Selman, M.H.J.
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, the Netherlands
Over the last decade the number of approved biopharmaceuticals harboring glycosylation has been rapidly growing. Glycans attached to therapeutic proteins affect their physical properties, stability and efficacy, and aberrant glycoforms may cause serious adverse effects. Detailed characterization of protein glycosylation is of utmost importance in pharmaceutical industry in order to maintain product quality and safety. Therapeutic immunoglobulins received much attention over the last years due to drastic enhancement of ADCC observed for antibodies lacking the core-fucoses on the Fc N-glycan.43,44 Therefore, IgG Fc N-glycosylation of biotechnologically produced IgGs is being designed in order to maximize the efficacy in e.g. anti-cancer therapy (next generation therapeutic antibodies).5,46
Glycan structural and linkage information of human IgG Fc N-glycans is well characterized and has been shown to be influenced by age, sex, pregnancy and health state.8-10,147 For a variety of infectious and autoimmune diseases, and cancer aberrant glycosylation has been reported.3,21,30-32 A common observation for many of these diseases is the lowered IgG Fc galactosylation and sialylation.147 Fc N-glycans have been shown to influence the biological activity of IgG by e.g. modulating Fcγ- receptor binding.19,35,38,39,43,44,146 Sensitive and detailed characterization of aberrant IgG Fc N-glycosylation profiles could provide valuable information on the regulatory mechanisms involved in a broad range of antibody mediated diseases. By implementing this knowledge into sensitive enzyme-linked immunosorbent assays capable of discriminating IgG with different glycosylation (e.g IgG with various levels of galactosylation but lacking the core-fucose), it might become possible to discriminate pathogenic from non-pathogenic situations. This would open the door for treating potentially harmful changes in glycosylation such as the decreased levels of core- fucosylation observed during pregnancy on IgG1 alloantibodies against the human platelet antigens of the fetus (causing fetoneonatal alloimmune thrombocytopenia)45 already in an early stage prior to the actual harm/damage.
Clearly, in view of the above mentioned interest in antibody glycosylation in the biopharmaceutical as well as in the clinical setting, analytical methods are needed to study these glycans. Detailed analysis of protein glycosylation is a challenging and time-consuming task due to the large structural variety in the attached glycans even for a single glycosylation site (microheterogeneity). In this thesis novel approaches for fast, miniaturized and high-throughput analysis of IgG Fc N-glycosylation are presented, and the utility of these methods has been demonstrated for clinically relevant research questions. Due to the complexity of human polyclonal IgG, Fc N-glycopeptide MS profiles might contain glycan information from a mixture of IgG subclasses. This is especially occurring with MALDI-MS approaches while obviously for LC-MS approaches it is a much less relevant issue. When analyzing biotechnologically produced therapeutic monoclonal antibodies, MALDI-MS and LC-MS both provide similar IgG Fc N-glycosylation information but differ in analysis speed. When analyzing monoclonal antibodies MALDI-MS offers the highest potential
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for accurate high-throughput IgG Fc N-glycosylation analysis. By contrast, LC-MS is better suited for accurate IgG Fc N-glycosylation analysis in complex biological samples such as plasma and serum. In general, LC-MS is less prone to in-source and metastable decay of highly sialylated glycopeptides than MALDI-MS. Nevertheless, the labile nature of glycoconjugates under standard ESI-MS proteomics settings requires several optimizations. Decay of glycoconjugates commonly occurs in the source and during the ion-transfer. Thus lowering of the potential differences during ESI (sprayer and MS inlet) and the ion-transfer (capillary and skimmer or lenses) is a good start to reduce glycan decay. Which potential differences have to be lowered, however, strongly depend on the type of mass spectrometer.
The IgG glycosylation analysis methods described in Chapter 2 and Chapter4 of this thesis are robust, can handle large sample cohorts, reveal glycosylation features such as galactosylation, sialylation, fucosylation, and the incidence of bisecting N-acetylglucosamine at an IgG subclass-specific level, and can be applied to analyze the Fc N-glycosylation of recombinantly produced IgG in great detail. Fast or high- throughput analysis was achieved by optimizing the entire analytical process, including sample preparation, glycopeptide detection and data processing. These techniques were then successfully applied for specific clinical research questions where IgG Fc N-glycosylation is believed to play a role (e.g. vaccination, autoimmune diseases). Other applications that one may envision involve longitudinal studies where total and autoantigen-specific IgG Fc N-glycosylation changes are evaluated to reveal associations between glycosylation features and e.g. disease severity. Furthermore, these sensitive fast profiling techniques are expected to prove their use for monitoring IgG Fc N-glycosylation changes during treatment. This would potentially provide information on the effectiveness of the treatment and offer the possibility to adjust the dose or treatment frequency for a specific patient. Within the pharmaceutical industry high-throughput glycopeptide profiling may prove its value during quality control and might be particularly useful for monitoring the fermentation process. This then allows early identification of harmful glycosylation changes and offers the possibility to interfere with the production process and restore product quality without losing an entire harvest.
