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US 12,571,797 B2Granted
Liquid chromatography assay for determining AAV capsid ratio
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsJonathan Wert, Li Zhi, Dingjiang Liu
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Abstract
Methods for determining the relative abundance of intact adeno-associated virus (AAV) capsid components in a sample of recombinant AAV particles are disclosed. In embodiments, the methods include a system regeneration process that minimizes or eliminates the presence of ghost peaks to maximize analytical accuracy and ensure product quality and consistency.
Claims20
A method for determining relative abundance of intact viral capsid components in a sample of adeno-associated virus (AAV) particles comprising a heterologous nucleic acid molecule, the method comprising:
- (a) performing a system regeneration process on a liquid chromatography system comprising a liquid chromatography (LC) instrument and an anion exchange column (AEX column), wherein the system regeneration process comprises:
- (i) washing the AEX column with at least 10 column volumes (CV) of mobile phase A;
- (ii) washing the AEX column with at least 10 CV of purified water;
- (iii) washing the AEX column with at least 20 CV of a wash solution comprising from 15% to 21% v/v ethanol in purified water;
- and (iv) washing liquid carrying components of the LC instrument with the wash solution at a flow rate of at least 0.5 mL/min for at least 30 minutes,
- wherein mobile phase A comprises from 15 mM to 25 mM bis-tris-propane, and from 1 mM to 3 mM magnesium chloride in purified water at a pH of from 8 to 9, and wherein the purified water has a resistivity of about 18.2 Mohm·cm at 25° C. and less than 5 ppb total organic carbon;
- (b) performing a system equilibration process on the liquid chromatography system, comprising purging the wash solution from the liquid carrying components of the LC instrument and the AEX column;
- (c) performing a sample separation process, wherein the sample separation process comprises:
- (i) introducing one or more blank samples into the liquid chromatography system, and running a separation gradient of mobile phase A, mobile phase B and mobile phase C through the liquid chromatography system;
- (ii) introducing one or more reference standard samples into the liquid chromatography system, and running a separation gradient of mobile phase A, mobile phase B and mobile phase C through the liquid chromatography system;
- and (iii) introducing one or more test samples of the AAV particles into the liquid chromatography system, wherein the one or more test samples comprises intact empty AAV capsids and intact full AAV capsids in a sample solution, and running a separation gradient of mobile phase A, mobile phase B and mobile phase C through the liquid chromatography system to separate the intact empty AAV capsids from the intact full AAV capsids,
- wherein mobile phase B comprises 15 mM to 25 mM bis-tris-propane, from 250 mM to 1 M tetramethylammonium chloride (TMAC), and from 1 mM to 3 mM magnesium chloride in purified water at a pH of from 8 to 9, and mobile phase C comprises 1.5 M to 2.5 M sodium chloride in purified water;
- and
- (d) identifying an amount of the intact empty AAV capsids and an amount of the intact full AAV capsids in each of the one or more test samples to determine the relative abundance of the intact viral capsid components in the sample of AAV particles.
11 more elements
A method for determining relative abundance of intact viral capsid components in a sample of adeno-associated virus (AAV) particles comprising a heterologous nucleic acid molecule, the method comprising:
- (a) performing a system regeneration process on a liquid chromatography system comprising a liquid chromatography (LC) instrument and an anion exchange column (AEX column), wherein the system regeneration process comprises:
- (i) washing the AEX column with at least 10 column volumes (CV) of mobile phase A;
- (ii) washing the AEX column with at least 10 CV of purified water;
- (iii) optionally reversing the orientation of the AEX column to reverse the flow within the AEX column;
- (iv) washing the AEX column with at least 20 CV of a wash solution comprising from 15% to 21% v/v ethanol in purified water;
- (v) removing the AEX column from the LC instrument;
- and (vi) washing liquid carrying components of the LC instrument with the wash solution at a flow rate of at least 0.5 mL/min for at least 30 minutes,
- wherein mobile phase A comprises from 15 mM to 25 mM bis-tris-propane, and from 1 mM to 3 mM magnesium chloride in purified water at a pH of from 8 to 9, and wherein the purified water has a resistivity of about 18.2 Mohm·cm at 25° C. and less than 5 ppb total organic carbon;
- (b) performing a system equilibration process on the liquid chromatography system, comprising:
- (i) reinstalling the AEX column into the LC instrument;
- and (ii) purging the wash solution from the liquid carrying components of the LC instrument and the AEX column;
- (c) performing a sample separation process, wherein the sample separation process comprises:
- (i) introducing one or more blank samples into the liquid chromatography system, and running a separation gradient of mobile phase A, mobile phase B and mobile phase C through the liquid chromatography system;
- (ii) introducing one or more reference standard samples into the liquid chromatography system, and running a separation gradient of mobile phase A, mobile phase B and mobile phase C through the liquid chromatography system;
- and (iii) introducing one or more test samples of the AAV particles into the liquid chromatography system, wherein the one or more test samples comprises intact empty AAV capsids and intact full AAV capsids in a sample solution, and running a separation gradient of mobile phase A, mobile phase B and mobile phase C through the liquid chromatography system to separate the intact empty AAV capsids from the intact full AAV capsids,
- wherein mobile phase B comprises 15 mM to 25 mM bis-tris-propane, from 250 mM to 1 M tetramethylammonium chloride (TMAC), and from 1 mM to 3 mM magnesium chloride in purified water at a pH of from 8 to 9, and mobile phase C comprises 1.5 M to 2.5 M sodium chloride in purified water;
- and
- (d) identifying an amount of the intact empty AAV capsids and an amount of the intact full AAV capsids in each of the one or more test samples to determine the relative abundance of the intact viral capsid components in the sample of AAV particles.
15 more elements
Dependencies are inferred from the claim text.