Antibody disulfide bonds analysis
- The number of disulfide bridges and free sulfhydryl groups
- The position of disulfide bridges in both non-reduced and reduced protein
- Combine with other characterization services
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Set up meetingCombining mass spec analyses for mAb characterization and batch-to-batch comparison
See moreProtein characterization for optimization of manufacturing processes.
See moreHigh-throughput characterization analysis of antibodies for fast quality assessment
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Mass spectrometry analysis is very useful for the characterization of disulfide bridges in biopharmaceutical proteins and mAb. You can use it both under reducing and non-reducing conditions. At Alphalyse we combine the results from intact mass analysis with peptide mapping, using one or more proteases.
Intact mass:
We use a Bruker Q-TOF Impact instrument for analysis of the protein/antibody. We thus analyze both by direct injection of a reduced and a non-reduced sample. The raw data is smoothed and de-convoluted using MaxEnt algorithm.
Peptide mapping:
Our analysts first digest the protein/antibody sample in two different ways; one half without reduction and the other half after reduction. An Agilent 1200 system then separates the peptide samples. The system is connected to a high-resolution Q-Tof mass spectrometer (Bruker Maxis Impact). It runs in Data Dependent Analysis mode (DDA), and switches between MS and MS/MS mode. Finally, we search the MS/MS data against a FASTA database with sequences of the protein/antibody, using MassAI software.
The final report includes a comparison of the intact mass data. We thus use this comparison to determine the total number of bridges in the protein/antibody. We also include a table with information about all identified disulfide-bridged peptides including relative quantity of each. Potentially scrambled disulfide bonds are also considered.
mAb characterization by mass spectrometry requires a pure sample in sufficient amounts to obtain good data. It is thus important that samples are prepared in a clean laboratory to avoid contamination with human keratin.
Samples can be submitted in liquid or lyophilized.