Typhimurium O-Antigen Glycoconjugate Vaccine - American Chemical

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Click chemistry applied to the synthesis of Salmonella Typhimurium O-antigen glycoconjugate vaccine on solid-phase with sugar recycle Francesca Micoli, Giuseppe Stefanetti, Allan J. Saul, and Calman A. MAcLennan Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00521 • Publication Date (Web): 09 Nov 2015 Downloaded from http://pubs.acs.org on November 14, 2015

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Bioconjugate Chemistry

Click chemistry applied to the synthesis of Salmonella Typhimurium O-antigen glycoconjugate vaccine on solid-phase with sugar recycle. Giuseppe Stefanetti,† Allan Saul, † Calman A. MacLennan, ‡,§ Francesca Micoli†* †

Sclavo Behring Vaccines Institute For Global Health (SBVGH) S.r.l., a GSK company (former

Novartis Vaccines Institute for Global Health NVGH), Via Fiorentina 1, 53100 Siena, Italy. ‡

Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

§

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK

* Corresponding author: F. Micoli, [email protected]

Key words: glycoconjugate vaccine, solid-phase conjugation, click chemistry, Salmonella Typhimurium

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Abstract A solid-phase conjugation method was developed and applied to the synthesis of an O-antigen based glycoconjugate vaccine against Salmonella Typhimurium, with CRM197 as the carrier protein. Copper-free click chemistry was used as the conjugation chemistry, after derivatizing the sugar and the protein components with alkyne and azido linkers, respectively. This chemistry has the advantage of not de-activating functional groups during the conjugation step, thereby allowing the recycling of unreacted components. The activated carrier protein was adsorbed to an anion exchange matrix and quantitatively conjugated to the O-antigen. The resulting conjugate was eluted from the resin free of unconjugated sugar which was previously removed by simple washing steps. Unreacted O-antigen was recycled by addition to a new batch of resin-CRM197 resulting in further quantitative protein conjugation. This process has advantages in relation to reduction of costs for production of conjugate vaccines, allowing unreacted sugar recovery and simplifying the purification of the glycoconjugate.


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Introduction Glycoconjugate vaccines, composed of a sugar antigen covalently linked (i.e. conjugated) to a carrier protein, are among the safest and most efficacious vaccines developed during the last 30 years.1 The conjugation strategy used is important as it can affect efficiency of conjugation, saccharide to protein ratio, and structure and size of the resulting conjugate, with consequent impact on immunogenicity.2 Click chemistry3,

4

has been recently applied to the synthesis of

glycoconjugate vaccines.5-8 One of the most important properties of the click chemistry Huisgen 1,3-dipolar cycloaddition9 is its bio-orthogonality. The azide moiety is absent in almost all naturally existing compounds, lacks reactivity with natural biomolecules and undergoes ligation only with a limited set of functionalities, such as alkyne groups. Despite some concerns about the stability of azide derivatives, possible immunogenicity of the spacers used,5, 7 and the need for a toxic metal in some applications, this reaction is likely to be used extensively in the design of future drugs and biomolecules due to its specificity and effectiveness10. Recently, we have applied the strain promoted azide-alkyne cycloaddition chemistry to the synthesis of O-antigen (OAg)-based glycoconjugate vaccines to protect against nontyphoidal Salmonella (NTS) serovar Typhimurium, with CRM197 as carrier protein8. Resulting OAg-CRM197 conjugates were able to induce high titers of anti-OAg IgG bactericidal antibodies in mice. NTS is the commonest cause of invasive bacteremia in Africa,11, 12 particularly affecting young children and HIV-infected adults. S. Typhimurium is the main serogroup responsible for invasive NTS disease in Africa.12 A vaccine against S. Typhimurium is not currently available. Antibodies directed against the OAg of NTS lipopolysaccharide mediate killing13-15 and confer protection against infection in animal models.16, 17 Therefore OAg-based glycoconjugates represent a leading approach for the development of a vaccine against NTS.17-20 Azido and alkyne groups involved in click chemistry are not de-activated during the conjugation reaction. Hence we tested further the potential of this conjugation approach by evaluating the possibility to recycle and re-conjugate unreacted components. The terminal KDO sugar of the OAg chain was derivatized with an alkyne linker8 and multiple azido groups were introduced on CRM197 (Figure 1). Conjugation conditions selected resulted in linkage of all protein in the reaction and free OAg was recovered and successfully reused for conjugation. Synthesis on solid-phase was then established, with the advantage of recovering unconjugated sugar while obtaining a conjugate pure from free saccharide without further purification steps.



