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Improving analytical characterization of glycoconjugate vaccines through combined high-resolution MS and NMR: Application to Neisseria meningitidis serogroup B oligosaccharide-peptide glycoconjugates Huifeng Yu, Yanming An, Marcos Daniel Battistel, John F. Cipollo, and Darón I. Freedberg Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04748 • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

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Analytical Chemistry

Improving Analytical Characterization of Glycoconjugate Vaccines Through Combined High-Resolution MS and NMR: Application to Neisseria meningitidis Serogroup B Oligosaccharide-Peptide Glycoconjugates Huifeng Yu, Yanming An, Marcos D. Battistel, John F. Cipollo, Darón I. Freedberg* Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, FDA, Silver Spring, Maryland 20993 Corresponding author: [email protected]

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Abstract Conjugate vaccines are highly heterogeneous in terms of glycosylation sites and linked oligosaccharide length. Therefore, the characterization of conjugate vaccines' glycosylation state is challenging. However, improved product characterization can lead to enhancements in product control and product quality. Here, we present a synergistic combination of high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy for the analysis of glycoconjugates. We use the power of this strategy to characterize model polysaccharide conjugates to demonstrate a detailed level of glycoproteomic analysis. These are first steps on model compounds will help untangle the details of complex product characterization in conjugate vaccines. Ultimately, this strategy can be applied to enhance the characterization of polysaccharide conjugate vaccines. In this study we lay the groundwork for the analysis of conjugate vaccines. To begin this effort, oligosaccharide-peptide conjugates were synthesized by periodate oxidation of an oligosaccharide of a defined length, α,2-8 sialic acid trimer (Sia3), followed by a reductive amination, and linking the trimer to an immunogenic peptide from tetanus toxoid. Combined mass spectrometry and nuclear magnetic resonance were used to monitor each reaction and conjugation products. Complete nuclear magnetic resonance peak assignment and detailed mass spectrometry information of oxidized oligosialic acid and conjugates are reported. These studies provide a deeper understanding of the conjugation chemistry process and products, which can lead to a better controlled production process.


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Introduction Several bacterial conjugate vaccines that protect against bacterial meningitis caused by Neisseria meningitidis serogroups A, C W and Y, Streptococcus pneumoniae and Haemophilus influenzae serogroup B (Hib) 1,2,3 are available in the United States. Neisseria meningitidis has 13 clinically significant serogroups, classified according to their antigenic primary structure and elemental composition of their polysaccharide (PS) capsule. Of the 13 known serogroups, six (serogroups A, B, C, X, Y and W135) are responsible for most of the meningococcal disease worldwide.4,5,6 Three meningococcal conjugate vaccines are licensed in the United States: Menactra® (MenACWY-DT) and Menveo® (MenACWY-CRM) and MenHibrix® (Hib-MenCY-TT). These are immunogenic PS covalently attached to DT (diphtheria toxoid), CRM (a cross reactive nontoxic diphtheria toxin mutant) or TT (tetanus toxoid), respectively.7,8,9 Conjugation of a PS to a protein carrier profoundly alters the immunological properties of the PS, converting it from a thymic-independent to a thymic-dependent antigen, fundamental for providing T cells epitopes and consequently eliciting a memory response of the immune system against the saccharide.10,11,12 One method to synthesize PS-conjugate vaccines is reductive amination in which aldehydes, generated from vicinal diols in PSs or oligosaccharides (OSs), are attached to the amino groups in lysine side chains. Since there may be many reactive lysines in a given protein, it is possible that activated (aldehyde containing) PSs will not homogeneously attach to lysines in a given conjugation reaction, thus contributing to heterogeneity of glycoconjugates. Different length activated OSs may also react with the protein at a given lysine site, further contributing to heterogeneity. The heterogeneity in a given sample makes characterization of the resulting glycoconjugates extremely difficult. We propose to reduce this difficulty using a combination of mass spectrometry (MS) and NMR, two extremely powerful analytical techniques. Recently a combination of NMR spectroscopy and advanced MS has been proposed for glycan, glycoprotein and metabolomics analysis; the combination can overcome the limitations of the individual techniques.13-18 MS can be performed in a rapid and sensitive manner with less material needed (as low as 0.1 µM), whereas NMR is less sensitive, with limits of detection on the order of 10µM or a few nmol at high fields using



