Protein-sugar conjugation with sodium cyanoborohydride

Abstract

The present specification discloses a method and use for conjugating sugar to a protein carrier.The present specification also discloses an exemplary conjugate prepared according to the method and use.In addition, the present specification discloses a method for quantifying the amount of sodium borohydride in sodium cyanoborohydride reagent.The present specification also discloses a vaccine composition and related methods and uses.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 329,547, filed April 11, 2022, and European Patent Application Publication No. 22172626.8, filed May 10, 2022, both of which are incorporated by reference in their entireties. [Background technology]

[0002] Sodium cyanoborohydride (NaCNBH 3 ) is used in a variety of chemical reactions. For example, sodium cyanoborohydride reagent is often used for the conjugation of protein carriers to sugars. Sodium borohydride (NaBH 4 ) is a stronger reducing agent than sodium cyanoborohydride, so the presence of sodium borohydride in the reaction may cause impurities in the final product.

[0003] However, it is unclear what amount of sodium borohydride in the sodium cyanoborohydride reagent would potentially interfere with the conjugation of the protein carrier to the sugar, and existing approaches for detecting sodium borohydride do not provide for quantification of the amount of sodium borohydride in a sample of sodium cyanoborohydride, presenting additional challenges. For example, 11 B NMR is proton NMR (also known as 1 H NMR) is less commonly available than 11 This is because B NMR requires tunable broadband probes. Furthermore, typical NMR tubes used in the analysis, and sometimes the NMR probe itself, are made of borosilicate glass. However, this type of glass is 11 In B NMR spectra, it can give extensive background signals that interfere with the baseline of the NMR spectrum, which can prevent accurate integration and quantification.

[0004] It has been described herein that even a small amount of sodium borohydride in sodium cyanoborohydride can adversely affect the conjugation reaction of sugar and protein carrier. Therefore, the present disclosure provides a method for conjugating at least one activated sugar to at least one protein carrier, comprising reacting at least one activated sugar with at least one protein carrier in the presence of a sodium cyanoborohydride reagent containing about 0.7% or less sodium borohydride to obtain a conjugate. Furthermore, 1 It has been reported that H NMR can distinguish between sodium cyanoborohydride and sodium borohydride in a sample and can be used for relative quantification of sodium borohydride contained in a sample of sodium cyanoborohydride.

[0005] According to the description, the method and use of conjugating sugar to protein carrier is disclosed herein.The exemplary conjugate prepared according to the method and use is also disclosed herein.In addition, the method of quantifying the amount of sodium borohydride in sodium cyanoborohydride reagent is also disclosed herein.Furthermore, vaccine compositions and related methods and uses are also disclosed herein. Summary of the Invention [Means for solving the problem]

[0006] Accordingly, the following embodiments are provided: Embodiment 1 is a method of conjugating at least one activated sugar to at least one protein carrier, the method comprising: (a) determining the amount of sodium borohydride in a sodium cyanoborohydride reagent; and (b) reacting the at least one activated sugar with at least one protein carrier in the presence of the sodium cyanoborohydride reagent containing about 0.7% or less sodium borohydride to obtain a conjugate.

[0007] Embodiment 2 is the use of a sodium cyanoborohydride reagent containing about 0.7% or less sodium borohydride as a reagent in conjugating at least one activated sugar to at least one protein carrier by reductive amination, the sodium cyanoborohydride reagent being determined to contain no more than 0.7% sodium borohydride.

[0008] Embodiment 3 is the use of a sodium cyanoborohydride reagent containing about 0.7% or less sodium borohydride as a reagent in conjugating at least one activated sugar to at least one protein carrier by reductive amination, comprising: (a) determining the amount of sodium borohydride in the sodium cyanoborohydride reagent; and (b) reacting the at least one activated sugar with at least one protein carrier in the presence of the sodium cyanoborohydride reagent containing about 0.7% or less sodium borohydride to obtain a conjugate.

[0009] Embodiment 4 is the method or use according to any one of the previous embodiments, wherein the method further comprises activating the at least one sugar with an activating agent to obtain at least one activated sugar.

[0010] Embodiment 5 is the method or use according to any one of the preceding embodiments, wherein the sodium cyanoborohydride reagent contains no more than about 0.6% sodium borohydride.

[0011] Embodiment 6 is the method or use according to the immediately preceding embodiment, wherein the sodium cyanoborohydride reagent contains no more than about 0.5% sodium borohydride.

[0012] Embodiment 7 is a method or use according to any one of the previous embodiments, wherein the at least one activated sugar comprises an activated form of a cell or viral surface carbohydrate.

[0013] Embodiment 8 is a method or use according to any one of the previous embodiments, wherein the at least one activated saccharide comprises an activated form of a bacterial capsular polysaccharide.

[0014] Embodiment 9 is a method or use according to any one of the previous embodiments, wherein the at least one activated saccharide comprises an activated form of bacterial capsular polysaccharide from Haemophilus influenzae.

[0015] Embodiment 10 is the method or use according to any one of embodiments 1 to 8, wherein the at least one activated saccharide comprises an activated form of bacterial capsular polysaccharide from Streptococcus pneumoniae.

[0016] Embodiment 11 is the method or use according to any one of embodiments 1 to 8, wherein the at least one activated saccharide comprises an activated form of bacterial capsular polysaccharide from Neisseria meningitidis.

[0017] Embodiment 12 is the method or use according to the immediately preceding embodiment, wherein the Neisseria meningitidis is serogroup C, A, W-135, or Y.

[0018] Embodiment 13 is a method or use according to any one of the previous embodiments, wherein the at least one protein carrier comprises recombinant exoprotein alpha (REP A), outer membrane protein complex (OMPC), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C fragment.

[0019] Embodiment 14 is a method or use according to any one of the previous embodiments, wherein the at least one protein carrier comprises diphtheria toxoid, CRM197, or tetanus toxoid.

[0020] Embodiment 15 is a method or use according to any one of the previous embodiments, wherein the at least one protein carrier comprises a tetanus toxoid.

[0021] Embodiment 16 is the method or use according to any one of the preceding embodiments, wherein the pH of the reaction is within the range of about 7 to about 10.

[0022] Embodiment 17 is a method or use according to any one of the previous embodiments, wherein the pH of the reaction is about 8.

[0023] Embodiment 18 is the method or use according to any one of embodiments 1 to 16, wherein the pH of the reaction is about 9.

[0024] Embodiment 19 is the method or use of any one of the immediately preceding embodiments, wherein determining the amount of sodium borohydride comprises performing NMR on the sodium cyanoborohydride reagent.

[0025] Embodiment 20 is the method or use of the immediately preceding embodiment, wherein the NMR comprises one-dimensional proton NMR.

[0026] Embodiment 21 is a method of quantifying the amount of sodium borohydride in sodium cyanoborohydride reagent, the method including: (a) subjecting a sample of the sodium cyanoborohydride reagent to one-dimensional proton NMR to obtain NMR data; and (b) determining the amount of sodium borohydride in the sample from the NMR data.

[0027] Embodiment 22 is the method or use according to any one of embodiments 19 to 21, wherein the NMR is carried out in a deuterated solvent comprising at least one deuterated form of dimethylsulfoxide, chloroform, or methylene chloride.

[0028] Embodiment 23 is the method or use according to embodiments 19 to 22, wherein the NMR comprises at least one scan with a relaxation delay of about 41 to 50 seconds.

[0029] Embodiment 24 is the method or use according to embodiment 23, wherein the NMR comprises at least one scan with a waiting time of about 43 to 48 seconds.

[0030] Embodiment 25 is the method or use according to embodiment 24, wherein the NMR comprises at least one scan with a waiting time of about 45 seconds.

[0031] Embodiment 26 is the method or use according to any one of embodiments 19 to 25, wherein the NMR is carried out at a temperature ranging from about 20°C to about 45°C.

[0032] Embodiment 27 is the method or use according to embodiment 26, wherein the NMR is carried out at a temperature ranging from about 28°C to about 40°C.

[0033] Embodiment 28 is the method or use according to embodiment 27, wherein the NMR is carried out at a temperature ranging from about 30°C to about 35°C.

[0034] Embodiment 29 is the method or use according to embodiment 28, wherein the NMR is carried out at a temperature of about 30°C.

