Methods for making saccharide-protein glycoconjugates

Abstract

The invention provides a process for the reductive amination of a carbonyl group at the reducing terminus of a polysaccharide, wherein the reductive amination is carried out at a pH between 4 and 5. The invention also provides a process for preparing a conjugate of a polysaccharide and a carrier molecule, comprising the steps of: (a) coupling the polysaccharide to a linker, to form a polysaccharide-linker compound in which the free terminus of the linker is an ester group; and (b) reacting the ester group with a primary amine group in the carrier molecule, to form a polysaccharide-linker-carrier molecule conjugate in which the linker is coupled to the carrier molecule via an amide linkage. The invention also provides a process for reducing contamination of a polysaccharide-linker compound with unreacted linker, comprising a step of precipitating unreacted linker under aqueous conditions at a pH of less than 5. The invention also provides polysaccharide-linker-carrier molecule conjugates and intermediate compounds obtained or obtainable by these processes.

Claims

1. A process for coupling a polysaccharide to a linker, comprising: combining the polysaccharide with an additional linker comprising a primary amine group in the presence of a reducing agent, wherein the polysaccharide comprises a carbonyl group at the reducing terminus, reacting the carbonyl group with the primary amine group by reductive amination to form a polysaccharide-additional linker intermediate, wherein the reductive amination is carried out at a pH between 4 and 5, coupling the polysaccharide-additional linker intermediate to the linker, to form a polysaccharide-linker compound, and precipitating unreacted linker under aqueous conditions at a pH of less than 5.

2. The process of claim 1, wherein the polysaccharide comprises a core domain from a lipopolysaccharide of a Gram-negative bacterium, and an O-antigen from a lipopolysaccharide of a Gram-negative bacterium linked to the core domain.

3. The process of claim 2, wherein the lipopolysaccharide is from a Salmonella bacterium.

4. The process of claim 3, wherein the lipopolysaccharide is from S. Paratyphi A, S. Typhimurium or S. Enteritidis.

5. The process of claim 1, wherein the polysaccharide is a bacterial capsular polysaccharide.

6. The process of claim 5, wherein the bacterial capsular polysaccharide is from N. meningitidis serogroup X.

7. The process of claim 1, wherein the reductive amination comprises reacting the carbonyl group with Y.sub.1 in the additional linker having the formula Y.sub.1-L-Y.sub.2, to form the polysaccharide-additional linker intermediate compound in which the polysaccharide is coupled to the additional linker via a CN linkage, wherein: Y.sub.1 comprises a primary amine group that can react with the carbonyl group in the polysaccharide; Y.sub.2 comprises a second reactive group; and L is a linking moiety.

8. The process of claim 7, wherein the Y.sub.1 and Y.sub.2 are both NHNH.sub.2 groups.

9. The process of claim 7, wherein L has formula -L-L.sup.2-L-, where L is carbonyl and L.sup.2 is a straight chain alkyl with 1 to 10 carbon atoms.

10. The process of claim 8, wherein L.sup.2 is (CH.sub.2).sub.4.

11. The process of claim 8, wherein L is carbonyl.

12. The process of claim 7, wherein Y.sub.2 comprises a primary amine group.

13. The process of claim 7, wherein Y.sub.2 comprises a SH group.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIGS. 1A-C illustrate published structures of the repeating units of the O-antigens from (FIG. 1A) Salmonella Paratyphi A; (FIG. 1B) Salmonella Typhimurium; and (FIG. 1C) Salmonella Enteritidis.

(2) FIG. 2 illustrates the structure of the Salmonella Paratyphi A O-antigen repeating unit and core domain.

(3) FIG. 3 shows the separation of O:2-CRM.sub.197 from unconjugated components by Sephacryl S300HR.

(4) FIG. 4 shows the immunogenicity of different O:2-CRM.sub.197 conjugates, as measured by an ELISA immunoassay for anti-O:2 antibodies.

(5) FIG. 5 is a schematic of a conjugation method of the invention applied to O-antigen-core.

(6) FIG. 6 illustrates a large scale method for the conjugation of O-antigen from Salmonella Paratyphi A to CRM.sub.197.

(7) FIG. 7 is a schematic of a conjugation method of the invention applied to the capsular polysaccharide from N. meningitidis serogroup X.

(8) FIG. 8 shows an SDS-PAGE analysis of conjugates of capsular polysaccharide from N. meningitidis serogroup X and CRM.sub.197.