cryoprobes or specialty coils.14,

17

However, NMR can provide details such as anomeric

configurations in a straightforward manner, while MS cannot easily yield this information. Additionally, as opposed to NMR, MS has difficulty in determining the connectivity of monosaccharides in the glycan, the linkages for each glycosidic bond (α or β anomeric position) and isomers without extensive fragmentation analysis where chemical derivatization is often required. On the other hand, NMR spectroscopy provides crucial structural information and overcomes all these problems with great reproducibility, but cannot yield mass and the elemental composition of a molecule as can MS methods. Together, the two methods are highly complementary and it is this complementarity we wish to exploit. The application of this combination of analytical methods is not new however although it has not been applied to the in depth characterization of conjugate vaccines.19-22 This combination of analytical methods is not new however it has not been applied to characterize conjugate vaccines. In the present study we use a combination of NMR spectroscopy with MS and show that this combination improves PS conjugate vaccine characterization that allows us to achieve a new, more detailed level of glycoproteomic analysis. We use a simplified OS-TT conjugate as a model system to enable us to gauge the feasibility of combining NMR and MS to achieve enhanced analytical characterization. As we show, even this simplified glycoconjugate is extremely difficult to characterize. Recent research indicates that glycan-protein connectivity impacts the structure and immunogenicity of glycoconjugate vaccines, which further suggests that combined use of NMR spectroscopy and MS for analysis of the glycoconjugate vaccine is important to elucidate crucial structural information.23 We aim to approach PS-conjugate vaccine characterization in a stepwise fashion. In the present study, we use an OS of a defined length, α, 2-8 sialic acid trimer (Sia3), rather than a polydispersed PS. Although Sia3 is a small and well-defined system, two different aldehydes can be form upon reaction with periodate. The number of repeating units in Sia3 remains constant after activation and MS can easily detect the mass change in the trisaccharide. This approach provides two main advantages: a) it provides a homogeneous starting material of a defined molecular weight, significantly decreasing the potential heterogeneity of the product and b) it enables data collection at atomic resolution required for detailing the connectivity and structure of the saccharide portion of the conjugate. Second, as a carrier protein model, we use an


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antigenic 15-residue antigenic peptide from tetanus toxoid (TT830-844), containing only two potential glycosylation sites (two lysine residues), which further simplifies analysis by reducing product heterogeneity. We oxidize Sia3 and then conjugate it to TT830-844, using reductive amination (Figure 1) with sodium cyanoborohydride (NaBH3CN).24,

25

We also quantitatively

demonstrate that for Sia3, the aldehyde group can be generated at either the III-7 or III-8 position of the end sialic acid ring containing three adjacent alcohols,26 depending on the NaIO4:saccharide ratio.

Figure 1. Oxidation reactions of Sia3 and subsequent conjugation to TT830-844. The aldehyde group is generated at the III-7 or III-8 position of the terminal sialic acid ring, which contains three adjacent OH groups. Lactonization of Sia3 on target in the presence of phosphoric acid was performed for MALDI measurement. Chemical reactions of oxidized Sia3 with lysine of TT830-844 are shown and marked with mass change.



Materials and Methods What follows is a general Materials and Methods section. Due to space constraints, a more detailed Materials and Methods write up is included in SI. Materials. An immunogenic 15residue peptide from tetanus toxoid 830-844 (TT830-844) QYIKANSKFIGITEL was prepared by solid-phase synthesis in the Facility for Biotechnology Resources (FBR) at the Center for Biologics Evaluation and Research, FDA. α(2,8)-linked N-acetylneuraminic acid trimer (Sia3) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Sodium periodate, sodium cyanoborohydride and all other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA) and used without further purification, unless otherwise noted. Bio-gel P2 gel (cat.1504115) was from Bio-Rad Laboratories, Inc. (California, USA) and the column was packed according to manual instruction. Sia3 conjugation to tetanus toxoid TT830-844 peptide Sia3 was oxidized with sodium periodate (NaIO4, freshly prepared in water) at a NaIO4 to Sia3 ratio of 1:1 (NaIO4:Sia3=1.1) in water or 5:1 molar ratio in 0.1M sodium acetate, pH 5.5 on ice for 30 min in the dark to avoid unwanted photo-activated radical reactions.27 For coupling of OSs to TT830-844, oxidized (activated) Sia3 were mixed with TT830-844 at a 1:2 molar ratio and incubated for 30 min at room temperature in 0.8 mL of water with or without DMSO. Sodium cyanoborohydride was directly added to the solution and the conjugation reactions were carried out at 37 °C for 1, 2 and 3 days. Subsequently, the reaction mixture was evaluated using MALDI-TOF-MS. On day 3, the reaction mixture was directly dialyzed against water and lyophilized for NMR and nanoscale liquid chromatography coupled to tandem mass spectrometry (NanoLC–MS/MS) analysis. Conjugates were further purified using C18 column (Agilent technologies, CA, USA) for NMR assignment. MALDI spot preparation and MALDI-TOF-MS analysis Sia3, activated Sia3 or glycoconjugate were loaded onto a stainless steel MALDI-TOF target and allowed to dry. On target lactonization of sialic acid was performed as reported by Galuska et al.28 MALDI-TOF-MS analysis was performed on an Autoflex instrument (Bruker-Daltonik, Bremen, Germany) equipped with a nitrogen laser and controlled by FlexControl 2.0 software. The instrument was operated in positive–ion mode in both linear and reflectron configurations.