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Figure 1. Conjugation scheme used for the synthesis of OAg-CRM197 glycoconjugate vaccine. The terminal KDO unit of the OAg chain (1) was activated with ADH (2) and then linked to BCN NHS I (3) to introduce the alkyne functionality. Controlled conjugation to the more surface exposed lysines of CRM197 (4) by activation of the amine groups with NHS-PEG4-N3 led to the introduction of an average of 12 azido functionalities per CRM197 (5). The activated OAg (3) was then conjugated with the azido-derivatized CRM197 (5) via copper-free click chemistry (6).

Results Synthesis of OAg-CRM197 conjugate in homogenous phase with fresh and recycled OAg An average of 12 azido linkers were introduced per molecule of CRM197 and the two steps of OAg activation resulted in >90% derivatization of sugar chains (Figure 1). Recoveries >80% were obtained for both protein and OAg intermediates. HPLC-SEC profiles of sugar and protein after derivatization were identical to the underivatised products, indicating no degradation or aggregation during the activation reactions.21 Conjugation conditions used allowed complete linkage of the protein as verified by absence of free CRM197-N3 in the HPLC-SEC profile of the conjugation mixture (Figure 2a, solid line). Unreacted sugar was recovered by HIC and the purified conjugate was fully characterized (Table 1).


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Figure 2. HPLC-SEC profiles of a) OAg-CRM197 conjugates obtained in homogeneous phase with fresh (solid line) and recycled OAg (dotted line); b) recycled OAg from HIC purification of the conjugate in homogeneous phase; c) OAg-CRM197 conjugates obtained in solid-phase with fresh (solid line) and recycled OAg (dotted line); d) recycled OAg from conjugation in solid-phase; e) CRM197 standard; f) OAg standard. OAg samples detected by refractive index; conjugates and CRM197 by fluorescence emission.

Table 1. Characterization of OAg-CRM197 conjugates generated in homogenous and solid-phase OAg/CRM197

OAg/CRM197

Recovery*

Kd

(w/w)

(mol/mol)

(%)

(HPLC-SEC)

OAg-CRM197

2.01

5.72

79.9

0.50

Recycled OAg-CRM197

2.04

5.82

73.3

0.47

Solid-phase OAg-CRM197

3.39

9.65

85.7

0.50

Recycled solid-phase OAg-CRM197

1.01

2.89

78.2

0.54

Conjugate

Kd values were calculated on a TSKgel 6000 PW + 5000 PW, 0.5 mL/min, 100 mM NaCl 100 mM NaH2PO4 5% CH3CN pH 7.2; Vtot (NaN3) 49.004 min; V0 (DNA) 24.382 min; Kd of free CRM197: 0.69; Kd of free OAg: 0.64. *Calculated based on protein content.



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Flow through fractions from HIC contained only unreacted sugar, with no traces of free or conjugated protein, as verified by HPLC-SEC (fluorescence emission profile not shown). Sugar recovered in the flow through was 50% of the initial amount supplied for the reaction and its structural integrity was verified by HPLC-SEC (Figure 2b) and HPAEC-PAD sugar composition analysis.22 Flow through fractions of OAg were desalted against water and re-conjugated to CRM197-N3 using the same conjugation conditions adopted for the synthesis of the first batch. Conjugate formation was verified by HPLC-SEC and once again no traces of free protein were detected in the reaction mixture (Figure 2a, dotted line). The OAg-CRM197 and recycled OAg-CRM197 showed similar HPLC-SEC profiles (Figure 2a), and OAg to protein ratio (Table1). This indicates that the recycling procedure does not alter the characteristics of the conjugate and therefore avoids wastage of large amounts of sugar.

Identification of conditions for conjugation on solid-phase As a first step in transferring the conjugation reaction to solid-phase, the best conditions for attaching CRM197 to the resin while avoiding the linking of the sugar were identified, by running a physical mixture of CRM197 and OAg on HiTrap 1mL Q FF column. We speculated that the behavior on the resin of OAg/CRM197 and of OAg-alkyne/CRM197-N3 would be similar, since structural modifications introduced in derivatised samples is modest considering the interaction with this kind of resin. Loading the mixture in 0.1 M NaH2PO4 pH 8.0, resulted in attachment of CRM197 to the resin, while OAg was recovered in the flow through. We verified that the protein could be eluted by increasing the concentration of NaCl (Figure 3).


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Figure 3. OAg and CRM197 physical mixture separated by HiTrap 1mL Q FF column. From 0 to 100% buffer B in 20 CV linear gradient. Buffer A: 100 mM NaH2PO4 pH 8.0; buffer B: 100 mM NaH2PO4 1 M NaCl pH 8.0. Inserts report HPLC-SEC profiles of the two peaks collected from the column: OAg fractions detected by refractive index (dRI); CRM197 fractions by fluorescence emission (em).