[0035] Embodiment 30 is the method or use according to any one of embodiments 19 to 29, wherein NMR is carried out with the parameters - temperature: about 30° C.; pulse width: pw90; spectral width: 7000 Hz; waiting time: about 45 seconds.

[0036] Embodiment 31 is the method or use according to any one of embodiments 19 to 30, wherein determining the amount of sodium borohydride comprises using the peak area at about 0.57 ppm for the sodium borohydride resonance referenced from the solvent resonance.

[0037] Embodiment 32 is the method or use of embodiment 31, wherein the solvent resonance comprises a dimethylsulfoxide resonance at about 2.5 ppm.

[0038] Embodiment 33 is the method or use according to any one of embodiments 19 to 32, wherein determining the amount of sodium borohydride uses an external standard curve of sodium borohydride, optionally wherein the external standard curve of sodium borohydride is about 20 to 120 μg / ml.

[0039] Embodiment 34 is the method or use according to embodiment 33, wherein the external standard curve comprises a 6-point external standard curve.

[0040] Embodiment 35 is a conjugate produced according to the method or use according to any one of embodiments 1 to 20 and 22 to 34.

[0041] Embodiment 36 is a vaccine composition comprising at least one protein-conjugated saccharide obtained according to any one of embodiments 1 to 20 and 22 to 34.

[0042] Embodiment 37 is the vaccine composition of embodiment 36, further comprising a pharma- ceutically acceptable buffer.

[0043] Embodiment 38 is the vaccine composition of embodiment 36 or 37, further comprising a pharma- ceutically acceptable salt.

[0044] Embodiment 39 is a vaccine composition of any one of embodiments 36-38, formulated for intramuscular administration.

[0045] Embodiment 40 is a method of vaccinating a subject comprising administering a single dose of the vaccine composition of any one of embodiments 36-39.

[0046] Embodiment 41 is the use of a vaccine composition according to any one of embodiments 36 to 39 for immunizing a subject.

[0047] Embodiment 42 is the use of a vaccine composition according to any one of embodiments 36 to 39 for the manufacture of a medicament for immunizing a subject.

[0048] Embodiment 43 is the method or use according to any one of embodiments 40 to 42, wherein the subject is immunized against Haemophilus influenzae.

[0049] Embodiment 44 is the method or use according to any one of embodiments 40 to 42, wherein the subject is immunized against Neisseria meningitidis.

[0050] Embodiment 45 is the method or use according to any one of embodiments 40 to 42, wherein the subject is immunized against Streptococcus pneumoniae.

[0051] Embodiment 46 is the method or use of any one of embodiments 40 to 45, wherein the vaccine composition is administered intramuscularly.

[0052] Embodiment 47 is a method for preparing a vaccine composition, comprising conjugating at least one activated sugar to at least one protein carrier according to the method or use of any one of embodiments 1 to 20 or 22 to 34; and formulating the conjugate into a vaccine composition.

[0053] Embodiment 48 is a method of preparing at least one vaccine composition comprising a conjugate, the method comprising: (a) determining an amount of sodium borohydride in a sodium cyanoborohydride reagent; (b) selecting a sodium cyanoborohydride reagent containing about 0.7% or less sodium borohydride; (c) providing at least one conjugate by reacting at least one activated sugar with at least one protein carrier in the presence of the selected sodium cyanoborohydride reagent; and (d) formulating the at least one conjugate into a vaccine composition.

[0054] Embodiment 49 is the method according to embodiment 48, further comprising activating at least one sugar with an activating agent to obtain at least one activated sugar.

[0055] Embodiment 50 is the method according to embodiment 48 or 49, wherein the sodium cyanoborohydride reagent selected contains no more than about 0.6% sodium borohydride.

[0056] Embodiment 51 is the method according to the immediately preceding embodiment, wherein the sodium cyanoborohydride reagent selected contains no more than about 0.5% sodium borohydride.

[0057] Embodiment 52 is the method according to any one of embodiments 48 to 51, wherein at least one activated sugar comprises an activated form of a cell or viral surface carbohydrate.

[0058] Embodiment 53 is the method according to any one of embodiments 48 to 52, wherein the at least one activated saccharide comprises an activated form of a bacterial capsular polysaccharide.

[0059] Embodiment 54 is the method according to any one of embodiments 48 to 53, wherein the at least one activated saccharide comprises an activated form of bacterial capsular polysaccharide from Haemophilus influenzae.

[0060] Embodiment 55 is the method according to any one of embodiments 48 to 53, wherein the at least one activated saccharide comprises an activated form of bacterial capsular polysaccharide from Streptococcus pneumoniae.

[0061] Embodiment 56 is the method according to any one of embodiments 48 to 53, wherein the at least one activated saccharide comprises an activated form of bacterial capsular polysaccharide from Neisseria meningitidis.

[0062] Embodiment 57 is the method according to the immediately preceding embodiment, wherein the Neisseria meningitidis is serogroup C, A, W-135, or Y.

[0063] Embodiment 58 is the method according to any one of embodiments 48 to 57, wherein at least one protein carrier comprises recombinant exoprotein alpha (REP A), outer membrane protein complex (OMPC), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C fragment.

[0064] Embodiment 59 is the method according to the immediately preceding embodiment, wherein the at least one protein carrier comprises diphtheria toxoid, CRM197, or tetanus toxoid.

[0065] Embodiment 60 is the method according to the immediately preceding embodiment, wherein the at least one protein carrier comprises a tetanus toxoid.

[0066] Embodiment 61 is a conjugate for use as a vaccine according to any one of embodiments 36 to 39.

[0067] Embodiment 62 is a vaccine according to any one of embodiments 36 to 39, for use in the prevention of Haemophilus influenzae, Neisseria meningitidis, or Streptococcus pneumoniae infection or disease.

[0068] Embodiment 63 is a vaccine according to any one of embodiments 36 to 39, for use in immunization against Haemophilus influenzae, Neisseria meningitidis, or Streptococcus pneumoniae.

[0069] Embodiment 64 is a conjugate for use in the manufacture of a vaccine according to any one of embodiments 36 to 39.

[0070] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the appended claims. [Brief description of the drawings]

[0071] [Figure 1] 1 shows 1-D proton NMR spectra of sodium borohydride and sodium cyanoborohydride, as discussed in Example 1. [Figure 2A] 1 shows the results of a temperature study on the 1-D proton NMR spectra of sodium borohydride and sodium cyanoborohydride, as discussed in Example 1. [Figure 2B] An overlay of the peaks corresponding to the borohydride from the NMR data shown in FIG. 2A is shown. [Diagram 3] 1 shows the results of a 1-D proton NMR study on various sodium cyanoborohydride samples discussed in Example 1. [Figure 4] 1 shows the integral value for the sodium borohydride peak discussed in Example 1. [Diagram 5] 1 shows an array of spectra from a sample of sodium cyanoborohydride spiked with sodium borohydride, as discussed in Example 1. [Figure 6] 1 shows a linear curve generated using sodium borohydride concentration versus peak area obtained from a sample of sodium borohydride in d-DMSO as discussed in Example 1. [Figure 7] 1 shows a linear curve generated using sodium borohydride concentration versus peak area obtained from samples of sodium cyanoborohydride spiked with varying amounts of sodium borohydride as discussed in Example 1. [Figure 8] 1 shows the linearity of the target (sodium borohydride) versus the observed sodium borohydride in the sample of sodium cyanoborohydride discussed in Example 2. [Figure 9] 1 shows an HPSEC chromatogram (absorbance 280 nm) of the conjugation reaction between activated intermediate serogroup W-135 polysaccharide and tetanus toxoid, as discussed in Example 3. [Figure 10] FIG. 2 shows an HPSEC chromatogram (absorbance 280 nm) of the conjugation reaction between activated intermediate serogroup Y polysaccharide and tetanus toxoid, as discussed in Example 3. [Figure 11] 11 shows an enlargement of a region of the conjugate chromatogram shown in FIG. [Figure 12] 1 shows an HPSEC chromatogram (absorbance 280 nm) of the conjugation reaction between intermediate serogroup C polysaccharide and tetanus toxoid, as discussed in Example 3. [Figure 13] 1 shows an HPSEC chromatogram (absorbance 280 nm) of the conjugation reaction between intermediate serogroup W-135 polysaccharide and tetanus toxoid discussed in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While embodiments of the invention will be described, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents of the embodiments described herein.