(9) FIG. 9 shows the result of Serum Bactericidal Activity (SBA) assays performed using day 42 pooled sera from mice immunized with 8 ug of conjugated or unconjugated O:2 and S. Paratyphi A strain. Data are presented as percentage of CFU recovered in test sera with active BRC (and in control serum with inactive BRC) compared with CFU present in negative control, per anti-O:2 ELISA antibody unit of each serum pool.

MODES FOR CARRYING OUT THE INVENTION

(10) Summary of Conjugate Production

(11) Different methods were compared for conjugating the O-antigen-core from S. Paratyphi to CRM.sub.197. In particular, conjugates obtained by activating the O:2 chain randomly, or specifically through the terminal KDO subunit, were compared. Theoretically, activation of only the KDO would not modify the structure of the repeating saccharides and would therefore result in a better defined and easier to characterize conjugate.

(12) Two methods were based on prior art methods. In the first of these methods, the O-antigen was randomly activated with ADH using CDAP and then conjugated to CRM.sub.197 using EDAC. In the second method, the core region of the O-antigen was activated with ADH (using the COOH group of KDO by EDAC) and then conjugated to CRM.sub.197 using EDAC.

(13) Alternative methods were also tested. A first method involved KOO activation with ADH through the ketone group by reductive amination, followed by activation with SIDEA, and then conjugation with CRM.sub.197. A second method substituted the ADH with CDH. A third method involved activation of O-antigen with SIDEA (without activation of KDO with ADH) using the pyrophosphoethanolamine group on the core region.

(14) Conjugation of O-antigen-core to CRM.sub.197

(15) (Comparative) Method A: Random Activation of the O-antigen-core Chain with ADH by CDAP and Conjugation with CRM.sub.197 by EDAC

(16) This conjugate was synthesized according to ref. 2, as described in detail below.

(17) O-antigen-core derivatisation with ADH through CDAP. 30 l of CDAP (100 mg/mL in acetonitrile) was added per ml of 10 mg/ml O-antigen-core in 150 mM NaCl at room temperature. The pH was maintained at 5.8 to 6.0 for 30 seconds, then 0.2 M TEA was added to increase the pH to 7.0 and the solution mixed at room temperature for 2 minutes. 1 ml of 0.8 M ADH in 0.5 M NaHC03 was then added per 10 mg of O-antigen-core. The reaction was carried out for 2 hours at room temperature, and pH maintained at 8.0 to 8.5 with 0.1 N NaOH. The reaction mixture was then desalted using a G-25 column against water and the product, designated as OAg-(CDAP)ADH, characterized.

(18) Conjugation of OAg-(CDAP)ADH with CRM.sub.197. The OAg-(CDAP)ADH was dissolved in 100 mM MES at pH 5.8. An equal weight of protein was added to give an O-antigen-core:CRM.sub.197 ratio of 1:1 by weight, with an O-antigen-core concentration of 5 mg/ml. The reaction mixture was placed on ice and EDAC added to a final concentration of 50 mM, The reaction was mixed on ice for a further 4 h. The resulting conjugate was designated OAg-(CDAP)ADH-CRM.sub.197.
(Comparative) Method B: Activation of Terminal KDO with ADH by EDAC and Conjugation with CRM.sub.197 by EDAC

(19) This conjugate was synthesized according to ref. 9.

(20) O-antigen-core derivatization with ADH at KDO through EDAC. The O-antigen-core was solubilized at 3 mg/ml in 100 mM MES at pH 5.8. ADH was then added at a w/w ratio ADH:O-antigen-core of 1.36, followed by EDAC to a final concentration of 3.7 mM. The reaction was mixed at room temperature for 4 hours. The reaction mixture was then desalted using a G-25 column against water and the product, designated as OAg-(EDAC)ADH, characterized.

(21) Conjugation of OAg-(EDAC)ADH with CRM.sub.197. The conjugate was prepared according to the method described in Method A above for OAg-(CDAP)ADH. The conjugate was designated as OAg-(EDAC)ADH-CRM.sub.197.

(22) Methods C and D: Activation of the Terminal KDO with ADH (Method C) or CDH (Method D) by Reductive Amination and Conjugation with CRM.sub.197 via SIDEA Linker

(23) O-antigen-core derivatization with ADH or CDH at KDO by reductive amination. After testing different conditions, an optimized protocol for the O-antigen-core derivatization was identified. O-antigen-core was solubilized at 40 mg/ml in 100 mM AcONa at pH 4.5. Either ADH or CDH was added at a w/w ratio of 1:2 with respect to the O-antigen-core. NaBH.sub.3CN was then added at a w/w ratio of 1:2 with respect to the O-antigen-core. The solution was mixed at 30 C. for 1 hour. The reaction mixture was then desalted using a G-25 column against water and the product, designated as OAg-ADH or OAg-CDH characterized.

