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Glycosylation analysis of mAb

Detailed glycosylation analysis of monoclonal antibodies

Glycosylation of monoclonal antibody therapeutics (mAbs) is a critical quality attribute. It has a great impact on the activity, efficacy and stability of the mAb. Hence, it is essential to determine the glycan structures and heterogeneity.

We offer different analytical strategies for a full characterization of mAb glycosylation. You can use it for batch comparison and process development samples. The analysis may be used in combination to profile both N-linked and O-linked mAb glycosylations.

Service 1: Separation and exact molecular weight of protein glycoforms

  • HILIC separation of mAb glycoforms
  • Online LC-MS to determine the mass of each protein isoform

Service 2: Separation and exact molecular weight of subunits

  • IdeS digestion and reduction to subunits
  • HILIC separation of LC, Fd’, single chain Fc (scFc) glycoforms
  • Online LC-MS to determine the mass of each subunit

Service 3: Glycopeptide mapping

  • Trypsin or Lys-C digestion
  • HILIC separation of glycopeptides
  • Online LC-MS/MS
  • Collection of glycopeptide fractions and offline MALDI TOF/TOF (optional)
  • Identification of glycopeptides based on MS/MS data

Service 4: Released N-glycan analysis

  • Glycan release, labelling and clean-up by Waters RapiFluor-MS N-Glycan kit
  • HILIC separation with fluorescence detection of labelled N-glycans
  • Online LC-MS/MS
  • Collection of the glycan fractions and offline MALDI TOF/TOF (Optional)
  • Identification of N-glycans based on MS and MS/MS data
Services/Products
Protein analysis by Alphalyse

Glycosylation analysis of intact mAb and subunits

  • IdeS cleavage and reduction into subunits
  • HILIC separation of mAb glycoforms & subunits
  • Exact MW by LC-MS of intact protein glycoforms and subunits
  • Quantitative ratio of glycoforms

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Quality analysis, presented in easy-to-understand reports
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Protein analysis by Alphalyse

Antibody glycopeptide analysis

  • Digest of antibody with trypsin or Lys-C
  • HILIC separation of glyco-peptides
  • LC-MS & MS/MS identification of different glyco-peptides
  • Optional glycopeptide profiling by MALDI MS

World Class Service
Quality analysis, presented in easy-to-understand reports
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Protein analysis by Alphalyse

Released N-glycan analysis

  • N-glycosylation profile
  • Exact MW of glycans and glycan type (G0, G0F, G1, G1F, G2F..)
  • Quantitative ratio of different glyco-forms
  • For comparison of batches and biosimilars versus originator

World Class Service
Quality analysis, presented in easy-to-understand reports
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Hydrophilic interaction chromatography

Hydrophilic interaction chromatography (HILIC) has been widely used as a tool for separating highly polar compounds. Recently, HILIC is employed in the sugar moiety separation as a function of their capacity to form hydrogen bonds with an amine bonded-silica phase. Several studies have demonstrated that HILIC with an amide-bonded stationary phase can provide complementary and highly resolving separations of glycoproteins, glycosylated peptides and glycans. Furthermore, HILIC, orthogonal to reversed phase, is compatible with both fluorescence and MS detection, which allows a simple and fast analysis.

 

ESI-MS and MS/MS

By ESI-MS, the samples 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. The mass of the sample is determined using a deconvolution algorithm that calculates the mass of the intact non-protonated antibody.

By MS/MS, glycopeptide and fluorescence-labelled glycan samples which have been separated in HILIC are further ionized and fragmented. The spectra of fragment ions will be searched against protein sequences and glycan database for structure identification.

Sample Preparation Guidelines

Protein analysis by mass spectrometry requires a pure protein in sufficient amounts to obtain good data. Avoid detergents and keep buffer concentration at a minimum.

For N-glycan profiling and identification, preferably no nucleophiles or SDS is contained in the sample buffer. Minimum protein concentration should be 1 mg/ml.

Protein samples can be submitted in liquid or lyophilized. Ship the samples with dry ice if possible.

Required minimum amount for each analysis: 50 µg

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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