We then investigated how the pH and the reaction time influence the adsorption of the protein on two different ion-exchange matrixes, Capto Q and Q FF, by working with the resin in batch. We compared pH 6, 7 and 8; and incubation times of 4, 5, 6 and 12h. Protein concentration was fixed at 0.83 mg/mL (0.21 mg per mL of resin) in NaH2PO4 0.02 M. In all conditions, no more than 2% protein was detected in the supernatant at the end of the incubation. We next evaluated optimal conditions for protein-resin washing. The use of NaH2PO4 20 mM pH 8 was found to be optimal as no traces of protein were found by micro BCA in the supernatant. Washing three times with PBS buffers (washing volume 2 times the resin volume), about 20% of the initial amount of protein was detected in the supernatant, presumably due to the higher ionic strength of the media. With regard to the elution of protein from the resin, the use of 20 mM NaH2PO4 1 M NaCl (elution volume 2 times the resin volume) caused the release of protein from the solid-phase. Comparing the two solid supports, Q FF resin allowed better elution of conjugate than Capto Q, as evidenced by the much higher area of the protein peak in the HPLC-SEC fluorescent emission profiles (data not shown). 7 ACS Paragon Plus Environment


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Synthesis of OAg-CRM197 conjugate in solid-phase with fresh and recycled OAg Conjugation on solid-phase was performed in batch. After linking CRM197-N3 to the resin, OAgalkyne was added and gently mixed at 4°C. After 4h, the supernatant was removed and the resin washed. Only free OAg (23% of the initial amount) could be detected in the supernatant. Decreasing amount of free OAg were recovered after each washing step with 0.02 M NaH2PO4 pH 8.0, reduced to around 3% of the initial OAg amount in the third washing solution. This value was further reduced to 0.3% after three washing steps with PBS, assuring much less than 20% free saccharide in the purified conjugate, the common limit value requested for licensed glycoconjugate vaccines. The conjugate was then eluted with 0.1 M NaH2PO4 1 M NaCl pH 8.0. HPLC-SEC analysis revealed that all the protein was conjugated with less than 10% free sugar (Figure 2c, solid line). 86% of protein was eluted from the resin. The supernatant was pooled with the washing solutions, and desalted through a PD 10 column containing 8.3 mL of SephadexTM G-25 Medium (PD 10) [GE Healthcare]; 75% of the sugar supplied to the reaction was recovered. This was added to a new batch of resin-bound CRM197 and a conjugate with same HPLC-SEC profile and OAg to CRM197 ratio as the first one was obtained. However, this methodology has the drawback of requiring more steps than just the addition of a supernatant solution to a new batch of CRM197-resin. In order to simplify the process, we attempted to recycle the OAg-containing supernatant (Figure 2d) and conjugate the OAg with fresh resinbound CRM197 directly, without performing any desalting or concentration steps (Figure 4).


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Figure 4. Click conjugation on solid-phase. The azido-derivatized CRM197 was adsorbed to an anion exchange matrix, and the immobilized protein was then conjugated to the OAg. Unreacted OAg was easily recovered by centrifugation and washing steps, while the conjugate was eluted from the resin, pure from both free protein and free sugar. Recovered OAg was used for conjugation with a new batch of immobilized CRM197. Although the amount of sugar added in this experiment was lower (molar ratio alkyne/N3 = 14 at a concentration of 14 mg/mL, compared to molar ratio alkyne/N3 = 22 at 50 mg/mL), conjugation was again quantitative with no free CRM197 detected by HPLC-SEC in the eluted conjugate (Figure 2c, dotted line). Also the recovery of the glycoconjugate product was high (78 %) and the final conjugate preparation contained less than 10% free OAg. This conjugate had a lower OAg to 9 ACS Paragon Plus Environment


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protein ratio than the first conjugate (Table 1), in agreement with the different conditions of OAg concentration and alkyne to azido molar ratio used during conjugation.