(24) OAg-ADH and OAg-CDH derivatization with SIDEA. Either OAg-ADH or OAg-CDH was dissolved in 1:9 (vol/vol) water/DMSO to a final O-antigen-core concentration of 50 mg/ml. Once the polysaccharide was in solution, TEA was added to give a molar ratio of TEA/total NH.sub.2 groups of 5 and then SIDEA to give a molar ratio of SIDEA/total NH.sub.2 groups of 12. The solution was mixed at room temperature for 3 hours. In preliminary attempts to purify the SIDEA-derivatised O-antigen-core, the O-antigen-core was precipitated by addition of AcOEt or dioxane (90% volume in the resulting solution) and washing the pellet times with the same organic solvent (ten times using of the volume added for the precipitation) in order to remove unreacted SIDEA. This process was then adapted to avoid the use of toxic AcOEt and dioxane reagents. A volume of 100 mM sodium citrate at pH 3 equal to two times the volume of the SIDEA-derivatised O-antigen-core reaction mixture was added and mixed at 4 C. for 30 min. Unreacted SIDEA was precipitated by the low pH and separated by centrifugation. The SIDEA-derivatised O-antigen-core was then recovered from the supernatant by precipitation with absolute ethanol (80% volume in the resulting solution). The pellet was washed with ethanol twice (using of the volume added for the precipitation) and dried. The product, designated as OAg-ADH-SIDEA or OAg-CDH-SIDEA was characterized.

(25) Conjugation of OAg-ADH-SIDEA and OAg-CDH-SIDEA with CRM.sub.197. The OAg-ADH-SIDEA or OAg-CDH-SIDEA was solubilized in NaH.sub.2PO.sub.4 buffer at pH 7.2 and CRM.sub.197 added to a final protein concentration of 20 mg/ml, final buffer capacity of 100 mM and molar ratio of active ester groups to CRM.sub.197 of 30 to 1. The reaction was mixed at room temperature for 2 hours.

(26) Method E: Direct Conjugation with CRM.sub.197 via SIDEA Linker

(27) The reaction conditions used in the above OAg-ADH and OAg-CDH derivatization with SIDEA was also applied to native O-antigen-core (i.e. O-antigen-core that had not previously been derivatized with ADH or CDH). The resulting product was designated as as OAg-SIDEA. The OAg-SIDEA was then conjugated to CRM.sub.197 by the reaction conditions used in the above Conjugation of OAg-ADH-SIDEA and OAg-CDH-SIDEA with CRM.sub.197.

(28) Purification of the O-antigen-core Conjugates

(29) Conjugates made according to methods A-E above were purified by size exclusion chromatography on a 1.6 cm90 cm S-300 HR column eluted at 0.5 ml/min in 50 mM NaH.sub.2PO.sub.4, 0.15 M NaCl at pH 7.2. Different pools were collected according to free O-antigen-core and free CRM.sub.197 profiles on the same column in the same eluting conditions (FIG. 3). The first pool at high molecular weight (corresponding to the purified conjugate) did not contain free saccharide or free protein.

(30) Analysis of Conjugates

(31) The conjugates were analysed by SDS-PAGE and showed an expected high molecular weight population smear compared to free CRM.sub.197. The conjugates were separated from free O:2 and CRM.sub.197 by Sephacryl S300HR size exclusion (1.690 cm column; 50 mM NaH.sub.2PO.sub.4, 150 mM NaCl pH 7.2; 0.5 mL/min flow). Results are shown in FIG. 3. Purified conjugates were then characterized by the phenol sulfuric assay of ref. 209 (total sugar), microBCA (total protein), HPAEC-PAD (sugar composition) and HPLC-SEC (size determination. Kd). Results are shown in Table 1 below.

