimilar. J. Pharm. The designation of disulfide bonds as CQAs is recognized by regulatory bodies including the FDA, EMA and ICH (Christl, XXXX; Lim, 2018; Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: quality issues (revision 1), 2014; Teasdale et al., 2018). 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. For proteins produced in mammalian cell lines, such as the CHO cell lines used to produce rituximab and bevacizumab, the endoplasmic reticulum acts a center for disulfide bond modulation, checking for the proper formation of bonds (Zhang et al., 2011; Cai et al., 2021). The fragment bands at 115kDa were consistent and prominent for rituximab. Mol. doi:10.1021/ACS.JPROTEOME.0C00823/SUPPL_FILE/PR0C00823_SI_001.XLSX, Dyck, Y. F. K., Rehm, D., Joseph, J. F., Winkler, K., Sandig, V., Jabs, W., et al. When they are shuffled, they can have detrimental effects on the proteins stability and, therefore, potentially its safety and potency as well. AB performed preliminary studies to show proof of concept. 22, 6914. 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. Full size image It was found that the weak charge interaction of site 1 occurs among Asn26, Asn27, Ser85.1 of CL . Data was reported as average % contribution of each peak type (monomer, aggregate and fragment) standard deviation. Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach. Structural Comparison of Fucosylated and Nonfucosylated Fc Fragments of Human Immunoglobulin G1. Then the gel was washed with SimplyBlue SafeStain (Invitrogen) for 1h with shaking and with water for 1h with shaking. Biomed. Additionally, an ICH guidance states that scrambled/exchanged disulfide bonds are a common protein degradation mechanism (Teasdale et al., 2018). (2016). Due to this, disulfide bonds are considered to be a subset of a cysteine form critical quality attribute (CQA) for biologics. Users can select whether an isotope or an averagine calculation is applied for the label-free quantitation of samples. 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. expected vs. shuffled) was based on FASTA protein sequences. HHS Vulnerability Disclosure, Help While the disulfide bond data indicated that bevacizumab has lower stability and, subsequently, a greater chance for degradation to occur, the SEC and SDS-PAGE data told their own version of the story. FIGURE 6. For example, the larger number and hinge region arrangement of disulfide bonds an IgG2 increases its potential for covalent dimerization, which leads to an increased binding avidity (Moritz and Stracke, 2017; Weinfurtner, 2018). We also confirmed that the MS1 plots were created by using both the most abundant isotopic peak (apex identified within the pink bar across the isotope on the isotope plot panel, Figure 2C) and the MSMS scan location in relation to the retention time (shown in the blue XIC intensity plot, Figure 2F). Other common shuffled bond locations included Cys194-Cys327 (intrachain) and Cys214-Cys206. 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%). The fragments at 115 and 85kDa were larger in the 0-week bevacizumab biosimilar sample compared to the 2 and 4-week sample, but this could be accounted for by differences in the protein concentration loaded on the gel. 73, 6975. 1184, 122976. doi:10.1016/j.jchromb.2021.122976. Because it contains this heterogeneous collection of antigen-binding immunoglobulins, an antibody purified from such a sample is called a polyclonal antibody. 2018 Sep 25;20(6):103. doi: 10.1208/s12248-018-0263-0. Non-enzymatic Hinge Region Fragmentation of Antibodies in Solution. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. doi:10.1093/glycob/cwr027, Gu, S., Wen, D., Weinreb, P. H., Sun, Y., Zhang, L., Foley, S. F., et al. 82, 52195226. There were fragments present in the bevacizumab samples, but those stayed relatively steady over time. This suggests that the biosimilar should follow similar disulfide shuffling trends compared to the ones we observed in the originator. doi:10.1080/19420862.2018.1512328, Shatat, S. M., Al-Ghobashy, M. A., Fathalla, F. A., Abbas, S. S., and Eltanany, B. M. (2021). 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). Bevacizumab lots were diluted from 25mg/ml down to 10mg/ml with water to match the aliquot concentration of rituximab. In comparing biosimilars with their originators, we generally saw similar trends. 