CN117836433A - Liquid chromatography assay for determining AAV capsid ratios - Google Patents
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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 method includes a system regeneration process that minimizes or eliminates the presence of false peaks to maximize analytical accuracy and ensure product quality and consistency.
Description
Technical Field
The present invention relates to methods for determining the relative abundance of intact adeno-associated virus (AAV) capsid components in a sample of AAV particles comprising a heterologous nucleic acid molecule using anion exchange chromatography.
Background
Adeno-associated virus is a non-enveloped, single-stranded DNA virus that has become an attractive therapeutic agent for delivering genetic material to host cells for gene therapy due to its ability to transduce a variety of substances and tissues in vivo, low risk of immune toxicity, and mild innate and adaptive immune responses. The complex nature of viral vectors (such as AAV) requires specific analytical methods for robust product testing and characterization.
Irrespective of the method of preparation, one feature of AAV production is the formation of empty capsids that are free of heterologous genes of interest (GOI). The level of empty capsids can vary greatly. While the effect of empty capsids on treatment outcome is not completely clear, it is necessary to closely monitor the number of empty capsids to ensure product quality and consistency.
Various methods of quantifying empty capsids in AAV samples have been described, and anion exchange chromatography (AEX) has been used for the purification and enrichment of complete AAV particles of serotypes 1, 2 and 8 (Wang, see below). Furthermore, AEX is atThe use of accurate quantification of AAV serotype 6.2 has been discussed in Wang et al, molecular Therapy: methods and Clinical Development,15:257-263,2019. In particular, wang reports that the structural integrity of AAV capsids under chromatographic conditions is a prerequisite for the separation of empty capsids from complete capsids, and 2mM MgCl 2 The presence of (2) results in the disappearance of broad peaks and an increase in peak intensity, but higher MgCl 2 No further improvement in concentration was provided. It is further reported by Wang that the use of tetramethylammonium chloride (TMAC) in the mobile phase improves the separation relative to NaCl, the separation is improved at higher pH values in the range of 7.5 to 9.0, the buffer has little effect on the degree of separation of empty capsids from complete capsids, and 1, 3-bis ((trimethylol) methylamino) propane (BTP) gives the best separation in the reagents tested.
Minimizing or eliminating potential sources of analytical error is critical to product quality and consistency, especially in the context of Good Manufacturing Practice (GMP) release assays. One such source of analytical error is the presence of "false peaks," commonly referred to as artifacts or false peaks, which are observed unexpectedly in chromatograms. Since such false peaks may be caused by impurities or artifacts within the liquid chromatography system, their presence may lead to non-compliant research requirements, or to inconsistent and inaccurate results, which are time consuming or unacceptable for GMP product determination.
Disclosure of Invention
The present disclosure relates to a method for determining the relative abundance of intact viral capsid components in a sample of adeno-associated virus (AAV) particles.
In one aspect, the present disclosure provides a method for determining the 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 (CVs) of mobile phase a; (ii) washing the AEX column with at least 10CV of purified water; (iii) Washing the AEX column with at least 20CV of a washing solution comprising a purified aqueous solution of 15% to 21% v/v ethanol; and (iv) washing the carrier liquid component of the LC instrument with the washing solution at a flow rate of at least 0.5mL/min for at least 30 minutes, wherein mobile phase a comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane and 1mM to 3mM magnesium chloride, at a pH of 8 to 9, and wherein the purified water has a resistivity of about 18.2mohm.cm at 25 ℃ and has less than 5ppb total organic carbon; (b) Performing a system equilibration process on the liquid chromatography system comprising purging the wash solution from the LC instrument and the carrier liquid component of 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 comprise intact empty AAV capsids and intact complete AAV capsids dissolved 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 complete AAV capsids, wherein mobile phase B comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane, 250mM to 1M tetramethyl ammonium chloride (TMAC), and 1mM to 3mM magnesium chloride, pH 8 to 9, and mobile phase C comprises a purified aqueous solution of 1.5M to 2.5M sodium chloride; and (d) identifying the amount of the intact empty AAV capsids and the amount of the intact complete 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.
In one aspect, the present disclosure provides a method for determining the relative abundance of intact viral capsid components in a sample of 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 (CVs) of mobile phase a; (ii) washing the AEX column with at least 10CV of purified water; (iii) Optionally reversing the orientation of the AEX column to reverse flow within the AEX column; (iv) Washing the AEX column with at least 20CV of a washing solution comprising a purified aqueous solution of 15% to 21% v/v ethanol; (v) removing the AEX column from the LC instrument; and (vi) washing the carrier liquid component of the LC instrument with the washing solution at a flow rate of at least 0.5mL/min for at least 30 minutes, wherein mobile phase a comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane and 1mM to 3mM magnesium chloride, at a pH of 8 to 9, and wherein the purified water has a resistivity of about 18.2mohm.cm at 25 ℃ and has less than 5ppb total organic carbon; (b) Performing a system balancing process on the liquid chromatography system, comprising: (i) reinstalling the AEX column into the LC instrument; and (ii) purging the wash solution from the LC instrument and the carrier liquid assembly of 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 comprise intact empty AAV capsids and intact complete AAV capsids dissolved 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 complete AAV capsids, wherein mobile phase B comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane, 250mM to 1M tetramethyl ammonium chloride (TMAC), and 1mM to 3mM magnesium chloride, pH 8 to 9, and mobile phase C comprises a purified aqueous solution of 1.5M to 2.5M sodium chloride; and (d) identifying the amount of the intact empty AAV capsids and the amount of the intact complete 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.
In some cases, the wash solution comprises a purified aqueous solution of 18% ± 1% v/v ethanol.
In any of the various embodiments of the methods discussed above or herein, the sample solution comprises 1mM to 100mM sodium chloride. In some cases, the sample solution comprises 60mM to 100mM sodium chloride.
In any of the various embodiments of the methods discussed above or herein, the sample solution comprises 0.003% w/v to 0.007% w/v surfactant. In some cases, the sample solution comprises 0.005% w/v±0.001% w/v surfactant. In some embodiments, the surfactant is poloxamer 188.
In some embodiments, the system balancing process comprises: (i) Purging the wash solution from the LC instrument and the carrier liquid component of the AEX column with purified water at a flow rate of at least 0.5mL/min for at least 10 minutes; (ii) washing the AEX column with at least 10CV of purified water; (iii) washing the AEX column with at least 10CV of mobile phase a; (iv) washing the AEX column with at least 20CV of mobile phase B; and (v) washing the AEX column with at least 20CV of mobile phase a.