[0073] Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, which may vary. It should be noted that, as used herein and in the appended claims, singular forms such as "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the phrase "at least one" should be understood to include one and more than one. Thus, for example, the phrases "conjugate" and "at least one conjugate" should both be understood to include one conjugate and more than one conjugate.

[0074] Numerical ranges are inclusive of the numbers defining the range. Measurements and measurable values ​​are understood to be approximate, taking into account significant digits and error associated with measurement. Also, the use of "comprise", "comprises", "comprising", "contain", "contains", "containing" and "include", "includes", "including" are not intended to be limiting. It is to be understood that both the general description and the detailed description set forth above are exemplary and explanatory only and are not restrictive of the teachings.

[0075] Unless otherwise indicated, embodiments herein that recite various components "comprising" are also contemplated as "consisting of" or "consisting essentially of" the recited components; embodiments herein that recite various components "consisting of" are also contemplated as "comprising" or "consisting essentially of" the recited components; embodiments herein that recite various components "consisting essentially of" are also contemplated as "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of such terms in the claims).

[0076] The section headings used herein are merely for organizational purposes and should not be construed as limiting the desired subject matter in any way. In the event that any document incorporated by reference conflicts with any term defined herein, the present specification shall control. Although the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0077] A.Definition Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings.

[0078] The terms "percent" or "%" with respect to the amount of sodium borohydride in the sodium cyanoborohydride reagent refer to percent by weight. For example, 5 μg sodium borohydride in a 5 mg sample of sodium cyanoborohydride reagent is 0.5%.

[0079] The term "or" is used in its inclusive sense, ie, equivalent to "and / or," unless the context requires otherwise.

[0080] The terms "linker" and "linkage" are used interchangeably and refer to a chemical moiety that comprises a chain of atoms that covalently bonds or attaches to an item such as a carrier protein or sugar.

[0081] "Linking moiety" means a chemically reactive group, substituent, or moiety, such as a nucleophile or electrophile, that can react with another molecule to form a covalent bond.

[0082] As used herein, "spacer" refers to a linker made of carbon atoms. For example, the spacer can contain 2 to 10 linear carbon atoms.

[0083] The term "sugar" includes both monosaccharides and polysaccharides.

[0084] The term "polysaccharide" refers to a chain of covalently linked monosaccharide units, which may be linear or branched. In some embodiments, the polysaccharide has a molecular weight (e.g., weight average molecular weight or number average molecular weight) in the range of 300 kDa to 1500 kDa.

[0085] The term "about" indicates a degree of variation that does not substantially affect the properties of the described subject matter, for example, within 10%, 5%, 2%, or 1%. Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the reported significant digits and by applying ordinary rounding techniques.

[0086] B. Exemplary methods and uses for conjugating at least one activated sugar to at least one protein carrier and exemplary conjugates produced according to the methods and uses In some embodiments, a method of conjugating at least one activated sugar to at least one protein carrier is provided. In some embodiments, the method comprises reacting at least one activated sugar with at least one protein carrier. In some embodiments, the method comprises reacting at least one activated sugar with at least one protein carrier in the presence of sodium cyanoborohydride reagent containing about 0.7% or less sodium borohydride to obtain a conjugate. Here and throughout, concentration percentages are w / w unless otherwise indicated.

[0087] In some embodiments, there is provided a use of a sodium cyanoborohydride reagent as a reagent in conjugating at least one activated sugar to at least one protein carrier by reductive amination. In some embodiments, the use comprises using a sodium cyanoborohydride reagent containing about 0.7% or less sodium borohydride as a reagent in conjugating at least one activated sugar to at least one protein carrier by reductive amination.

[0088] General characteristics of procedures for conjugating sugars to protein carriers using sodium cyanoborohydride are available, for example, in U.S. Pat. No. 4,365,170, U.S. Pat. No. 4,673,574, EP 0161188, EP 0208375, EP 0477508, and WO 2018 / 045286. These documents do not discuss limitations on the amount of sodium borohydride in the sodium cyanoborohydride reagent.

[0089] In some embodiments, the methods or uses disclosed herein comprise direct reductive amination.

[0090] In some embodiments, in the methods and uses disclosed herein, at least one activated sugar is directly conjugated to at least one carrier protein. In some embodiments, at least one activated sugar is indirectly conjugated to an amino group on at least one carrier protein via a linker group. In some embodiments, the linker group comprises a carbonyl, carbamate, spacer, amide, or hydrazide linker. In some embodiments, the linker group comprises a dihydrazide linker.

[0091] In some embodiments, the methods and uses disclosed herein further comprise activating at least one saccharide with an activating agent to obtain at least one activated saccharide. Depending on the structure of the at least one polysaccharide, different activation strategies can be applied to facilitate covalent attachment to a carrier protein. See, for example, Vaccine Analysis: Strategies, Principles, and Control; Nunnally, BK, Turula, VE, Sitrin, RD, Eds.; Springer: Berlin / Heidelberg, Germany, 2015; pp. 313-320.

[0092] Methods for activating sugars can be accomplished by procedures known in the art (see, for example, WO 2018 / 045286, which describes exemplary activation procedures). A general review of various activation strategies can be found in Vaccine Analysis: Strategies, Principles, and Control; Nunnally, BK, Turula, VE, Sitrin, RD, Eds.; Springer: Berlin / Heidelberg, Germany, 2015; pp. 313-320.

[0093] In some embodiments, activation is achieved by reacting the sugar with sodium periodate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), or carbonyldiimidazole (CDI). In some embodiments, the sugar is activated to introduce an aldehyde moiety to provide an activated sugar. In some embodiments, introduction of the aldehyde moiety is achieved by reacting the sugar with sodium periodate.

[0094] In some embodiments, an activated sugar is a sugar that includes a moiety suitable for conjugation to a protein carrier in a reaction, e.g., a reductive amination reaction, e.g., in the presence of a cyano hydride reagent. In some embodiments, the moiety suitable for conjugation to a protein carrier in a reaction is an ester, an aldehyde, -OCN, -CN, -OC(O)NH(CH 2 ) 3 NH 2 , -NH 2 , -NHNHC(O)(CH 2 ) n C(O)NHNH 2 , -NHC(O)(CH 2 ) n C(O)O-Pyrrolidine-2,5-dionyl, -NHC(O)CH 2 SH, and -NHC(O)(CH 2 ) nC≡CH, and in each instance n is CH in the chain 2 It is the number of units.

[0095] In some embodiments, the sodium cyanoborohydride reagent in the methods and uses disclosed herein contains about 0.6% or less sodium borohydride, hi some embodiments, the sodium cyanoborohydride reagent contains about 0.5% or less sodium borohydride.

[0096] Any known sugar can be used in the methods and uses disclosed herein (see, e.g., WO 2018 / 045286, WO 2008 / 135514, U.S. Pat. No. 7,862,823). In some embodiments, in the methods and uses disclosed herein, at least one activated sugar comprises an activated form of a cell or virus surface carbohydrate. Exemplary cell types include bacteria, protozoa, worms, fungi, and cancer cells. See, e.g., Astronomo et al., "Carbohydrate Vaccines: developing sweet solutions to sticky solutions?" Nat. Rev. Drug Discov., 2010 April, 9(4):10.1038 / nrd3012.

[0097] In some embodiments, in the methods and uses disclosed herein, at least one activated saccharide comprises an activated form of bacterial capsular polysaccharide. Capsular polysaccharide can be prepared according to known methods. See, for example, US Patent Publication No. 2003 / 0068336 in Example 1; US ​​Patent No. 6,933,137.

[0098] In some embodiments, the at least one activated saccharide comprises an activated form of bacterial capsular polysaccharide from Haemophilus influenzae. In some embodiments, the Haemophilus influenzae is Haemophilus influenzae type B. In some embodiments, the at least one activated saccharide comprises an activated form of bacterial capsular polysaccharide from Streptococcus pneumoniae. In some embodiments, the at least one activated saccharide comprises an activated form of bacterial capsular polysaccharide from Neisseria meningitidis. In some embodiments, the Neisseria meningitidis is serogroup C, A, W-135, or Y.

