US-12617873-B2 - Method of purifying polysaccharides

Abstract

The present disclosure provides a method of purifying polysaccharides from a cell lysate, comprising partially purifying the cell lysate comprising an impurity and a polysaccharide to obtain a clarified crude lysate; mixing the clarified crude lysate with a neutralization solution comprising a salt to form a neutralized lysate; mixing the neutralized lysate with a precipitation solution comprising cetyltrimethylammonium bromide to form a first supernatant and a first precipitate; and separating the first precipitate from the first supernatant, wherein the polysaccharide is located in the first supernatant. The present disclosure further provides a method of making a polysaccharide vaccine. Also provided are vaccines, delivery systems, compositions and polysaccharides made by the methods described herein.

Inventors

• Andreas Zurbriggen

Assignees

• LONZA LTD

Dates

Publication Date

20260505

Application Date

20201130

Claims (20)

1. 1 . A method of purifying polysaccharides from a cell lysate, the method comprising a. partially purifying the cell lysate comprising a polysaccharide and an impurity to obtain a clarified crude lysate; b. mixing the clarified crude lysate with a neutralization solution to form a neutralized lysate comprising about 100 mM to about 2 M salt; c. mixing the neutralized lysate with a precipitation solution comprising cetyltrimethylammonium bromide (CTAB) to form a first supernatant and a first precipitate, wherein the first supernatant comprises about 0.5% v/v to about 4% v/v CTAB; and d. separating the first precipitate from the first supernatant, wherein the polysaccharide is substantially located in the first supernatant; and wherein the method is capable of purifying positively charged, negatively charged, and neutral polysaccharides, and wherein the positively charged, negatively charged, and neutral polysaccharides are substantially located in the first supernatant.
2. 2 . The method of claim 1 , wherein the partially purifying the cell lysate comprises precipitation, centrifugation, filtration, or combination thereof.
3. 3 . The method of claim 2 , wherein the filtration comprises depth filtration, tangential flow filtration (TFF), sterile filtration, or combination thereof.
4. 4 . The method of claim 1 , wherein: the neutralized lysate comprises about 200 mM to about 1 M salt; and/or the salt is NaCl, KCl, CH 4 Cl, NH 4 Cl, MgCl 2 , CaCl 2 , Na 3 PO 4 , or combination thereof.
5. 5 . The method of claim 1 , wherein the mixing of the clarified crude lysate with the neutralization solution in (b) is performed simultaneously with or prior to a filtration step, wherein the filtration step is prior to (c).
6. 6 . The method of claim 1 , wherein the cell lysate comprises a positively charged polysaccharide, a neutral polysaccharide, a negatively charged polysaccharide, or combination thereof, wherein the positively charged polysaccharide, the neutral polysaccharide, the negatively charged polysaccharide, or combination thereof are substantially in the first supernatant, and wherein the impurity is substantially in the first precipitate.
7. 7 . The method of claim 1 , wherein the impurity comprises a polynucleotide.
8. 8 . The method of claim 1 , wherein the mixing in (b), (c), or both, is performed by shaking, stirring, or pumping; and/or wherein the separating in (d) is performed by centrifugation, filtration, or combination thereof.
9. 9 . The method of claim 1 , further comprising subjecting the first supernatant to ultrafiltration, diafiltration, or combination thereof to produce a retentate and a permeate, wherein the polysaccharide is substantially in the retentate, and the CTAB is substantially in the permeate.
10. 10 . The method of claim 9 , further comprising subjecting the retentate to carbon filtration, chromatography, or combination thereof.
11. 11 . The method of claim 1 , further comprising adding potassium iodide (KI) to the first supernatant to form a second precipitate and a second supernatant, wherein the polysaccharide is substantially in the second supernatant and the CTAB is substantially in the second precipitate.
12. 12 . The method of claim 11 , wherein the second supernatant comprises the KI and further comprises NaCl.
13. 13 . The method of claim 11 , wherein the method does not comprise a chromatography step prior to the adding of KI.
14. 14 . The method of claim 11 , further comprising separating the second supernatant from the second precipitate by centrifugation, filtration, or combination thereof; and subjecting the second supernatant to carbon filtration, chromatography, or combination thereof.
15. 15 . The method of claim 14 , further comprising performing ultrafiltration, diafiltration, or combination thereof following the chromatography, to concentrate the polysaccharide.
16. 16 . The method of claim 1 , wherein the method comprises at least one of: (i) the method does not comprise a first precipitate containing greater than 10% of the polysaccharides in the cell lysate during the purification; (ii) the positively charged polysaccharide, the neutral polysaccharide, the negatively charged polysaccharide, or the combination thereof are not substantially precipitated during the purification; (iii) the positively charged polysaccharide, the neutral polysaccharide, the negatively charged polysaccharide, or the combination thereof are not isolated from each other; (iv) greater than 90% (v/v) of negatively charged polysaccharides are in the first supernatant and less than 10% (v/v) of negatively charged polysaccharides are in the first precipitate; (v) the percent yield of polysaccharides is greater than 30% relative to the polysaccharides in the cell lysate.
17. 17 . The method of claim 1 , wherein the cell lysate is derived from a bacterial cell or a fungal cell.
18. 18 . The method of claim 17 , wherein the cell is a Streptococcus cell of serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20A, 22F, 23F, or 33F.
19. 19 . The method of claim 17 , wherein the cell lysate is derived from a Staphylococcus cell, Streptococcus cell, Salmonella cell, Neisseria cell, Mycobacterium cell, or Aureobasidium cell.
20. 20 . A method of purifying polysaccharides from a cell lysate, the method comprising a. partially purifying the cell lysate comprising an impurity and a polysaccharide to obtain a clarified crude lysate; b. mixing the clarified crude lysate with a neutralization solution to form a neutralized lysate comprising about 400 mM sodium chloride (NaCl); c. mixing the neutralized lysate with cetyltrimethylammonium bromide (CTAB) to form a first supernatant and a first precipitate, wherein the first supernatant comprises about 1.0% v/v CTAB; d. separating the first precipitate from the first supernatant; e. mixing the first supernatant with potassium iodide (KI) to form a second precipitate and a second supernatant; and f. separating the second precipitate from the second supernatant, wherein the polysaccharide is substantially located in the second supernatant; and wherein the method is capable of purifying positively charged, negatively charged, and neutral polysaccharides, and wherein the positively charged, negatively charged, and neutral polysaccharides are substantially located in the first supernatant.