In some embodiments, the system balancing process comprises: (i) Purging the wash solution from the carrier liquid component of the LC instrument with purified water at a flow rate of at least 0.5mL/min for at least 10 minutes; (ii) reinstalling the AEX column into the LC instrument; (iii) washing the AEX column with at least 10CV of purified water; (iv) washing the AEX column with at least 10CV of mobile phase a; (v) washing the AEX column with at least 20CV of mobile phase B; and (vi) washing the AEX column with at least 20CV of mobile phase a. In some cases, performing the system equilibration process further comprises washing the carrier liquid component of the LC instrument with purified water at a flow rate of 0.5mL/min for at least 10 minutes and then reinstalling the AEX column into the LC instrument. In some cases, the washing solution is purged from the carrier liquid component of the LC instrument with purified water at a flow rate of at least 0.5mL/min for at least 30 minutes.
In some cases, the at least 10CV includes 10 to 20CV. In some cases, the at least 20CV comprises 20 to 30CV.
In some embodiments, the sample separation process comprises: (i) Introducing three blank samples into the liquid chromatography system, followed by four reference standard samples, followed by two blank samples, followed by 1 to 10 test samples, followed by reference standard samples, followed by blank samples; and (ii) running the separation gradient of mobile phase a, mobile phase B, and mobile phase C through the liquid chromatography system separately for each sample.
In some embodiments, the separation gradient is run at a flow rate of 0.8mL/min for 36 minutes.
In some embodiments, the separation gradient comprises, in order: (i) 90% mobile phase a and 10% mobile phase B for 1 minute; (ii) Mobile phase a was reduced from 90% to 58% and mobile phase B was increased from 10% to 42% over a period of 20 minutes; (iii) 100% mobile phase C for 4 minutes; and (iv) 90% mobile phase a and 10% mobile phase B for at least 10 minutes.
In some embodiments, the method further comprises if the one or more test samples comprise ≡5×10 12 Each viral genome/mL (vg/mL), the test sample is diluted 1.5 to 3-fold (e.g., 2.3-fold) in dilution buffer.
In some embodiments (e.g., if the test sample contains ≡5X10) 12 vg/mL), each of the one or more test samples introduced into the liquid chromatography system comprising about 10 microliters. In some embodiments (e.g., if the test sample contains<5×10 12 vg/mL), introducing saidEach of the one or more test samples in the liquid chromatography system comprises about 20 microliters.
In some embodiments, the mobile phase a comprises a purified aqueous solution of 20mm±2mM 1, 3-bis ((trimethylol) methylamino) propane and 2mm±0.2mM magnesium chloride, at a pH of 8.5±0.1.
In some embodiments, the mobile phase B comprises a purified aqueous solution of 20mm±2mM 1, 3-bis ((trimethylol) methylamino) propane, 500mm±50mM TMAC, and 2mm±0.2mM magnesium chloride, at a pH of 8.5±0.1.
In some embodiments, the mobile phase C comprises a purified aqueous solution of 2m±0.2M sodium chloride.
In any of the various embodiments, the AAV particle has serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ/8, AAV-Rh10, AAV-retro, AAV-php.b, AAV8-php.eb, or AAV-php.s. In some cases, the AAV particle has serotype AAV1, AAV5, or AAV8. In some embodiments, the AAV particle has serotype AAV8.
In any of the various embodiments of the method, the residue from a previous sample run into a blank sample run in the liquid chromatography system is no more than 0.3% of the previous sample full capsid peak. In some cases, the previous sample is a reference standard sample. In some cases, the previous sample is a test sample. In some embodiments, the residue is no more than 0.15% of the previous sample full capsid peak.
In various embodiments, any features or components of the embodiments discussed above or herein may be combined, and such combinations are contemplated as being within the scope of the present disclosure. Any of the specified values discussed above or herein may be combined with another related value discussed above or herein to list a range, the values representing the upper and lower limits of the range, and such ranges are encompassed within the scope of the disclosure.
Other embodiments will become apparent upon reading the detailed description that follows.
Detailed Description
Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the terms "include" and "comprising" are intended to be non-limiting and may be understood to mean "comprising" and "including", respectively.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are herein incorporated by reference in their entirety.
Selected abbreviations
LC (liquid crystal): liquid chromatography
CV: column volume
rAAV: recombinant AAV particles or capsids
AAV: adeno-associated virus
AEX-anion exchange chromatography
GOI-target gene
Definition of the definition
"intact viral capsid component" refers to viral capsids (e.g., empty viral capsids, partially complete viral capsids, and/or complete viral capsids) that are intact (i.e., have not been denatured or broken down or disintegrated into their constituent parts (e.g., different viral proteins) and retain the structural characteristics of the viral capsids (e.g., the icosahedral conformation of the AAV capsids).
The term "empty viral capsid" or "empty capsid" refers to a capsid that does not contain a heterologous nucleic acid molecule (e.g., a therapeutic gene).
The term "partially complete viral capsid" or "partially complete capsid" refers to a capsid that contains only a portion of a heterologous nucleic acid molecule (e.g., a therapeutic gene).
The term "complete viral capsid" or "complete capsid" refers to a capsid containing all heterologous nucleic acid molecules (e.g., therapeutic genes or genes of interest).
As used herein, the term "sample" refers to a mixture of viral particles (e.g., AAV particles) comprising at least one viral capsid component (i.e., empty capsid, partially complete capsid, and/or complete capsid) that is manipulated according to the methods of the invention (including, e.g., isolation and analysis).
The terms "analysis" or "analysis" are used interchangeably and refer to any of a variety of methods of isolating, detecting, isolating, purifying, and/or characterizing a viral particle or component moiety of interest (e.g., an AAV capsid). Examples include, but are not limited to, liquid chromatography (e.g., AEX).
As used herein, "contacting" includes bringing together at least two substances in solution or solid phase, e.g., contacting a stationary phase of a chromatographic material with a sample (such as a sample containing viral particles or viral proteins).
As used herein, the term "liquid chromatography" refers to a process in which a chemical mixture carried by a liquid can be separated into components due to the differential distribution of chemical entities as they flow around or over a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include ion exchange chromatography (e.g., anion exchange chromatography).
An "adeno-associated virus" or "AAV" is a nonpathogenic parvovirus having single-stranded DNA, a genome of about 4.7kb, non-enveloped, and an icosahedral conformation. AAV was first discovered in 1965 as a contaminant of adenovirus preparations. AAV belongs to the genus dependoviridae and parvoviridae, and requires helper functions of either the herpes virus or adenovirus for replication. In the absence of helper virus, AAV can set latency by integrating into the 19q13.4 position of human chromosome 19. The AAV genome consists of two Open Reading Frames (ORFs), one open reading frame corresponding to each of the two AAV genes Rep and Cap. AAV DNA ends have an Inverted Terminal Repeat (ITR) of 145bp and 125 terminal bases are palindromic, yielding a characteristic T-shaped hairpin structure.