[0099] Any known protein carrier can be used in the methods and uses disclosed herein. Examples of protein carriers are discussed, for example, in Pichichero ME. "Protein carriers of conjugate vaccines: Characteristics, development, and clinical trials." Human Vaccines & Immunotherapeutics, 2013; 9(12):2505-2523. Doi:10.4161 / hv.26109 (incorporated herein by reference). In some embodiments, in the methods and uses disclosed herein, at least one protein carrier comprises recombinant exoprotein alpha (REP A), outer membrane protein complex (OMPC), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C fragment. In some embodiments, at least one protein carrier comprises diphtheria toxoid, CRM197, or tetanus toxoid. In some embodiments, the at least one protein carrier comprises a tetanus toxoid, hi some embodiments, the tetanus toxoid is a genetically detoxified tetanus toxoid.

[0100] In some embodiments, tetanus toxoid (TT) is prepared by extraction of toxin from cultures of Clostridium tetani (Harvard strain) grown in Mueller and Miller medium or modified Mueller and Miller medium, ammonium sulfate purification, and formalin inactivation. In some embodiments, TT is processed to reduce residual formaldehyde, concentrated in sodium chloride, and filter sterilized. In some embodiments, TT is purified by chromatography rather than ammonium sulfate purification. In some embodiments, modified Mueller and Miller medium does not contain beef heart infusion. In some embodiments, Clostridium tetani is grown in the medium described in Table 3 on page 16 of WO 2006 / 042542.

[0101] In some embodiments, in the methods and uses disclosed herein, the pH of the reaction is in the range of about 7 to about 10. In some embodiments, the pH is about pH 7, 8, 9, or 10. In some embodiments, the pH is about 7, 7.5, 8, 8.5, 9, 9.5, or 10. In some embodiments, the pH is about 7, 7.25, 7.5, 7.75, 8, 8.25, 8.50, 8.75, 9, 9.25, 9.5, 9.75, or 10. In some embodiments, the pH is about 8. In some embodiments, the pH is about 9.

[0102] In some embodiments, the methods and uses disclosed herein further comprise determining the amount of sodium borohydride in the sodium cyanoborohydride reagent. In some embodiments, the quantification comprises performing NMR on the sodium cyanoborohydride reagent. In some embodiments, the NMR comprises one-dimensional proton NMR.

[0103] In some embodiments, a conjugate of at least one activated sugar with at least one protein carrier is provided. In some embodiments, the conjugate is produced according to the methods and uses disclosed herein.

[0104] In some embodiments, the conjugate can be a Haemophilus influenzae polysaccharide conjugated to recombinant exoprotein alpha (REP A), outer membrane protein complex (OMPC), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C fragment. In some embodiments, the conjugate can be a Streptococcus pneumoniae polysaccharide conjugated to recombinant exoprotein alpha (REP A), outer membrane protein complex (OMPC), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C fragment. In some embodiments, the conjugate can be a Neisseria meningitidis polysaccharide conjugated to recombinant exoprotein alpha (REP A), outer membrane protein complex (OMPC), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C fragment. In some embodiments, the conjugate is a Neisseria meningitidis polysaccharide tetanus toxoid conjugate.

[0105] C. Exemplary Methods for Quantifying the Amount of Sodium Borohydride in Sodium Cyanoborohydride Reagent In some embodiments, a method is provided for quantifying the amount of sodium borohydride in a sodium cyanoborohydride reagent. In some embodiments, the method includes obtaining NMR data by subjecting a sample of the sodium cyanoborohydride reagent to one-dimensional proton NMR. In some embodiments, the method includes determining the amount of sodium borohydride in the sample from the NMR data.

[0106] In some embodiments, in the methods disclosed herein, NMR is performed in a deuterated solvent. In some embodiments, the deuterated solvent comprises at least one deuterated form of dimethylsulfoxide, chloroform, or methylene chloride. In some embodiments, NMR is performed in deuterated dimethylsulfoxide.

[0107] In some embodiments, in the methods disclosed herein, the NMR comprises at least one scan with a wait time of about 41 to 50 seconds. In some embodiments, the NMR comprises at least one scan with a wait time of about 43 to 48 seconds. In some embodiments, the NMR comprises at least one scan with a wait time of about 45 seconds.

[0108] In some embodiments, in the methods disclosed herein, NMR is performed at a temperature ranging from about 20° C. to about 45° C. In some embodiments, NMR is performed at a temperature ranging from about 28° C. to about 40° C. In some embodiments, NMR is performed at a temperature ranging from about 30° C. to about 35° C. In some embodiments, NMR is performed at a temperature of about 30° C.

[0109] In some embodiments, in the methods disclosed herein, NMR is performed with the parameters: temperature—about 30° C.; pulse width—pw90; spectral width—7000 Hz; and waiting time—about 45 seconds.

[0110] In some embodiments, in the methods disclosed herein, the determining step comprises using the peak area at about 0.57 ppm for the sodium borohydride resonance referenced from the solvent resonance. In some embodiments, in the methods disclosed herein, the solvent resonance comprises the dimethylsulfoxide resonance at about 2.5 ppm.

[0111] In some embodiments, in the methods disclosed herein, the determining step uses an external standard curve. In some embodiments, the external standard is about 20-120 μg / ml of sodium borohydride. In some embodiments, the external standard curve comprises a 6-point external standard curve.

[0112] D. Exemplary Vaccine Compositions and Related Methods and Uses In some embodiments, a vaccine composition is provided. In some embodiments, the vaccine composition comprises at least one protein-conjugated saccharide. In some embodiments, the at least one protein-conjugated saccharide is produced according to the methods and uses disclosed herein.

[0113] Formulation of the vaccine compositions disclosed herein can be accomplished using art-recognized methods. The selection of appropriate carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form.

[0114] In some embodiments, at least one protein-conjugated sugar is purified. One method for purification, including ultrafiltration in the presence of ammonium sulfate, is described in U.S. Pat. No. 6,146,902. Alternatively, the conjugate can be purified away from unreacted proteins and polysaccharides by any number of standard techniques, including size exclusion chromatography, density gradient centrifugation, hydrophobic interaction chromatography, mixed-mode resin chromatography, or ammonium sulfate fractionation, among others. See, for example, PW Anderson, et. Al. (1986). J. Immunol. 137: 1181-1186; also see HJ Jennings and C. Lugowski (1981) J. Immunol. 127: 1011-1018.

[0115] In some embodiments, the vaccine compositions disclosed herein further comprise a pharma- ceutically acceptable preservative, carrier, buffer excipient, etc. In one embodiment, the pharma- ceutically acceptable preservative, carrier, or excipient increases or prolongs the shelf life of the composition.

[0116] In some embodiments, the vaccine compositions disclosed herein include a buffer, such as a pharma- ceutically acceptable buffer. In some embodiments, the pharma- ceutically acceptable buffer includes a phosphate buffer, an acetate buffer, a borate buffer, a histidine buffer, or a succinate buffer. In some embodiments, the pharma- ceutically acceptable buffer includes sodium phosphate. In some embodiments, the pharma- ceutically acceptable buffer includes sodium acetate. In some embodiments, the buffer is present at a concentration ranging from 10 mM to 100 mM, e.g., from 10 mM to 70 mM, from 15 mM to 45 mM, from 20 mM to 40 mM, from 40 mM to 60 mM, or from 60 mM to 100 mM. In some embodiments, the buffer is present at 30 mM. In some embodiments, the buffer has a pH of 4.5 to 7.5, 4.5 to 7.0, 4.5 to 6.5, 4.5 to 6.0, 4.5 to 5.5, or 4.5 to 5.0. In some embodiments, the buffer has a pH ranging from 5.5 to 7.0, such as 5.75 to 6.25 or 6.25 to 6.75. In some embodiments, the buffer has a pH of 5.5 to 6.5. In some embodiments, the buffer has a pH of 5 or 6.

[0117] In some embodiments, the vaccine compositions disclosed herein further comprise a pharma- ceutically acceptable salt. In some embodiments, the vaccine compositions disclosed herein comprise saline. In some embodiments, the saline comprises or is NaCl. NaCl may be present at a concentration of 0.45 w / v% to 0.9 w / v%, e.g., 0.5 w / v% to 0.85 w / v%, or 0.6 w / v% to 0.8 w / v%, or 0.6 w / v%, 0.67 w / v%, 0.75 w / v%, 0.8 w / v%, 0.85 w / v%, or 0.9 w / v%. In some embodiments, NaCl may be present at a concentration of about 0.67%.