2022 Jan-Dec;14(1):2020082. doi: 10.1080/19420862.2021.2020082. JC and PK conducted optimized versions of the preliminary studies for digestion sample preparation for LC-MS/MS, SEC and SDS-PAGE. It could lead to a decrease of product purity and failure to meet the targeted product profile and/or specifications. It has been known that inter chain disulfide bonds are more susceptible to reduction than intra chain disulfide bonds. Monoclonal antibodies are especially useful as primary antibodies in applications that require single epitope specificity and an unchanging supply over many years of use. Unauthorized use of these marks is strictly prohibited. Upon completion of the run, the gel was washed 3 times with water, shaking each time for 5min. The presence of specific glycans can affect protein therapeutics potency by conferring stability, controlling conformation, altering target binding, and increasing clearance rate (Kanda et al., 2007; Matsumiya et al., 2007; Goetze et al., 2011; Zheng et al., 2011; Hmiel et al., 2015; Thomann et al., 2016; Pereira et al., 2018). government site. Clipboard, Search History, and several other advanced features are temporarily unavailable. We also saw fragments present at 115 and 85kDa in both proteins. doi:10.4161/MABS.4.1.18347, Liu-Shin, L., Fung, A., Malhotra, A., and Ratnaswamy, G. (2018). doi:10.1016/j.jchromb.2004.12.033, Dean, A. Q., Luo, S., Twomey, J. D., and Zhang, B. Analysis of Disulfide Bond Formation in Therapeutic Proteins, in Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering. Then samples were left at 37C overnight. 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. An official website of the United States government. In addition to disulfide bond shuffling, a rare modification called a trisulfide bond can occur in IgGs. Disulfide bonds are clearly important contributors to the proper functioning of a therapeutic IgG1. *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. 407, 7994. Distribution of reduced species detected, Distribution of reduced species detected in the ADCs with DAR of ~2, 4,, Average DAR of ADCs generated under alkaline (top) and acidic (bottom) conditions as, Chromatograms of partially-reduced mAbs (left, Chromatograms of partially-reduced mAbs (left column) and the resulting ADCs with average DAR, Disulfide isoform distribution of IgG2-A, Disulfide isoform distribution of IgG2-A (gray) and IgG2-B (black) mAb incubated in the, nrRP-UHPLC chromatograms of partially-reduced IgG2-A, nrRP-UHPLC chromatograms of partially-reduced IgG2-A (gray) and IgG2-B (black) mAbs generated by reactions, DSC thermograms of mAb (A) and ADC (B) with average DAR of 3.2, MeSH Federal government websites often end in .gov or .mil. When analyzing the frequency of specific disulfide bond locations, we normalized the number of times that each bond type was measured relative to the total number of disulfide bonds. Disulfide Bond Formation and its Impact on the Biological Activity and Stability of Recombinant Therapeutic Proteins Produced by Escherichia coli Expression System. Creative Commons Attribution License (CC BY). Determination of Critical Quality Attributes for Monoclonal Antibodies Using Quality by Design Principles. 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. Dan Zabetakis, 89, 48384846. doi:10.1080/19420862.2018.1553476, Zhang, E., Xie, L., Qin, P., Lu, L., Xu, Y., Gao, W., et al. Characterization of Disulfide Bonds in Bevacizumab Biosimilar Using A Q-TOF Mass Spectrometer. Singapore: National University of Singapore. Similarly, we noticed that disulfide bond shuffling trends matched those of protein degradation, as measured by SEC and SDS-PAGE. IgG2-A and IgG2-B antibodies generated under basic conditions yielded ADCs with comparable average drug-to-antibody ratios and conjugate distributions. Data was processed using the Byos disulfide bond workflow (Protein Metrics, Inc.), accounting for trypsin and Lys-C cleavage. To identify sites where intermolecular disulfide bond can be introduced into the Fab's constant. Basic & Clinical Immunology. Introduction Sulfhydryl-reactive crosslinker reactive groups Besides amine-reactive compounds, those having chemical groups that form bonds with sulfhydryls (-SH) are the most common crosslinkers and modification reagents for protein and other bioconjugate techniques. (2019). Antibody-drug conjugates (ADCs) that are formed using thiol-maleimide chemistry are commonly produced by reactions that occur at or above neutral pHs. Then again, sometimes unconventional disulfide bond formation can be detrimental. 