17 5


Inner Rota (IR)


Enrichment Enrichment Analysis Analysis


Outer Rotor (OR)


Inline


Bypass Inline


Bypass


Columns -


Nanoflow Pump PGC-SC PGC-SC


PGC-EC / PGC-SC PGC-EC / PGC-SC


Table 1: All possible rotor valve switching positions of the mAb-Glyco Chip


ACN [5 mM FA] were used as nanoflow pump mobile phases A and B, respectively. ACN was from Merck (Germany), water from a Milli-Q water purification system, FA from Sigma (USA). A detailed description of the analysis method is given in Table 2. Mass detection occurred with an Agilent 6520 Q-TOF operated in positive ion mode with Vcap = 1850V, drying gas flow = 3.5L/min at T = 360°C, and a fragmentor voltage of 160V. Data were acquired at 2GHz in MS only mode, range 450-3,000 m/z at a rate of 3 spectra/s. Internal mass calibration used m/z 922.0098. The MassHunter Workstation was used for data acquisition and processing.


mAb-Glyco Chip layout and operation Figure 1 illustrates the architecture of the mAb-Glyco Chip, which is made from inert, biocompatible polyimide. It integrates: (a) a 310nL enzyme reactor (ER), packed with immobilised PNGase F beads (Peptide-N4- (acetyl-ß-glucosaminyl)-asparagine amidase N-Glycosidase F), (b) a 160nL porous graphitised carbon enrichment column (PGC-EC), (c) a 43mm x 75µm ID PGC separation column (PGC-SC), that directly connects to the metalised nano electrospray tip (Figure 1 (d)). PGC particle size = 5µm. The Chip-Cube interface automatically positions the chip orthogonal to the MS inlet and makes the necessary electrical and hydraulic connections to the chip [4-6]. The


mAb-Glyco Chip uses the concentric rotor-in- rotor valve design of the Chip-Cube. The stator-chip-rotor sandwich creates an outer 10-port valve (OR) and an inner 6-port valve (IR). This unique design allows for switching ER and PGC-EC independently into or out of the sample loading flow path. Table 1 summarises all possible chip valve positions. Initial designs used a 6-port valve setup with the enzyme reactor in flow through mode, i.e. ER directly in-line with PGC-EC and thus residence time of the mAb on the enzyme reactor (which determines the deglycosylation efficiency) was a function of flow used to load the mAb onto the chip. Data have shown that a period of 6 seconds obtained at 1µL/min loading flow could efficiently deglycosylate the majority of many mAbs studied on the chip [e.g. 7]. However, some did not react quantitatively and required further lowering the flow rate. This was detrimental for analysis speed.


Description of the on-chip workflow 1) Data acquisition Figure 2 illustrates the on-chip workflow comprising five automated steps: 1. Sample injection: Outer rotor is set to bypass (enzyme reactor bypass), inner rotor to analysis. A volume of antibody sample is injected and loaded onto the chip using deglycosylation buffer.


2. Enzyme reactor fill: Outer rotor switches enzyme reactor to inline


Columns -


Capillary Pump ER / PGC-EC PGC-EC ER


None


position, which cuts a piece from the heart of the injected sample plug.


3. Deglycosylation: Outer rotor switches enzyme reactor back to bypass. The PNGase F reacts with the mAb and cleaves off the N-linked glycans while the capillary pump flushes the system.


4. Glycan transfer: Both, inner and outer rotors turn simultaneously switching both, enzyme reactor and enrichment column into the loading pump flow path. N-glycans become trapped on the PGC-EC.


5. Glycan separation/detection: Inner rotor turns into analysis position. Both, PGC-EC and PGC-SC are in the nanopump flow path. A reversed phase gradient is used to chromatographically separate glycans prior to TOF-detection. During analysis the outer rotor keeps the inline position for cleaning and re- equilibration of the ER with deglycosylation buffer.


Figure 3 shows results obtained from the analysis of the Kit’s Antibody Standard using the HPLC-Chip method outlined in Table 2. Figure 3 shows time segments of the workflow and the nanoflow pump gradient. At a 3µL/min capillary pump flow rate, sample injection requires 1 minute and ER fill 6 seconds. Reaction time was 4 minutes, which was determined to result in complete deglycosylation of antibodies (this time may be shortened and the method optimised for a particular mAb of interest). The transfer of cleaved N-glycans from the ER to the PGC- EC takes 1 min and the separation of the enriched glycans on the PGC-SC including a column flushing step and re-equilibration, takes 6 min. This makes a total of 12 minutes for the entire on-chip workflow including


Figure 2: Valve switching scheme for automated on-chip deglycosylation of mAbs, enrichment, separation and MS-detection of N-glycans.


Figure 3: Analysis speed. The whole workflow time is 12 min. Sample: Antibody Standard, 75ng on-column.


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