Integrated microfluidic LC–MS workflow
Current glycan characterization methods are proven, but are time-consuming and labor-intensive. As discussed and shown in
Figure 1, these methods use lengthy solution-phase enzyme cleavage of glycans, fluorescent labeling for CE and LC analysis,
and sample cleanup for MALDI, LC–MS, or CE–MS detection. A reliable, rapid method, simplified by automated sample preparation
and separation, combined with the specificity of MS detection would be very useful in all stages of glycoprotein development
Figure 2: Glycan profiling workflow using an integrated microfluidic chip. The time required for glycan cleavage, sample preparation,
and glycan analysis is reduced to 10 min. In comparison, current methods require at least 6 h and involve many sample handling,
cleanup, and labeling steps. LC-LIF is laser-induced fluorescence. MALDI–MS is matrix-assisted laser desorption–mass spectrometry.
LC–MS is liquid chromatography–mass spectrometry. mAb is monoclonal antibody. HPLC is high-performance liquid chromatography.
TOF MS detector is time of flight–mass spectrometry.
Recent developments in microfluidics technology enable a new approach that integrates and automates established glycan analysis
sample preparation and characterization steps onto a single microfluidic chip coupled to a TOF-MS (see Figure 2) (13). These
steps are as follows:
- N-glycan release with immobilized PNGase F
- Removal of deglycosylated protein
- Capture of released glycans
- Separation by PGC chromatography
- Nanoelectrospray ionization for subsequent detection by TOF-MS.
As shown in Figure 3, the integrated microfluidic chip is composed of three biocompatible polyimide microfluidic chips constructed
using patented laser ablation and lamination technologies developed by Agilent Laboratories, the central research laboratory
for Agilent Technologies (Santa Clara, CA) (14). Laser ablation of polyimide film is used to fabricate the chip's surface
structures, and lamination of multiple layers together is used to create multifunction chips.
Figure 3: Diagram of the three-layer integrated microfluidic liquid chromatography–mass spectrometry (LC–MS) chip. The three
chips are fixed between a stator and a rotor on an Agilent HPLC Chip Cube MS. The chip components are constructed using patented
polyimide laser ablation and lamination technology developed by Agilent Technologies Agilent Laboratories. HPLC is high-performance
liquid chromatography. (FIGURE 3 IS COURTESY REID BRENNEN, AGILENT TECHOLOGIES AGILENT LABORATORIES)
The first chip, on top, has a reactor chamber packed with PNGase F-immobilized silica beads. The second chip, in the middle,
is packed with 5µm reverse-phase C8 beads to capture deglycosylated protein. The third chip, at the bottom, contains a sample
enrichment column, an LC separation column packed with PGC and a nanospray tip for nanoelectrospray ionization. The chips
are stacked, aligned, and sealed to each other, and then fixed between a stator on the top and a rotor on the bottom of an
Agilent HPLC-Chip Cube MS interface.
Figure 4 shows the sample flow during chip operation. In the first-rotor valve position (i.e., the sample-preparation configuration),
a transfer capillary attached to the stator delivers intact antibody sample into the enzyme reactor, and the glycans are cleaved
from the antibody. The deglycosylated antibody and the released glycans flow together into the C8 bead-packed retention column
where the protein is trapped. The glycans are trapped and concentrated on the PGC enrichment column. In the second-rotor valve
position (i.e., the sample-analysis configuration), the aqueous to organic gradient flow delivered by a nanoflow pump (Agilent
1200 Series, Agilent Technologies) elutes the glycans from the enrichment column and separates them on the LC column. Finally
the glycans are sprayed through the nanoelectrospray tip into the TOF-MS.
Figure 4: Microfluidic chip operation and flow path during sample preparation and analysis. In the valve configuration and
flow path shown in Figure 4 (a) (i.e., the sample-preparation configuration), the intact glycoprotein sample travels to the
peptide-N4-(N-acetyl-β-D-glucosaminy-I) asparagine amidase F (PNGase F) enzyme reactor where it is deglycosylated. Both the
deglycosylated protein and the free glycans travel together into the C8 bead-packed retention column where the proteins are
retained. Free glycans continue, via a rotor groove shown in red, to the enrichment column. Figure 4 (b) illustrates the valve
configuration and flow path for sample analysis. In the sample-analysis configuration, the nanopump delivers an aqueous-to-organic
gradient to elute the glycans from the enrichment column and to separate them on the porous graphitized chromatography column.
PGC is porous graphitized carbon. MS is mass spectrometry.
The microfluidic LC–MS chip workflow completes all analysis steps, from antibody injection to LC–MS results, in 10 min and
is completely automated once sample is introduced.