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US 12,552,831 B2Granted
Saponin purification
AssigneeGlaxosmithkline Biologicals SA
InventorsCarine Berthe Ghislaine De Kesel, Chad Austin Farrenburg, Jeri Kay Sandvick, Jeb Yeatts Vandenburg
Life of this patent
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Abstract
Saponin extracts containing at least 88% QS-21 main peak and >3% to 10% 2018 component by UV absorbance at 214 nm, methods for making said extracts, their use as vaccine adjuvants and related aspects.
Claims20
A composition comprising:
- (i) a saponin extract and (ii) liposomes or an emulsion;
- the liposomes comprising cholesterol;
- the emulsion comprising a toll-like receptor 4 (TLR-4) agonist;
- the saponin extract comprising:
- (i) at least 88% of a QS-21 main peak and (ii) greater than 3% but less than or equal to 10% of a 2018 component;
- the at least 88% of the QS-21 main peak and the greater than 3% but less than or equal to 10% of the 2018 component being determined by:
- (a) injecting at a retention time of 0 minutes 10 μL of a candidate saponin extract that is in an injection solution into an ultra-performance liquid chromatography (UPLC) column at a flow rate of 0.6 mL per minute of a first mobile phase solution until a retention time of 6.23 minutes and a flow rate of 0.6 mL per minute of a second mobile phase solution at retention times from 6.23 minutes to 9.0 minutes, thereby obtaining a candidate saponin extract eluant,
- (1) the injection solution consisting of 0.2% by g acetic acid per mL of:
- (A) 70% by volume water and (B) 30% volume acetonitrile,
- (2) the first mobile phase solution consisting of 0.025% by g trifluoracetic acid per mL of:
- (A) 5% by volume isopropyl alcohol, (B) 20% by volume acetonitrile, and (C) 75% by volume water,
- (3) the second mobile phase solution consisting of:
- (A) 23% by volume the first mobile phase solution and (B) 77% by volume of a first solution,
- (4) the first solution consisting of:
- 0.025% by g trifluoroacetic acid per mL of:
- (A) 18% by volume isopropyl alcohol, (B) 72% by volume acetonitrile, and (C) 10% by volume water, and
- (5) the UPLC column having an inner diameter of 2.1 mm, a length of 100 mm, and 1.7 μm diameter ethylene bridged hybrid C18 particles and being 55° C.;
- (b) measuring ultraviolet (UV) absorbance at a wavelength of 214 nm of the candidate saponin extract eluant during (a), thereby obtaining a raw candidate saponin extract UV absorbances versus the retention times;
- the raw candidate saponin extract UV absorbances versus retention times comprising a solvent front and a plurality of raw candidate saponin extract absorbance peaks;
- the plurality of raw candidate saponin extract absorbance peaks comprising a raw candidate QS-21 main absorbance peak and a raw candidate 2018 component absorbance peak;
- the raw candidate QS-21 main absorbance peak having a retention time of approximately 4.4 minutes;
- and the raw candidate 2018 component absorbance peak having a retention time of approximately 4.5 minutes;
- (c) measuring the monoisotopic weights of the candidate saponin extract eluant during (a) with a quadrupole mass spectrometer under a scanning range of monoisotopic weights from 1400 to 2040, thereby obtaining raw candidate saponin extract monoisotopic weights versus the retention times;
- the raw candidate saponin extract monoisotopic weights versus the retention times comprising a plurality of monoisotopic weight peaks;
- the plurality of monoisotopic weight peaks comprising a candidate QS-21 main monoisotopic weights peak and a candidate 2018 component monoisotopic weights peak;
- the candidate QS-21 main monoisotopic weights peak having the retention time of approximately 4.4 minutes;
- the candidate 2018 component monoisotopic weights peak having the retention time of approximately 4.5 minutes;
- (d) injecting at a blank retention time of 0 minutes of 10 μL of the injection solution into the UPLC column at a flow rate of 0.6 mL per minute of the first mobile phase solution until the blank retention time of 6.23 minutes followed by a flow rate of 0.6 mL per minute of the second mobile phase solution at the blank retention times of from 6.23 minutes to 9.0 minutes, thereby obtaining a blank eluant;