(32) TABLE-US-00001 TABLE 1 Total sugar, Presence Protein Wt/wt ratio Kd Conjugate g/mL of free O:2 g/mL O:2/CRM.sub.197 (HPLC-SEC) O:2 yes 0.549 CRM.sub.197 0.690 O:2-(CDAP)ADH-CRM.sub.197 pool 1 32.88 no 62.98 0.52 0.439 O:2-(CDAP)ADH-CRM.sub.197 pool 2 101.09 yes 60.44 1.67 0.534 O:2-CDH-SIDEA-CRM.sub.197 Lot A 82.82 no 36.67 2.26 0.403 O:2-CDH-SIDEA-CRM.sub.197 Lot B 167.61 no 38.19 4.39 2 peaks 0.128 0.413 O:2-ADH-SIDEA-CRM.sub.197 Lot A 51.54 no 29.56 1.74 2 peaks 0.115 0.388 O:2-ADH-SIDEA-CRM.sub.197 Lot B 215.13 no 50.89 4.23 2 peaks 0.118 0.421 O:2-(EDAC)ADH-CRM.sub.197 50.19 yes 31.31 1.60 0.52 O:2-SIDEA-CRM.sub.197, pool 1 108.57 no 52.79 2.06 0.376 O:2-SIDEA-CRM.sub.197, pool 2 185.31 yes 68.18 2.72 0.457
Immunogenicity Studies

(33) An ELISA immunoassay was used to detect anti-O:2 antibodies elicited by O:2-CRM.sub.197 immunized mice. For the assay, MaxiSorp microtiter plates were coated with 15 g/mL O:2 in a carbonate coating buffer (pH 9.6) overnight at 4 C.

(34) The O:2-CRM.sub.197 conjugates were compared to unconjugated O:2 antigen. Briefly, groups of female CDI mice (8 per group at 5 weeks of age) were injected subcutaneously with 200 L of conjugates as set out in Table 2. Mice received immunizations on days 0, 14 and 28. Sera were collected from the mice during the course of the study and tested by the ELISA assay.

(35) TABLE-US-00002 TABLE 2 Group Vaccine O:2 antigen dose, g 1 O:2-(CDAP)ADH-CRM.sub.197, pool 1 1 2 8 3 O:2-(CDAP)ADH-CRM.sub.197, pool 2 1 4 8 5 O:2-CDH-SIDEA-CRM.sub.197 1 6 8 7 O:2-ADH-SIDEA-CRM.sub.197 1 8 8 9 O:2-(EDAC)ADH-CRM.sub.197 1 10 8 11 Unconjugated O:2 antigen 8

(36) ELISA results through day 42 of the study showed that all the conjugates, except O:2-(EDAC)ADH-CRM.sub.197 were able to elicit high serum levels of anti-O:2 IgG antibodies in mice when delivered at the 1 g dose (FIG. 4). Increases in antibody were observed following the second vaccination with conjugate, whereas repeated immunization with 8 g of unconjugated O:2-antigen did not result in specific IgG antibodies. Delivery of 8 g doses was no better than 1 g doses at generating a humoral immune response in mice (FIG. 4).

(37) Serum Bactericidal Activity

(38) Serum Bactericidal Activity (SBA) assays were performed using day 42 pooled sera from mice immunized with 8 ug of conjugated or unconjugated O:2 and S. Paratyphi A (see Table 2). The result is shown in FIG. 9. Inhibition of S. Paratyphi A growth in vitro correlated with increasing anti-O:2 ELISA units present in the sera pools. The strongest growth inhibition was observed with those conjugates produced using a selective chemistry; both SIDEA conjugates and O:2-(EDAC)ADH-CRM197 resulted in increased inhibition compared to O:2-(CDAP)ADH-CRM197, whose O:2 was randomly modified prior to conjugation. Even unconjugated O:2, although far from reaching high levels of bacterial growth inhibition due to the lower amount of antibody present in the sera, presented an inhibition profile similar to the conjugates prepared with unmodified O:2 chains. No bacterial growth inhibition was detected using control serum in the presence of iBRC (the same sera in the presence of active BRC is shown as control), indicating a role for complement mediated killing.

(39) Larger-scale Processing

(40) Based on the immunogenicity study and ease of conjugate characterization, the conjugation method based on O:2-activation with ADH and then SIDEA and reaction with CRM.sub.197 (method C, FIG. 5) was selected for further development. Conjugates made by this method were more immunogenic than conjugates made by the method B, for example. Although conjugates made by method A gave comparable ELISA titers (FIG. 4), they resulted in considerably lower Serum Bactericidal Activity (see Example and FIG. 9). Furthermore these conjugates had a cross-linked structure, with multiple possible points of linkage on the polysaccharide chain to one or more protein molecules, a with disadvantages in terms of reproducibility and characterization of the product. In contrast, the conjugates of method C contain more polysaccharide chains per protein molecule, with only one point of linkage on the polysaccharide chain. The remainder of the polysaccharide chain is left unchanged. Derivatization of the O-antigen-core through CDAP (method A) can result in crosslinking of the sugar chains and the overall conjugation method was more complicated. The use of EDAC in methods A and B can also result in cross-linking of the protein.