As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of any length, i.e., ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The backbone of the nucleic acid may comprise sugar and phosphate groups (as may be typically present in RNA or DNA), or modified or substituted sugar or phosphate groups.
"recombinant AAV particle" refers to an adeno-associated viral particle comprising one or more heterologous sequences (e.g., non-AAV-derived nucleic acid sequences) that may flank at least one (e.g., two) AAV Inverted Terminal Repeats (ITRs). Such rAAV particles can replicate and package when present in host cells that have been infected with a suitable helper virus (or express a suitable helper function) and express AAV Rep and Cap gene products (i.e., AAV Rep and Cap proteins).
By "viral particle" is meant a viral particle consisting of at least one viral capsid protein and an encapsulated viral genome.
"heterologous" means derived from an entity that is genotype-wise different than the remainder of the entity being compared or introduced or incorporated. For example, nucleic acids introduced into different cell types by genetic engineering techniques are heterologous nucleic acids (and may encode heterologous polypeptides when expressed). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral particle is a heterologous nucleotide sequence with respect to the viral particle.
An "inverted terminal repeat" or "ITR" sequence is a relatively short sequence at the end of the viral genome that is in the opposite orientation. An "AAV Inverted Terminal Repeat (ITR)" sequence is a sequence of about 145 nucleotides present at both ends of a single stranded AAV genome.
As used herein, the term "isolated" refers to a biological component (such as a nucleic acid, peptide, protein, lipid, viral particle, or metabolite) that has been substantially separated from, produced from, or purified from other biological components in the cells of an organism in which the component naturally occurs or is expressed in the cells of the organism.
As used herein, "vector" refers to a recombinant plasmid or virus comprising a nucleic acid to be delivered to a host cell in vitro or in vivo.
"recombinant viral vector" refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-viral origin).
The term "anion exchange chromatography" or AEX is intended to include a process of separating a substance based on the charge of the substance using an ion exchange resin containing positively charged groups, such as diethyl-aminoethyl groups. In solution, the resin is coated with positively charged counterions.
The term "carrier liquid assembly" of a liquid chromatography instrument refers to the following elements: for example, the sample and mobile phase are carried from the sample point (either manually or by an autosampler) to the column, and from the column to an HPLC or UPLC instrument of the detector.
General description
The present disclosure provides liquid chromatography methods that provide sensitive and quantitative characterization of viral capsid components of a sample of viral particles (e.g., AAV particles). Characterization of the viral capsid component of the viral particle composition (such as the viral capsid component of a sample of AAV particles) is necessary to ensure product quality and consistency to maintain the safety and efficacy of the composition.
Recombinant viral vector compositions (e.g., AAV vector compositions) can contain varying levels of empty capsid components and complete capsid components produced during production. The present methods provide analytical techniques for identifying and quantifying the ratio of viral capsid components in a sample of viral particles.
Method for determining the relative abundance of viral capsid components
Aspects of the disclosure relate to methods for determining the relative abundance of intact viral capsid components in a sample of AAV particles comprising a heterologous gene of interest. Generally, the methods involve separating AAV capsid components using anion exchange chromatography, and determining the relative abundance of empty capsid components and full capsid components from the peak areas of the separated components.
In some cases, the method comprises: (a) Performing a system regeneration process on a liquid chromatography system comprising a Liquid Chromatography (LC) instrument and an anion exchange column (AEX column) to reduce or eliminate the percentage of residue carried into a blank sample from a reference standard sample or a previous test sample that may affect the accuracy and reproducibility of the analysis of the test sample (e.g., less than 0.15% of the area count of the reference standard full capsid peak); (b) Performing a system equilibration process on the liquid chromatography system to purge wash material from the regeneration process from the LC instrument and the carrier liquid component of the AEX column; and (c) performing a sample isolation procedure on one or more test samples containing empty AAV capsids and full AAV capsids to determine the relative abundance of each capsid component in the one or more test samples.
In some cases, the method comprises: (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 (CVs) of mobile phase a; (ii) washing the AEX column with at least 10CV of purified water; (iii) Washing the AEX column with at least 20CV of a washing solution comprising a purified aqueous solution of 15% to 21% v/v ethanol; and (iv) washing the carrier liquid component of the LC instrument with the washing solution at a flow rate of at least 0.5mL/min for at least 30 minutes, wherein mobile phase a comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane and 1mM to 3mM magnesium chloride, at a pH of 8 to 9, and wherein the purified water has a resistivity of about 18.2mohm.cm at 25 ℃ and has less than 5ppb total organic carbon; (b) Performing a system equilibration process on the liquid chromatography system comprising purging the wash solution from the LC instrument and the carrier liquid component of 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 comprise intact empty AAV capsids and intact complete AAV capsids dissolved 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 complete AAV capsids, wherein mobile phase B comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane, 250mM to 1M tetramethyl ammonium chloride (TMAC), and 1mM to 3mM magnesium chloride, pH 8 to 9, and mobile phase C comprises a purified aqueous solution of 1.5M to 2.5M sodium chloride; and (d) identifying the amount of the intact empty AAV capsids and the amount of the intact complete 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.
In some cases, the method comprises: (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 (CVs) of mobile phase a; (ii) washing the AEX column with at least 10CV of purified water; (iii) Optionally reversing the orientation of the AEX column to reverse flow within the AEX column; (iv) Washing the AEX column with at least 20CV of a washing solution comprising a purified aqueous solution of 15% to 21% v/v ethanol; (v) removing the AEX column from the LC instrument; and (vi) washing the carrier liquid component of the LC instrument with the washing solution at a flow rate of at least 0.5mL/min for at least 30 minutes, wherein mobile phase a comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane and 1mM to 3mM magnesium chloride, at a pH of 8 to 9, and wherein the purified water has a resistivity of about 18.2mohm.cm at 25 ℃ and has less than 5ppb total organic carbon; (b) Performing a system balancing process on the liquid chromatography system, comprising: (i) reinstalling the AEX column into the LC instrument; and (ii) purging the wash solution from the LC instrument and the carrier liquid assembly of 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 comprise intact empty AAV capsids and intact complete AAV capsids dissolved 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 complete AAV capsids, wherein mobile phase B comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane, 250mM to 1M tetramethyl ammonium chloride (TMAC), and 1mM to 3mM magnesium chloride, pH 8 to 9, and mobile phase C comprises a purified aqueous solution of 1.5M to 2.5M sodium chloride; and (d) identifying the amount of the intact empty AAV capsids and the intact complete 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.