- (e) measuring the UV absorbance at a wavelength of 214 nm of the blank eluant during (d), thereby obtaining blank UV absorbances versus blank retention times;
- the blank UV absorbances versus blank retention times comprising a plurality of blank absorbance peaks;
- the plurality of blank saponin extract absorbance peaks comprising a blank candidate QS-21 main absorbance peak and a blank candidate 2018 component absorbance peak;
- the blank candidate QS-21 main absorbance peak having a blank retention time of approximately 4.4 minutes;
- and the blank candidate 2018 component absorbance peak having a blank retention time of approximately 4.5 minutes;
- (f) subtracting the blank UV absorbance for each of the blank retention times from the raw candidate saponin extract UV absorbance for each of the respective retention times, thereby obtaining blank-subtracted candidate saponin extract absorbances versus retention times;
- the blank-subtracted candidate saponin extract absorbances versus retention times comprising a plurality of blank-subtracted candidate saponin extract absorbance peaks;
- the plurality of blank-subtracted candidate saponin extract absorbance peaks comprising a blank-subtracted candidate QS-21 main absorbance peak and a blank-subtracted candidate 2018 component absorbance peak;
- the blank-subtracted candidate QS-21 main absorbance peak thereby being obtained by subtracting the blank candidate QS-21 main absorbance peak for each blank retention time of approximately 4.4 minutes from the raw candidate QS-21 main absorbance peak for each of the respective retention times of approximately 4.4 minutes;
- and the blank-subtracted candidate 2018 component absorbance peak thereby being obtained by subtracting the blank candidate 2018 component absorbance peak for each of the blank retention times of approximately 4.5 minutes from the raw candidate 2018 component absorbance peak for each of the retention times of approximately 4.5 minutes;
- (g) confirming that:
- (1) the candidate QS-21 main monoisotopic weights peak comprises triterpenoid glycosides that have monoisotopic weights of 1855.9, 1987.9, and 2001.9, thereby, with the obtaining the blank-subtracted candidate QS-21 main absorbance peak of (f), obtaining the QS-21 main peak;
- and
- (2) the candidate 2018 component monoisotopic weights peak comprises a triterpenoid glycoside that has a monoisotopic weight of 2017.9, thereby, with the obtaining the blank-subtracted candidate 2018 component absorbance peak of (f), obtaining the 2018 component;
- (h) obtaining:
- (1) an area of the plurality of blank-subtracted saponin extract absorbance peaks that elude from the later of the solvent front and the retention time of 0.5 minutes and until the retention time of 5.5 minutes, thereby obtaining a total peaks area;
- (2) an area that is of the blank-subtracted candidate QS-21 main absorbance peak, thereby obtaining a QS-21 main peak area;
- and
- (3) an area that is of the blank-subtracted candidate 2018 component absorbance peak, thereby obtaining a 2018 component peak area;
- (i) dividing:
- (1) the QS-21 main peak area by the total peaks area, thereby obtaining a fraction of the QS-21 main peak;
- and
- (2) the 2018 component peak area by the total peaks area, thereby obtaining a fraction of the 2018 component;
- (j) multiplying:
- (1) the fraction of the QS-21 main peak by 100, thereby obtaining a percentage of the QS-21 main peak;
- and
- (2) the fraction of the 2018 component by 100, thereby obtaining a percentage of the 2018 component;
- and
- (k) confirming that:
- (1) the percentage of the QS-21 main peak is at least 88% and, thereby obtaining the at least 88% of the QS-21 main peak;
- and
- (2) the percentage of the 2018 component is greater than 3% but less than or equal to 10% and thereby obtaining the greater than 3% but less than or equal to 10% of the 2018 component.