As already mentioned the glycosylation of therapeutic proteins has to be human- like to prevent adverse effects. Therefore, prior to marketing complete structural characterization of protein glycosylation is mandatory for every biopharmaceutical. Here, a strategy based on MS can be problematic as no glycan linkage information is obtained and discrimination of isomers is virtually impossible even when cross ring fragment information is obtained.191,248 To obtain such structural and linkage information MS may be combined with lectin chromatography, and/or endo- and exoglycosidase treatment. High-throughput protein glycosylation analysis by MS with analysis techniques such as those developed in this thesis is, therefore, only useful with sufficient pre-knowledge on the glycosylation features of the specific protein. It should
be kept in mind that hitherto no technique is available which is capable of elucidating the entire structural complexity of protein glycosylation in a single analysis, and for detailed structural analysis the information from several (complementary) strategies has to be combined.
To our knowledge, the Fc N-glycosylation sites of human polyclonal IgG are fully occupied. In cases where only partial occupation of N-glycosylation sites occurs (e.g. recombinant expressed proteins)189 it might be required to monitor both glycosylated peptides and non-glycosylated variants of the peptides carrying the N-glycosylation consensus sequence. For these situations RP-SPE is the most suitable purification method as glycosylated and non-glycosylated variants of the peptides tend to co-elute. However, care should be taken with such an approach as MS glycopeptide signals are easily suppressed by co-eluting peptides which often possess higher proton affinities. Moreover, the acidic conditions applied during elution of (glyco)peptides from reversed phase stationary phases can quickly lead to partial desialylation of the glycopeptides. By contrast, HILIC-SPE is highly suited for glycoproteins with a high level of sialylation as elution from HILIC stationary phases can be performed with water. Moreover, HILIC purifications such as those described in Chapter 3 and Chapter4 of this thesis allow very selective glycoconjugate enrichment and at the same time the removal of salts, most non-glycosylated peptides, and detergents from glycoconjugate samples.
In this thesis we predominantly performed targeted analyses where we selectively studied Fc N-glycosylation of purified IgG samples and did not gather other potentially useful information. With this strategy we effectively determined IgG Fc N-glycosylation changes occurring during pregnancy (Chapter 2), aging (Chapter 4), vaccination (Chapter 6) and two autoimmune diseases (Chapter 7). Our results of clinical cohorts confirmed the previously reported IgG glycosylation changes with age, sex and pregnancy,4,8-10,13,14 and revealed specific glycosylation changes with vaccination and the two autoimmune diseases (i.e. LEMS and MG). However, no conclusive information on the regulatory mechanisms involved in IgG Fc N-glycosylation changes or on the immunological role and implications of these alterations have been obtained. While effector immune mechanisms of IgG are modulated by the Fc N-glycosylation, binding of the antigen is influenced by glycosylation of the Fab portion as has already been shown for asymmetric antibodies in pregnancy.212 By developing high-throughput techniques capable of simultaneously analyzing IgG Fc and Fab glycosylation more conclusive information might be obtained on the immune-regulatory and functional aspects of IgG glycosylation. Moreover, by broadening the range of simultaneously monitored blood glycoproteins (e.g. circulating lysosomal hydrolases,249 erythropoietin, fibrinogen, apolipoproteins, tissue plasminogen activator, fetuin, transferrin etc.) additional information on disease related glycosylation changes may be revealed. For complex biological samples the use of LC-MS is preferred over MALDI as the complexity of the mass spectra is reduced by the LC separation, and the retention time provides an additional criterion for the identification of glycopeptides apart from their
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m/z. The more sophisticated ultra high pressure LC separation systems coupled to high resolution mass spectrometers are expected to improve chromatographic resolution and confidence with glycopeptide identification, and will allow analysis of more complex glycopeptide samples.
With current technologies, targeted analysis and de novo glycan sequencing in complex biological samples remains a challenging and tedious task due to the lack of computational tools (e.g. algorithms, databases) capable of automated interpretation of high-throughput MS and MS/MS data from glycoconjugates. Hitherto, computational tools (e.g. GlycoPep DB,250 GlycoSpectrumScan,251 GlycoPeptideSearch) are available which facilitate compositional assignment for glycopeptides by comparing experimentally measured masses to calculated glycopeptide masses from carbohydrate databases. However, MS/MS spectra often do not reveal the complete structure information of the glycan. Furthermore, the quality of glycopeptide fragmentation spectra strongly depends on the experimental settings and can substantially differ between different MS instruments. Therefore, intensive manual evaluation remains required for correct assignment. To overcome these limitations a large database is required that contains good quality (high resolution) glycopeptide fragmentation mass spectra (preferably obtained with various instruments) of different biological and clinical samples. Together with a standardization of mass spectrometric procedures and settings, this would offer the possibility to match experimentally obtained MS/ MS spectra with spectra in the database and might allow the association of specific glycosylation changes with a particular biological state in a more automated high- throughput fashion.