[0118] In some embodiments, the vaccine compositions disclosed herein contain one or more adjuvants. Examples of adjuvants include, but are not limited to, aluminum adjuvants, Freund's adjuvants, BAY, DC-chol, pcpp, monophosphoryl lipid A, CPG, QS-21, cholera toxin, and formylmethionyl peptide. See, for example, Vaccine Design, the Subunit and Adjuvant Approach, 1995 (MF Powell and MJ Newman, eds., Plenum Press, NY). When present, the adjuvant can be an aluminum adjuvant, such as aluminum hydroxide or aluminum phosphate. In some embodiments, the vaccine compositions disclosed herein do not include an adjuvant. In some embodiments, the vaccine compositions disclosed herein include an adjuvant.

[0119] In some embodiments, the vaccine compositions disclosed herein are formulated for intramuscular administration. In some embodiments, administration is subcutaneous, intradermal, intraperitoneal, parenteral, or intravenous. The compositions may be in admixture with a suitable carrier, diluent, or excipient, such as sodium acetate buffered saline, sterile water, saline. The compositions disclosed herein may also be lyophilized. The compositions disclosed herein may contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, and the like, depending on the route of administration and the desired preparation. The preparation of suitable preparations without undue experimentation may be accomplished using the methods described in "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th issue of the American College of Pharmacology. th edition, 1985, which is incorporated herein by reference.

[0120] In some embodiments, the vaccine composition disclosed herein is provided as a liquid formulation.In some embodiments, the liquid formulation is provided in a syringe, for example, a pre-filled syringe and / or a silicone-free syringe.In some embodiments, such a syringe is commercially packaged for sale and / or distribution.

[0121] In some embodiments, the vaccine compositions disclosed herein can be administered as a single dose, or sequentially (i.e., with a "booster"), or as a booster following a previous administration of a different vaccine, such as a Neisseria meningitidis capsule sugar conjugate vaccine. For example, a child can receive a single dose early in life, as currently recommended for other vaccines to prevent childhood diseases, followed by a booster dose up to 10 years later. In some embodiments, a single dose of the vaccine disclosed herein is administered 2 months to 10 years after the previously administered vaccine, such as 2 months to 4 months, 4 months to 6 months, 6 months to 12 months, 1 year to 2 years, 2 years to 3 years, 3 years to 4 years, 4 years to 5 years, 5 years to 6 years, 6 years to 7 years, 7 years to 8 years, 8 years to 9 years, or 9 years to 10 years after the previously administered vaccine.

[0122] In some embodiments, the vaccine compositions disclosed herein are formulated as a single unit dose. In some embodiments, the single unit dose comprises about 2 μg to about 15 μg of saccharide. In some embodiments, the single unit dose comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μg of saccharide. In some embodiments, the single unit dose comprises about 2 μg of saccharide. In some embodiments, the single unit dose comprises about 4 μg of saccharide. In some embodiments, the single unit dose comprises about 10 μg of saccharide. In some embodiments, where the vaccine is a multivalent vaccine, the single unit dose comprises about 2 to about 15 μg of each of the multiple saccharides.

[0123] In some embodiments, the carrier protein is present in an amount of about 15 μg to about 80 μg in a single unit dose. In some embodiments, the carrier protein is present in an amount of about 15, 20, 30, 40, 50, 60, 70, or 80 μg in a single unit dose. In some embodiments, the carrier protein is present in an amount of about 15 μg to about 30 μg in a single unit dose. In some embodiments, the carrier protein is present in an amount of about 30 μg to about 45 μg in a single unit dose. In some embodiments, the carrier protein is present in an amount of about 45 μg to about 65 μg in a single unit dose. In some embodiments, the carrier protein is present in an amount of about 25 μg in a single unit dose. In some embodiments, the carrier protein is present in an amount of about 55 μg in a single unit dose.

[0124] In some embodiments, a method of vaccinating a subject is provided. In some embodiments, the method comprises administering a single dose of a vaccine composition disclosed herein. In some embodiments, a use of a vaccine composition for immunizing a subject is provided. In some embodiments, the use comprises administering a single dose of a vaccine composition disclosed herein. In some embodiments, a use of a vaccine composition for the manufacture of a medicament for immunizing a subject is provided. In some embodiments, the use comprises administering a vaccine composition disclosed herein.

[0125] In some embodiments, in the methods or uses disclosed herein, the subject will be immunized against Haemophilus influenzae. In some embodiments, the Haemophilus influenzae is Haemophilus influenzae type B. In some embodiments, the subject will be immunized against Neisseria meningitidis. In some embodiments, the subject will be immunized against Streptococcus pneumoniae.

[0126] In some embodiments, in the methods or uses disclosed herein, the vaccine composition is administered intramuscularly. In some embodiments, the administration is subcutaneous, intradermal, intraperitoneal, parenteral, or intravenous. The composition may be in admixture with a suitable carrier, diluent, or excipient, such as sodium acetate buffered saline, sterile water, saline. The compositions disclosed herein may also be lyophilized. The compositions disclosed herein may contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, and the like, depending on the route of administration and the desired preparation. The preparation of suitable preparations can be accomplished without undue experimentation using the methods described in "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th issue of the National Academy of Sciences. th edition, 1985, which is incorporated herein by reference.

[0127] In some embodiments, a method for preparing a vaccine composition is disclosed herein. In some embodiments, the method comprises conjugating at least one activated sugar to at least one protein carrier. In some embodiments, conjugating at least one activated sugar to at least one protein carrier is carried out according to the method or use disclosed herein. In some embodiments, the method further comprises formulating the conjugate into a vaccine composition.

[0128] In some embodiments, a method of preparing at least one vaccine composition comprising a conjugate is provided, the method comprising: determining the amount of sodium borohydride in a sodium cyanoborohydride reagent; selecting a sodium cyanoborohydride reagent containing about 0.7% or less sodium borohydride; reacting at least one activated sugar with at least one protein carrier in the presence of the selected sodium cyanoborohydride reagent to provide at least one conjugate; and formulating the at least one conjugate into a vaccine composition.

[0129] In some embodiments, the methods disclosed herein further comprise activating at least one saccharide with an activating agent to obtain at least one activated saccharide. As mentioned above, activation methods are known in the art.

[0130] In some embodiments, in the methods disclosed herein, the sodium cyanoborohydride reagent selected contains about 0.6% or less sodium borohydride, hi some embodiments, the sodium cyanoborohydride reagent selected contains about 0.5% or less sodium borohydride.

[0131] In some embodiments, in the methods disclosed herein, the at least one activated saccharide comprises an activated form of a cell or viral surface carbohydrate. In some embodiments, the at least one activated saccharide comprises an activated form of a bacterial capsular polysaccharide. In some embodiments, the at least one activated saccharide comprises an activated form of a bacterial capsular polysaccharide from Haemophilus influenzae. In some embodiments, the Haemophilus influenzae is Haemophilus influenzae type B. In some embodiments, the at least one activated saccharide comprises an activated form of a bacterial capsular polysaccharide from Streptococcus pneumoniae. In some embodiments, the at least one activated saccharide comprises an activated form of a bacterial capsular polysaccharide from Neisseria meningitidis. In some embodiments, the Neisseria meningitidis is serogroup C, A, W-135, or Y.

[0132] In some embodiments, in the methods disclosed herein, at least one protein carrier comprises recombinant exoprotein alpha (REP A), outer membrane protein complex (OMPC), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C fragment. In some embodiments, at least one protein carrier comprises diphtheria toxoid, CRM197, or tetanus toxoid. In some embodiments, at least one protein carrier comprises tetanus toxoid. In some embodiments, at least one protein carrier comprises genetically detoxified tetanus toxoid. EXAMPLES

[0133] The following are examples of the methods, uses, conjugates, and compositions disclosed herein. In view of the above general and detailed descriptions, it is understood that various other embodiments may be implemented. The following examples should not be construed as limiting the scope of the present disclosure or the claims.