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Masses were annotated and processed with FlexAnalysis 2.0. External calibration of mass spectra was carried out using maltoheptaose. Reverse phase NanoLC– MSE analysis Characterization of glycoconjugates was performed via a Waters SYNAPT G2 HDMS mass spectrometer (Waters Corporation, Milford, MA) equipped with nano-spray source and nanoAcquity LC system. The data produced during the experiment are used to reconstruct MS/MS spectra without the bias associated with data-dependent ion scanning.29 Before analysis, the conjugate

mixture was dialyzed against water using a 1000 MWCO dialysis for buffer exchange and removal of small chemical products. NMR measurements Sia3 samples were dissolved and recorded in 20 mM phosphate buffer pH 6.0 in 100% D2O or 90%/10% H2O/D2O. TT830-844, glycoconjugate TT830-844-Sia3 were reconstituted in 20 mM phosphate buffer at pH=5.7 with 10% D2O. All NMR measurements were performed on Avance III 500 MHz spectrometer equipped with a z-gradient QXI probe.



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Results and Discussion Assignment of oligosialic acid 1

H NMR chemical shifts of monomer, dimer and trimer measured at 300 K in D2O were reported

previously.30,31 There are difficulties in assigning the H6 and H7 1H’s due to the small 3JH6-H7, and spectral overlap, which prohibit direct assignments in normal two-dimensional spectra such as COSY, TOCSY, HSQC and HMBC.31, 32, 33 These problems were circumvented by recording an HSQC-TOCSY and HSQC experiments that alleviate spectra overlap by providing spectral dispersion in the

13

C dimension and aid in making assignments.30,34 Thus, we use the 1H-13C

HSQC-TOCSY to separately assign the H8-H7 correlations and H5-H6 correlations. 1H-13C HSQC-TOCSY gives through-bond correlations between a 1

13

C attached 1H to all other coupled

H. In our present study, HSQC-TOCSY spectra were recorded with different mixing times (15

and 90 ms). Shorter mixing times are useful for assigning 1H resonances on adjacent carbons since only vicinal 1H-1H correlations are observed. Moreover, shorter mixing times reduce spectral crowding because fewer correlations are observed. However, longer mixing times are still needed to facilitate the assignment of all 1H’s in one ring of the saccharide's spin system. To explore the minimal set of experiments required to characterize larger sialic acid oligosaccharides we first characterized monomeric sialic acid and Sia2 by NMR. In solution, monomeric Sia is found mainly as the β anomer (~93% in β, ~7% in α).35 However, for Sia2 or larger oligosaccharides the first residue (free OH at C2) is almost exclusively in the β form. Due to their distinct NMR spectra, both α and β forms of sialic acid can be readily identified in a 1H1

H TOCSY spectrum (data not shown). We seek full characterization of a glycoconjugate

including the anomer and linkage determination of the saccharide. Therefore, we opted for NMR experiments for oligosaccharide characterization at this stage. We found that 1H-13C HSQC and 1

H-13C HSQC-TOCSY afforded full assignment of oligosaccharides. 1H-13C HSQC and 1H-13C

HSQC-TOCSY spectra of Sia3 and assignments of all resonances are shown in Figure 1S. The 1H and 13C chemical shifts for Sia3 are general agreement with previous reports (Table S1).31-32 Oxidation of oligosialic acid Sia3 Mild sodium periodate (e.g.1-2mM) selectively oxidizes only the glycerol chain of the terminal (III) sialic acid residue of Sia3 at III-7 or III-8 position to yield reactive aldehyde groups.26,36 To


