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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). We can also determine the exact attachment site(s) of the drug molecule in your 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.

Our service for determination of drug molecule attachment sites includes:

  • Peptide mapping by nano LC-MS/MS or UV LC-MS/MS.
  • Comparison with expected amino acid sequence, including modifications.
  • You receive a total sequence coverage map, both for heavy chain and light chain.
  • Finally, you receive information about which tryptic peptides are conjugated to the drug molecule.

Innovation collaboration supported by Eurostars.

Alphalyse is collaborating 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/Products
Protein analysis by Alphalyse

DAR distribution of intact ADC

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

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Protein analysis by Alphalyse

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

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Protein analysis by Alphalyse

Drug conjugation sites in ADC

  • Peptide mapping by LC-MS/MS
  • Protease cleavage with 1 enzyme
  • Peptide coverage maps for heavy chain and light chain
  • Determination of drug-conjugated peptides

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Quality analysis, presented in easy-to-understand reports
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DAR Distribution
By ESI-MS, antibody-drug-conjugate (ADC) sample in liquid phase are sprayed through a capillary at high voltage into the MS instrument where the mass over charge ratio (m/z) is measured. For ADC’s the electrospray process results in multiply protonated molecules with a distribution of ion species at m/z range from 1-3000. The mass of the ADC is determined using a deconvolution algorithm that calculates the mass of the intact non-protonated antibody. The ESI process requires that the sample preparation is quite pure without interfering salts or detergents.

The sample is purified by reversed phase HPLC (Agilent 1200 system) using a short C8 column before being analyzed on a Q-TOF mass spectrometer (Bruker Maxis Impact system). The mass of the sample is determined 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 ADC’s the sample is analyzed in the intact form and after reduction with DTT.

From the deconvoluted data the drug-to-antibody ratio (DAR) distributions can be determined.

Peptide mapping

In Mass spectrometric Peptide Mapping the ADC is cleaved into smaller peptides using a specific protease. The peptides are analyzed by mass spectrometry and the observed peptides correlated to the antibody amino acid sequence.

The identified peptides in the peptide map thus confirms 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 will typically give a sequence coverage of 30-90%. By using two or more proteases this coverage can be increased to 95-100%.

Detailed characterization of an ADC’s requires careful investigation of the antibody sequence to select the best proteases, and choice of nano-flow LC-MS/MS or standard flow UV LC MS/MS to observe the peptides. The difference between the two techniques is related 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 include a UV-trace of the chromatogram.

nano-flow LC-MS/MS
The peptide mapping using nanoLC-MS/MS is performed on a Dionex 3000 system running at 300 nl/min equipped with C18 capilary column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument is running 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 provide a Base peak chromatogram, and all MS/MS data is collected for peptides with mass/charge ratio from 200-1600 dalton.

high-flow LC-MS/MS with UV
The peptide mapping using high-flow LC-MS/MS is performed on a Agilent 1200 system running at 0.2 ml/min equipped with C18 column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument is running 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 all MS/MS data is collected for peptides with mass/charge ratio from 200-1600 dalton.

Sample Preparation Guidelines
Antibody-Drug-Conjugate (ADC) samples can be submitted 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 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.
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 ADC’s by mass spectrometry CANNOT be conducted on samples from gels or PVDF membranes!

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

Order analysis now
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