HIGH THROUGHPUT GLYCAN ANALYSIS
N-linked oligosaccharide mapping is routinely performed as an in-process test for recombinant glycoproteins derived from mammalian
cell lines. Reasons include the complexities associated with processing Asparagine-linked (N-linked) oligosaccharides and
the sensitivity of the enzymes involved, to even subtle changes in cell culture conditions during manufacturing. In addition,
oligosaccharide mapping as a potential component of product lot release is increasingly requested from regulatory agencies
Standard methodologies contain several bottlenecks with respect to throughput. First, the sample preparation required for
analysis is both laborious and time-consuming, requiring multiple steps of hands-on manipulation by the analyst. Second, the
typical separation methods used have cycle times ranging from 90 to 180 minutes.17,18 In instances where multiple cell culture conditions are being screened or large numbers of in-process samples are being tested,
the resulting analysis sequence can take days. In addition, the mobile phases typically used in the separation of oligosaccharides
are not compatible with online MS analysis, requiring the collection of multiple fractions and additional sample manipulation
before MS analysis, providing another bottleneck in the characterization stage.
We have implemented methodologies to address each of these throughput constraints. Through the use of robotic liquid handling,
automated sample preparation, and rapid resolution reverse phase chromatography (RRRP–HPLC), we are able to completely process
30 samples per 24 hour period for oligosaccharide analysis, from the point of initial enzymatic digestion through full MS
characterization of species accounting for as little as 0.1% of the oligosaccharide moiety.19,20 A traditional sample preparation scheme involves removal of deglycosylated protein by porous graphitized carbon (PGC) following
PNGase F digestion, vacuum centrifugation before labeling with the fluorophore, and finally, removal of excess fluorophore
and labeling reaction components using a cellulose phase matrix (S-cartridge) with subsequent vacuum centrifugation. We have
implemented methodologies that replace these manually operated PGC and S-cartridges with PhyTip columns (Phynexus, Inc.) packed
with Carbopack B and DPA-6S resins that are compatible with robotic platforms.
A total of six identical PNGase F digestions were performed on 500 ug aliquots of a recombinant IgG. Three of the digests
were processed following traditional protocols and the remaining samples were processed following the automated method. Samples
analysis was performed by traditional high pH anion exchange chromatography (HPAEC). An overlay of the resulting chromatograms
is shown in Figure 2. No significant differences were observed in the chromatograms obtained by the two preparation methods.
In addition to requiring limited analyst manipulation during preparation, the samples prepared following the automated protocol
were ready for analysis at the end of Day 1, whereas samples prepared following the standard protocol were not ready for analysis
until the start of Day 3.
Figure 2. Comparison of samples prepared by automated and standard methods and separated by high pH anion exchange chromatography
The most commonly used separation methods for oligosaccharide analysis are HPAEC and normal phase chromatography (NP–HPLC).
The mobile phases used for these separations are not compatible with online MS characterization. Reverse phase (RP–HPLC) separations
have been described previously, and provide superior resolution of species compared to HPAEC and NP–HPLC methods.18 However, with cycle times of three hours, they are not suitable for routine analysis. As seen in Figure 4, we have developed
a method that takes advantage of new small particle-size resins available from column manufacturers. A batch of 30 samples
from cell culture screening conditions can be prepared, separated, and characterized in 24 h using this approach with the
chromatographic cycle time dramatically reduced relative to traditional RP–HPLC. This RRRP–HPLC method has a cycle time of
35 minutes, provides comparable resolution to standard RP–HPLC, and is compatible with online ESI–MS/MS detection with a limit
of detection (LOD) of <20 fmol by fluorescence and ~1 pmol by MS. Using this approach, full characterization of the oligosaccharide
map, including species accounting for as little as 0.1% of the total moiety, can be achieved in a timeframe that is competitive
with basic profile fingerprinting achieved by standard capillary zone electrophoresis (CZE) methods. An example separation
for an rIgG is shown in Figure 4. For this particular rIgG, a total of 36 unique species were identified from a single sample
Figure 3. Comparison of flow schemes for standard and automated methodologies for oligosaccharide analysis
Through the combination of improvements in sample preparation and chromatographic cycle time, we are now able to perform complete
characterization of an oligosaccharide map in a single day, providing a minimum of a five-fold reduction in process time.
In addition, because of the short cycle time required by the RRRP–HPLC and compatibility with online MS detection, thorough
MS analysis can be incorporated as part of routine sample analysis.
Figure 4. Typical separation by reverse-phase of an N-linked oligosaccharide pool from an rIgG