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further clarify how to produce aldehydes at C7, C8 and to produce a homogeneous glycoconjugate, we used different NaIO4:Sia3 molar ratios. As expected, we found that at a 1:1 molar ratio of NaIO4:Sia3, aldehyde groups were produced both at III-7 (Sia3-III-7CHO) and III8 (Sia3-III-8CHO) positions (Figure 2A); whereas a 5:1 molar ratio of NaIO4:Sia3 yielded an aldehyde only at III-7 (Sia3-III-7CHO) (Figure 2B). The remainder is not oxidized. 1D 1H spectra of a 1:1 molar ratio of NaIO4:Sia3 are shown in Figure 2A. The NMR peak at 9.68 ppm corresponds to the aldehyde at III-8 position and the peak at 5.19 ppm corresponds to a hydrated aldehyde at III-8 position (as per HSQC-TOCSY assignment, Figure 2 and Table S3). Under these conditions, three molecular species can be identified in yields of 77%, 18% and 5%, based on quantitative NMR. The compound in 77% (1H chemical shift at 5.19 ppm) is assigned to a hydrate form of the aldehyde at C8 (Figure 2). The compound present in 18% was assigned to the hemiacetal formed between III-C7 (where residue III is ring-inverted) and II-9 (for details of assignment, see below, Figure 2. The 1H chemical shift at 9.68 ppm corresponds to an aldehyde at C8 (5%). For quantitative NMR, 1D proton experiments were recorded without presaturation and water suppression (Bruker pulse sequence zgpr). It is known that in aqueous solution, water adds rapidly to the carbonyl function of aldehydes, establishing a reversible equilibrium with a hydrate in the presence of an acid or a base.37 The chemical shift for III-7 (5.9 ppm) is somewhat higher than expected for a hydrate. This peak was shown to arise from the formation of an interring hemiacetal between III-7 position and II-9.32 These findings agree with the MS data (Figure 3). Moreover, it provides more detailed structural information that cannot be addressed by MS data. Detailed assignments are shown in Figure 2S A-C for Sia3-III-7CHO, 2D-F for Sia3-III8CHO based on 1H-13C HSQC-TOCSY experiments.



2A

2C

2E

2B

2D

2F

Figure 2. A, B. 1D 1H spectra of Sia3 with different ratios of sodium periodate. The peak at 9.68 indicates an aldehyde at III-8 position and a peak at 5.19 ppm indicates the aldehyde’s hydrate in aqueous solution. The peak at 5.90 ppm indicates hemiacetal formation between III-7 and II-9. C, D. Expanded overlays of two-dimensional 1H-13C HSQC spectra of Sia3 and Sia3-III-7CHO (sodium periodate: Sia3 at a molar ratio of 5:1) and assignments of Sia3-III-7CHO. E, F. Expanded overlays of two-dimensional 1 H-13C HSQC spectra of Sia3 and Sia3-III-8CHO (sodium periodate: Sia3 at a 1:1 molar ratio) and assignments of Sia3-III-8CHO.

Terminal Sia residues in biomolecules are known to form aldehydes at C7 or at C8 depending on the concentration of NaIO4 utilized in the oxidation reaction. Though this was reported previously and the oxidation chemistry is similar for oligosialic acids, NMR spectral assignments for the oxidized mucins were not reported.38 NMR assignments for the different oxidation products show that different molar ratios of NaIO4:Sia3 yield dissimilar products including C7 hemiacetal (Figures S1 and S2, Tables S2 and S3), C8 aldehyde, and C8 hydrate. As stated above, a 5:1 molar ratio of NaIO4:Sia3 did not yield an aldehyde at III-8 and only the peak corresponding to a hemiacetal at III-7 was observed (Figure 2A, Table S5). However, at a 1:1 molar ratio of NaIO4:Sia3, NMR peaks corresponding to C7 hemiacetal, C8 aldehyde, and C8 hydrate were observed. These observations were confirmed with the following MS results, 1:1


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molar ratio of NaIO4:Sia3, aldehyde groups were produced at III-8 (824.46 amu) and III-7 (794.44 amu), whereas at NaIO4:Sia3 5:1 molar ratio, aldehyde group only at III-7 was generated. A comparison of Sia3 and Sia3-III-7CHO 1H-13C HSQC spectra (Figure 2C) reveals the changes that occur upon Sia3 oxidation (NaIO4: Sia3 = 5:1). Because an aldehyde is produced only at III7, cross peaks of III9, III9’ and III8 disappeared in the spectrum of Sia3-III-7CHO. Hemiacetal formation between III-7 and II-9 results in a pronounced chemical shift change of II-8 and II-9 was observed. The assignment of Sia3-III-7CHO shown in Figure 1, Figure 2C and Table S2 is in agreement with previous research.32 As shown in Figure 2D, aldehyde formation at III-8 was observed when the NaIO4:Sia3 molar ratio was 1:1. Discrimination between Sia3-III-7CHO and Sia3-III-8CHO can be easily accomplished by NMR because these compounds display different 1

