How iGlycoMAb can make a difference
Glycans exhibit a great deal of diversity and the analysis of these mixtures often entail the use of separation techniques with high resolving powers, such as capillary electrophoresis (CE) (35) or liquid chromatography (LC). While both of these techniques have demonstrated the ability to resolve glycan structural and positional isomers (36-39), the glycan complexity leads to some glycans co-eluting, which makes it difficult if not impossible to quantitate these glycans based only on a chromatogram using fluorescence detection. Identification of glycans using these strategies is also problematic since this depends on the availability of standards, which are uncommon, and the use of exoglycosidases. Consequently, these strategies do not permit the identification of unknown or unique structures on a routine basis. The inability to identify the composition of unknown and co-eluting peaks has led to the increased use of these separation strategies being combined with mass spectrometry (MS), which also offers the potential to quantitate co-eluting glycans if the masses of these compounds or their fragments are different.
error are encountered that lead to difficulty when using MS for quantitation (40). Sources of these errors include the sample matrix, the instrument response, and instrument-to-instrument performance (40-42). Variability in instrument performance is particularly problematic when samples are analyzed at distant points in time, as might be the case when performing a batch-to-batch comparison of a therapeutic produced today to data obtained several weeks/months/years ago. Furthermore, different MS systems can produce different ion intensities and different ratios of ion abundances from the same sample, which is particularly true when different MS configurations from different vendors are compared and can limit cross-laboratory reproducibility.
The difficulty with multi-laboratory comparisons is apparent in the Human Proteome Organization (HUPO) on glycoprotein glycans profiling without the use internal standards (7). This study found that the variation between laboratories was rather large, particularly for minor components. For example, the level of IgG glycans with galactosylation ranged from 2.2% to 14%, nearly a 7-fold difference, while the level of fucosylated glycans attached to a serum transferrin sample ranged from not detectable to 9.2%. Consequently the approaches that do not utilize internal standards appear to be incapable of accurately quantitating the low level glycans that at times have the largest impact.
The use of internal standards is the accepted strategy to enable quantitation via MS. (42) In general, the closer the chemical properties of an internal standard to its analyte, the better it compensates for the various sources of error, and thus the optimal internal standard is typically an isotopically labeled analyte itself (42). Here, the mass analyzer resolves the isotopic pair, permitting their relative abundances to be determined by comparing the signal intensity of each analyte ion to that from its isotopically labeled form. Until this proposal, it was thought to be too difficult and/or expensive to obtain glycoproteins with isotopically labeled glycans, which led to the development of isotopic labeling procedures. Here, the glycans in one sample are modified with a “light” tag while the other sample is derivatized with a “heavy” tag. (43-50) These labeling procedures offer a significant improvement over the several hundred percent errors reported for the label free approaches (7, 8). These strategies are not ideal because they are only applicable to released glycans and thus are several steps removed from the actual analyte, i.e., the glycoprotein. Hence, differential errors can accumulate during the parallel processing of the two samples prior to the isotopically labeled glycans being mixed. The use of these approaches may also cause researchers to change their analytical sample flow, i.e., change their derivatization protocol or their separation strategy to accommodate the introduction of different tagging agent. These approaches also provide relative, not absolute, quantitation. Hence, despite isotopic labeling being an improvement, there is clear room for improvement.
Because the optimal internal standard is an isotopically labeled version of the analyte, we propose to develop an isotopically labeled mAb (iGlycoMAb), which we envision will be an ideal internal standard for the analysis of glycans on recombinant mAbs and/or native IgGs. We have selected a monoclonal antibody (mAb) because of the widespread use of these as therapeutic agents coupled with the need for glycan quantification by various regulatory agencies. We predict that a single iGlycoMAb will be a suitable internal standard for a wide range of recombinant mAbs and native IgGs because the glycan structures on native IgGs are fairly similar across most mammals (51) and those on mAbs are also similar, albeit with a lower diversity. This discussion concerns only that IgGs from various sources have some glycans in common, not that their levels are similar. Consequently the iGlycoMAb we have generated from a mouse hybridoma line contains many of the typical glycans that are commonly observed on therapeutic mAbs, as demonstrated by the LC-MS chromatograms show in Figure 2. In addition, the amino acid sequence of the Fc region surrounding the glycosylation site are similar for Abs, and thus the kinetics of glycan release are expected to be the same. This rationale leads us to firmly anticipate that iGlycoMAb will be virtually identical to other mAbs and IgGs from a glycoanalysis perspective.
We feel that the benefits of iGlycoMAb over other methodologies for glycan quantitation include the following.
(1) No need for laboratories to alter their workflow to benefit from the internal standard, provided they utilize MS detection, which many already do.
(2) Enable different researchers at different locations to obtain comparable results despite using different instruments. iGlycoMAb is added directly to the sample before any processing; hence we predict that this approach is capable of compensating for a wide range of systematic errors that make cross laboratory comparisons difficult.
(3) iGlycoMAb is expected to be the first approach to allow the absolute quantitation of glycans attached to glycoprotein. Prior to iGlycoMAb becoming a commercial product (although outside of the scope here) we will quantify the amount of each abundant glycan on iGlycoMAb, which will enable researchers to obtain the absolute quantitation of these glycans on their Ab samples.