Discussion Click chemistry has been identified as a promising method for the synthesis of glycoconjugates using short synthetic oligosaccharides with few points of attachment targeted on the protein. 6, 23 We have recently applied click chemistry to the linkage of longer sugar chains such as S Typhimurium 2192 OAg (20.5 kDa), selectively activated on the terminal KDO unit, which were then conjugated to just one amino acid position on CRM197.8 The strain promoted azide-alkyne cycloaddition variant was preferred to the copper-catalyzed variant because it gave better conjugation yields5, 24, 25 and avoids the use of a toxic metal. Here we have further evaluated the potential of this conjugation approach. Unreacted OAg, with alkyne groups not de-activated during conjugation, was successfully recycled for conjugation. In addition, the ability to perform conjugation in solid-phase was verified (Figure 4). CRM197 was adsorbed to an anion exchange matrix using a batch procedure, and the immobilized protein was then conjugated to the OAg. Unreacted OAg was easily recovered, while the conjugate was eluted from the resin, pure from both free protein and free sugar. The absence of free protein in the eluted conjugate confirmed the efficiency of this conjugation strategy. In this way, the purification of the conjugate is reduced to a simple washing/elution procedure, where unreacted sugar is removed from the resin-bound conjugate, and the glycoconjugate is then simply eluted and can be used for immunization. When the conjugates are intended for use as vaccines, removal of unconjugated saccharide is particularly important in order to obtain consistent products reproducibly thereby minimizing vaccine-related variation in the immune response elicited.26,

27

Several methods are in use for the separation of free saccharide from conjugate

vaccines. These often involve complex steps, and some of them may result in the selection of subpopulations of conjugates. Thus there is a need for improved processes for purifying saccharideprotein conjugates, and particularly for processes that are simple, requiring the minimum number of steps, with no selection of sub-populations or risk of altering vaccine immunogenicity. The possibility of recycling the unreacted sugar for further conjugation improves the cost-effectiveness of the process. Solid-phase synthesis of peptides directly to carrier proteins,28 of cysteine-containing peptides to carrier proteins pre-adsorbed on aluminum hydroxide,29 and of glutathione to pre-activated ovalbumin adsorbed to an anion exchange matrix30 have been described. No application of solid phase methodologies to the synthesis of glycoconjugate vaccines have been reported so far. The advantages of this methodology are the possibility of using a large excess of reagents to obtain fast 10 ACS Paragon Plus Environment

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and efficient reactions, the possibility to perform multiple reaction steps on the resin with simple wash purification of intermediates, and the ability to release conjugates from the solid-phase under mild conditions. The process described here could be further simplified by performing also the step of CRM197 derivatization with the azido linker on the resin. In order to obtain quantitative protein conjugation using solid-phase, higher OAg concentrations in solution and higher alkyne to azido groups ratios were used compared with those used for conjugation in solution. We reasoned that these conditions can account for why the resulting conjugate was characterized by a higher OAg to protein ratio compared with the conjugate obtained in homogeneous phase. However, by performing conjugation with recycled OAg, we verified that 100% of the protein was conjugated even working with lower OAg concentrations and alkyne to azido groups ratios, resulting in a purified conjugate with a lower OAg to protein ratio. This indicates that the OAg concentration and ratio of alkyne to azido groups can be modified in order to modulate the characteristics of the corresponding conjugates, such as sugar to protein ratio and size, allowing for the selection of the most appropriate conditions in relation to cost and production of a vaccine with optimal efficacy. S. Typhimurium conjugates obtained by this chemistry have been already tested in mice, showing their ability to induce anti-OAg specific IgG antibodies with serum bactericidal activity against an endemic strain8. We have also shown that for conjugates obtained by randomly targeting a different number of lysine residues on CRM197, the OAg to protein ratio does not impact on immunogenicity. In conclusion, we have identified a method for performing conjugation on solid-phase, with the possibility of recovering unreacted sugar and simplifying the purification process. This has the potential to reduce the cost of production of glycoconjugate vaccines. Conjugation on solid phase can be extended to the synthesis of glycoconjugate vaccines of other polysaccharides/proteins and by using different conjugation chemistries further then click chemistry. The chemistry used needs to be efficient not to have residual unconjugated protein and possibly involving reactive groups on the sugar that are not deactivated during the conjugation itself so to allow sugar recycle.

Experimental procedures

Reagents The following chemicals were used in this study: triethylamine (TEA), sodium acetate (AcONa), phosphate buffer solution (PBS), adipic acid dihydrazide (ADH), sodium cyanoborohydride (NaBH3CN), dimethyl sulfoxide (DMSO), sodium chloride (NaCl), sodium phosphate monobasic (NaH2PO4) [Sigma]; acetonitrile (CH3CN) [LC-MS Chromasolv]; NHS-PEG4-N3 [Pierce], ClickeasyTM BCN N-hydroxysuccinimide ester I (BCN NHS I) [Berry & Associates], Q Separose Fast 11 ACS Paragon Plus Environment


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Flow (Q FF) resin, Capto Q resin [GE Healthcare]. CRM197 was obtained from Novartis Vaccines, Siena.

OAg purification and characterization S. Typhimurium OAg was purified as previously described,21 following fermentation of the animalderived isolate 2192, obtained from the University of Calgary. OAg resulted pure from proteins (

Typhimurium O-Antigen Glycoconjugate Vaccine - American Chemical

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