H and

13

C resonances corresponding to positions III-3, III-4, III-5, III-6 and III-7 and III-8

(Figure 2D). When the aldehyde is produced at III-8, resonances of I-Sia and II-Sia were still in agreement with literature values for unoxidized Sia residues.39,34,40 The chemical shift for oxidized Sia3 agrees with that of an aldehyde at III-8 in its hydrated form according to our assignment. The 1H and 13C chemical shift Sia3-III-8CHO are shown in Table S5. 1H-13C HSQCTOCSY experiments of Sia3-III-7CHO and Sia3-III-8CHO with assignments are shown in Figure S2A-E. The initial assignments of NMR spectra are time consuming; nevertheless, NMR provides extraordinarily detailed information for molecules in solution. NMR spectra are highly reproducible and once spectra of fragments are present in databases their use is straightforward. Subsequently, the Sia3 sample was analyzed by MALDI-TOF-MS by on-target lactonization (under acidic conditions) as previously reported.28,41 Compared to NMR data, MS data are more straightforward to interpret as the primary output is mass because there are databases for MSbased molecular fragments; in contrast, similar NMR databases are non-existent. Therefore, we provide our logic in the following. The MALDI-TOF MS of Sia3 (C33H53N3O25 891.2968 amu) contains a peak corresponding to singly charged ion at m/z 878.32. A loss of 14 amu was observed by MS, which corresponds to lactone formation, equivalent to the loss of two H2O molecules and addition of a sodium ion (Figures 1, 3). Several peaks corresponding to oxidized Sia3 were observed in the MS spectrum. After Sia3 oxidation, molecular ions with m/z 824.46 (Na, -22.99 amu, addition of H+, 1.009 amu and aldehyde at III-8 positon -CH4O, -32.04 amu,) and 794.44 (-Na+, -22.99 amu, addition of H+, 1.009 amu aldehyde at III-7 position -C2H6O, -



62.07 amu) were observed. MS data confirm that at a 1:1 molar ratio of NaIO4:Sia3, aldehyde groups were produced at III-8 and III-7, whereas at NaIO4:Sia3 5:1 molar ratio, an aldehyde resulted solely at III-7 (Figures 3A, 3C); relative ratios of III-8:III-7 aldehydes in MS are 4:1, in agreement with 1D 1H NMR integration data. The corresponding mass is 816.91 amu (794.457 + Na+, 22.99 – H+, 1.009) for the aldehyde at III-7. As opposed to our NMR data, MS data did not provide any evidence for hydrated aldehyde at III-7 and III-8 formation in aqueous solution. This result is not surprising since there is no mass change upon hemiacetal formation between III-7 and II-9. Thus, it was impossible to determine whether there is a hemiacetal formation between III-7 and II-9 by MS alone.

Figure 3. MALDI-TOF-MS spectra of Sia3 and its oxidized forms. Molecular ions were observed at m/z 878.327 (Sia3 + Na+), 824.43 (from Sia3 + Na+ - 3H2O), 794.44 (Sia3 + Na+ - 3H2O - COH2) and 778.408 (Sia3 + Na+ - 2H2O – CH3OH. (B) and its oxidized forms aldehyde at III-8 positon (-CH4O, -32.04 amu) and III-7 position (C2H6O, -62.07 amu). At a 1:1 molar ratio of sodium periodate:Sia3, the aldehyde groups were produced predominantly at III-C8 (A), whereas at a 5:1 molar ratio of sodium periodate:Sia3, the aldehyde group is only generated at III-7 816.913 (Sia2-CHO + Na+), 794.871 (Sia2-CHO) and 776.912 (Sia2-CHO-H2O) (C).

The above results illustrate how NMR and MS complement each other to yield additional information. MS together with the atomic level insight and reproducibility of NMR data provide great benefit for characterization of oxidized OS. Several peaks were observed in the MS spectrum of oxidized Sia3. Although there were peaks corresponding to aldehyde formation, we