12 intra-chain (red) and 4 inter-chain (orange) bonds are typical for IgG1. HHS Vulnerability Disclosure, Help 2017;114(6): . Glycobiology. doi:10.1080/19420862.2017.1368602, Ouellette, D., Alessandri, L., Chin, A., Grinnell, C., Tarcsa, E., Radziejewski, C., et al. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. DSC thermograms of mAb (A) and ADC (B) with average DAR of 3.2 (A isoform) and 3.4 (B isoform). The https:// ensures that you are connecting to the Disulfide bonds form between different amino acids of a protein chain and the help to stabilize and maintain a distinct three dimensional form. However, since minimal disulfide bond shuffling is expected when samples are treated at pH 7 or below (Sung et al., 2016; Dong et al., 2021), as ours were, seeing these general upwards trends in shuffling supports our hypothesis that disulfide shuffling occurs more frequently as a protein is exposed to stress and begins degrading. 109, 621. 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. Coupling of Trastuzumab Chromatographic Profiling with Machine Learning Tools: A Complementary Approach for Biosimilarity and Stability Assessment. MAbs. Cambridge: Royal Society of Chemistry, 81, 98. doi:10.1039/9781788013253-00081, Xu, Y., Wang, D., Mason, B., Rossomando, T., Li, N., Liu, D., et al. After all, certain modifications can be indicators of protein degradation, immunogenicity, improper manufacturing conditions, etc. While we recognize that correlation is not causation and other factors could be influencing IgG1 degradation propensity, this initial study justifies our further exploration into how disulfide bond shuffling and protein degradation may be linked. 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). PMC Similarly, Nie et al. 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). Bookshelf Differences in heavy chain polypeptides allow these immunoglobulins to function in different types of immune responses and at particular stages of the immune response. 8600 Rockville Pike Isotope Selection in Label-free Quantitation and its Effects in Biopharmaceutical CharacterizationSummit, NJ and Cupertino, CA: Celgene Corp. and Protein Metrics Inc. Matsumiya, S., Yamaguchi, Y., Nagano, H., Otaki, S., Satoh, M., Shitara, K., et al. A trisulfide bond is described as the insertion of a third sulfur between the cysteines of a disulfide bond. ADC; IgG2; antibody-drug conjugate; conjugation process; conjugation profile; disulfide isoform; disulfide scrambling; mAb; monoclonal antibody. Inter-chain disulfide bonds of IgG mAbs are crucial to maintaining IgG integrity. Each heavy chain has about twice the number of amino acids and molecular weight (~50,000 Da) as each light chain (~25,000 Da), resulting in a total immunoglobulin monomer molecular weight of approximately 150,000 Da. J Chromatogr A. According to our results, the bevacizumab biosimilar 0-week samples had the highest level of shuffled bonds (1.62 0.78%) which is unexpected given other trends, but this can be explained by analytical variability at such low levels as well as the relatively small sample size (n = 3). (2016) have studied disulfide bond shuffling in bevacizumab under different pH and enzymatic conditions. Garland Publishing, Inc., New York, NY. (2021). Position 224 is normally involved in an interchain bond, which may be why it was participating in the most prominent shuffled bond. The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in Escherichia coli. Moreover, the Ig-fold domains are characterized by a buried intra-domain disulfide bond, which is important for its structural stability. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (2020). The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation. The degree of scrambling (S x) of disulfide bonds involving C x was calculated as shown in Equation 1. doi:10.1007/s00216-014-8108-x, Kanda, Y., Yamada, T., Mori, K., Okazaki, A., Inoue, M., Kitajima-Miyama, K., et al. Label free quantitation was used to create an extracted ion chromatogram (XIC) from the summation of the MS1 isotope area(s) over an elution time range for the peptides resulting after digestion. (2016). We studied this to see how protein residue exposure and unfolding may differ after varying incubation times. Mass spectrometry instrumentation companies such as Waters and Shimadzu have also characterized disulfide bonds on biosimilar and originator IgG1 therapeutics to showcase how they can detect any product/batch variability on their latest platforms (Lu et al., 2020; Shion et al., 2016). 818, 115121. Epub 2005 May 3. The samples were analyzed using an Acquity LC (Waters) interfaced to an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher). Low levels of shuffled disulfide bonds were detected in both antibodies. Unable to load your collection due to an error, Unable to load your delegates due to an error. The Properties of Cysteine-Conjugated Antibody-Drug Conjugates Are Impacted by the IgG Subclass. MAbs. These were most commonly found at position Cys22-Cys96 in the variable region of the antibody, which is an expected disulfide bond location (Figure 4B). Authors LV and SJS were employed by the company Protein Metrics. NCI CPTC Antibody Characterization Program. For example, IgG1 is more closely related to IgG2, IgG3 and IgG4 than to IgA, IgM, IgD, or IgE. For the MS, a data dependent HCD mode was used with MS at a 60,000 FWHM resolution and MS/MS at a 15,000 FWHM resolution. However, a complete ranking of the susceptibility of disulfide bonds in IgG1 molecules is lacking. Annotated diagram of immunoglobulin structure. Am. We also compared originator vs. biosimilar versions of the two therapeutics to determine if there were notable variations in the disulfide shuffling and overall degradation between originator and biosimilar drug products. The percent monomer for the rituximab originator changed from 92.48 0.26% to 91.03 0.26% and the biosimilar changed from 90.79% 0.01 to 89.57 0.50% over the course of 4 weeks. 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. Copyright 2006-2023 Thermo Fisher Scientific Inc. All rights reserved, Spectroscopy, Elemental and Isotope Analysis, Antibody labeling and immobilization sites, Introduction to antibody production & purification, How to choose and select secondary antibodies, Antibody isotyping and characterization products, Distribution: intravascular and secretions, Distribution: basophils and mast cells in saliva and nasal secretions. Automated Disulfide Bond Mapping in Comparing Innovator and Biosimilar mAbs Using UNIFI Software | Waters. We confirmed that the samples marked as true positives and false positives were indeed properly labeled or changed them to true or false positives based on our manual analysis. 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. Consequently, antibody-binding proteins (e.g., Protein A or Protein G) and most secondary antibodies used in immunodetection methods cross-react with multiple subclasses but usually not multiple classes of Ig. Search Samples were run in triplicate. Federal government websites often end in .gov or .mil. Heavy and light chains are held together by a combination of non-covalent interactions and covalent interchain disulfide bonds, forming a bilaterally symmetric structure. Sample plates for the digestion reaction were purchased through Agilent. 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.) Conversely, we observed that the bevacizumab samples had more degradation in the form of aggregates. 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. Anal. Lipovsek D, Lippow SM, Hackel BJ, Gregson MW, Cheng P, Kapila A, Wittrup KD. Disulfide isoform distribution of IgG2-A (gray) and IgG2-B (black) mAb incubated in the reaction buffer for 3hours at 37C. All antibodies secreted by a B cell clone are identical, providing a source of homogeneous antibody having a single defined specificity. 400, 8998. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). MAbs. The fragment bands at 85kDa increased, becoming darker, across the 0, 2 and 4-week samples for rituximab. doi:10.1016/j.biotechadv.2011.07.013, Zheng, K., Bantog, C., and Bayer, R. (2011). Nevertheless, IgG1 therapeutics are not fully immune to disulfide bond shuffling.

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