In various embodiments of the method (including those embodiments of surfactant concentration discussed below), the sample solution may contain 2mM to 100mM sodium chloride. In some cases, the sample solution contains 60mM to 100mM sodium chloride.
In various embodiments of the method, including those embodiments of sodium chloride concentration discussed above, the sample solution may contain 0.003% w/v to 0.007% w/v surfactant. In some cases, the sample solution may contain 0.005% w/v.+ -. 0.001% w/v surfactant. In some embodiments, the surfactant is a poloxamer (e.g., poloxamer 188).
In various embodiments of the method, the liquid chromatography system may be a High Performance Liquid Chromatography (HPLC) system or an ultra high performance liquid chromatography (UPLC) system. HPLC and UPLC refer to forms of column chromatography that pumps a sample mixture or analyte in a solvent (referred to as the mobile phase) through a chromatography column with a chromatographic packing material or matrix (stationary phase) at high pressure. The sample is carried by a moving carrier gas stream (e.g., helium or nitrogen). The stationary phase column consists of a chromatographic medium or resin that interacts with a mobile phase mixture or analyte. While still manually injectable, typical HPLC/UPLC systems are fully automated and computer controlled. A sample injector or an autosampler connected to a device housing the column hardware may be employed, which device is additionally connected to a detector. The types of HPLC and UPLC are well known in the art. UPLC typically operates at higher pressures than HPLC and may employ a chromatographic column with smaller particle sizes relative to HPLC. HPLC and UPLC instruments are well known in the art and will be familiar to the skilled person.
In various embodiments of the method, for example, one or more mobile phases comprise a buffer composition such as 1, 3-bis ((trimethylol) methylamino) propane (BTP). The mobile phase buffer concentration may vary. For example, a concentration of about 1mM to about 100mM (e.g., 1 to 50 mM) may be used. In various embodiments, the buffer concentration may be 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, or 100mM. In some cases, the buffer concentration (e.g., concentration of BTP) may be 10mM to 30mM or 15mM to 25mM. In some cases, the buffer concentration (e.g., concentration of BTP) may be 20mm±2mM. In some cases, the buffer concentration (e.g., concentration of BTP) may be about 20mM.
In various embodiments of the method, one or more mobile phases may contain one or more salts, for example, magnesium chloride, sodium chloride, and/or tetramethyl ammonium chloride (TMAC). In some embodiments of the present invention, in some embodiments, the salt may be present at a concentration of about 0.1mM to about 5mM, or about 0.2mM, about 0.3mM, about 0.4mM, about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.25mM, about 1.5mM, about 1.75mM, about 2mM, about 2.25mM, about 2.5mM, about 2.75mM, about 3mM, about 3.25mM, about 3.5mM, about 3.75mM, about 4mM, about 4.25mM, about 4.5mM, about 4.75mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 50mM, about 100mM, about 150mM, about 200mM, about 250mM, about 300mM, about 350mM, about 400mM, about 450mM, about 500mM, about 550mM, about 600mM, about 650mM, about 700M, about 5.5M, about 50mM, about 50M, about 700M, about 5.5M, about 50M, about 5M, about 50M, about 5.5M, about 3.25mM, about 5M, about 50M. Mobile phases, mobile phases (e.g., mobile phase a and mobile phase B) may contain about 1mM to about 3mM MgCl 2 . In some cases, the mobile phase (e.g., mobile phase a and mobile phase B) may contain about 1.5mM to about 2.5mM MgCl 2 . In some cases, the mobile phase (e.g., mobile phase a and mobile phase B) may contain about 2mM MgCl 2 . In some cases, the mobile phase (e.g., mobile phase C) may contain about 1M to about 3MNaCl. In some cases, the mobile phase (e.g., mobile phase C) may contain about 1.5M to about 2.5M NaCl. In some cases, the mobile phase (e.g., mobile phase C) may contain about 2m naci. In some cases, the mobile phase (e.g., mobile phase B) may contain about 100mM to about 900mM TAnd (3) MAC. In some cases, the mobile phase (e.g., mobile phase B) may contain about 250mM to about 750mM TMAC. In some cases, the mobile phase (e.g., mobile phase B) may contain about 400mM to about 600mM TMAC. In some cases, the mobile phase (e.g., mobile phase B) may contain about 500mM TMAC.
In various embodiments of the method, the one or more mobile phases contain a buffer and/or a purified aqueous solution of salt having a pH ranging from about 7.5 to about 9.5. The pH of the one or more mobile phases may be 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5. In some cases, the pH is about 8.0 to about 9.0. In some cases, the pH is about 8.3 to about 8.7. In some cases, the pH is about 8.5. The purified water may be Milli-Q water. Milli-Q water is purified water produced by the Milli-Q water purification System (Sigma-Aldrich) familiar to those skilled in the art. Milli-Q water has a resistivity of about 18.2mohms.cm at 25 ℃ and has less than about 5ppb total organic carbon.
The system regeneration process discussed herein includes a wash solution comprising a purified aqueous solution of about 18% v/v ethanol. In various embodiments, the ethanol concentration may be about 10% v/v to about 26% v/v. In some cases, the ethanol concentration may be about 15% v/v to about 21% v/v. In some embodiments, the ethanol concentration of a wash solution dissolved in purified water (e.g., milli-Q water) is about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, or about 26% v/v.
In one embodiment, the system regeneration process discussed herein comprises (i) washing the AEX column with at least 10 Column Volumes (CVs) of mobile phase a; (ii) washing the AEX column with at least 10CV of purified water; (iii) Washing the AEX column with at least 20CV of a washing solution comprising a purified aqueous solution of 15% to 21% v/v ethanol; and (iv) washing the carrier liquid component of the LC instrument with the washing solution at a flow rate of at least 0.5mL/min for at least 30 minutes. In another embodiment, the system regeneration process discussed herein comprises (i) washing the AEX column with at least 10 Column Volumes (CVs) of mobile phase a; (ii) washing the AEX column with at least 10CV of purified water; (iii) Optionally reversing the orientation of the AEX column to reverse flow within the AEX column; (iv) Washing the AEX column with at least 20CV of a washing solution comprising a purified aqueous solution of 15% to 21% v/v ethanol; (v) removing the AEX column from the LC instrument; and (vi) washing the carrier liquid component of the LC instrument with the washing solution at a flow rate of at least 0.5mL/min for at least 30 minutes. In some cases, mention of at least 10CV corresponds to 10CV to 20CV. In some cases, mention of at least 10CV corresponds to 10CV, 11CV, 12CV, 13CV, 14CV, 15CV, 16CV, 17CV, 18CV, 19CV, 20CV, 21CV, 22CV, 23CV, 24CV, 25CV, 30CV, 35CV, or 40CV. Similarly, in some embodiments, mention of at least 20CV may correspond to 20CV to 30CV. In some cases, mention of at least 20CV corresponds to 20CV, 21CV, 22CV, 23CV, 24CV, 25CV, 26CV, 27CV, 28CV, 29CV, 30CV, 31CV, 32CV, 33CV, 34CV, 35CV, 36CV, 37CV, 38CV, 39CV, or 40CV. Similarly, in some embodiments, reference to a flow rate of at least 0.5mL/min associated with a system regeneration process for at least 30 minutes may correspond to a flow rate of 0.5mL/min to 1mL/min or 0.5mL/min to 0.8 mL/min. In some embodiments, reference to a flow rate of at least 0.5mL/min for at least 30 minutes means a flow rate of 0.5mL/min, 0.6mL/min, 0.7mL/min, 0.8mL/min, 0.9mL/min, or 1mL/min for 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes. Similarly, reference to duration of at least 10 minutes means, for example, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, or 29 minutes.