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were unsure whether any additional reaction occurred. If the aldehydes at III-7 and III-8 were hydrated in aqueous solution, MS might not easily distinguish these due to addition or loss of water during ionization, but this could have important structural and immunogenic consequences. Moreover, there is a hemiacetal formation between aldehyde at III-C7 and II-C9 as evidence by NMR. However, since there is no mass gain or loss, MS could not address this issue. On the other hand, MS data aided in interpreting NMR data, especially for the product mixture obtained from oxidation using 1:1 NaIO4:Sia3 molar ratio which yielded a mixture III-7 and III-8 aldehyde groups. Therefore, our combined use of NMR spectroscopy and MS for analysis of oxidation OS for drug conjugation, provided insight into OS reactivity, experimental reproducibility and its conjugation chemistry. Analysis of TT830-844 and glycoconjugate TT830-844 - Sia3 by mass spectrometry In MALDI analysis, the analyte is ionized through the matrix, usually to a single ion, whereas in ESI and nano-spray, the analyte in solution is converted to an aerosol by the electrospray and ions are usually multiply charged. TT830-844-Sia3 has two predicted masses 2567.23 amu and 2537.23 amu due to conjugation of the two different aldehyde-containing isomers at III-8 and III-7 position (Figure 1). Because TT830-844 has two lysine residues, depending on the oligosaccharide activation method was used, the conjugation reaction can yield up to 4 isomeric di-glycosylated peptides, namely: two Sia3 linked to the peptide through III-7 (7,7); two Sia3 linked through III-8 (8,8); or one Sia3 through III-7 and or one through III-8 (7,8 and 8,7). Moreover, singly glycosylated products are also possible yielding four additional glycoforms. However, the degree of glycosylation can be tuned by tuning the activated oligosaccharide to TT830-844 molar ratio. We found that when we use TT830-844:Sia3 at 2:1 molar ratio, most of the conjugated product has only one Sia3 linked to TT830-844. Real time measurements of the conjugation reaction were performed by monitoring product formation at 0, 1, 2 and 3 days using MALDI-TOF (Figure S3). A comparison of product formation on day 3 and day 2, revealed that the ratio of the conjugate peak's intensity at 2500 amu vs. peptide peak did not increase, indicating at day 2, the reaction was complete. The mass spectrum of TT830-844 (1724 amu) contained a peak corresponding to singly charged (M + H)+ molecular ions at m/z 1725, whereas TT830-844 - Sia3 contains peaks corresponding to singly charged (M + H)+ molecular ions (ca.



2500 amu indicated by an arrow Figure S3). There are several peaks in MS spectra indicating the presence of a mixture of different compound. The ESI mass spectrum of TT830-844 displayed peaks corresponding to triply (M + 3H)+3 charged molecular ions at m/z 575.64, in agreement with the expected mass for TT830-844 (1724 amu) (Figure S4A). As shown in Figure S4B, the ESI mass spectrum of TT830-844-SiA3 contains peaks corresponding to triply (M + 3H)+3 charged molecular ions m/z 856.71, in agreement with the mass of TT830-844-SiA3 (2567.23 amu due to aldehyde group at III-8). One triply (M + 3H)+3 charged molecular ions m/z 846.71 with low intensity is in agreement with the mass of TT830-844SiA3 (2537.23 amu due to aldehyde group at III-7 position). As expected, NaIO4:Sia3 at 5:1 molar ratio only the m/z 846.74 was observed molecular ion which corresponds to Sia3III-7 (Figure S4C). Peaks at m/z 850.71 and 840.73 indicate the loss of water, when compared to peaks at m/z 856.71 and 846.71. Even a mono-glycosylated conjugate synthesized from homogeneous activated oligosaccharide species has the potential to yield two different glycosylated peptides. Therefore, to characterize site occupancy for each of the potential glycosylation sites in TT830-844, MS/MS fragmentation was performed. Peptide fragmentation was observed using nanoelectrospray (nanoESI). nLCESI-MSEdata of TT830-844 and TT830-844-Sia3 are shown in Figure S5. Molecular ions m/z at 2259.125 (309 amu), 1968.03 (291 amu), and 1707.92 (260 amu) indicated the fragmentation of sialic acids of TT830-844-Sia3 at 2568.23amu, whereas molecular ions m/z at 2229.10 (309 amu), and 1938.02(291 amu) indicated the fragmentation of sialic acids of TT830-844-Sia3 at 2538.23 amu (Figure S5B). At a 5:1 molar ratio of NaIO4:Sia3, only TT830-844-Sia3 at 2538.23 amu was observed. Molecular ions m/z at 2229.10 (309 amu), 1938.02 (291 amu), and 1707.92 (230 amu) were observed due to the aldehyde at III-7 (Figure S5C). Since only one lysine was occupied by Sia3, based on fragmentation data of C18 purified TT830-844-Sia3 the percentage occupancy of Lys4 and Lys8 was 54% and 45% respectively. Without more advanced technology and fragmentation such as ion mobility spectrometry combined with collision induced dissociation (CID) fragmentation, the linkage types such as α-2,8 sialic acid cannot be separated or identified with its standard

42

. However, the type of linkage of the oligosaccharide for TT830-844-Sia3 was

easily confirmed by NMR analysis, as we describe below. Analysis of TT830-844 and TT830-844-Sia3 by NMR


