Characterization of the N-glycosylation Pattern of Antibodies by Complementary Use of ESI and MALDI Mass Spectrometry

Antibodies represent one of the most important classes of glycoproteins playing a central role in the immune response of living organisms and therefore there is a growing interest in recombinant antibodies as potential biotherapeutic agents. The detailed analysis of the N-glycosylation pattern present on antibodies is challenging due to the heterogeneous structure of this posttranslational modification. The glycan structure is highly dependent on the process by which the recombinant glycoprotein is generated, such as host organism and growth conditions. Due to the fact that changes to the glycosylation pattern can significantly alter biological function, characterization of the N-glycosylation pattern is very important. Various mass spectrometric techniques have been developed and may be applied to analyze either the intact glycoprotein or the N-glycopeptides obtained from enzymatic digestion to study these glycosylation patterns. We describe here the in-depth characterization of the N-glycosylation pattern, in various antibody samples, by utilizing two complementary MS based analysis techniques:

• Fast top-down analysis based on LC-MS accurate mass measurement of the intact antibody and the released heavy chain; and
• Comprehensive bottom-up characterization of the N-glycosylation patterns by nanoLC-MALDI-TOF/TOF analysis of N-glycopeptides obtained from enzymatically digested antibodies.

Experimental

The analyses described here were performed on three different samples: a human IgG1 expressed in Chinese hamster ovary (CHO) cells, a commercially available bovine IgG, and a sheep IgG from prefractionated sheep plasma. The human IgG was reduced and alkylated to release the glycosylated heavy chain (HC) and an aliquot of the sample was separated by SDS-PAGE. The gel-separated heavy chain was then subjected to enzymatic in-gel digestion by trypsin. The bovine IgG was digested in-solution after reduction and alkylation. The sheep IgG was part of a pre-fractionated plasma sample that had been reduced, alkylated using methyl methanethiosulfonate (MMTS), and, after tryptic digestion, was labeled with the iTRAQ4plex label.

The following experimental setup was used for the LC-ESI-UHR-TOF measurements on the intact glycoprotein:

The LC-MALDI-TOF/TOF analyses were performed under the following conditions:

Annotation of glycan structures and glycan specific fragments on MS and MS/MS spectra was carried out using Glycoworkbench software (www.eurocarbdb.org).

Results

Rapid assignment of major glycosylation isoforms

Figure 1 shows the data obtained from fast LC-MS analysis of the intact human IgG1. The ability of the ESI-UHR-TOF instrument to generate high-resolution, highly accurate mass data of intact proteins allows a rapid assignment of major glycosylation isoforms of the analyzed human IgG1. Maximum Entropy deconvolution of the data yields a mass for the major glycosylation form, which fits very well to the expected average mass range based on a calculation of atomic weights from organic sources according to Zhang et al. (deviation between measured mass and expected mass based on average atomic masses from organic sources: 2 ppm). Further glycosylation isoforms were assigned based on characteristic mass distances of 162 Da indicating a rising number of galactose units.

The released heavy chain of the intact human IgG was also analyzed by ESI-UHR-TOF (see Figure 2). The measured mass was found to be in excellent agreement with the expected mass, showing a 2 ppm deviation from the calculated mass. In addition, the high-definition data provided allows unambiguous assignment of chemical artifacts which were generated during sample preparation. In the example shown , spectral peaks that did not match the masses of expected protein sequence/glycosylation structures (denoted with * in Figure 2) could be assigned to non-desired by-products originating from the alkylation step, leading to an over-alkylation, yielding additional peaks shifted by +57 Da in mass.

In-depth characterization of the N-glycosylation pattern of various antibodies

Applying multiple enzymes (trypsin, chymotrypsin, GluC, LysC), LC-MALDI analysis yielded, on average, a sequence coverage of 100% for the light chain and more than 98% for the heavy chain. As the glycan moiety does not contribute significantly to the specific retention of N-glycopeptides on a reversed phase column (data not shown), N-glycopeptides appear at a relatively early retention time. This helps to separate the glycopeptides from non-glycosylated tryptic peptides, particularly those with masses greater than 2500 Da.

