GEN-MKT-18-7897-A
May 11, 2016 | Biopharma, Blogs | 0 comments
Fast LC-MS acquisition and automated data processing will help you speed up peptide mapping of your biotherapeutic, including critical disulfide bond and post-translational modification characterization. SCIEX helps you untangle the complexity of disulfide bonds, speeding up your characterization process.
See the full article by downloading the compendium >
Accurate disulfide bond mapping is essential for correctly establishing structure-function relationships as well as for monitoring the structural integrity of recombinant monoclonal antibodies (mAbs) throughout their production. Inappropriate disulfide bonds can affect a mAb’s stability, potency, aggregation, and may also signal errors in the cell culture or purification process. By following a biotherapeutic’s disulfide patterns over time, manufacturers can quickly detect production problems and then correct them as early as possible.
Correctly assigning disulfide bonds in a mAb, or ADC can be challenging and time-consuming due to the heterogeneity, large size, and multiple cysteine residues found in these biomolecules. Traditional approaches for disulfide mapping are based on fast liquid chromatography-mass spectrometry (LC-MS) analysis; however, these methods can be inefficient and usually involve digestion with multiple enzymes, tedious data processing, and intensive manual inspection of chromatograms for the identification of any possible disulfide linkages.
As the biotherapeutics industry develops and expands, there is an urgent need for software tools that can rapidly facilitate and accelerate the higher-order structural characterization of biopharmaceutical products. To meet these requirements, SCIEX has developed BioPharmaView™ Software, a data processing suite that can reduce the complexity of the massive data sets generated during biotherapeutic analysis. BioPharmaView Software uses rapid processing tools to accelerate critical characterization assays–such as peptide mapping and disulfide bond identification– by automating peak assignments, simplifying data processing, and streamlining the reporting process.
Peak Assignment Reduces Time for Peptide Mapping ExperimentsTo identify and match peptides, BioPharmaView Software automatically scores b- and y- ions from the high-resolution MS/MS spectra; and then the highest scoring experimental peaks are compared to a list of theoretical masses automatically generated by the software. The peak assignment process is further enhanced by predicting the theoretical fragment ion masses for non-reduced, disulfide-linked peptides before comparison with experimental data. Including other criteria in the ion selection process–such as MS/MS scoring, multiple charge states, and a retention time (RT) filter–can also help reduce the time needed for peptide mapping experiments. This enables manufacturers to meet regulatory requirements more quickly during the production and marketing of a new biotherapeutic product.
The ResultsIn this article, we successfully developed an efficient and automated workflow that comprehensively identified every disulfide linkage in the Fab region of an mAb. The use of the high-speed, TripleTOF® LC-MS System contributed to time-savings during disulfide analysis by permitting accurate mass MS and MS/MS information to be collected simultaneously, providing the high-resolution data necessary for differentiating closely related species and confirming structural assignments. And by using BioPharmaView Software to process the dataset, identifying the location of five disulfide linkages in the Fab region of an mAb was completed in a fast and automated fashion.
See the results in the full article by downloading the Biologics Analytical Characterization Compendium >
Finding the right information shouldn’t slow you down. Whether you’re troubleshooting your mass spec, learning something new, or optimizing performance, access to the right resources at the right moment makes all the difference.
As an analytical strategy, middle-down mass spectrometry (MS) workflows characterize biotherapeutic proteins by analyzing large, digested protein fragments or defined subunits, rather than fully intact proteins (top-down) or digested peptides (bottom-up). A middle-down strategy combines the strengths of top-down and bottom-up approaches by delivering high sequence coverage and structural specificity while maintaining relatively simple sample preparation. In practice, middle-down analysis enables accurate mass measurement, rapid sequence confirmation, and localization of key post-translational modifications (PTMs) on protein subunits that are directly relevant to product quality.
In biopharmaceutical development, sequence variants (SV) are considered an inherent risk of producing complex proteins in living systems. Sequence variants are unintended changes to the amino acid sequence of a biotherapeutic and can be caused by errors in transcription or translation in the host cell, or cell culture and process conditions. Detailed analysis of SVs is important in process and product development to ensure the drug’s safety and efficacy. Even low‑level sequence variants can have significant implications for product quality, safety, and efficacy, making their accurate detection and characterization a critical requirement across development, process optimization, and regulatory submission.
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