[0134] Abbreviations used:

[0135] [Table 1]

[0136] Example 1. Development of a quantitative one-dimensional proton NMR method for the determination of sodium borohydride in raw sodium cyanoborohydride NaCNBH 4 NaBH in the reagent 4 was performed using 1-D proton NMR. The method used an external six-point standard curve of sodium borohydride from about 20-120 μg / mL. The quantification range was about 30-300 μg / mL, with a limit of quantification of about 30 μg / mL.

[0137] Quantitation was achieved by linear regression using the peak area sodium borohydride peak at a chemical shift value of approximately -0.57 pm referenced from the dimethylsulfoxide resonance at 2.5 ppm.

[0138] Early method development To evaluate the feasibility of the method, samples consisting of 50 mg / ml sodium cyanoborohydride and 0.050 mg / ml sodium borohydride were prepared in d-DMSO. NMR analysis was performed using the instrumental parameters in Table 1 below.

[0139] [Table 2]

[0140] Proton NMR of borohydride compounds The resonances in the proton spectra for sodium borohydride and sodium cyanoborohydride show splitting patterns due to binding to boron. Boron is a species of two magnetically active isotopes, 11 B and 10 B, the properties of which are shown in Table 2 below. In the proton spectrum, the signals were split by coupling to the boron nucleus. 11 Four equally intense single peaks were observed due to B and spin=3 / 2. See Figure 1. Conversely, the lower natural abundance 10 B and spin=3 give rise to a seven-line multiplet at much lower intensity.

[0141] The splitting patterns were similar for both sodium borohydride and sodium cyanoborohydride, but the chemical shifts and line widths for sodium cyanoborohydride were different due to the presence of the cyano group, see Figure 1.

[0142] [Table 3]

[0143] temperature research For accurate integration, baseline separation of the peaks of interest is necessary. Especially if an internal standard is employed, the overlap of the sodium borohydride and sodium cyanoborohydride resonances introduces a large source of error when integration is performed over the entire resonance, i.e., over all peaks of the analyte. As shown in Figure 1, there is an overlap between sodium borohydride and sodium cyanoborohydride. By changing the temperature, the sodium cyanoborohydride resonances showed a slight variation in the chemical shift values, but no baseline separation was achieved even at high temperatures. See Figure 2. Therefore, an acquisition temperature of 30 °C was maintained.

[0144] Sodium borohydride-free materials In order to find more material that does not contain measurable amounts of sodium borohydride, various lots of sodium cyanoborohydride were prepared for NMR analysis, see Figure 3. Lot 4 did not show any measurable amounts of sodium borohydride, so this material was selected for method development studies.

[0145] Quantitative peaks As shown above in Figure 1, the overlap of the sodium cyanoborohydride resonance with some of the sodium borohydride peaks will result in errors in quantification. Figure 4 shows the integrals for all sodium borohydride peaks in Lot 4 spiked with 50 μg / mL sodium borohydride. The presence of the large sodium cyanoborohydride resonance contributes to baseline distortion for the leftmost sodium borohydride resonance. The resonance at -0.57 ppm exhibits a flat baseline and is well resolved from the adjacent peaks. Therefore, this peak was selected for quantification.

[0146] Determination of spin-lattice relaxation time Inversion recovery experiments are performed to determine the spin-lattice relaxation times (T 1 ) was determined. 1 is a measure of the time required for each nucleus to reach equilibrium with its surroundings in the magnetic field. T 1An accurate estimate of N ensures an adequate wait time (d2) between scans to allow the magnetization to return to equilibrium. For experiments in which multiple scans are collected (nt>1), if a value of d2 that is too short is used, the magnetization will not fully recover to the equilibrium value before the start of the next scan, resulting in errors in the integration.

[0147] In the inversion recovery experiment, the magnetization (M 0 ) is inverted based on the 180° pulse, and then a time τ (d2) is applied to allow relaxation. The magnetization after time τ is measured by applying a 90° observation pulse. In the experiment, the value of τ (or d2) is arranged.

[0148] A 50 mg / mL sodium cyanoborohydride sample from lot 4 spiked with 0.05 mg / mL sodium borohydride was used for the experiment. Acquisition parameters were 16 scans (45 s wait time, 90 pulse width of 8.3 microseconds at 57 dB transmitter power, 180 pulse width of 16.6 microseconds, temperature of 30 °C, acquisition time of 8.194 seconds) with d2 sequenced with 15 points between 0.010 seconds and 60 seconds interpulse time. The sweep width was 7998.4 Hz and 128K data points were acquired.

[0149] Peak height and T 1 The analysis was carried out according to the following formula: M = M 0 (1-2e -τ / T 1 ) using Varian software T 1 Analysis was carried out using.

[0150] The waiting time (d2), i.e., the time delay between pulse scans in the experiment, was the longest T 1 The T for the sodium borohydride resonance of about 8 seconds is at least five times 1 The value of (i.e., 5×T 1 = approximately 40 seconds), a value of 45 seconds for d1 is an appropriate wait time between pulse scans.

[0151] T 1The sequence spectrum from the experiment is shown in FIG. 5 and the results of the analysis are shown in Table 3 below.

[0152] [Table 4]

[0153] Early Recovery Studies Sodium borohydride in dimethylsulfoxide-d6 - A 4.7 mg / ml stock solution of sodium borohydride in DMSO-d6 was prepared. 750 ml of sample ranging from 6 to 295 ppm was prepared. NMR analysis was performed using the following instrument parameters: temperature 30 °C, receiver gain 30, 90° pulse width (8.3 μs), spectral width 7998.4 Hz, and 64K acquisition points. After four steady-state scans, 32 scans were acquired with a wait time of 45 seconds. A linear curve was generated using sodium borohydride concentration versus peak area for the resonance at -0.57 ppm. See Figure 6.

[0154] Sodium Borohydride in Sodium Cyanoborohydride Sample Matrix - A 50 mg / mL stock solution of sodium cyanoborohydride was prepared. Using the 4.7 mg / mL sodium borohydride stock, samples of various concentrations of sodium borohydride were prepared for NMR analysis. Figure 7 shows the data table and concentration vs. peak area plot for the experiment.

[0155] Example 2. Evaluation method Accuracy, precision and linearity A 50 mg / ml solution of sodium cyanoborohydride was prepared. Five levels of sodium borohydride were prepared in this sample matrix at 31, 104, 183, 261, and 313 μg / mL. Three samples were analyzed at each level. A six-point external calibration curve was used.

[0156] Table 4 below shows the results of the test. Level 1 corresponds to 31 μg / mL; Level 2 corresponds to 104 μg / mL; Level 3 corresponds to 183 μg / mL; Level 4 corresponds to 261 μg / mL; Level 5 corresponds to 313 μg / mL. For accuracy, 80-120% is considered good, closer to 100% is best. For precision, less than 15% is acceptable, the lower the better. As shown below, the method shows good accuracy and precision (relative standard deviation %). Figure 8 shows the linearity of the target (i.e., expected borohydride) versus observed sodium borohydride in sodium cyanoborohydride.

[0157] [Table 5]

[0158] Example 3. Threshold at which sodium borohydride contamination adversely affects conjugation reactions The objective of this study was to determine the threshold at which sodium borohydride contamination adversely affects the conjugation reaction.

[0159] Efficacy of polysaccharide activation prior to conjugation A reducing activity test was performed on the activated polysaccharide intermediates (sodium periodate-activated N. meningitidis polysaccharide serogroups C, W-135, and Y) to determine the effectiveness of polysaccharide activation. The assay is colorimetric and uses Cu +2 Cu obtained by lowering the glycoactivity of the ion + The reduction activity test was based on the formation of a purple complex with bicinchoninic acid (BCA) in alkaline solution with ions. This test is an index used to determine the ability of activated polysaccharides to conjugate with tetanus protein in a conjugation reaction. A review of the reduction activity results showed that the polysaccharides were effectively activated and suitable for conjugation.

[0160] Tetanus toxoid compatibility A review of protein content was completed for the lot of tetanus toxoid used for conjugation. More specifically, the BCA (bicinchoninic acid) protein assay was adapted from the BCA colorimetric assay for proteins described by Smith, et al., Analytical Biochemistry 1985;150(1):76-85. When combined with sample protein, Cu from the BCA working reagent was +2 Cu +1 Each Cu +1 The ion combines with two BCA molecules to form BCA-Cu +1 A complex is formed which produces a purple color with strong absorbance at 562 nm. The protein concentration was found to be suitable for conjugation.

