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Antibody-drug conjugates

Detailed characterization of ADCs

Do you want to know the exact number and position of drug molecules attached to the antibody in your ADC (antibody-drug conjugate)?

Antibody-drug conjugates (ADCs) are site-directed cancer drugs with great potential. They consist of an antibody with one or more cytotoxic drug molecules attached (conjugated) to it. The antibody has a very specific binding to cancer cells. It uses this ability to direct the drug molecule to the exact target of the treatment. Most often, an antibody-drug conjugate is a mixtures of antibodies with 0, 1, 2, 3, or more attached drug molecules in various positions.

Alphalyse offers analytical services in order to determine the exact distribution of drug-to-antibody ratio (DAR) in your antibody-drug conjugate (ADC).

Our service for determination of DAR distribution includes:

  • First, exact masses are determined by UV LC-ESI-MS.
  • Then the exact mass distribution of intact ADC is determined.
  • We include the exact mass distribution of de-glycosylated ADC.
  • You receive calculated DAR distribution in percentage based on MS peak intensity.

Innovation collaboration supported by Eurostars.

Alphalyse collaborates with ChromaCon in Switzerland. Together, we develop purification technologies and analytical services for separation and detailed characterization of antibody-drug conjugate (ADC) with various drug-to-antibody ratio (DAR) distributions. This innovation collaboration is supported by Eurostars.

Services
Antibody drug conjugate analysis

Drug-to-antibody ratio of antibody-drug conjugate

  • Analysis by UV LC-ESI-MS
  • Exact mass distribution of ADC
  • Percentage of each DAR form

$3,100.00

Convenient Service
Will be delivered in 10-20 days
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Antibody drug conjugate analysis

DAR distribution of deglycosylated ADC

  • Analysis by UV LC-ESI-MS
  • Deglycosylation by PNGase f treatment
  • Exact mass distribution of deglycosylated ADC
  • Percentage of each DAR form

Customized Service
Quality analysis, presented in easy-to-understand reports
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DAR Distribution

ESI-MS sprays antibody-drug-conjugate (ADC) sample in liquid phase through a capillary at high voltage into the MS instrument. This measures the mass over charge ratio (m/z). For ADCs the electrospray process results in multiply protonated molecules with a distribution of ion species at m/z range from 1-3000. A deconvolution algorithm calculates the mass of the intact non-protonated antibody to determine the mass of the ADC.

The ESI process requires a quete pure preparation of the sample, without interfering salts or detergents. Reversed phase HPLC (Agilent 1200 system) using a short C8 column purifies the sample. Then a Q-TOF mass spectrometer (Bruker Maxis Impact system) performs the analysis. We determine the mass of the sample by deconvolution of the obtained raw spectra using the MaxEnt algorithm.

The high resolution and accuracy of the Q-Tof instrument result in very accurate mass determination within 0-3 Da of the theoretical mass. For ADCs we analyze the sample in the intact form and after reduction with DTT. The drug-to-antibody ratio (DAR) distributions is then determined from the deconvoluted data.

Peptide mapping

A specific protease cleaves the ADC into smaller peptides. These are then analyzed by mass spectrometry and the observed peptides correlated to the antibody amino acid sequence.

The identified peptides in the peptide map thus confirm the specific amino acid sequences covered by the peptide map, as well as the attachment sites for Drug-conjugates. Peptide mapping using a single protease typically gives a sequence coverage of 30-90%. Two or more proteases increases the coverage to 95-100%.

Detailed characterization of ADCs requires careful investigation of the antibody sequence. This is done to select the best proteases, and choose between nano-flow LC-MS/MS or standard flow UV LC MS/MS to observe the peptides. The difference between the two techniques relates to the amount of material needed for the analysis. Nano-flow can be conducted on low microgram antibody whereas the high-flow analysis requires at least 20 microgram per analysis. The high-flow analysis includes a UV-trace of the chromatogram.

Nano-flow LC-MS/MS

Peptide mapping by nanoLC-MS/MS is performed on a Dionex 3000 system running at 300 nl/min equipped with C18 capilary column. It connects to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument runs either with a short or long gradient. The Q-TOF is in data dependent analysis mode (DDA), switching between MS and MS/MS mode.

The analysis provides a Base peak chromatogram. In addition, we collect all MS/MS data for peptides with mass/charge ratio from 200-1600 dalton.

High-flow LC-MS/MS with UV

We perform peptide mapping using high-flow LC-MS/MS on a Agilent 1200 system. It runs at 0.2 ml/min equipped with C18 column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument runs either with a short or long gradient, and the Q-TOF set to data dependent analysis mode (DDA), switching between MS and MS/MS mode.

The analysis provides both a UV- and a Base peak chromatogram, and collects all MS/MS data for peptides with mass/charge ratio from 200-1600 dalton.

Sample Preparation Guidelines

Submit Antibody-Drug-Conjugate (ADC) samples in liquid or as lyophilized material.

The chromatographic purity should be >90%

Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS  analysis most often works on samples containing small amounts of salts, urea or detergent, but you obtain the best result with low buffer strength in volatile solvents without detergents.

Minimum amount ~ 100 micrograms of ADC

Put the ADC into microcentrifuge tube:

  • Use Eppendorf Safe Lock tubes, or similar tubes from your lab.
  • Lyophilize or speed vac the ADC to a solid sample to ensure stability during shipment.
  • Alternatively, freeze the liquid sample for cold shipment.

Attention

Please note that MW determination of intact ADCs by mass spectrometry CANNOT be conducted on samples from gels or PVDF membranes!

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.
299 California Avenue, Ste 200
Palo Alto, CA 94306
USA
Att: Thomas Kofoed
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220 Odense SO
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
Order analysis now
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