In various embodiments, the system regeneration process is performed such that residues of capsid components from a previous sample (e.g., a reference standard sample or a previous test sample) into a blank sample are reduced or eliminated. In some cases, the blank sample contains less than 0.3% of the area count of the reference standard full capsid peak or the full capsid peak of the previous test sample. In some cases, the blank sample contains less than 0.25% of the area count of the reference standard full capsid peak or the full capsid peak of the previous test sample. In some cases, the blank sample contains less than 0.2% of the area count of the reference standard full capsid peak or the full capsid peak of the previous test sample. In some cases, the blank sample contains less than 0.15% of the area count of the reference standard full capsid peak or the full capsid peak of the previous test sample.
The system equilibration process discussed herein is typically designed to remove wash material (e.g., ethanol-containing wash solution) from the carrier liquid component of the LC instrument and AEX column, and prepare the column for sample separation. In some cases, the system balancing process includes: (i) Purging the wash solution from the carrier liquid component of the LC instrument with purified water at a flow rate of at least 0.5mL/min for at least 30 minutes; (ii) Reinstalling the AEX column into the LC instrument (if previously removed); (iii) washing the AEX column with at least 10CV of purified water; (iv) washing the AEX column with at least 10CV of mobile phase a; (v) washing the AEX column with at least 20CV of mobile phase B; and (vi) washing the AEX column with at least 20CV of mobile phase a. In some cases, mention of at least 10CV corresponds to 10CV to 20CV. In some cases, mention of at least 10CV corresponds to 10CV, 11CV, 12CV, 13CV, 14CV, 15CV, 16CV, 17CV, 18CV, 19CV, 20CV, 21CV, 22CV, 23CV, 24CV, 25CV, 30CV, 35CV, or 40CV. Similarly, in some embodiments, mention of at least 20CV may correspond to 20CV to 30CV. In some cases, mention of at least 20CV corresponds to 20CV, 21CV, 22CV, 23CV, 24CV, 25CV, 26CV, 27CV, 28CV, 29CV, 30CV, 31CV, 32CV, 33CV, 34CV, 35CV, 36CV, 37CV, 38CV, 39CV, or 40CV. Similarly, in some embodiments, reference to a flow rate of at least 0.5mL/min associated with a system balancing process for at least 30 minutes may correspond to a flow rate of 0.5mL/min to 1mL/min or 0.5mL/min to 0.8 mL/min. In some embodiments, reference to a flow rate of at least 0.5mL/min for at least 30 minutes means a flow rate of 0.5mL/min, 0.6mL/min, 0.7mL/min, 0.8mL/min, 0.9mL/min, or 1mL/min for 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes. Similarly, reference to duration of at least 10 minutes means, for example, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, or 29 minutes.
In some embodiments, the separation process includes one or more mobile phases that operate as a gradient. For example, if two, three or more mobile phases are used, a gradient of the mobile phases may be used. In the gradient, the concentration or percentage of the first mobile phase may decrease and the concentration or percentage of the second mobile phase increases during the chromatographic run. In some cases, during chromatographic operation, the concentration of the first and second mobile phases may be reduced while the concentration or percentage of the third mobile phase is increased (e.g., temporarily). For example, the percentage of the first mobile phase may be reduced from about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 50%, about 45%, or about 40% to about 0%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% during a portion or the entire course of the chromatographic run. As another example, the percentage of the second mobile phase may be increased from about 0%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% to about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 50%, about 45%, or about 40% during a portion or throughout the same run. As another example, the percentage of the first mobile phase and the second mobile phase may be reduced from about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 50%, about 45%, or about 40% to about 0%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% during a portion or throughout the same operation, and the percentage of the third mobile phase may be increased from about 0%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% to about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 50%, about 45%, or about 40% during a portion or throughout the same operation. In certain aspects, during chromatographic runs, the proportion of mobile phase a decreases and then increases over time. In certain aspects, during chromatographic runs, the proportion of mobile phase B increases, then decreases, and then increases again over time. In certain aspects, during chromatographic operation, the proportion of mobile phase C increases and then decreases over time. Optionally, the concentrations or percentages of the first mobile phase and the second mobile phase may be returned to their starting values at the end of the chromatographic run. The percentages may be varied gradually in a linear gradient or in a non-linear (e.g., stepwise) manner. For example, the gradient may be multi-phase, e.g., biphasic, triphasic, etc.
In some cases, the separation gradient comprises, in order: (i) 85% to 95% mobile phase a and 5% to 15% mobile phase B for a period of time (e.g., 1-5 minutes); (ii) Reducing mobile phase a from 85% -95% to 5070% and increasing mobile phase B from 5% -15% to 30% -50% over a period of time (e.g., 10 to 30 minutes); (iii) 100% mobile phase C for a period of time (e.g., 1 to 15 minutes); and (iv) 85% to 95% mobile phase a and 5% to 15% mobile phase B for a period of time (e.g., 5 to 15 minutes). In one embodiment, the separation gradient comprises, in order: (i) 90% mobile phase a and 10% mobile phase B for 1 minute; (ii) Mobile phase a was reduced from 90% to 58% and mobile phase B was increased from 10% to 42% over a period of 20 minutes; (iii) 100% mobile phase C for 5 minutes; and (iv) 90% mobile phase a and 10% mobile phase B for 10 minutes.