[0161] Suitability of sodium cyanoborohydride used in conjugation The goal of this study was to perform an initial screen to determine the threshold at which sodium borohydride contamination adversely affects the conjugation reaction. Four experiments were performed with sodium borohydride concentrations of 0.5%, 1.0%, 1.5%, and 2.0%.

[0162] Sodium cyanoborohydride was used in the conjugation reaction during the manufacture of N. meningitidis polysaccharide tetanus toxoid conjugate concentrates for serogroups C, W-135, and Y. The pH of the conjugation reaction for serogroup C was 8 and for serogroups W-135 and Y was 9.

[0163] During the conjugation reaction, polysaccharide was added in excess to tetanus to ensure complete utilization of the tetanus. Conjugate concentrates were produced by covalent attachment of depolymerized / activated polysaccharide to tetanus toxoid carrier protein. This attachment occurred via reductive amination of aldehyde groups on the polysaccharide chain to amine groups present on the tetanus toxoid carrier protein. In this reaction, sodium cyanoborohydride specifically reduced the Schiff base intermediate formed between the polysaccharide aldehyde groups and the protein amine groups to form a stable secondary amine bond. The resulting conjugate had the polysaccharide chain attached to the protein carrier. The reactions were allowed to mix for 16-24 hours. After the mixing time, the conjugation reaction was capped by adding sodium borohydride. Sodium borohydride inhibited further conjugation by reducing remaining aldehyde groups to hydroxyl groups. If trace amounts of sodium borohydride are present during the conjugation reaction, it will reduce the amount of aldehyde groups available for attachment to the protein carrier, resulting in free protein.

[0164] HPSEC analysis was performed to determine the size difference of the conjugate molecules. Larger molecules flow faster through a SEC column than smaller molecules. Thus, a shift to a longer retention time indicates a smaller conjugate size, which corresponds to an effect on the reaction efficiency.

[0165] (1) N. meningitidis polysaccharide-tetanus toxoid conjugate concentrate for serogroup W: The starting materials used were activated intermediate serogroup W-135 polysaccharide and tetanus toxoid. For the purposes of this study, pharmaceutical grade 97.5% purity sodium cyanoborohydride was used. Sodium borohydride was then spiked into the sodium cyanoborohydride stock solution at various concentrations and allowed to mix for 24 hours. After 24 hours, the completed samples were pulled and run on a HPSEC column to determine the reaction efficiency. The chromatograms (absorbance at 280 nm) are shown in FIG. 9.

[0166] The results of this study indicate that concentrations > 1.0% have a detrimental effect on the conjugation reaction, i.e. there is a large amount of free protein in the reaction.

[0167] (2) N. meningitidis polysaccharide-tetanus toxoid conjugate concentrate for serogroup Y: The starting materials used were activated intermediate serogroup Y polysaccharide and tetanus toxoid. Pharmaceutical grade 97.5% purity sodium cyanoborohydride was used for the purpose of this study, except for the control arm, where reagent grade sodium cyanoborohydride was used. Sodium borohydride was then spiked into the sodium cyanoborohydride stock solution at various concentrations (0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%) and then mixed for 24 hours. After 24 hours, the completed samples were pulled and run on a size exclusion chromatography column to determine the reaction efficiency. The HPSEC chromatograms (absorbance at 280 nm) are shown in Figures 10 and 11. Similar results are expected for serogroup W polysaccharide (similar structure and same conjugation process). The results of this study indicate that concentrations > 0.7% adversely affect the conjugation reaction.

[0168] Free Protein Results A total of eight runs (see Table 5 below) of N. meningitidis polysaccharide tetanus toxoid conjugate concentrate were successfully produced following the methods described in WO 2018 / 045286 to complete the evaluation of the impact of residual sodium borohydride present in sodium cyanoborohydride.

[0169] The experiment consisted of four control arms and four experimental arms spiked with sodium borohydride at concentrations of 0.5% and 1.0%. For the purpose of this study, pharmaceutical grade and reagent grade sodium cyanoborohydride were used. The appropriate amount of sodium borohydride was spiked into sodium cyanoborohydride stock solution at the predetermined concentration to provide for the conjugation reaction.

[0170] [Table 6]

[0171] The material was then processed to completion via diafiltration, hydrophobic interaction chromatography purification, and final diafiltration. HPSEC samples were pulled from the crude conjugate mixture after completion of the 16-24 hour retention times. These samples were pulled to determine if there was a shift in the chromatogram towards longer retention times. Longer retention times indicate a potential effect on the conjugation reaction.

[0172] The serogroup C chromatograms (Figure 12) showed no significant differences between the pharmaceutical grade and reagent grade controls. The serogroup C chromatograms showed a slight shift in the 0.5% sodium borohydride sample and a large shift in the 1.0% sodium borohydride sample. The large shift in the 1% chromatogram indicates that the 1% sodium borohydride content may have a negative effect on reaction efficiency.

[0173] Serogroup W-135 chromatograms (FIG. 13) did not show significant differences between runs 1, 2, and 3. However, serogroup W-135 (1.0% sodium borohydride) showed a large shift, indicating that the presence of 1% sodium borohydride content may have a detrimental effect on reaction efficiency.

[0174] Lots of sodium cyanoborohydride containing 0.5% sodium borohydride have been shown to have no adverse effect on the free protein content of the final bulk concentrate, however lots containing at least 1% sodium borohydride content may cause an increase in the free protein content.

[0175] Equivalent The above specification is believed to be sufficient to enable one skilled in the art to practice the embodiments. The above description and examples detail certain embodiments and explain the best mode contemplated by the inventors. However, no matter how detailed the above specification appears in text, it will be understood that the embodiments may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0176] The term about, as used herein, refers to numerical values, including, for example, integers, ratios, and percentages, whether or not expressly stated. The term about generally refers to a range of numerical values ​​(e.g., + / - 5 to 10% of the stated range) that one of ordinary skill in the art would consider equivalent to the stated value (e.g., having the same function or result). When a term such as at least and about precedes a list of numerical values ​​or ranges, the term modifies all of the values ​​or ranges set forth in the list. In some cases, the term about may include numerical values ​​that are rounded to the nearest significant figure.

Claims

1. A method for conjugating at least one activated sugar to at least one protein carrier: a) To determine the amount of sodium borohydride in the sodium cyanoborohydride reagent; b) Reacting the at least one activated sugar with the at least one protein carrier in the presence of a sodium cyanoborohydride reagent containing approximately 0.7% or less sodium borohydride to obtain a conjugate. A method that includes this.

2. The use of a sodium cyanoborohydride reagent containing approximately 0.7% or less sodium borohydride as a reagent for conjugating at least one activated sugar to at least one protein carrier by reductive amination, wherein the sodium cyanoborohydride reagent is determined to contain 0.7% or less sodium borohydride.

3. The use of a sodium cyanoborohydride reagent containing approximately 0.7% or less sodium borohydride as a reagent for conjugating at least one activated sugar to at least one protein carrier by reductive amination, wherein: a) To determine the amount of sodium borohydride in the sodium cyanoborohydride reagent; b) Reacting the at least one activated sugar with the at least one protein carrier in the presence of a sodium cyanoborohydride reagent containing approximately 0.7% or less sodium borohydride to obtain a conjugate. Use including.

4. The method further comprises activating at least one sugar with an activator to obtain the at least one activated sugar, and / or The aforementioned sodium cyanoborohydride reagent contains approximately 0.6% or less sodium borohydride, and in some cases contains approximately 0.5% or less sodium borohydride, according to the claim. The method or use described in any one of items 1 to 3.

5. The at least one activated sugar comprises an activated form of a surface carbohydrate of a cell or virus, In some cases, the at least one activated sugar comprises an activated form of a bacterial capsule polysaccharide, and in some cases, the activated form of the bacterial capsule polysaccharide is selected from Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis. In some cases, the activated polysaccharide comprises an activated form of a bacterial capsule polysaccharide derived from Neisseria meningitidis serogroup C, A, W-135, or Y, according to or use of claim 4.

6. The method or use according to any one of claims 1 to 3, wherein the at least one protein carrier comprises recombinant exoprotein alpha (REP A), outer membrane protein complex (OMPC), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C fragment, and / or the pH of the reaction is in the range of about 7 to about 10, and optionally the pH is about 8 or about 9.