Description

FIELD OF THE INVENTION The present disclosure provides a method of purifying polysaccharides from a cell and/or cell lysate. The present disclosure further provides a method of making a polysaccharide vaccine. Also provided are vaccines, delivery systems, compositions, and polysaccharides made by the methods described herein. BACKGROUND Pathogenic bacteria often have a cell surface capsule coated with polysaccharides. In a similar manner, pathogenic fungi can have a cell wall containing polysaccharides. Upon infection, these bacterial and fungal polysaccharides interfere with the immune response by preventing host antibodies from attaching to the cells. These polysaccharides have been discovered to be effective vaccines against the bacterial or fungal pathogens. In certain instances, multiple polysaccharides are conjugated into one vaccine formulation to increase protection against multiple serotypes or strains, and/or to improve the effectiveness of the vaccine. Additionally, polysaccharides have been used as non-antigen specific vaccine adjuvants to enhance the immunogenicity of vaccines. Polysaccharides used in vaccine formulations can be isolated from the bacteria or fungi after culturing the organism, then lysing and purifying the polysaccharides from undesired impurities. Typical impurities can include cellular proteins, nucleic acids, other cellular components, and components of the culture medium. In general, polysaccharides can be charged, e.g., positively charged or negatively charged, or neutral. Separation of each type of polysaccharide from impurities may require different processes, e.g., different reagents and concentrations, separation procedure, etc. For example, U.S. Pat. No. 5,714,354 utilizes two separate processes for the purification of negatively charged polysaccharides and neutral polysaccharides, each purification process being a multi-day procedure that involves several sub-processes. Furthermore, the use of different reagents in each of the separate purification procedures can lead to reduced consistency in yield and quality. Moreover, use of different processes can increase burden for manufacture compliance, since each step and reagent may need to be evaluated separately during process characterization, resulting in lower efficiency and higher costs. Thus, purification of polysaccharides often can be a cumbersome, complex, time consuming, and inefficient endeavor. SUMMARY OF THE INVENTION In some embodiments, the disclosure provides a method of purifying polysaccharides from a cell lysate, the method comprising (a) partially purifying the cell lysate comprising a polysaccharide and an impurity to obtain a clarified crude lysate; (b) mixing the clarified crude lysate with a neutralization solution comprising about 100 mM to about 2 M salt to form a neutralized lysate; (c) mixing the neutralized lysate with a precipitation solution comprising about 0.1% v/v to about 6% v/v cetyltrimethylammonium bromide (CTAB) to form a first supernatant and a first precipitate; and (d) separating the first precipitate from the first supernatant, wherein the polysaccharide is substantially located in the first supernatant. In some embodiments, the partially purifying the cell lysate comprises precipitation, centrifugation, filtration, or combination thereof. In some embodiments, the filtration comprises depth filtration, tangential flow filtration (TFF), sterile filtration, or combination thereof In some embodiments, the neutralization solution comprises about 200 mM to about 1 M salt. In some embodiments, the neutralization solution comprises about 250 mM to about 650 mM salt. In some embodiments, the neutralization solution comprises about 300 mM to about 500 mM salt. In some embodiments, the salt is NaCl, KCl, CH4Cl, NH4Cl, MgCl2, CaCl2, Na3PO4, or combination thereof. In some embodiments, the neutralization solution comprises about 440 mM NaCl. In some embodiments, the mixing of the clarified crude lysate with the neutralization solution in (b) is performed simultaneously with or prior to a filtration step, wherein the filtration step is prior to (c). In some embodiments, the mixing in (b) is performed simultaneously with the filtration step. In some embodiments, the filtration comprises tangential flow filtration (TFF). In some embodiments, the precipitation solution comprises about 0.1% v/v to about 10% v/v CTAB. In some embodiments, the precipitation solution comprises about 0.2% v/v to about 5% v/v CTAB. In some embodiments, the precipitation solution comprises about 0.5% v/v to about 4% v/v CTAB. In some embodiments, the precipitation solution comprises about 1% v/v to about 3% v/v CTAB. In some embodiments, the cell lysate comprises a positively charged polysaccharide, a neutral polysaccharide, a negatively charged polysaccharide, or combination thereof. In some embodiments, the positively charged polysaccharide, the neutral polysaccharide, the negatively charged polysaccharide, or combin