In some exemplary embodiments, the one or more mobile phases may have a flow rate through the liquid chromatography column of about 0.05mL/min to about 1mL/min or about 0.05mL/min to about 0.08 mL/min. In some cases, the flow rate is about 0.05mL/min, about 0.06mL/min, about 0.07mL/min, about 0.08mL/min, about 0.09mL/min, about 0.1mL/min, about 0.15mL/min, about 0.2mL/min, about 0.25mL/min, about 0.3mL/min, about 0.35mL/min, about 0.4mL/min, about 0.45mL/min, about 0.5mL/min, about 0.55mL/min, about 0.6mL/min, about 0.65mL/min, about 0.7mL/min, about 0.75mL/min, about 0.8mL/min, about 0.85mL/min, about 0.9mL/min, about 0.95mL/min, or about 1mL/min. In some cases, the flow rate is about 0.8mL/min. In some embodiments, the run time of the one or more mobile phases (whether run in a gradient or not) is between about 1 minute and 60 minutes. In other embodiments, the one or more mobile phases are run for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, or about 45 minutes.
In various embodiments, the sample introduced into or into the liquid chromatography system is limited to about 20 μl per sample. In some cases, the sample volume is about 5 μl to about 20 μl. In some cases, the sample volume is about 5 μl, about 6 μl, about 7 μl, about 8 μl, about 9 μl, about 10 μl, about 11 μl, about 12 μl, about 13 μl, about 14 μl, about 15 μl, about 16 μl, about 17 μl, about 18 μl, about 19 μl, or about 20 μl.
The sample may be diluted in a sample dilution buffer, depending on the viral titer in any particular test sample. In some cases, the sample dilution buffer comprises a purified aqueous solution of 10mM sodium phosphate and 0.005% w/v poloxamer 188, pH 7.3. For example, where the viral titer is greater than or equal to 5E+12vg/mL, the test sample may be diluted 1.5 to 3 times with the sample dilution buffer and then injected or introduced into the liquid chromatography system. In some cases, the test sample is diluted 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, or 3-fold with the sample dilution buffer. In various embodiments, the test sample (whether diluted based on viral titer or not) may be contained in a sample solution comprising about 0.001% to about 0.01% w/v surfactant. In some cases, the sample solution comprises 0.004% to 0.006% w/v surfactant. In some cases, the sample solution comprises about 0.005% w/v surfactant. In various embodiments, the surfactant may be a poloxamer, such as poloxamer 188.
In various embodiments, the test sample (e.g., sample solution) that is injected or introduced into the liquid chromatography system comprises about 2mM to about 100mM sodium chloride. In some cases, the test sample comprises about 60mM to about 100mM. In various embodiments, the test sample comprises about 1.8mM, about 1.9mM, about 2mM, about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, or about 100mM sodium chloride.
In various embodiments of the methods discussed herein, a liquid chromatography system may be used with fluorescence detection. In some embodiments, the excitation and emission wavelengths are set to 280nm 20nm and 350nm 20nm, respectively. In some embodiments, the response time of the detector is set to 0.5 seconds. In some embodiments, the response time is set to generate at least 20 data points on the chromatographic peak. In some embodiments, the response time is set between about 0.1 seconds and 1.0 seconds.
Virus particles
In certain aspects, the viral particles are AAV particles, and the disclosed methods can be used to determine the relative abundance of viral capsid components in a sample of AAV particles. The AAV particles may be recombinant AAV (rAAV) particles. The rAAV particles comprise AAV vectors encoding heterologous transgenes or heterologous nucleic acid molecules.
In certain aspects, the AAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAV11 capsid, an AAV12 capsid, or variants thereof. In certain aspects, the AAV particle has serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ/8, AAV-Rh10, AAV-retro, AAV-PHP.B, AAV8-PHP.eB, or AAV-PHP.S. In some embodiments, the AAV particle has serotype AAV1, AAV5, or AAV8. In some embodiments, the AAV particle has serotype AAV8.
In some aspects, the viral particles (e.g., AAV particles) contain a heterologous nucleic acid molecule (e.g., a therapeutic gene or gene of interest). In some aspects, the heterologous nucleic acid molecule is operably linked to a promoter. Exemplary promoters include, but are not limited to, the Cytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, the simian virus 40 (SV 40) promoter, and the CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline-responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; a cytomegalovirus enhancer/chicken beta-actin/rabbit beta-globin promoter and an elongation factor 1-alpha promoter (EF 1-alpha) promoter. In some aspects, the promoter comprises a human β -glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken β -actin (CBA) promoter. The promoter may be a constitutive, inducible or repressible promoter. In some aspects, the invention provides recombinant vectors comprising a nucleic acid encoding a heterologous transgene of the present disclosure operably linked to a CBA promoter. In some cases, a native promoter of the transgene, or a fragment thereof, will be used. Where expression of the transgene is desired to mimic natural expression, a natural promoter may be used. A natural promoter may be used when expression of the transgene must be regulated in time or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In another aspect, other natural expression control elements (such as enhancer elements, polyadenylation sites, or Kozak consensus sequences) may also be used to mimic natural expression.
In various aspects, the viral particles may be obtained by any known production system, such as mammalian cell AAV production systems (e.g., those based on 293T or HEK293 cells) and insect cell AAV production systems (e.g., those based on sf9 insect cells and/or those using baculovirus helper vectors). The viral particles may be purified from the cell culture by using well known techniques such as discontinuous cesium chloride density gradients or other processes such as column-based downstream processes.
Examples
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees celsius and pressure is at or near atmospheric pressure.
Example 1: isolation and identification of relative abundance of empty and full AAV capsids
Samples containing empty AAV8 capsids and full AAV8 capsids (containing the gene of interest) were separated by Anion Exchange (AEX) chromatography and the relative abundance of capsid components was determined. AEX Separations were performed using BIA Separations, CIMac AAV empty/full 0.1mL analytical column (1.3 μm pore size, 5.2mm diameter. Times.4.95 mm long) (catalog number 110.8503-1.3) on an ACQUITY UPLC H-Class system (Waters Corporation) equipped with a fluorescence detector. Mobile Phase A (MPA) contains 20mM 1, 3-bis ((trimethylol) methylamino) propane and 2mM MgCl 2 Milli-Q water, pH 8.5, mobile Phase B (MPB) contains 20mM 1, 3-bis ((trimethylol) methylamino) propane, 500mM tetramethylammonium chloride (TMAC) and 2mM MgCl 2 In Milli-Q water, pH 8.5, mobile Phase C (MPC) contained 2M NaCl/Milli-Q water. The AEX flow rate was 0.8mL/min and the gradient was as shown in Table 1 below.
