The document discusses glycosylation analysis methods for monoclonal antibodies. It states that glycosylation is a critical post-translational modification that affects characteristics like solubility, stability, and efficacy. It also discusses different analytical techniques for glycosylation analysis, including HPLC, CE, and HPAEC-PAD. For each technique, it provides details on labeling methods, separation mechanisms, advantages, and limitations.

1. - by Sada siva

2.  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.

3.  Glycan attachment site identification
 Glycan site occupancy
 Glycan forms distribution

6.  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

7.  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).

8.  (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)

9.  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.

10.  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.

11.  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

12.  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.

14. Release
2-24 hrs
•Intact/Reduce
•PngaseF Treatment/ Chemical
Purify
2-4 hrs
•SPE /HILIC cartridges
•Acetone precipitation, GFC, Centricon
Label
2-4 hrs
•2 AB label is commonly used (2-3 hr)
•2 AA (MS), APTS (CE) and new advanced commertial dyes
Purify
2-4 hrs
•SPE /HILIC catridges

15.  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

16.  Initial gradient optimization
 Gradient slope
 Flow rate
 Temperature
 Ion pairing
 Concentration&pH of buffer (charged glycans)

17.  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

21. Questions ?
Thanks you very much.