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The 1D 1H NMR experiment is the most common and simplest NMR experiment to execute. For these glycopeptides, 1D 1H NMR experiments yield sharp signals quickly and efficiently that can be used as fingerprints to report changes on both conjugates and glycan. The HPLC fraction containing TT830-844-Sia3 at 2537.23 amu was analyzed by NMR after purification on a C18 column. Compared to 1D proton spectra of Sia3-III-7CHO, TT830-844 and TT830-844-Sia3, combined peaks of peptide and Sia3-III-7CHO are easily observed in the conjugated TT830-844Sia3 sample (Figure 4A). A sharp peak corresponding to N-acetyl CH3 (at ~2 ppm) of Sia3-III7CHO was found in TT830-844-Sia3 spectrum. As shown in Figure 4B, the peak at 5.90 disappeared after Sia3-III-7CHO was conjugated to TT830-844, indicating that a chemical reaction occurred between the aldehyde at III-7 and TT830-844.

Figure 4. A-B. 1H NMR spectra of Sia3-III-7CHO, TT830-844 and TT830-844-Sia3. A sharp peak corresponding to the N-acetyl CH3 (about 2 ppm) of Sia3-III-7CHO was found in TT830-844-Sia3 spectrum. C. 1H-1H TOCSY spectra overlay of TT830-844 and TT830-844-Sia3 D. Assignment of TT830-844-Sia3.

In order to assign peaks in the 1D spectrum, 2D 1H-1H TOCSY, 1H-13C HSQC and 1H-13C HSQC-TOCSY NMR spectra of TT830-844 and TT830-844-Sia3 were recorded. The assignment of



TT830-844 can be found in Figure S6 and Table S2. Overlaid 1H-1H TOCSY of TT830-844 and TT830844-Sia3

show that there is not a significant change in the 1H chemical shift of most amino acids

upon conjugation, except for a slight change for Lys-4 (K4), Lys-8 (K8) and Ile-3 (I3). The chemical shift change of K4, K8 and I3 upon conjugation is consistent with the modification of TT830-844 by reductive amination. (Figure 4C). After conjugation, new cross peaks corresponding to Sia3 were found in 2D NMR spectra. Assignment of TT830-844-Sia3 and the proton chemical shift of TT830-844-Sia3 are shown in Figure 4D and Table S4. Bold letters indicate 1H chemical shift change of amino acids including lysine, isoleucine, after the peptide was coupled to Sia3III7. However, the covalent bond between TT830-844 Lysine residues to Sia3III-7 could not be directly detected by NMR due to amine group (NH) proton rapid exchange with water. In contrast, based on MS fragmentation data, we could easily distinguish which lysine is occupied by Sia3 and the percentage of occupancy for each K4 and K8 site. Applied MS for glycoconjugate analysis, in low abundance samples (as low as 0.1 µM), can rapidly determine the mass of the glycoconjugate and also display all possible conjugate products in a single reaction. Therefore, MS-based glycoconjugate analysis is very helpful for real time conjugation process monitoring. But for more detailed structural information such as isomer identification and glycosidic linkage determination we had to resort to NMR measurement. A recent report established that glycan conjugation to lysine residues of carrier protein can potentially impact the immunogenicity of a glycoconjugate vaccine due to an antigenic structural change upon conjugation.23 When glycans are conjugated to carrier proteins, unexpected structural changes can occur. In this regard, NMR provides unique and detailed structural information if antigen fingerprints are identified. Moreover, since NMR is non-destructive, analyzed samples remain intact and can be re-used for different analysis. Although NMR could be used to probe for structural changes after conjugation, valuable structural data can be lost when analyzing complex molecules unless glycoconjugates fingerprints are uncovered. When reference data are not available, MS analysis is the method of choice. In summary, combining NMR with MS can overcome the limitations of each and result in great advantages for glycoconjugates analysis. Conclusion


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Analytical quantitative characterization of PS conjugate vaccines is surely a daunting task. We have begun to approach this task with simpler model systems than PS conjugate vaccines in order to identify the pitfalls and address the challenges of the methods. We anticipate that in the future, conjugate vaccines may be enzymatically digested to yield some form of glycopeptides, which will resemble the model systems presented herein. Although we believe the real PS conjugate vaccines will be more complex than these systems, we consider this a necessary fundamental step.

Characterizations of whole conjugate vaccines will most certainly be more complicated than the glycopeptides in the present study. Furthermore, this study is not intended to establish the specific oligosaccharide-conjugates in conjugate-vaccines. Nor do we believe that in conjugate vaccines, the characterizable fragments will contain merely 3 oligosaccharide residues. This study highlights some of the difficulties present in even small molecular fragments that are likely to be encountered as a part of conjugate vaccine characterization.