Table 1: AEX separation gradient
| Time (minutes) | Salt concentration (mM) |
| 0 | 50TMAC |
| 1 | 50TMAC |
| 21 | 210TMAC |
| 22 | 2000NaCl |
| 26 | 2000NaCl |
| 26.1 | 50TMAC |
| 36 | 50TMAC |
Each sample was diluted to 80mM NaCl with a sample dilution buffer containing 10mM sodium phosphate and 0.005% w/v poloxamer 188/Milli-Q water, pH7.3, and 10 microliter volumes were injected into the UPLC system. Data were recorded using fluorescence detectors with excitation (Ex) and emission (Em) wavelengths of 280nm and 350nm, respectively.
The isolation protocol demonstrates linearity and accuracy over a range of viral genome loads as shown in table 2 below.
Table 2: method linearity and precision
Each sample was the average of three replicates
* Coefficient of variation
The different parameters (individual changes are shown in Table 3 below) were evaluated using this method to confirm the use of a mobile phase containing TMAC at pH 8.5, an initial TMAC concentration of 50mM, an initial retention time of 1 minute, a gradient slope of 8mM TMAC/minute, 2mM MgCl 2 The concentration achieves the optimal separation.
Table 3: method parameters of evaluation
| Method parameters | Parameter values tested |
| Mobile phase pH | 8、8.5、9、10 |
| Initial salt concentration | 0、20、50mM |
| Hold time at initial conditions | 1. 3, 5 minutes |
| Gradient slope | 8 and 10mM/min |
| MgCl 2 Concentration of | 0、1、2、5mM |
| Mobile phase salts | NaCl、TMAC |
Sample volumes greater than 20 microliters (e.g., 25 μl and 30 μl) resulted in peak shape degradation, resulting in poor separation of empty and complete capsid components (data not shown).
Example 2: assessment of sodium chloride concentration in sample solutions
The performance of the method was evaluated over a series of test samples using the separation protocol detailed in example 1, but varying the concentration of sodium chloride in the test sample solution. AAV8-GOI samples at 1.05E+14vg/mL were diluted to test sample solutions containing 180, 140, 100, 90, 80, 70, 60, 30, 10 and 1.8mM sodium chloride. Samples with sodium chloride concentrations ranging from 1.8mM to 100mM did not affect the performance of the method (similar area/vg of these samples), but samples containing sodium chloride concentrations greater than 100mM (140 mM and 180 mM) had a negative impact on the separation of the sample capsids (improved separation of empty capsids from complete capsids was observed with 100mM NaCl relative to 140mM and 180 mM). The results are shown in table 4 below.
Table 4: influence of NaCl concentration in sample solution
The absence of sodium chloride can negatively impact separation (data not shown).
Example 3: evaluation of surfactant concentration in sample solutions
To assess whether surfactant was required in the sample solution (i.e., the test sample composition injected into the LC system), 1.05e+14vg/mL of AAV8-GOI was 50-fold diluted with 10mM phosphate, 180mM sodium chloride (ph 7.3), with (0.005% w/v) and without (0.0001% w/v) poloxamer 188. After dilution, two samples containing 2.1E+12vg/mL were incubated for 1 day at 37℃and diluted to contain 80mM sodium chloride for AEX chromatography.
Reducing poloxamer 188 concentration from 0.005% w/v to 0.0001% w/v resulted in significant loss (> 20% loss) of AAV material recovered by AEX chromatography (data not shown), confirming the need for surfactant in the sample solution.
Example 4: evaluation of% residue entering blank sample by System regeneration procedure
The separation protocol detailed in example 1 was used to evaluate the effect of system regeneration. The reference standard was diluted 2.3-fold in sample dilution buffer and loaded into the UPLC system, followed by blank sampling. The blank area count was 0.35% of the area count of the reference standard full capsid peak. This procedure was repeated in a second experiment with a blank area count of 1.33% of the area count of the reference standard full capsid peak.
After significant false peaks are identified in the blank sample injection, a system regeneration process is performed, including: (i) Washing the AEX column with at least 10 Column Volumes (CVs) of mobile phase a; (ii) washing the AEX column with at least 10CV of purified water; (iii) Reversing the orientation of the AEX column to reverse flow within the AEX column; (iv) Washing the AEX column with at least 20CV of a washing solution comprising a purified aqueous solution of 15% to 21% v/v ethanol; (v) removing the AEX column from the LC instrument; and (vi) washing the carrier liquid component of the LC instrument with the washing solution at a flow rate of 0.5mL/min for 10 minutes. After regeneration, the wash solution was purged from the LC instrument and AEX column and the column was equilibrated with Milli-Q water, mobile phase a and mobile phase B. As described above, the reference standard was diluted 2.3-fold in sample dilution buffer and loaded into the UPLC system, followed by blank injection. After this second reference standard sample injection, the blank area count was 0.11% of the area count of the reference standard full capsid peak. This procedure was repeated in a second experiment with a blank area count of 0.08% of the area count of the reference standard full capsid peak. As demonstrated by this data, the system regeneration process significantly reduced the residue entering the blank sample, and the reproducibility of the results showed low variability and the ability to control the residue using the system regeneration process.
The scope of the invention is not limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
Claims (30)
1. A method for determining the 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 (CVs) of mobile phase a; (ii) washing the AEX column with at least 10CV of purified water; (iii) Washing the AEX column with at least 20CV of a washing solution comprising a purified aqueous solution of 15% to 21% v/v ethanol; and (iv) washing the carrier liquid component of the LC instrument with the washing solution at a flow rate of at least 0.5mL/min for at least 30 minutes,
wherein mobile phase a comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane and 1mM to 3mM magnesium chloride, having a pH of 8 to 9, and wherein the purified water has a resistivity of about 18.2mohm.cm at 25 ℃ and has less than 5ppb total organic carbon;
(b) Performing a system equilibration process on the liquid chromatography system comprising purging the wash solution from the LC instrument and the carrier liquid component of 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 comprise intact empty AAV capsids and intact complete AAV capsids dissolved 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 complete AAV capsids,
wherein mobile phase B comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane, 250mM to 1M tetramethylammonium chloride (TMAC) and 1mM to 3mM magnesium chloride, pH 8 to 9, and mobile phase C comprises a purified aqueous solution of 1.5M to 2.5M sodium chloride; and
(d) Identifying the amount of the complete empty AAV capsid and the amount of the complete AAV capsid in each of the one or more test samples to determine the relative abundance of the complete viral capsid component in the sample of the AAV particles.