Figure 3 shows the averaged LC-MALDI-MS spectrum of bovine IgG covering the N-glycopeptide retention time range (27-31min). The MALDI-TOF/TOF instrument used typically achieves resolution values between 30,000 and 45,000 in the mass range of interest for the analysis of N-glycopeptides. This is beneficial so as to avoid false annotations due to partially or non-resolved overlapping peaks, and contributes considerably to improving the significance of the results.

Figure 4 shows a MALDI-MS/MS spectrum of a bovine IgG N-glycopeptide at a parent m/z of 2780 Da. The displayed spectrum clearly illustrates a unique feature of N-glycopeptide MS/MS spectra generated on a TOF/TOF system: Detailed structural information is provided on both the peptide as well as the glycan moiety in a single MS/MS spectrum. Most importantly, the mass of the peptide moiety can be read out directly from the spectrum. Using this mass as a “pseudo” parent mass, the peptide sequence can even be identified by means of a database search using MASCOT or alternative search engines.  The sequence annotation of the spectrum was carried out using Bruker´s BioTools software.

Furthermore, the fragment peak that corresponds to the peptide moiety is part of a pattern consisting of four peaks occurring in subsequent distances of 17/83/120 Da. This peak pattern is highly specific to TOF/TOF spectra of N-glycopeptides, and originates from the fragmentation of the core HexNac unit that is attached to the peptide´s N-glycosylation site. This N-glycospecific fragmentation pattern can be used as the subject of a query to specifically extract (from an extensive LC-MALDI-MS/MS dataset) all those MS/MS spectra which potentially originate from N-glycopeptides. The structural annotation of the glycan fragments was performed using the “Glycoworkbench”, a software tool developed and made available to the public by the EurocarbDB project (http://www.eurocarbdb.org).

Based on the LC-MALDI-MS and – MS/MS data, the detected patterns of N-glycopeptides can now be assigned in detail. This is shown in Figure 5 for three different trypsin digested antibody samples, a bovine IgG, a sheep IgG, and a human IgG1, respectively. The three samples described here, according to their individual origins, yield N-glycosylation patterns of very different complexity, including complex and high mannose glycan structures. These patterns consist of at least two overlapping series of glycopeptides, one of them containing a missed trypsin cleavage site. When comparing the human IgG data generated by either top-down ESI or bottom-up LC-MALDI analysis (see Figure 2 and Figure 5c, respectively), the relative abundance of the major glycosylation isoforms are in very close agreement with one another.

Conclusions

LC coupled to either ESI-UHR-TOF or MALDI-TOF/TOF represent complementary techniques offering unique capabilities for the characterization of the N-glycosylation pattern present in antibodies. The methods described here serve as powerful tools for rapid quality control and comprehensive characterization of glycoprotein drugs. LC-ESI-UHR-TOF is a fast top-down method to provide exact mass data (mass accuracy in low ppm range) of antibodies in both, intact and reduced form. This data enables an instant assignment of major N-glycosylation isoforms, including the characterization of processing artifacts (e.g. non-desired protein over-alkylation).In a complementary fashion, MALDI-TOF/TOF is a superior platform for the in-depth analysis of N-glycopeptides from digested antibodies. The extraordinarily high resolution provided by the instrument is of great benefit to tackle the complex heterogeneity of N-linked glycans. LC-MALDI-TOF/TOF provides information-rich, easy-to-interpret MS and MS/MS spectra of N-glycopeptides, yielding a detailed picture of the N-glycosylation pattern. MALDI-TOF/TOF data of N-glycopeptides simultaneously provides information on both the peptide sequence and the glycan structure in the same spectrum.

Novel features have been designed into today’s MALDI software suites to support selective filtering of MS/MS spectra of N-glycopeptides out of large LC-MALDI-MSMS datasets, and to enable efficient subsequent analysis of this data to ultimately allow the annotation of highly heterogeneous N-glycosylation patterns.