57 more elements
A composition comprising:
- (i) a saponin extract and (ii) liposomes or an emulsion;
- the liposomes comprising cholesterol;
- the emulsion comprising a TLR4 agonist;
- the saponin extract comprising:
- (i) at least 88% in sum of one or more of a compound (a), a compound (b), and a compound (c) and (ii) greater than 3% but less than or equal to 10% of a compound (d);
- wherein:
- and the at least 88% in sum of the one or more of the compound (a), the compound (b), and the compound (c) and the greater than 3% but less than or equal to 10% of the compound (d) is determined by:
- (a) injecting at a retention time of 0 minutes 10 μL of a candidate saponin extract that is in an injection solution into a UPLC column at a flow rate of 0.6 mL per minute of a first mobile phase solution until a retention time of 6.23 minutes and a flow rate of 0.6 mL per minute of a second mobile phase solution at retention times from 6.23 minutes to 9.0 minutes, thereby obtaining a candidate saponin extract eluant,
- (1) the injection solution consisting of 0.2% by g acetic acid per mL of:
- (A) 70% by volume water and (B) 30% volume acetonitrile,
- (2) the first mobile phase solution consisting of 0.025% by g trifluoracetic acid per mL of:
- (A) 5% by volume isopropyl alcohol, (B) 20% by volume acetonitrile, and (C) 75% by volume water,
- (3) the second mobile phase solution consisting of:
- (A) 23% by volume the first mobile phase solution and (B) 77% by volume of a first solution,
- (4) the first solution consisting of:
- 0.025% by g trifluoroacetic acid per mL of:
- (A) 18% by volume isopropyl alcohol, (B) 72% by volume acetonitrile, and (C) 10% by volume water, and
- (5) the UPLC column having an inner diameter of 2.1 mm, a length of 100 mm, and 1.7 um diameter ethylene bridged hybrid C18 particles and being 55° C.;
- (b) measuring UV absorbance at a wavelength of 214 nm of the candidate saponin extract eluant during (a), thereby obtaining raw candidate saponin extract UV absorbances versus the retention times;
- the raw candidate saponin extract UV absorbances versus retention times comprising a solvent front and a plurality of raw candidate saponin extract absorbance peaks;
- the plurality of raw candidate saponin extract absorbance peaks comprising a raw candidate compounds (a)-(c) absorbance peak and a raw candidate compound (d) absorbance peak;
- (c) measuring the monoisotopic weights of the candidate saponin extract eluant during (a) with a quadrupole mass spectrometer under a scanning range of monoisotopic weights from 1400 to 2040, thereby obtaining raw candidate saponin extract monoisotopic weights versus the retention times;
- the raw candidate saponin extract monoisotopic weights versus the retention times comprising a plurality of monoisotopic weight peaks;
- the plurality of monoisotopic weight peaks comprising a candidate compounds (a)-(c) monoisotopic weights peak and a candidate compound (d) monoisotopic weights peak;
- the candidate compounds (a)-(c) monoisotopic weights peak having a retention time equal to the retention time of the raw candidate compounds (a)-(c) absorbance peak;
- and the candidate compound (d) monoisotopic weights peak having a retention time equal to the retention time of the raw candidate compound (d) absorbance peak;
- (d) confirming that:
- (1) the candidate compounds (a)-(c) monoisotopic weights peak comprises a triterpenoid glycoside that has a monoisotopic weight of 1855.9 or at least one first triterpenoid glycoside that has a monoisotopic weight of 1987.9;
- the confirming that the candidate compounds (a)-(c) monoisotopic weights peak comprises the at least one first triterpenoid glycoside that has a monoisotopic weight of 1987.9 further comprising confirming that the candidate compounds (a)-(c) monoisotopic weights peak comprises:
- (i) the compound (a), (ii) the compound (b), or (iii) the compound (a) and the compound (b) and the confirming that the candidate compounds (a)-(c) monoisotopic weights peak comprises the triterpenoid glycoside that has a monoisotopic weight of 1855.9 further comprising confirming that the candidate compounds (a)-(c) monoisotopic weights peak comprises the compound (c), thereby obtaining the one or more of the compound (a), the compound (b), and the compound (c), and thereby confirming the raw candidate compounds (a)-(c) absorbance peak is a raw compounds (a)-(c) absorbance peak;
- and
- (2) the candidate compound (d) monoisotopic weights peak comprises a triterpenoid glycoside that has a monoisotopic weight of 2017.9 and further confirming that the triterpenoid glycoside that has a monoisotopic weight of 2017.9 is the compound (d), thereby obtaining the compound (d) and thereby confirming the raw candidate compound (d) absorbance peak is a raw compound (d) absorbance peak;
- (e) injecting at a blank retention time of 0 minutes of 10 μL of the injection solution into the UPLC column at a flow rate of 0.6 mL per minute of the first mobile phase solution until the blank retention time of 6.23 minutes followed by a flow rate of 0.6 mL per minute of the second mobile phase solution at the blank retention times of from 6.23 minutes to 9.0 minutes, thereby obtaining a blank eluant;
- (f) measuring the UV absorbance at a wavelength of 214 nm of the blank eluant during (d), thereby obtaining blank UV absorbances versus blank retention times;
- the blank UV absorbances versus blank retention times comprising a plurality of blank absorbance peaks;
- the plurality of blank saponin extract absorbance peaks comprising a blank compounds (a)-(c) absorbance peak and a blank compound (d) absorbance peak;
- the blank compounds (a)-(c) absorbance peak having a blank retention time equal to the retention time of the raw compounds (a)-(c) absorbance peak;
- the blank compound (d) absorbance peak having a blank retention time equal to the retention time of the raw compound (d) absorbance peak;
- (g) subtracting the blank UV absorbance for each of the blank retention times from the raw candidate saponin extract UV absorbance for each of the respective retention times, thereby obtaining blank-subtracted saponin extract absorbances versus retention times;
- the blank-subtracted saponin extract absorbances versus retention times comprising a plurality of blank-subtracted saponin extract absorbance peaks;
- the plurality of blank-subtracted saponin extract absorbance peaks comprising a blank-subtracted compounds (a)-(c) absorbance peak and a blank-subtracted compound (d) absorbance peak;
- (h) obtaining:
- (1) an area of the plurality of blank-subtracted saponin extract absorbance peaks that elude from the later of the solvent front and the retention time of 0.5 minutes and until the retention time of 5.5 minutes, thereby obtaining a total peaks area;
- (2) an area that is of the blank-subtracted compounds (a)-(c) absorbance peak, thereby obtaining a compounds (a)-(c) peak area;
- and
- (3) an area that is of the blank-subtracted compound (d) absorbance peak, thereby obtaining a compound (d) peak area;
- (i) dividing:
- (1) the compounds (a)-(c) peak area by the total peaks area, thereby obtaining a fraction of the compounds (a)-(c) peak;
- and
- (2) the compound (d) peak area by the total peaks area, thereby obtaining a fraction of the compound (d);
- (j) multiplying:
- (1) the fraction of the compounds (a)-(c) peak by 100, thereby obtaining a percentage of the compounds (a)-(c) peak;
- and
- (2) the fraction of the compound (d) by 100, thereby obtaining a percentage of the compound (d);
- and
- (k) confirming that:
- (1) the percentage of the compounds (a)-(c) peak is at least 88% and, thereby obtaining the at least 88% in sum of the one or more of the compound (a), the compound (b), and the compound (c);
- and
- (2) the percentage of the compound (d) is greater than 3% but less than or equal to 10% and thereby obtaining the greater than 3% but less than or equal to 10% of the compound (d).
56 more elements
Dependencies are inferred from the claim text.