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Disulfide bridge analysis

Determination of disulfide bridges in drug substance protein

Want to know number and position of disulfide bridges in your drug substance protein?

We offer an accurate determination of the number and position of protein disulfide bridges in your drug substance by disulfide mapping. Disulfide mapping consist of two parts as explained below.

The ICH Q6B Guidelines (Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) requires determination of number and position of protein disulfide bridges.

Our disulfide bridge Service includes:

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

  • Exact molecular weight of the native protein is  determined by UV-LC ESI-MS, ie. non-reduced protein with intact disulfide bridges.
  • Exact molecular weight of reduced protein determined by UV-LC ESI-MS, ie disufide bridges converted to free cysteine thiol groups.
  • The number of disulfide bridges and free thiol groups is calculated from the Mw shift between the native and the reduced protein.

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

  • Non-reduced protein is digested with 2 enzymes and subjected to peptide mapping by nano LC-MS/MS.
  • Reduced protein is digested with 2 enzymes and subjected to peptide mapping by nano LC-MS/MS.

Send us a request to discuss your project.

Services/Products
Protein analysis by Alphalyse

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

 

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Quality analysis, presented in easy-to-understand reports
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Introduction
Mass spectrometry analysis under reducing and non-reducing conditions, is very useful for the characterization of disulfide bridges in biopharmaceuticals. At Alphalyse we combine the results from intact mass analysis with peptide mapping using one or more proteases.

Analysis procedure
Intact mass:
The protein is analyzed on a Bruker Q-Tof Impact instrument by direct injection of a reduced and a non-reduced sample. The raw data is smoothed and de-convoluted using MaxEnt algorithm.

Peptide mapping:
The protein sample is digested in two different ways; one half is digested without reduction and the other half is digested after reduction. The peptide samples are separated on an Agilent 1200 system connected to a high-resolution Q-Tof mass spectrometer (Bruker Maxis Impact). The MS instrument is running in Data Dependent Analysis mode (DDA), switching between MS and MS/MS mode. The MS/MS data are searched against a FASTA database containing the sequences of the protein using MassAI software.

Report:
The final report will include a comparison of the intact mass data to determine the total number of disulfide bridges in the protein and a table containing information about all identified disulfide-bridged peptides including relative quantity of each. Potentially scrambled disulfide bonds are also considered.

Introduction
Protein analysis by mass spectrometry requires a pure protein in sufficient amounts to obtain good data. It is important that samples are prepared in a clean laboratory to avoid contamination with human keratin.

Protein samples can be submitted in liquid or lyophilized.

  1. Purify the protein/peptide

    1. The chromatographic protein/peptide 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, but the best result are obtained with low buffer strength in volatile solvents without detergents
    3. Minimum amount ~ 0.5 mg
  2. Put the protein sample into microcentrifuge tube

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

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.

  • 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
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.
  • 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.

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. 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!

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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