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Disulfide bridge analysis

Determination of disulfide bridges in biopharmaceutical proteins and mAbs

The ICH Q6B Guidelines requires disulfide bridge analysis in biopharmaceutical proteins and antibodies. This include both determination of the number and the position of disulfide bridges.

Service includes:

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

  • Determination of exact molecular weight (MW) of the native protein by UV-LC ESI-MS. This means non-reduced protein with intact disulfide bridges.
  • Determination of exact molecular weight of reduced protein by UV-LC ESI-MS. This means disulfide bridges converted to free cysteine thiol groups.
  • Calculation of the number of both disulfide bridges and free thiol groups. We do this from the MW shift between the native and the reduced protein.

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

  • Digestion of non-reduced protein/antibody. We use 2-4 enzymes followed by peptide mapping.
  • Digestion of reduced protein/antibody. Here we also use 2-4 enzymes, followed by peptide mapping.

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

Do you want to combine this analysis with other characterization analyses? Then check out our pages on Molecular weight determination • De-novo sequencing of mAb  • Peptide mapping.

Send us a request, so we can discuss your project and customize our analysis.

Services
post translational modifications analysis

Disulfide bridge mapping of drug substance

  • Number of disulfide bridges by intact mass before and after reduction
  • Position of disulfide bridges by peptide mapping
  • Comparison with Drug Substance sequence
  • Customized analysis based on specific protein property

 

Customized Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote

Introduction
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.

Analysis procedure
Intact mass:
We use a Bruker Q-TOF Impact instrument for analysis of the protein/antibody. We 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 disulfide 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.

Introduction
Protein/Antibody analysis by mass spectrometry requires a pure sample in sufficient amounts to obtain good data. It is 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 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.

shipping
Ship your protein samples by a courier company of your own choice

Place your samples into micro tubes and pack in a suitable transport box. Remember to print and include the sample submission form.

  • Contact a courier company of your choice (FedEx, UPS, DHL, TNT, World Courier etc.). Use the following sentence to describe the content: “Protein material – Non-toxic, non-hazardous, for research purpose only“.
  • Use cool packs or cooling units if your samples should be stored below 5°C/40°F
  • Ship the samples to the office closest to you (Canadian customers, please ship to Europe):
Customers in the USA
Alphalyse, Inc.
200 Page Mill Road, Suite 100
Palo Alto, CA, 94306
USA
Att: Jette Williams
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220, Odense
DENMARK
Att: Anette Rasmussen
Tel: +45 6310 6500
shipping
Order a prepaid FedEx Air Waybill < 1 lb./ 500g

A prepaid FedEx Air Waybill can be used for fast and easy shipment of samples in packages up to 1 lb./ 500g.
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack in suitable transport box. Remember to print and include the sample submission form.
  • Print the PDF document and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to send your samples stored at room temperature, this option is usually the right choice. If you fill your box with cooling packs, the weight will most often exceed 1 lb./500g.

shipping
Order a prepaid FedEx Air Waybill < 10 lb./5kg (e.g. for packages with cooling packs)

This prepaid FedEx Air Waybill can be used for shipment of samples in packages up to 10 lb./ 5kg, with or without cooling packs (do not use dry ice!).
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack them in a suitable transport box. Remember to print and include the sample submission form.
  • Fill with cooling units/cold packs (do not use dry ice!).
  • Print the PDF and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to fill your transportation box with cooling packs, select this shipping option. If you plan to use dry ice, you cannot use this Waybill!

shipping
Get a quote for shipment by World Courier

The World Courier service can be used for shipment of samples of all types. If you select this option, we will contact you to provide a quotation based on location and shipping conditions. You will receive a prepaid Air Waybill which will be invoiced together with your analysis results. This is usually a good option if FedEx does not service your location.
With the prepaid World Courier service you get:

  • GxP compliant processes to ensure maximal security and traceability
  • Door-to-door solution on a dedicated vehicle
  • Possibility of providing qualified packaging, temperature monitoring and dry ice
  • Shortest possible transit-time through “next flight out”-procedure
  • Customs clearance and help with paperwork
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