2. A method for determining the 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 (CVs) of mobile phase a; (ii) washing the AEX column with at least 10CV of purified water; (iii) Optionally reversing the orientation of the AEX column to reverse flow within the AEX column; (iv) Washing the AEX column with at least 20CV of a washing solution comprising a purified aqueous solution of 15% to 21% v/v ethanol; (v) removing the AEX column from the LC instrument; and (vi) washing the carrier liquid component of the LC instrument with the washing solution at a flow rate of at least 0.5mL/min for at least 30 minutes,
Wherein mobile phase a comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane and 1mM to 3mM magnesium chloride, having a pH of 8 to 9, and wherein the purified water has a resistivity of about 18.2mohm.cm at 25 ℃ and has less than 5ppb total organic carbon;
(b) Performing a system balancing process on the liquid chromatography system, comprising: (i) reinstalling the AEX column into the LC instrument; and (ii) purging the wash solution from the LC instrument and the carrier liquid assembly of 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 comprise intact empty AAV capsids and intact complete AAV capsids dissolved 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 complete AAV capsids,
Wherein mobile phase B comprises a purified aqueous solution of 15mM to 25mM 1, 3-bis ((trimethylol) methylamino) propane, 250mM to 1M tetramethylammonium chloride (TMAC) and 1mM to 3mM magnesium chloride, pH 8 to 9, and mobile phase C comprises a purified aqueous solution of 1.5M to 2.5M sodium chloride; and
(d) Identifying the amount of the complete empty AAV capsid and the amount of the complete AAV capsid in each of the one or more test samples to determine the relative abundance of the complete viral capsid component in the sample of the AAV particles.
3. The method of claim 1 or 2, wherein the wash solution comprises a purified aqueous solution of 18% ± 1% v/v ethanol.
4. A method according to any one of claims 1 to 3, wherein the sample solution comprises 1mM to 100mM sodium chloride.
5. The method of claim 4, wherein the sample solution comprises 60mM to 100mM sodium chloride.
6. The method of any one of claims 1 to 5, wherein the sample solution comprises 0.003% w/v to 0.007% w/v surfactant.
7. The method of claim 6, wherein the sample solution comprises 0.005% w/v ± 0.001% w/v surfactant.
8. The method of claim 6 or 7, wherein the surfactant is poloxamer 188.
9. The method of any of claims 1 and 3-8, wherein the system balancing process comprises: (i) Purging the wash solution from the LC instrument and the carrier liquid component of the AEX column with purified water at a flow rate of at least 0.5mL/min for at least 10 minutes; (ii) washing the AEX column with at least 10CV of purified water; (iii) washing the AEX column with at least 10CV of mobile phase a; (iv) washing the AEX column with at least 20CV of mobile phase B; and (v) washing the AEX column with at least 20CV of mobile phase a.
10. The method of any of claims 2 to 8, wherein the system balancing process comprises: (i) Purging the wash solution from the carrier liquid component of the LC instrument with purified water at a flow rate of at least 0.5mL/min for at least 10 minutes; (ii) reinstalling the AEX column into the LC instrument; (iii) washing the AEX column with at least 10CV of purified water; (iv) washing the AEX column with at least 10CV of mobile phase a; (v) washing the AEX column with at least 20CV of mobile phase B; and (vi) washing the AEX column with at least 20CV of mobile phase a.
11. The method of any one of claims 2 to 11, wherein performing the system equilibration process further comprises washing the carrier liquid component of the LC instrument with purified water at a flow rate of at least 0.5mL/min for at least 10 minutes, then reinstalling the AEX column into the LC instrument.
12. The method of claim 11, wherein the washing solution is purged from the carrier liquid component of the LC instrument with purified water at a flow rate of at least 0.5mL/min for at least 30 minutes.
13. The method of any one of claims 1 to 12, wherein the at least 10CV comprises 10 to 20CV.
14. The method of any one of claims 1 to 13, wherein the at least 20CV comprises 20 to 30CV.
15. The method of any one of claims 1 to 14, wherein the sample separation process comprises: (i) Introducing three blank samples into the liquid chromatography system, followed by four reference standard samples, followed by two blank samples, followed by 1 to 10 test samples, followed by reference standard samples, followed by blank samples; and (ii) running the separation gradient of mobile phase a, mobile phase B, and mobile phase C through the liquid chromatography system separately for each sample.
16. The method of any one of claims 1 to 15, wherein the separation gradient is run at a flow rate of 0.8mL/min for 36 minutes.
17. The method of any one of claims 1 to 16, wherein the separation gradient comprises, in order: (i) 90% mobile phase a and 10% mobile phase B for 1 minute; (ii) Mobile phase a was reduced from 90% to 58% and mobile phase B was increased from 10% to 42% over a period of 20 minutes; (iii) 100% mobile phase C for 4 minutes; and (iv) 90% mobile phase a and 10% mobile phase B for at least 10 minutes.
18. The method of any one of claims 1 to 17, further comprising if the one or more test samples comprise ≡5×10 12 Each viral genome/mL (vg/mL), the test sample is diluted 1.5 to 3-fold in dilution buffer.
19. The method of claim 18, wherein each of the one or more test samples introduced into the liquid chromatography system comprises about 10 microliters.
20. The method of any one of claims 1 to 17, wherein each of the one or more test samples introduced into the liquid chromatography system comprises about 20 microliters.
21. The process of any one of claims 1 to 20, wherein the mobile phase a comprises a purified aqueous solution of 20mM ± 2mM 1, 3-bis ((trimethylol) methylamino) propane and 2mM ± 0.2mM magnesium chloride, at pH 8.5 ± 0.1.
22. The method of any one of claims 1 to 21, wherein the mobile phase B comprises a purified aqueous solution of 20mM ± 2mM 1, 3-bis ((trimethylol) methylamino) propane, 500mM ± 50mM TMAC and 2mM ± 0.2mM magnesium chloride, at pH 8.5 ± 0.1.
23. The method of any one of claims 1 to 22, wherein the mobile phase C comprises a purified aqueous solution of 2M ± 0.2M sodium chloride.
24. The method of any one of claims 1-23, wherein the AAV particle has serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ/8, AAV-Rh10, AAV-retro, AAV-php.b, AAV8-php.eb, or AAV-php.s.
25. The method of claim 24, wherein the AAV particle has serotype AAV1, AAV5, or AAV8.
26. The method of claim 25, wherein the AAV particle has serotype AAV8.
27. The method of any one of claims 1 to 26, wherein the residue from a previous sample run into a blank sample run in the liquid chromatography system is no more than 0.3% of the previous sample full capsid peak.
28. The method of claim 27, wherein the previous sample is a reference standard sample.
29. The method of claim 27, wherein the previous sample is a test sample.
30. The method of any one of claims 27 to 29, wherein the residue is no more than 0.15% of the previous sample full capsid peak.
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| US63/359,554 | 2022-07-08 | ||
| US63/359,557 | 2022-07-08 | ||
| PCT/US2022/036728 WO2023287725A1 (en) | 2021-07-12 | 2022-07-11 | Liquid chromatography assay for determining aav capsid ratio |
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