18


August/September 2011 Chip Cube Description Time [min]


Sample Injection


Enzyme Reactor Fill


Deglycosylation Glycan


Transfer Glycan


Analysis and Detection


0.00 1.00


1.10


5.00 6.00 7.50 8.00 9.00 9.01


12.00 Table 2: Optimised acquisition method.


sample preparation and analysis. Figure 3 also shows the corresponding chromatogram (inset = zoom out). The red peaks are glycans that result from mAb deglycosylation and the black peaks are from the internal standard that is spiked into the Antibody Standard. The internal standard is a free reducing end glycan that serves for functional checkout of the HPLC-Chip/MS system. For instance, on an intact chip the ratio between the antibody glycans and the internal standard should be similar to that shown in Figure 3.


Taking a closer look at the glycan chromatogram of the antibody standard,


which is shown in Figure 4 including structure assignments*: Why is the same glycan structure assigned to three distinct peaks? Figure 4 illustrates that during enzymatic deglycosylation by PNGase F, glycosylamines are released from the polypeptide backbone of the antibody. These are reactive intermediates that hydrolyse under acidic conditions to free reducing end glycans [8]. Due to the anomeric equilibrium at the carbon on the reducing end, each glycosylamine has two corresponding free glycan structures. The chromatogram on the right shows that these diastereomeric species can be resolved on the PGC separation column so that each cleaved N-


Enrichment Inline Analysis 32


85 85 2


IR OR Capillary Pump Nanoflow Pump Flow [µl/min] Bypass Analysis


Inline Bypass


3 0 0.5


Solvent B [%]


Flow [µl/min]


Solvent B [%]


glycan results in three peaks. Since the workflow with the mAb-Glyco Chip occurs within a short time frame, the predominately detected species are glycosylamines. Yet, exposure to the acidic nanoflow pump gradient converts some amount of glycoslyamine to free reducing end glycans (between 5% and 10%).


*The peaks for the internal standard were removed for reasons of simplicity.


2


2)Data processing Data obtained from the analysis of N-glycans cleaved off mAbs can be complex. Therefore an automated data analysis and reporting procedure was developed to facilitate interpretation. Figure 5 provides an overview of the steps that convert a TOF raw data file (TIC) to a report of identified glycans with information on structure, retention time, mass error, abundance (volume) and relative abundance.


Extraction and identification The (Q)-TOF data file is processed using the Molecular Feature Extractor (MFE) that is part of MassHunter. MFE utilises an algorithm taking into account LC retention time, accurate mass, charge states, adducts and the generation of oligomers (up to trimers, which might typically occur in the ESI process, in particular when the concentration increases) to extract unique compounds from the TIC data set. Furthermore, comparisons are made between the compound masses detected and those stored in the accurate mass mAb-Glyco database. The search of the


(Q)TOF Data File (TIC) EXTRACTION


Extracted glycosylamines and free reducing end glycans


IDENTIFICATION (optional)


Structure Database


Identified glycosylamines and free reducing end glycans with structural information


Mass Database Molecular Feature Extrac


Addition of all


volumes from same glycosylamine species


Addition of all volumes


from the same free reducing end glycan species


Multiplication of volume by ionization efficiency factor


REPORTING


Addition of glycosylamine and free reducing end glycan volume to total volume


Deletion all free reducing glycan entries (redundant information)


Glycosylamine isomers visualized by colour coding


Figure 4: Left: Chemistry of the enzymatic PNGase F cleavage of N-linked glycans from a mAb. Glycosylamine intermediate hydrolyses to two anomeric free reducing end glycans. Right: mAb-Glyco Chip predominately detects glycoslyamines. Sample: Antibody Standard, 75ng on-column.


Identified glycans reported with volume and relative ration information


Figure 5: Schematic of the automated glycan extraction, identification, clustering and reporting procedure.


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