Glycan Analysis of mAb’sIndustry Practices , Orthogonal methods & Advances

- by Sada siva

Glycosylation is a critical post-translational modification because it may affect mAbs characteristics such as solubility, stability, pharmacokinetic and pharmacodynamics properties, as well as in vivo efficacy.

Due to its various functional implications, the glycosylation pattern of a therapeutic antibody may represent a critical quality attribute, and therefore may require close monitoring during bioprocess development and routine manufacturing.

What is glycosylaion…

Glycan attachment site identification Glycan site occupancy Glycan forms distribution

What information…

How to do it…

NP-HPLC Simple and straight forward HPLC gives a high resolution and a detailed

profile of fluorescence-labeled N-glycans Glycan separation using HILIC is dominated

by the physical properties of the glycan moiety. The label (2-AB and 2 AA) often has little influence on the separation.

HPLC’s & resources are common & available in all labs

This indicates the overall glycosylation pattern and allows you to monitor:

(a) specific glycoforms important for maintaining standard drug potency (for example glycoforms with bisecting GlcNAc or non-fucosylated oligosaccharides that have controlling influence on activation of the immune response) and

(b) aberrant glycoforms that could cause adverse reactions (such as those containing the potentially immunogenic nonhuman Gallili antigen Galα1,3Gal).

NP HPLC

(HPAEC-PAD) has proved a useful tool for the separation of sialylated glycans, as well as uncharged oligosaccharides.

Underivatized glycans can be monitored by pulsed amperometric detection (PAD) at low picomole concentrations,

But this method is nonspecific for carbohydrates PAD - Response factors are different for each Glycan forms. However, this detection PAD method is not very selective, and compounds from the

sample matrix such as amino acids also give rise to signals. It has some Reproducibility issues complete separation of all structures present in a mixture is

rarely achieved in a single chromatographic step. Less Compatibility to MS for further analysis (NaOH)

HPAEC-PAD

In CE-based separations the charge is necessary to provide the glycan with an effective electrophoretic

mobility for migration. Glycan labeling with APTS and ANTS is often applied for CE:

with their three sulfonic acid groups, both labels provide a nearly pH independent high anionic charge, giving rise to low analysis times.

Laserinduced- fluorescence detection is applied at 488 and 325 nm, respectively

This approach offers a unique ability to differentiate isomeric glycan structures.However, only a limited number of glycan standards is available for use in making the assignment of a glycan structure to a corresponding CE peak signal.

CE -LIF

Glycans do not absorb ultraviolet (UV) light refractive index pulsed amperometric detectors, labeling glycans with radioactive isotopes, Chemical derivatization is now the most

common method used for labeling glycans at their reducing ends by reductive amination.

Dyes

2-AA and 2-AB are hardly retained 2 AB is prefered more LC 2 AA is preferred for CE , LC & MS APTS label used for CE-LIF

Common Fluorescent dyes…

2-AB is a label that lacks negative charges and is widely applied in chromatographic analysis.

An extensive database has been developed which uses the standardized elution positions of 2-Ablabeled glycans in hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection for structural assignment.Not compatible with LC MS because of low signal

The 2-AA label carries one negative charge, which makes it very versatile. It is used in HPLC and capillary electrophoresis (CE) separations as well as in positive-mode and negative-mode matrix-assisted laser desorption/ionization (MALDI) analysis, allowing detection of both neutral and sialylated glycan species

APTS has three negative charges and is therefore very suitable for CE and capillary gel electrophoresis However, the analysis of APTS-labeled oligosaccharides by MALDI appears to be difficult.

` Few other dyes are available apart from above.

2-AB , 2-AA and APTS dyes

Glycan Work flow

Release2-24 hrs

•Intact/Reduce•PngaseF Treatment/ Chemical

Purify2-4 hrs

•SPE /HILIC cartridges •Acetone precipitation, GFC, Centricon

Label2-4 hrs

•2 AB label is commonly used (2-3 hr)•2 AA (MS), APTS (CE) and new advanced commertial dyes

Purify2-4 hrs

•SPE /HILIC catridges

Samples preparation work flow

HPLC with Fluriscence Detector Column: Luna 3μ NH2 100A ,4.6 × 150 mm, 3 μm, Eluent 1: Acetonitrile Eluent 2: 15 mM Ammonium acetate pH

5.2 Flow: 1.5 mL/min Time : 120 minutes Ex: 330nm Em: 420 nm

Separation

Initial gradient optimization Gradient slope Flow rate Temperature Ion pairing Concentration&pH of buffer (charged

glycans)

Imp Method development facors

Peaks identification is done by any one or combination of below method

By coupling with MS By the use of Labeled Glycan standard By use of selective and sequential

enzymatic treatment By using Dextrin ladder indices

Peak Idenification/Assignment

Advances …

Questions ?

Thanks you very much.