We have shown by NMR that using different molar ratios of NaIO4 and Sia3 we can selectively target different oxidized Sia3 products. At a 1:1 molar ratio of NaIO4:Sia3, III-7-hemiacetal, III-8 aldehyde, as well as III-8 hydrate forms were produced. A 5:1 molar ratio of NaIO4:Sia3 yielded solely III-7-hemiacetal. To our knowledge, this is the first quantitative report that shows that the NaIO4:oligosaccharide ratio impacts the product formed. After conjugation of the oxidized Sia3 with TT830-844, the conjugated products with expected mass and fragmentation were confirmed by MS. Once the intermediate substances and the conjugated product have been characterized, 1D and 2D NMR spectra with assignments for oxidized Sia3 and TT830-844, TT830-844-Sia3 can be used as reference fingerprint spectra which will improve intermediate and product quality control. Together, the high sensitivity of MS plus the excellent reproducibility of NMR provide great benefit for real-time monitoring conjugation and characterization of glycoconjugates. Combined use of MS and NMR could overcome the separate limitations of each method and provide reliability, and reproducibility in for glycoconjugate analysis. These can be applied to analyze and characterize new and next generation glycoconjugate vaccines analysis. Acknowledgements



We are grateful to Dr. W. F. Vann, Dr. R. Lee, Dr. Hugo Azurmendi, Flora Lichaa, Dr. Lisa Parsons and Dr. Aaron Marcella for helpful discussions. This work was supported by intramural funds from CBER/FDA.

Supporting Information Supporting information available: Detailed Materials and Methods; Chemical shift assignment tables for all molecules; Assigned MS and 2D heteronuclear NMR spectra of starting materials, oxidized intermediates and conjugates; and chemical structures of activated intermediates.


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Figure 1. Oxidation reactions of Sia3 and subsequent conjugation to TT830-844. The aldehyde group is generated at the III-7 or III-8 position of the terminal sialic acid ring, which contains three adjacent OH groups. Lactonization of Sia3 on target in the presence of phosphoric acid was performed for MALDI measurement. Chemical reactions of oxidized Sia3 with lysine of TT830-844 are shown and marked with mass change. 190x131mm (300 x 300 DPI)


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Figure 2. A, B. 1D 1H spectra of Sia3 with different ratios of sodium periodate. The peak at 9.68 indicates an aldehyde at III-8 position and a peak at 5.19 ppm indicates the aldehyde’s hydrate in aqueous solution. The peak at 5.90 ppm indicates hemiacetal formation between III-7 and II-9. C. Overlay of two-dimensional 1 H-13C HSQC spectra of Sia3 and Sia3-III-7CHO (sodium periodate: Sia3 at a molar ratio of 5:1) and assignments of Sia3-III-7CHO. D. Overlay of two-dimensional 1H-13C HSQC spectra of Sia3 and Sia3-III-8CHO (sodium periodate: Sia3 at a 1:1 molar ratio) and assignments of Sia3-III-8CHO. 269x202mm (300 x 300 DPI)



Figure 3. MALDI-TOF-MS spectra of Sia3 and its oxidized forms. Molecular ions were observed at m/z 878.327 (Sia3 + Na+), 824.43 (Sia3 + Na+ - 3H2O), 794.44 (Sia3 + Na+ - 3H2O - COH2) and 778.408 (Sia3 + Na+ - 2H2O - CH3OH. (B) and its oxidized forms aldehyde at III-8 positon (-CH4O, -32.04 amu) and III-7 position (-C2H6O, -62.07 amu). At a 1:1 molar ratio of sodium periodate:Sia3, the aldehyde groups were produced predominantly at III-C8 (A), whereas at a 5:1 molar ratio of sodium periodate:Sia3, the aldehyde group is only generated at III-7 816.913 (Sia2-CHO + Na+), 794.871 (Sia2-CHO) and 776.912 (Sia2-CHOH2O) (C). 88x70mm (300 x 300 DPI)


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Figure 4. A-B. 1H NMR spectra of Sia3-III-7CHO, TT830-844 and TT 830-844 -Sia3. A sharp peak corresponding to the N-acetyl CH3 (about 2 ppm) of Sia3-III-7CHO was found in TT 830-844 -Sia3 spectrum. C. 1H-1H TOCSY spectra overlay of TT 830-844 and TT 830-844 -Sia3 D. Assignment of TT 830-844 -Sia3. 127x104mm (300 x 300 DPI)


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