(41) A scaled-up process was developed for method C for the production of greater amounts of conjugate (FIG. 6). In particular, the step of O:2 reductive amination with ADH was optimized to reduce the reaction time to 1 hour (instead of 7-14 days as previous reported for this kind of reaction [13]) with good % of O:2 activation (>65%). This step was scaled to 300 mg of O:2 and repeated several times with reproducible results in terms of yield and derivatisation degree. Recovery was >75% after tangential flow filtration, with >70% of O-antigen-core activation (calculated as molar ratio of linked ADH groups per GlcNAc groups on the O-antigen-core) and good purity (ratio of free ADH/linked ADH<1%). The stability of the O:2-ADH intermediate in aqueous solution at 4 C. was verified to be >1 month. The inventors envisage replacing the drying step by O:2-ADH precipitation in 85% ethanol.

(42) The reaction of O:2-ADH with SIDEA was optimized to avoid the use of AcOEt during O:2-ADH-SIDEA purification. Removal of free SIDEA by precipitation at pH 4-5 and then precipitation of O:2-ADH-SIDEA in 85% EtOH showed better precipitate formation that was easier to wash compared with AcOEt. Recoveries >85% were obtained working on a 100 mg scale with activated NH.sub.2 groups >80%.

(43) The inventors have found that in addition to O:2 from S. Paratyphi A the conjugation method based on O-antigen-core activation with ADH and then SIDEA and reaction with CRM.sub.197 (method C, FIG. 5), works equally well for O-antigen-core from S. Typhimurium and O-antigen-core from S. Enteritidis.

(44) Conjugation of MenX Capsular Polysaccharide to CRM.sub.197

(45) Method C was also applied to the conjugation of capsular polysaccharide from N. meningitidis serogroup X (FIG. 7), resulting in conjugate formation with no free protein in the reaction mixture (FIG. 8).

(46) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

(47) Effect of pH and Temperature on the Reductive Amination of S. Paratyphi A O-antigen-core with ADH as Linker

(48) Experiments summarized in Table 3 were performed working with S. Paratyphi A O-antigen-core. The effect of different pH and different temperatures was evaluated. The best activation was obtained at lower pH and was temperature independent. Reactions were performed in 100 mM buffer, with O-antigen-core concentration of 20 mg/mL and a ratio of ADH to O-antigen-core and NaBH.sub.3CN to O-antigen-core both of 1.2 to 1 (w/w). ADH and NaBH.sub.3CN were added at the same time and solutions were mixed for 1 hour.

(49) TABLE-US-00003 TABLE 3 Reductive amination of O:2-KDO with ADH is pH dependent and temperature independent. Buffer Temperature % activated O:2 Sodium acetate pH 4.5 30 C. 65.8 Sodium acetate pH 4.5 50 C. 65.4 Sodium acetate pH 4.5 60 C. 68.3 MES pH 6.0 30 C. 43.9 Phosphate pH 8.0 30 C. 26.2
Derivation of S. Typhimurium O-antigen-core with ADH by Reductive Amination Comparing Different Reaction Conditions (Table 4).

(50) Reaction conditions described in this application (Table 4, method 1) for performing the reductive amination with ADH were compared with the traditional method reported in literature [13] (Table 4, method 2), working with S. Typhimurium O-antigen-core (strain D23580). Results are summarized in Table 5, showing that the process at lower pH is faster and also more efficient.

(51) TABLE-US-00004 TABLE 4 Reaction conditions used for performing reductive amination with ADH comparing NVGH procedure with the classical procedure reported in literature. OAg ADH NaBH.sub.3CN Temperature Method (mg/mL) (mg/mL) (mg/mL) Buffer ( C.) 1 40 48 48 AcONa 30 100 mM pH 4.5 2 20 100 100 NaHCO.sub.3 37 100 mM pH 8.3

(52) TABLE-US-00005 TABLE 5 Reductive amination of O:4,5-KDO with ADH using method 1 is more efficient and faster than using the classical method reported in literature (method 2). Reaction % activated Method time O:4,5 1 3 h 88 2 3 h 31.6 2 24 h 28.5 2 5 d 28.5

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