Introduction
Disulfide bonds (SS bonds) are formed by the oxidation of sulfhydryl groups (-SH) on two cysteines in proteins, which
is an important post-translational modification of protein therapeutics. Monoclonal antibodies (mAbs), the fastest
growing class of biotherapeutics, have a wide range of therapeutic and diagnostic applications. The higher-order
structure of mAbs plays a crucial role in efficacy and safety. For example, the number of disulfide bonds and their
location are critical quality attributes (CQA) of mAbs, as incorrect disulfide bond formation can lead to loss of
biological activity and even elicit an immune response in the host. Therefore, the confirmation of disulfide bonds
plays a very important role in the process of confirming the structure of antibody drugs.
Fig.
1. Workflow for Analysis of Disulfide Bonds by Multienzyme Digestion Combined with LC-MS/MS. (Lam AK, et
al., 2022)
Our Services
Disulfide linkage assignment, localization, and monitoring are important to ensure process consistency and product
integrity during biotherapeutic drug development. In addition, regulatory agencies have specific requirements for
mapping disulfide bonds in biotherapeutics such as monoclonal antibodies (mAbs). Here, Creative
Proteomics introduces a comprehensive protein disulfide bond analysis solution to help you in your
research, including normal, dense, and mismatch disulfide bond analysis, etc.
According to ICH Q6B guidelines, the biotherapeutics should determine the number and location of any free sulfhydryl
and/or disulfide bonds whenever possible. Peptide mapping (under reducing and non-reducing conditions), mass
spectrometry, or other appropriate techniques may be useful for this assessment. We introduce a highly sensitive
HPLC-MS/MS platform for this purpose to analysis disulfide bonds in multiple samples from different sources.
Here, our expert team provides you with comprehensive disulfide bond analysis services, including analysis of unknown
disulfide bonds, verification of disulfide bond mismatches in proteins, and disulfide bond mapping, etc.
Creative Proteomics provides one-stop disulfide bond analysis services, including sample
preparation, protein purification, enzymatic digestion, LC-MS/MS identification and bioinformatics analysis. You
just need to tell us your experiment purpose and send us your samples, we will take care of all follow-up matters of
the project.
Experiment Process
- Sample preparation: We have developed well-established sample handling techniques, such as alkylation of free
thiol side chains in the native protein state by adding reagents such as IAM, IAA or NEM, avoiding the formation
of artificial disulfides during subsequent proteolytic digestion bond, and maximizing the preservation of native
protein structure.
- Enzymatic digestion: Specific enzymes, usually trypsin, pepsin, and Glu-C are used to form abundant peptides.
- LC-MS/MS identification: We use LC-MS/MS to accurately detect all peptides of a protein and identify multiple
post-translational modifications of these peptides and map disulfide bonds.
- Bioinformatics analysis: Disulfide linkage and free sulfur analysis are achieved using different software such
as pLink-SS and SlinkS, and bioinformatics analysis will be performed.
- Detailed report: A report you received containing experimental procedures, MS raw data files, liquid
chromatography and mass spectrometer parameters, disulfide bond and cysteine analysis results, and
bioinformatics analysis.
Creative Proteomics is a reliable biopharmaceutical partner. Our professional team can provide
customers with comprehensive biopharmaceutical disulfide bond analysis service. Our services guarantee accurate and
reliable results, at quick turnaround time! If you would like more information about specific aspects of our
services, feel free to contact us and we will be happy to answer any
questions.
References
- Huang L J, Chiang C W, Chen S L, et al. (2019) Complete mapping of disulfide linkages for etanercept
products by multi-enzyme digestion coupled with LC-MS/MS using multi-fragmentations including CID and ETD. J
Food Drug Anal. 27(2):531-541.