7. The method or use according to claim 4, wherein the at least one protein carrier comprises recombinant exoprotein alpha (REP A), outer membrane protein complex (OMPC), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C fragment, and / or the pH of the reaction is in the range of about 7 to about 10, and optionally the pH is about 8 or about 9.

8. The method or use of claim 5, wherein the at least one protein carrier comprises recombinant exoprotein alpha (REP A), outer membrane protein complex (OMPC), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C fragment, and / or the pH of the reaction is in the range of about 7 to about 10, and optionally the pH is about 8 or about 9.

9. Determining the amount of sodium borohydride in a sodium cyanoborohydride reagent involves performing NMR on the sodium cyanoborohydride reagent. In some cases, the NMR includes a one-dimensional proton NMR. In some cases, the NMR is performed in a deuterated solvent containing dimethyl sulfoxide, chloroform, or at least one deuterium form of methylene chloride. In some cases, the NMR includes at least one scan with a latency of approximately 41 to 50 seconds, and in some cases, at least one scan with a latency of approximately 43 to 48 seconds, or even more in some cases, approximately 45 seconds. In some cases, the NMR is performed at temperatures ranging from approximately 20°C to approximately 45°C, in some cases ranging from approximately 28°C to approximately 40°C, or from approximately 30°C to approximately 35°C, and / or In some cases, the NMR parameter: -Temperature: Approximately 30℃ - Pulse width: pw90 - Spectral width: 7000 Hz - Waiting time: Approximately 45 seconds The method or use according to any one of claims 1 to 3, as performed by [the specified method].

10. Determining the amount of sodium borohydride in a sodium cyanoborohydride reagent is This includes performing NMR on the sodium borohydride reagent. In some cases, the NMR includes a one-dimensional proton NMR. In some cases, the NMR is performed in a deuterated solvent containing dimethyl sulfoxide, chloroform, or at least one deuterium form of methylene chloride. In some cases, the NMR includes at least one scan with a latency of approximately 41 to 50 seconds, and in some cases, at least one scan with a latency of approximately 43 to 48 seconds, or even more in some cases, approximately 45 seconds. In some cases, the NMR is performed at temperatures ranging from approximately 20°C to approximately 45°C, in some cases ranging from approximately 28°C to approximately 40°C, or from approximately 30°C to approximately 35°C, and / or In some cases, the NMR parameter: -Temperature: Approximately 30℃ - Pulse width: pw90 - Spectral width: 7000 Hz - Waiting time: Approximately 45 seconds The method or use of claim 4, as performed by [the specified method].

11. Determining the amount of sodium borohydride in a sodium cyanoborohydride reagent involves performing NMR on the sodium cyanoborohydride reagent. In some cases, the NMR includes a one-dimensional proton NMR. In some cases, the NMR is performed in a deuterated solvent containing dimethyl sulfoxide, chloroform, or at least one deuterium form of methylene chloride. In some cases, the NMR includes at least one scan with a latency of approximately 41 to 50 seconds, and in some cases, at least one scan with a latency of approximately 43 to 48 seconds, or even more in some cases, approximately 45 seconds. In some cases, the NMR is performed at temperatures ranging from approximately 20°C to approximately 45°C, in some cases ranging from approximately 28°C to approximately 40°C, or from approximately 30°C to approximately 35°C, and / or In some cases, the NMR parameter: -Temperature: Approximately 30℃ - Pulse width: pw90 - Spectral width: 7000 Hz - Waiting time: Approximately 45 seconds The method or use of claim 8, as performed by [the specified method].

12. A method for quantifying the amount of sodium borohydride in a sodium cyanoborohydride reagent, wherein the method is: (a) Subject a sample of sodium borohydride reagent to one-dimensional proton NMR and thereby obtain NMR data; and (b) Determining the amount of sodium borohydride in the sample from NMR data, In some cases, the NMR is performed in a deuterated solvent containing dimethyl sulfoxide, chloroform, or at least one deuterium form of methylene chloride. In some cases, the NMR includes at least one scan with a latency of approximately 41 to 50 seconds, and in some cases, at least one scan with a latency of approximately 43 to 48 seconds, or even more in some cases, approximately 45 seconds. In some cases, the NMR is performed at temperatures ranging from approximately 20°C to approximately 45°C, in some cases ranging from approximately 28°C to approximately 40°C, or from approximately 30°C to approximately 35°C, and / or In some cases, the NMR parameter: -Temperature: Approximately 30℃ - Pulse width: pw90 - Spectral width: 7000 Hz - Waiting time: Approximately 45 seconds The method that is executed.

13. Determining the amount of sodium borohydride is This includes using a peak area at approximately 0.57 ppm for the sodium borohydride resonance referenced from the solvent resonance, and optionally the solvent resonance includes a dimethyl sulfoxide resonance at approximately 2.5 ppm, and / or The method or use of claim 12, wherein the method comprises using an external standard curve for sodium borohydride, wherein the external standard curve for sodium borohydride is approximately 20 to 120 μg / ml, and wherein the external standard curve comprises a six-point external standard curve.

14. Determining the amount of sodium borohydride is This includes using a peak area at approximately 0.57 ppm for the sodium borohydride resonance referenced from the solvent resonance, and optionally the solvent resonance includes a dimethyl sulfoxide resonance at approximately 2.5 ppm, and / or The method or use of claim 9, wherein the method comprises using an external standard curve for sodium borohydride, wherein the external standard curve for sodium borohydride is approximately 20 to 120 μg / ml, and wherein the external standard curve comprises a six-point external standard curve.

15. A method for preparing a vaccine composition, comprising: conjugating at least one activated sugar to at least one protein carrier according to the method or use described in any one of claims 1 to 3; and formulating the conjugate into a vaccine composition.

16. A method for preparing a vaccine composition, comprising: conjugating at least one activated sugar to at least one protein carrier according to the method or use described in claim 4; and formulating the conjugate into a vaccine composition.

17. A method for preparing a vaccine composition, comprising: conjugating at least one activated sugar to at least one protein carrier according to the method or use described in claim 5; and formulating the conjugate into a vaccine composition.

18. A method for preparing a vaccine composition, comprising: conjugating at least one activated sugar to at least one protein carrier according to the method or use described in claim 6; and formulating the conjugate into a vaccine composition.

19. A method for preparing a vaccine composition, comprising: conjugating at least one activated sugar to at least one protein carrier according to the method or use described in claim 7; and formulating the conjugate into a vaccine composition.

20. A method for preparing a vaccine composition, comprising: conjugating at least one activated sugar to at least one protein carrier according to the method or use described in claim 8; and formulating the conjugate into a vaccine composition.

21. A method for preparing a vaccine composition, comprising: conjugating at least one activated sugar to at least one protein carrier according to the method or use described in claim 9; and formulating the conjugate into a vaccine composition.

22. A method for preparing a vaccine composition, comprising: conjugating at least one activated sugar to at least one protein carrier according to the method or use described in claim 11; and formulating the conjugate into a vaccine composition.

23. A method for preparing at least one vaccine composition containing a conjugate: a) A step of determining the amount of sodium borohydride in the sodium cyanoborohydride reagent, b) A step of selecting a sodium cyanoborohydride reagent containing approximately 0.7% or less sodium borohydride, possibly approximately 0.6% or less sodium borohydride, or approximately 0.5% or less sodium borohydride, c) A step of preparing at least one conjugate by reacting at least one activated sugar with at least one protein carrier in the presence of the selected sodium cyanoborohydride reagent, d) A step of formulating the at least one conjugate into a vaccine composition. A method comprising, in some cases, activating at least one sugar with an activator to obtain the at least one activated sugar.

24. The method according to claim 23, wherein the at least one activated sugar comprises an activated form of a surface carbohydrate of a cell or virus, optionally an activated form of a bacterial capsule polysaccharide, optionally the bacterial capsule polysaccharide being derived from Haemophilus influenzae, Streptococcus pneumoniae, or Neisseria meningitidis, optionally the Neisseria meningitidis being serogroup C, A, W-135, or Y.

25. The method according to claim 23 or 24, wherein the at least one protein carrier comprises recombinant exoprotein alpha (REP A), outer membrane protein complex (OMPC), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C fragment.

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