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Typical Glycan Mapping HILIC Conditions

The standard analytical methodology employed is a HILIC separation followed by fluorescence detection (FLD) of the labeled glycans.  The common labels typically employed in glycan analysis - 2-AB or procainamide – are both chromotags and flurotags, indicating they can be used in conjunction with a UV detector or an FLD.  (Figure 1).

Figure 1: Labeled Glycans

The benefit of using the FLD over a UV detector is that as fewer things fluoresce, sensitivity tends to be higher as compared with UV detection where a great number of species absorb UV light.  Remember, sensitivity is defined as the signal-to-noise ratio, and the sensitivity is governed in the part by the strength of the signal detected, but in the main by the background noise.  When employing mass spectrometric detection, as is often the case during development and characterization, one may be tempted to forgo the labeling step as the unlabeled glycans readily ionize in negative mode electrospray ionization via the numerous terminal hydroxyl groups.  However, it is always recommended to perform the labeling as the unlabeled, or free, glycan can exist in two anomeric forms, an alpha and a beta, and this can lead to peak splitting (Figure 2).

Peak splitting in LC-MS

Figure 2: Peak splitting in LC-MS of the two anomeric forms of unlabeled glycans

The benefits of labeling glycans is not merely from a detection perspective, the labeled glycans are also more easily retained and separated too.  Whilst there has been a lot of development in particle morphology and size and in glycan liberation and labeling in recent times, the method has remained almost unchanged since it was first published in 1996 [2]. The paper was titled 'A Rapid High Resolution HPLC Method for Separating Glycan Mixtures and Analyzing Oligosaccharide Profiles'. The benchmark in terms of resolution has been raised over the last couple of decades with the introduction sub-2 µm fully porous particles and sub-3 µm superficially porous particles, and we now know the method as HILIC.  Rather interestingly the method was defined as being normal phase even though there was a significant portion of ammonium formate in the mobile phase.  However, a close resemblance to this method is maintained to this day and it very much remains the gold standard approach for glycan mapping.  The method consists of a shallow acetonitrile / ammonium formate gradient run through a column packed with porous silica particles coated with an amide stationary phase.  The method is a HILIC method and as such more polar species are typically retained longer.  This is the case with glycan analysis as with an increase in polymerization/polar glycan units, often cited as an increase in the number of glycosidic bonds, retention increases. Typical chromatographic conditions are shown in Table 1.
 
Instrument: (U)HPLC
Mobile Phase A: 100 mM ammonium formate pH 4.4
Column: 150 mm L x 2.1 mm ID x < 2 µm FPP Amide
Mobile Phase B: Acetonitrile
Flow Rate: 400 µL/min
Gradient:

0-25 min. 80-60%B

25-30 min. 60-20%B~

Column Temperature: 55 °C
Injection Vol: 5 µL
Sample solvent: 1:1 v/v Water: Acetonitrile
FLD Settings*: Excitation = 260 nm, Emission = 430 nm

Table 1: Typical glycan mapping LC conditions

~ Clean up step – not part of the qualitative or quantitative separation
* The FLD settings, in particular the excitation wavelength, often require empirical optimization as they tend to be specific to the fluorescence detector employed

The chromatogram shown in Figure 3 is of a 2-AB labeled dextran ladder, an integer increase in glucose units from 2 up to 15, and demonstrates the expected retention and separation as the number of glucose units/glycosidic bonds increases.

Typical Separation of a 2-AB Labelled Dextran Ladder

Figure 3: Typical separation of a 2-AB labeled dextran ladder

Not only can this dextran ladder be used in order to try and help identify unknown glycans, or at least the number of associated glucose units, but it can also be used to optimize gradient conditions such that separation of glycans of a specific size is enhanced.  It is a useful tool to ensure that acceptable chromatography is being generated and the instrument, method, and column are performing satisfactorily. 

The main and most common complex type glycans, labelled with 2-AB, found associated with mAbs are shown in Figure 10.  The approximate glucose unites are indicated across the upper x-axis.  What is of particular noteworthiness is the baseline separation of the G1F isomers, retention times of 10.8 and 11.2 respectively, when the conditions detailed in Table 1 are utilized.

Typical Separation of a 2-AB Labelled Dextran Ladder

Figure 4: Typical separation of common 2-AB labeled mAb glycans

Herceptin glycoprofile

Figure 5: Herceptin glycoprofile

 

 

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