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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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Map the disulfide bridges in biopharmaceutical proteins and mAbs

Problems with protein stability and activity often correlate with an abnormal pattern of disulfide bonds. For many proteins and mAbs, disulfide bridges are essential for optimal biologic function.

Thus, the ICH Q6B Guidelines requires an analysis of the disulfide bonds for biopharmaceutical proteins and antibodies. This include both determination of the numbe...

Map the disulfide bridges in biopharmaceutical proteins and mAbs

Problems with protein stability and activity often correlate with an abnormal pattern of disulfide bonds. For many proteins and mAbs, disulfide bridges are essential for optimal biologic function.

Thus, the ICH Q6B Guidelines requires an analysis of the disulfide bonds for biopharmaceutical proteins and antibodies. This include both determination of the number and positions of free sulfhydryl-groups and disulfide bridges.

Our disulfide bridge analysis includes:

Part 1: Determination of number of disulfide bonds and free sulfhydryl groups

  • Determination of the exact molecular weight (MW) of the native protein by UV-LC ESI-MS. Analysis of non-reduced protein with intact disulfide bonds
  • Determination of the exact molecular weight of reduced protein by UV-LC ESI-MS
  • Calculate the number of both disulfide bridges and free thiol groups

Part 2: Position of disulfide bonds determined by peptide mapping of  non-reduced and reduced protein

  • Digestion of non-reduced protein/antibody using 2-4 enzymes followed by peptide mapping
  • Digest of the reduced protein/antibody with 2-4 enzymes.
  • Compiling data in detailed report

We conduct the analysis at conditions that keep scrambling effects to a minimum. 

Not exactly what you are looking for? Send us a request so we can adjust the analysis to meet your specific needs.

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The process:

  • 1 Contact us to discuss your project and receive a project proposal
  • 2 We determine the number and position of disulfide bonds and free sulfhydryl groups
  • 3 You receive a report with a clear presentation of the findings
  • 4 Follow-up by phone or email

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  • "With the results from the extensive characterization, we identified a scale-up problem and could optimize the process to increase mAb stability."

    Combining mass spec analyses for mAb characterization and batch-to-batch comparison

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We would like to help you as much as possible with your project and therefore provide several kinds of customer support:

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Technical details

Introduction to disulfide bridge mapping

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.

Antibody disulfide bonds Analysis procedure

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.

Report:

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.

Disulfide bridge mapping of mAb

Identification of disulfide bridges in protein

Molecular weight determination of antibody

Identification of disulfide bridges in monoclonal antibody

Sample preparation

Sample requirements for disulfide bonds analysis

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.

  1. Purify the protein/antibody

    1. The chromatographic protein/antibody purity should be >90%.
    2. Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS can most often be done on samples containing small amounts of salts, urea or detergent. However, you obtain the best result with low buffer strength in volatile solvents without detergents.
    3. Minimum amount ~ 0.5 mg.

  2. Put the protein/antibody sample into a microcentrifuge tube

    1. Use a Eppendorf Safe Lock tube supplied, or similar tubes from your lab.
    2. Lyophilize or speed vac the protein/antibody to a solid sample to ensure stability during shipment.
    3. Alternatively, freeze or refrigerate to +4 oC for cold shipment of the liquid sample.
Related services

You can combine this analysis with other characterization analyses such as:

Meet the experts

For this type of analysis our experts include:

Do you need help?
Li Peng Lundgren

PhD in Protein Chemistry

Read more
Do you need help?
Stine Thyssen

PhD in Health Science

Read more
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